Secondary literature sources for YaeQ

The following references were automatically generated.

Ouellette M, Jackson L, Chimileski S, Papke RT
Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.
Front Microbiol. 2015; 6: 251-251
Display abstract
Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: C(m4)TAG and GCA(m6)BN6VTGC. Analysis of the DeltaHVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

Kodavali PK, Dudkiewicz M, Pikula S, Pawlowski K
Bioinformatics analysis of bacterial annexins--putative ancestral relatives of eukaryotic annexins.
PLoS One. 2014; 9: 85428-85428
Display abstract
Annexins are Ca(2+)-binding, membrane-interacting proteins, widespread among eukaryotes, consisting usually of four structurally similar repeated domains. It is accepted that vertebrate annexins derive from a double genome duplication event. It has been postulated that a single domain annexin, if found, might represent a molecule related to the hypothetical ancestral annexin. The recent discovery of a single-domain annexin in a bacterium, Cytophaga hutchinsonii, apparently confirmed this hypothesis. Here, we present a more complex picture. Using remote sequence similarity detection tools, a survey of bacterial genomes was performed in search of annexin-like proteins. In total, we identified about thirty annexin homologues, including single-domain and multi-domain annexins, in seventeen bacterial species. The thorough search yielded, besides the known annexin homologue from C. hutchinsonii, homologues from the Bacteroidetes/Chlorobi phylum, from Gemmatimonadetes, from beta- and delta-Proteobacteria, and from Actinobacteria. The sequences of bacterial annexins exhibited remote but statistically significant similarity to sequence profiles built of the eukaryotic ones. Some bacterial annexins are equipped with additional, different domains, for example those characteristic for toxins. The variation in bacterial annexin sequences, much wider than that observed in eukaryotes, and different domain architectures suggest that annexins found in bacteria may actually descend from an ancestral bacterial annexin, from which eukaryotic annexins also originate. The hypothesis of an ancient origin of bacterial annexins has to be reconciled with the fact that remarkably few bacterial strains possess annexin genes compared to the thousands of known bacterial genomes and with the patchy, anomalous phylogenetic distribution of bacterial annexins. Thus, a massive annexin gene loss in several bacterial lineages or very divergent evolution would appear a likely explanation. Alternative evolutionary scenarios, involving horizontal gene transfer between bacteria and protozoan eukaryotes, in either direction, appear much less likely. Altogether, current evidence does not allow unequivocal judgement as to the origin of bacterial annexins.

Kazrani AA, Kowalska M, Czapinska H, Bochtler M
Crystal structure of the 5hmC specific endonuclease PvuRts1I.
Nucleic Acids Res. 2014; 42: 5929-36
Display abstract
PvuRts1I is a prototype for a larger family of restriction endonucleases that cleave DNA containing 5-hydroxymethylcytosine (5hmC) or 5-glucosylhydroxymethylcytosine (5ghmC), but not 5-methylcytosine (5mC) or cytosine. Here, we report a crystal structure of the enzyme at 2.35 A resolution. Although the protein has been crystallized in the absence of DNA, the structure is very informative. It shows that PvuRts1I consists of an N-terminal, atypical PD-(D/E)XK catalytic domain and a C-terminal SRA domain that might accommodate a flipped 5hmC or 5ghmC base. Changes to predicted catalytic residues of the PD-(D/E)XK domain or to the putative pocket for a flipped base abolish catalytic activity. Surprisingly, fluorescence changes indicative of base flipping are not observed when PvuRts1I is added to DNA substrates containing pyrrolocytosine in place of 5hmC (5ghmC). Despite this caveat, the structure suggests a model for PvuRts1I activity and presents opportunities for protein engineering to alter the enzyme properties for biotechnological applications.

Horton JR et al.
Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.
Nucleic Acids Res. 2014; 42: 7947-59
Display abstract
AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by approximately 70 A, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines approximately 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

Dias-Lopes C et al.
Identification of new sphingomyelinases D in pathogenic fungi and other pathogenic organisms.
PLoS One. 2013; 8: 79240-79240
Display abstract
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

Bauman JD et al.
Crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors.
ACS Chem Biol. 2013; 8: 2501-8
Display abstract
Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase. A high-resolution crystal form of pandemic 2009 H1N1 influenza polymerase acidic protein N-terminal endonuclease domain (PAN) was engineered and used for fragment screening leading to the identification of new chemical scaffolds binding to the PAN active site cleft. During the course of screening, binding of a third metal ion that is potentially relevant to endonuclease activity was detected in the active site cleft of PAN in the presence of a fragment. Using structure-based optimization, we developed a highly potent hydroxypyridinone series of compounds from a fragment hit that defines a new mode of chelation to the active site metal ions. A compound from the series demonstrating promising enzymatic inhibition in a fluorescence-based enzyme assay with an IC50 value of 11 nM was found to have an antiviral activity (EC50) of 11 muM against PR8 H1N1 influenza A in MDCK cells.

Cimini D, De Rosa M, Carlino E, Ruggiero A, Schiraldi C
Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide.
Microb Cell Fact. 2013; 12: 46-46
Display abstract
BACKGROUND: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. RESULTS: E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g L(-1) of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. CONCLUSIONS: The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.

Wang Y, Deng D, Zhang R, Wang S, Bian Y, Yin Z
Systematic analysis of plant-specific B3 domain-containing proteins based on the genome resources of 11 sequenced species.
Mol Biol Rep. 2012; 39: 6267-82
Display abstract
B3 domain-containing proteins constitute a large transcription factor superfamily. The plant-specific B3 superfamily consists of four family members, i.e., LAV (LEC2 [LEAFY COTYLEDON 2]/ABI3 [ABSCISIC ACID INSENSITIVE 3] - VAL [VP1/ABI3-LIKE]), RAV (RELATED to ABI3/VP1), ARF (AUXIN RESPONSE FACTOR) and REM (REPRODUCTIVE MERISTEM) families. The B3 superfamily plays a central role in plant life, from embryogenesis to seed maturation and dormancy. In previous research, we have characterized ARF family, member of the B3 superfamily in silico (Wang et al., Mol Biol Rep, 2011, doi:10.1007/s11033-011-0991-z). In this study, we systematically analyzed the diversity, phylogeny and evolution of B3 domain-containing proteins based on genomic resources of 11 sequenced species. A total of 865 B3 domain-containing genes were identified from 11 sequenced species through an iterative strategy. The number of B3 domain-containing genes varies not only between species but between gene families. B3 domain-containing genes are unevenly distributed in chromosomes and tend to cluster in the genome. Numerous combinations of B3 domains and their partner domains contribute to the sequences and structural diversification of the B3 superfamiy. Phylogenetic results showed that moss VAL proteins are related to LEC2/ABI3 instead of VAL proteins from higher plants. Lineage-specific expansion of ARF and REM proteins was observed. The REM family is the most diversified member among the B3 superfamily and experiences a rapid divergence during selective sweep. Based on structural and phylogenetic analysis results, two possible evolutional modes of the B3 superfamily were presented. Results presented here provide a resource for further characterization of the B3 superfamily.

Zou SB et al.
Loss of elongation factor P disrupts bacterial outer membrane integrity.
J Bacteriol. 2012; 194: 413-25
Display abstract
Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.

Stern A, Sorek R
The phage-host arms race: shaping the evolution of microbes.
Bioessays. 2011; 33: 43-51
Display abstract
Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. Here, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. The commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits.

Atkinson GC, Tenson T, Hauryliuk V
The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life.
PLoS One. 2011; 6: 23479-23479
Display abstract
RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the "stringent response" to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.

Reguera J, Weber F, Cusack S
Bunyaviridae RNA polymerases (L-protein) have an N-terminal, influenza-like endonuclease domain, essential for viral cap-dependent transcription.
PLoS Pathog. 2010; 6: 1001101-1001101
Display abstract
Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 A resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4-6 segments), and orthomyxoviruses (6-8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation.

Wu K, Rao CV
The role of configuration and coupling in autoregulatory gene circuits.
Mol Microbiol. 2010; 75: 513-27
Display abstract
Autoregulatory gene circuits can be physically encoded within the genome in a number of different configurations. By physical encoding, we mean the orientation and relative proximity of the genes within the circuit. In this work, we quantified the behaviour of an inducible, negatively autoregulated gene circuit arranged in different transcriptional configurations using the tetRA circuit from Tn10 as our basis. Mathematical modelling predicted that circuits arranged in configurations where the expression of the transcription factor is decoupled from its target genes afforded more flexibility relative to configurations where expression is coupled. We found that these decoupled configurations reduced the concentration of transcription factor needed to regulate inducible expression from the circuit. As lower concentrations of transcription factor were required, these decoupled configurations could also be activated at much lower concentrations of the inducer. We experimentally validated these predictions in Escherichia coli by comparing the response of synthetic circuits based on the tetRA circuit arranged in different configurations. Collectively, these results provide one example of how the arrangement of a gene circuit within the genome can affect its behaviour.

Bertrand L, Leiva-Torres GA, Hyjazie H, Pearson A
Conserved residues in the UL24 protein of herpes simplex virus 1 are important for dispersal of the nucleolar protein nucleolin.
J Virol. 2010; 84: 109-18
Display abstract
The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39 degrees C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.

Dehal PS et al.
MicrobesOnline: an integrated portal for comparative and functional genomics.
Nucleic Acids Res. 2010; 38: 396400-396400
Display abstract
Since 2003, MicrobesOnline (http://www.microbesonline.org) has been providing a community resource for comparative and functional genome analysis. The portal includes over 1000 complete genomes of bacteria, archaea and fungi and thousands of expression microarrays from diverse organisms ranging from model organisms such as Escherichia coli and Saccharomyces cerevisiae to environmental microbes such as Desulfovibrio vulgaris and Shewanella oneidensis. To assist in annotating genes and in reconstructing their evolutionary history, MicrobesOnline includes a comparative genome browser based on phylogenetic trees for every gene family as well as a species tree. To identify co-regulated genes, MicrobesOnline can search for genes based on their expression profile, and provides tools for identifying regulatory motifs and seeing if they are conserved. MicrobesOnline also includes fast phylogenetic profile searches, comparative views of metabolic pathways, operon predictions, a workbench for sequence analysis and integration with RegTransBase and other microbial genome resources. The next update of MicrobesOnline will contain significant new functionality, including comparative analysis of metagenomic sequence data. Programmatic access to the database, along with source code and documentation, is available at http://microbesonline.org/programmers.html.

Margelevicius M, Laganeckas M, Venclovas C
COMA server for protein distant homology search.
Bioinformatics. 2010; 26: 1905-6
Display abstract
SUMMARY: Detection of distant homology is a widely used computational approach for studying protein evolution, structure and function. Here, we report a homology search web server based on sequence profile-profile comparison. The user may perform searches in one of several regularly updated profile databases using either a single sequence or a multiple sequence alignment as an input. The same profile databases can also be downloaded for local use. The capabilities of the server are illustrated with the identification of new members of the highly diverse PD-(D/E)XK nuclease superfamily. AVAILABILITY: http://www.ibt.lt/bioinformatics/coma/

Thrash JC, Cho JC, Vergin KL, Morris RM, Giovannoni SJ
Genome sequence of Lentisphaera araneosa HTCC2155T, the type species of the order Lentisphaerales in the phylum Lentisphaerae.
J Bacteriol. 2010; 192: 2938-9
Display abstract
Information on the genome content of deeply branching phyla with very few cultured members is invaluable for expanding understanding of microbial evolution. Lentisphaera araneosa HTCC2155(T) was isolated from the Oregon coast using dilution-to-extinction culturing. It is a marine heterotroph found in surface and mesopelagic waters in both the Pacific and Atlantic oceans and has the unusual property of producing a net-like matrix of secreted exopolysaccharide. Here we present the genome sequence of L. araneosa HTCC2155(T), importantly, one of only two sequenced members of the phylum Lentisphaerae.

Merighi M et al.
Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium.
BMC Microbiol. 2009; 9: 42-42
Display abstract
BACKGROUND: The Salmonella PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of Escherichia coli. In both Salmonella and E. coli, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the Salmonella enterica serovar Typhimurium (S. Typhimurium) pmrAB operon, which encodes an important virulence-associated TCS. RESULTS: To determine the PreA/PreB regulon in S. Typhimurium, we performed DNA microarrays comparing the wild type strain and various preA and/or preB mutants in the presence of ectopically expressed preA (qseB). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (yibD, pmrAB, cptA, etc.) or were genes located in the local region around preA, including the preAB operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of preA-preB, mdaB-ygiN, and ygiW-STM3175. Several putative virulence-related phenotypes were examined for preAB mutants, resulting in the observation of a host cell invasion and slight virulence defect of a preAB mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on S. Typhimurium with regard to bacterial motility. CONCLUSION: This work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in E. coli.

Nascimento R, Dias JD, Parkhouse RM
The conserved UL24 family of human alpha, beta and gamma herpesviruses induces cell cycle arrest and inactivation of the cyclinB/cdc2 complex.
Arch Virol. 2009; 154: 1143-9
Display abstract
The conserved murine gammaherpesvirus 68 ORF20 has recently been demonstrated to induce G2 cell cycle arrest followed by apoptosis in human and mouse cells. Here, we demonstrate that its homologues UL24 in HSV-1, ORF20 in KSHV and UL76 in HCMV are also inducers of cell cycle arrest followed by apoptosis in both human and mouse cells. The mechanism of action is similar to that reported for MHV-68 ORF20, inactivating the mitotic complex cyclinB/Cdc2.

Zhao C et al.
Nucleoside monophosphate complex structures of the endonuclease domain from the influenza virus polymerase PA subunit reveal the substrate binding site inside the catalytic center.
J Virol. 2009; 83: 9024-30
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Highly pathogenic influenza virus strains currently in circulation pose a significant risk of a global pandemic. Following the reported crystal structure of the endonuclease domain from the avian influenza virus polymerase PA subunit, here we report the results of a systematic X-ray crystallographic analysis of its complex with adenosine, uridine, and thymidine nucleoside monophosphates (NMPs). Electron density corresponding to the monophosphate moiety of each nucleotide was apparent in each NMP complex and bound to the catalytic metal. A hydrophobic site was found to contribute to nucleoside binding. The NMP complex structures should represent the conformation of the bound product after nuclease cleavage. Moreover, one solvent molecule was found to occupy an equivalent position to the second reported Mn(2+) ion, where it mediates the interaction between bound NMPs and the N-terminal PA domain in the presence of the Mg(2+) ion. The results presented here indicate a possible cleavage mechanism and identify a distinct nucleotide binding pocket. The identification of this binding pocket opens a new avenue for anti-influenza drug discovery, targeting the cap-dependent endonuclease, in response to the worldwide threat of influenza.

Lewis C et al.
Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection.
Microbiology. 2009; 155: 873-81
Display abstract
HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on sigma(E) (RpoE). S. Typhimurium htrA also possesses a sigma(E)-dependent promoter; however, we found that the absence of sigma(E) had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was sigma(E) dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor sigma(70). These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 degrees C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

Kim MJ, Lim S, Ryu S
Molecular analysis of the Salmonella typhimurium tdc operon regulation.
J Microbiol Biotechnol. 2008; 18: 1024-32
Display abstract
Efficient expression of the Salmonella Typhimurium tdcABCDEG operon involved in the degradation of Lserine and L-threonine requires TdcA, the transcriptional activator of the tdc operon. We found that the tdcA gene was transiently activated when bacterial growth condition was changed from aerobic to anaerobic, but this was not observed if Salmonella was grown anaerobically from the beginning of the culture. Expression kinetics of six tdc genes after anaerobic shock demonstrated by a real-time PCR assay showed that the tdcCDEG genes were not induced in tdcA mutant but tdcB maintained its inducibility by anaerobic shock even in the absence of tdcA, suggesting that an additional unknown transcriptional regulation may work for the tdcB expression. We also investigated the effects of nucleoid-associated proteins by primer extension analysis and found that H-NS repressed tdcA under anaerobic shock conditions and fis mutation delayed the peak expression time of the tdc operon. DNA microarray analysis of genes regulated by TdcA revealed that the genes involved in Nacetylmannosamine, maltose, and propanediol utilization were significantly induced in a tdcA mutant. These findings suggest that Tdc enzymes may play a pivotal role in energy metabolism under a sudden change of oxygen tension.

Neely RK, Roberts RJ
The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.
BMC Mol Biol. 2008; 9: 48-48
Display abstract
BACKGROUND: Restriction and modification enzymes typically recognise short DNA sequences of between two and eight bases in length. Understanding the mechanism of this recognition represents a significant challenge that we begin to address for the BsaHI restriction-modification system, which recognises the six base sequence GRCGYC. RESULTS: The DNA sequences of the genes for the BsaHI methyltransferase, bsaHIM, and restriction endonuclease, bsaHIR, have been determined (GenBank accession #EU386360), cloned and expressed in E. coli. Both the restriction endonuclease and methyltransferase enzymes share significant similarity with a group of 6 other enzymes comprising the restriction-modification systems HgiDI and HgiGI and the putative HindVP, NlaCORFDP, NpuORFC228P and SplZORFNP restriction-modification systems. A sequence alignment of these homologues shows that their amino acid sequences are largely conserved and highlights several motifs of interest. We target one such conserved motif, reading SPERRFD, at the C-terminal end of the bsaHIR gene. A mutational analysis of these amino acids indicates that the motif is crucial for enzymatic activity. Sequence alignment of the methyltransferase gene reveals a short motif within the target recognition domain that is conserved among enzymes recognising the same sequences. Thus, this motif may be used as a diagnostic tool to define the recognition sequences of the cytosine C5 methyltransferases. CONCLUSION: We have cloned and sequenced the BsaHI restriction and modification enzymes. We have identified a region of the R. BsaHI enzyme that is crucial for its activity. Analysis of the amino acid sequence of the BsaHI methyltransferase enzyme led us to propose two new motifs that can be used in the diagnosis of the recognition sequence of the cytosine C5-methyltransferases.

Zaros C et al.
Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.
Nucleic Acids Res. 2007; 35: 634-47
Display abstract
Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5-Rpb1 binding and the Rpb9-Rpb1 interaction is to hold the Bridge helix, the Rpb1-alpha44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.

Shi S, Zhong Y, Majumdar I, Sri Krishna S, Grishin NV
Searching for three-dimensional secondary structural patterns in proteins with ProSMoS.
Bioinformatics. 2007; 23: 1331-8
Display abstract
MOTIVATION: Many evolutionarily distant, but functionally meaningful links between proteins come to light through comparison of spatial structures. Most programs that assess structural similarity compare two proteins to each other and find regions in common between them. Structural classification experts look for a particular structural motif instead. Programs base similarity scores on superposition or closeness of either Cartesian coordinates or inter-residue contacts. Experts pay more attention to the general orientation of the main chain and mutual spatial arrangement of secondary structural elements. There is a need for a computational tool to find proteins with the same secondary structures, topological connections and spatial architecture, regardless of subtle differences in 3D coordinates. RESULTS: We developed ProSMoS--a Protein Structure Motif Search program that emulates an expert. Starting from a spatial structure, the program uses previously delineated secondary structural elements. A meta-matrix of interactions between the elements (parallel or antiparallel) minding handedness of connections (left or right) and other features (e.g. element lengths and hydrogen bonds) is constructed prior to or during the searches. All structures are reduced to such meta-matrices that contain just enough information to define a protein fold, but this definition remains very general and deviations in 3D coordinates are tolerated. User supplies a meta-matrix for a structural motif of interest, and ProSMoS finds all proteins in the protein data bank (PDB) that match the meta-matrix. ProSMoS performance is compared to other programs and is illustrated on a beta-Grasp motif. A brief analysis of all beta-Grasp-containing proteins is presented. Program availability: ProSMoS is freely available for non-commercial use from ftp://iole.swmed.edu/pub/ProSMoS.

Miller DJ, Shuvalova L, Evdokimova E, Savchenko A, Yakunin AF, Anderson WF
Structural and biochemical characterization of a novel Mn2+-dependent phosphodiesterase encoded by the yfcE gene.
Protein Sci. 2007; 16: 1338-48
Display abstract
Escherichia coli YfcE belongs to a conserved protein family within the calcineurin-like phosphoesterase superfamily (Pfam00149) that is widely distributed in bacteria and archaea. Superfamily members are metallophosphatases that include monoesterases and diesterases involved in a variety of cellular functions. YfcE exhibited catalytic activity against bis-p-nitrophenyl phosphate, a general substrate for phosphodiesterases, and had an absolute requirement for Mn2+. However, no activity was observed with phosphodiesters and over 50 naturally occurring phosphomonoesters. The crystal structure of the YfcE phosphodiesterase has been determined to 2.25 A resolution. YfcE has a beta-sandwich architecture similar to metallophosphatases of common ancestral origin. Unlike its more complex homologs that have added structural elements for regulation and substrate recognition, the relatively small 184-amino-acid protein has retained its ancestral simplicity. The tetrameric protein carries two zinc ions per active site from the E. coli extract that reflect the conserved di-Mn2+ active site geometry. A cocrystallized sulfate inhibitor mimics the binding of phosphate moeities in known ligand/phosphatase complexes. Thus, YfcE has a similar active site and biochemical mechanism as well-characterized superfamily members, while the YfcE phosphodiester-containing substrate is unique.

Song J, Markley JL
Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold.
J Mol Biol. 2007; 366: 155-64
Display abstract
Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.

Skowronek KJ, Kosinski J, Bujnicki JM
Theoretical model of restriction endonuclease HpaI in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis.
Proteins. 2006; 63: 1059-68
Display abstract
Type II restriction enzymes are commercially important deoxyribonucleases and very attractive targets for protein engineering of new specificities. At the same time they are a very challenging test bed for protein structure prediction methods. Typically, enzymes that recognize different sequences show little or no amino acid sequence similarity to each other and to other proteins. Based on crystallographic analyses that revealed the same PD-(D/E)XK fold for more than a dozen case studies, they were nevertheless considered to be related until the combination of bioinformatics and mutational analyses has demonstrated that some of these proteins belong to other, unrelated folds PLD, HNH, and GIY-YIG. As a part of a large-scale project aiming at identification of a three-dimensional fold for all type II REases with known sequences (currently approximately 1000 proteins), we carried out preliminary structure prediction and selected candidates for experimental validation. Here, we present the analysis of HpaI REase, an ORFan with no detectable homologs, for which we detected a structural template by protein fold recognition, constructed a model using the FRankenstein monster approach and identified a number of residues important for the DNA binding and catalysis. These predictions were confirmed by site-directed mutagenesis and in vitro analysis of the mutant proteins. The experimentally validated model of HpaI will serve as a low-resolution structural platform for evolutionary considerations in the subgroup of blunt-cutting REases with different specificities. The research protocol developed in the course of this work represents a streamlined version of the previously used techniques and can be used in a high-throughput fashion to build and validate models for other enzymes, especially ORFans that exhibit no sequence similarity to any other protein in the database.

Durr H, Flaus A, Owen-Hughes T, Hopfner KP
Snf2 family ATPases and DExx box helicases: differences and unifying concepts from high-resolution crystal structures.
Nucleic Acids Res. 2006; 34: 4160-7
Display abstract
Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA-protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases.

Desai BV, Morrison DA
An unstable competence-induced protein, CoiA, promotes processing of donor DNA after uptake during genetic transformation in Streptococcus pneumoniae.
J Bacteriol. 2006; 188: 5177-86
Display abstract
Natural genetic transformation in Streptococcus pneumoniae entails transcriptional activation of at least two sets of genes. One set of genes, activated by the competence-specific response regulator ComE, is involved in initiating competence, whereas a second set is activated by the competence-specific alternative sigma factor ComX and functions in DNA uptake and recombination. Here we report an initial characterization of CoiA, a ComX-dependent gene product that is induced during competence and is required for transformation. CoiA is widely conserved among gram-positive bacteria, and in streptococci, the entire coiA locus composed of four genes is conserved. By use of immunoblot assay, we show that, similar to its message, CoiA protein is transient, appearing at 10 min and largely disappearing by 30 min post-competence induction. Using complementation analysis, we establish that coiA is the only gene of this induced locus needed for transformability. We find no indication of CoiA having a role in regulating competence. Finally, using 32P- and 3H-labeled donor DNA, we demonstrate that a coiA mutant can internalize normal amounts of donor DNA compared to the wild-type strain but is unable to process it into viable transformants, suggesting a role for CoiA after DNA uptake, either in DNA processing or recombination.

Nagy G, Dobrindt U, Grozdanov L, Hacker J, Emody L
Transcriptional regulation through RfaH contributes to intestinal colonization by Escherichia coli.
FEMS Microbiol Lett. 2005; 244: 173-80
Display abstract
The Escherichia coli regulatory protein RfaH contributes to efficient colonization of the mouse gut. Extraintestinal pathogenic (ExPEC) as well as non-pathogenic probiotic E. coli strains rapidly outcompeted their isogenic rfaH mutants following oral mixed infections. LPS-core and O-antigen side-chain as well as capsular polysaccharide synthesis are among the E. coli virulence factors affected by RfaH. In respect of colonization, deep-rough LPS mutants (waaG) but not capsular (kps) mutants were shown to behave similarly to rfaH mutants. Furthermore, alteration in the length of O-antigen side-chains did not modify colonization ability either indicating that it was the regulatory effect of RfaH on LPS-core synthesis, which affected intestinal colonization. Loss of RfaH did not significantly influence adhesion of bacteria to cultured colon epithelial cells. Increased susceptibility of rfaH mutants to bile salts, on the other hand, suggested that impaired in vivo survival could be responsible for the reduced colonization capacity.

McGregor N et al.
The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage.
Structure. 2005; 13: 1341-51
Display abstract
We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.

Feder M, Bujnicki JM
Identification of a new family of putative PD-(D/E)XK nucleases with unusual phylogenomic distribution and a new type of the active site.
BMC Genomics. 2005; 6: 21-21
Display abstract
BACKGROUND: Prediction of structure and function for uncharacterized protein families by identification of evolutionary links to characterized families and known structures is one of the cornerstones of genomics. Theoretical assignment of three-dimensional folds and prediction of protein function even at a very general level can facilitate the experimental determination of the molecular mechanism of action and the role that members of a given protein family fulfill in the cell. Here, we predict the three-dimensional fold and study the phylogenomic distribution of members of a large family of uncharacterized proteins classified in the Clusters of Orthologous Groups database as COG4636. RESULTS: Using protein fold-recognition we found that members of COG4636 are remotely related to Holliday junction resolvases and other nucleases from the PD-(D/E)XK superfamily. Structure modeling and sequence analyses suggest that most members of COG4636 exhibit a new, unusual variant of the putative active site, in which the catalytic Lys residue migrated in the sequence, but retained similar spatial position with respect to other functionally important residues. Sequence analyses revealed that members of COG4636 and their homologs are found mainly in Cyanobacteria, but also in other bacterial phyla. They undergo horizontal transfer and extensive proliferation in the colonized genomes; for instance in Gloeobacter violaceus PCC 7421 they comprise over 2% of all protein-encoding genes. Thus, members of COG4636 appear to be a new type of selfish genetic elements, which may fulfill an important role in the genome dynamics of Cyanobacteria and other species they invaded. Our analyses provide a platform for experimental determination of the molecular and cellular function of members of this large protein family. CONCLUSION: After submission of this manuscript, a crystal structure of one of the COG4636 members was released in the Protein Data Bank (code 1wdj; Idaka, M., Wada, T., Murayama, K., Terada, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S.: Crystal structure of Tt1808 from Thermus thermophilus Hb8, to be published). Our analysis of the Tt1808 structure reveals that we correctly predicted all functionally important features of the COG4636 family, including the membership in the PD-(D/E)xK superfamily of nucleases, the three-dimensional fold, the putative catalytic residues, and the unusual configuration of the active site.

Baxter MA, Jones BD
The fimYZ genes regulate Salmonella enterica Serovar Typhimurium invasion in addition to type 1 fimbrial expression and bacterial motility.
Infect Immun. 2005; 73: 1377-85
Display abstract
An important step in Salmonella enterica serovar Typhimurium virulence is the ability to invade the intestinal epithelium. The invasion process requires a large number of genes encoded on Salmonella pathogenicity island 1 (SPI-1) at centisome 63 as well as genes located in other positions throughout the chromosome. Expression of the invasive phenotype is tightly regulated by environmental cues that are processed by a complex regulatory scheme. A central player in the invasion regulatory pathway is the HilA protein, which is transcriptional activator belonging to the OmpR/ToxR family. A number of positive regulators (hilC, hilD, fis, sirA/barA, csrAB, phoBR, fadD, envZ/ompR, and fliZ) and negative regulators (hha, hilE, lon, ams, phoPc and pag) have been identified that are able to alter expression of hilA transcription. Recent work has found that hilA transcription requires the HilD protein for activation. Other work has emphasized the importance of HilE as a negative regulator of hilA. Overexpression of hilE superrepresses hilA transcription, as well as the invasive phenotype. Two-hybrid experiments suggest that HilE exerts its regulatory influence on hilA through protein-protein interactions with HilD as the protein does not bind to the hilA promoter nor does it affect hilD transcription. As it seems likely that hilE plays an important role in translating environmental signals into invasion gene regulation, we have attempted to identify how the hilE gene itself is regulated. Our results indicate that the fimYZ genes, response regulatory proteins involved in type 1 fimbrial gene expression and recently implicated in motility gene regulation, are important activators of hilE expression. These findings indicate that invasion gene expression is coregulated with motility and adherence and provide experimental evidence that the expression of these virulence phenotypes is a subset of the overall regulation of bacterial physiology.

Leipe DD, Koonin EV, Aravind L
STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer.
J Mol Biol. 2004; 343: 1-28
Display abstract
Using sequence profile analysis and sequence-based structure predictions, we define a previously unrecognized, widespread class of P-loop NTPases. The signal transduction ATPases with numerous domains (STAND) class includes the AP-ATPases (animal apoptosis regulators CED4/Apaf-1, plant disease resistance proteins, and bacterial AfsR-like transcription regulators) and NACHT NTPases (e.g. NAIP, TLP1, Het-E-1) that have been studied extensively in the context of apoptosis, pathogen response in animals and plants, and transcriptional regulation in bacteria. We show that, in addition to these well-characterized protein families, the STAND class includes several other groups of (predicted) NTPase domains from diverse signaling and transcription regulatory proteins from bacteria and eukaryotes, and three Archaea-specific families. We identified the STAND domain in several biologically well-characterized proteins that have not been suspected to have NTPase activity, including soluble adenylyl cyclases, nephrocystin 3 (implicated in polycystic kidney disease), and Rolling pebble (a regulator of muscle development); these findings are expected to facilitate elucidation of the functions of these proteins. The STAND class belongs to the additional strand, catalytic E division of P-loop NTPases together with the AAA+ ATPases, RecA/helicase-related ATPases, ABC-ATPases, and VirD4/PilT-like ATPases. The STAND proteins are distinguished from other P-loop NTPases by the presence of unique sequence motifs associated with the N-terminal helix and the core strand-4, as well as a C-terminal helical bundle that is fused to the NTPase domain. This helical module contains a signature GxP motif in the loop between the two distal helices. With the exception of the archaeal families, almost all STAND NTPases are multidomain proteins containing three or more domains. In addition to the NTPase domain, these proteins typically contain DNA-binding or protein-binding domains, superstructure-forming repeats, such as WD40 and TPR, and enzymatic domains involved in signal transduction, including adenylate cyclases and kinases. By analogy to the AAA+ ATPases, it can be predicted that STAND NTPases use the C-terminal helical bundle as a "lever" to transmit the conformational changes brought about by NTP hydrolysis to effector domains. STAND NTPases represent a novel paradigm in signal transduction, whereby adaptor, regulatory switch, scaffolding, and, in some cases, signal-generating moieties are combined into a single polypeptide. The STAND class consists of 14 distinct families, and the evolutionary history of most of these families is riddled with dramatic instances of lineage-specific expansion and apparent horizontal gene transfer. The STAND NTPases are most abundant in developmentally and organizationally complex prokaryotes and eukaryotes. Transfer of genes for STAND NTPases from bacteria to eukaryotes on several occasions might have played a significant role in the evolution of eukaryotic signaling systems.

van der Straaten T, Janssen R, Mevius DJ, van Dissel JT
Salmonella gene rma (ramA) and multiple-drug-resistant Salmonella enterica serovar typhimurium.
Antimicrob Agents Chemother. 2004; 48: 2292-4
Display abstract
MarA and its homologue, RamA, have been implicated in multidrug resistance (MDR). RamA overexpression in Salmonella enterica serovar Typhimurium and Escherichia coli conferred MDR independently of marA. Inactivation of ramA did not affect the antibiotic susceptibilities of wild-type S. enterica serovar Typhimurium or 15 unrelated clinical MDR isolates. Thus, ramA overexpression is not a common MDR mechanism in Salmonella.

Xu QS, Kucera RB, Roberts RJ, Guo HC
An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site.
Structure. 2004; 12: 1741-7
Display abstract
Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other Type IIP enzyme reported to date, an MspI monomer and not a dimer binds to a palindromic DNA sequence. The enzyme makes specific contacts with all 4 base pairs in the recognition sequence, by six direct and five water-mediated hydrogen bonds and numerous van der Waal contacts. This MspI-DNA structure represents the first example of asymmetric recognition of a palindromic DNA sequence by two different structural motifs in one polypeptide. A few possible pathways are discussed for MspI to cut both strands of DNA, either as a monomer or dimer.

Bittner M, Saldias S, Altamirano F, Valvano MA, Contreras I
RpoS and RpoN are involved in the growth-dependent regulation of rfaH transcription and O antigen expression in Salmonella enterica serovar Typhi.
Microb Pathog. 2004; 36: 19-24
Display abstract
We reported earlier that the production of O antigen lipopolysaccharide (LPS) by Salmonella enterica serovar Typhi (Salmonella typhi) increases at the onset of stationary phase and correlates with a growth-regulated expression of the rfaH gene under the control of the alternative sigma factor RpoN (Microbiology 148 (2002) 3789). In this study, we demonstrate that RpoS also modulates rfaH promoter activity as revealed by the absence of growth-dependent regulation of an rfaH-lacZ transcriptional fusion and O antigen production in a S. typhi rpoS mutant. Introduction of a constitutively expressed rpoN gene into the rpoS mutant restored increased production of O antigen during stationary phase, suggesting that constitutive production of RpoN could overcome the RpoS defect. Similar results were observed when an rpoS rpoN double mutant was transformed with the intact rpoN gene. Thus, we conclude that both RpoS and RpoN control the rfaH promoter activity and concomitantly, the production of O-specific LPS in S. typhi.

Huai Q, Liu Y, Francis SH, Corbin JD, Ke H
Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity.
J Biol Chem. 2004; 279: 13095-101
Display abstract
Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.

Vicari D, Artsimovitch I
Virulence regulators RfaH and YaeQ do not operate in the same pathway.
Mol Genet Genomics. 2004; 272: 489-96
Display abstract
The expression of virulence factors such as hemolysin and lipopolysaccharides in Proteobacteria is regulated by the transcription elongation factor RfaH. RfaH reduces pausing and termination at intergenic sites, and thus allows RNA polymerase to conclude transcription of the distal genes in long virulence operons. The yaeQ gene of Salmonella enterica sv. Typhimurium has been identified as a high-copy-number suppressor of the hemolytic defect in an rfaH deletion strain, leading to speculation regarding a direct role of YaeQ in the transcriptional control of bacterial virulence. In order to evaluate this hypothesis, yaeQ genes from Escherichia coli and S. enterica sv. Typhimurium were cloned and expressed. Their products, purified YaeQ proteins, displayed no antitermination effects in in-vitro transcription assays over a wide range of concentrations, neither by themselves nor in competition with RfaH. When overexpressed in vivo, plasmid-borne E. coli and S. enterica sv. Typhimurium yaeQ genes also failed to restore hemolytic activity in an rfaH deletion strain under conditions in which episomal E. coli rfaH and its orthologs exhibited full complementation of the genomic rfaH deletion. Taken together, our findings do not support the hypothesis of YaeQ involvement in RfaH-dependent regulation of virulence, even in stoichiometric excess in vitro or upon overexpression in vivo.

Zambonelli C, Casali M, Roberts MF
Mutagenesis of putative catalytic and regulatory residues of Streptomyces chromofuscus phospholipase D differentially modifies phosphatase and phosphodiesterase activities.
J Biol Chem. 2003; 278: 52282-9
Display abstract
Phospholipase D from Streptomyces chromofuscus (sc-PLD) is a member of the diverse family of metallo-phosphodiesterase/phosphatase enzymes that also includes purple acid phosphatases, protein phosphatases, and nucleotide phosphodiesterases. Whereas iron is an essential cofactor for scPLD activity, Mn2+ is also found in the enzyme. A third metal ion, Ca2+, has been shown to enhance scPLD catalytic activity although it is not an essential cofactor. Sequence alignment of scPLD with known phosphodiesterases and phosphatases requiring metal ions suggested that His-212, Glu-213, and Asp-389 could be involved in Mn2+ binding. H212A, E213A, and D389A were prepared to test this hypothesis. These three mutant enzymes and wild type scPLD show similar metal content but considerably different catalytic properties, suggesting different roles for each residue. His-212 appears involved in binding the phosphate group of substrates, whereas Glu-213 acts as a ligand for Ca2+. D389A showed a greatly reduced phosphodiesterase activity but almost unaltered ability to hydrolyze the phosphate group in p-nitrophenyl phosphate suggesting it had a critical role in aligning groups at the active site to control phosphodiesterase versus phosphatase activities. We propose a model for substrate and cofactor binding to the catalytic site of scPLD based on these results and on sequence alignment to purple acid phosphatases of known structure.

Winfield MD, Groisman EA
Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli.
Appl Environ Microbiol. 2003; 69: 3687-94

Lawhon SD, Frye JG, Suyemoto M, Porwollik S, McClelland M, Altier C
Global regulation by CsrA in Salmonella typhimurium.
Mol Microbiol. 2003; 48: 1633-45
Display abstract
CsrA is a regulator of invasion genes in Salmonella enterica serovar Typhimurium. To investigate the wider role of CsrA in gene regulation, we compared the expression of Salmonella genes in a csrA mutant with those in the wild type using a DNA microarray. As expected, we found that expression of Salmonella pathogenicity island 1 (SPI-1) invasion genes was greatly reduced in the csrA mutant, as were genes outside the island that encode proteins translocated into eukaryotic cells by the SPI-1 type III secretion apparatus. The flagellar synthesis operons, flg and fli, were also poorly expressed, and the csrA mutant was aflagellate and non-motile. The genes of two metabolic pathways likely to be used by Salmonella in the intestinal milieu also showed reduced expression: the pdu operon for utilization of 1,2-propanediol and the eut operon for ethanolamine catabolism. Reduced expression of reporter fusions in these two operons confirmed the microarray data. Moreover, csrA was found to regulate co-ordinately the cob operon for synthesis of vitamin B12, required for the metabolism of either 1,2-propanediol or ethanolamine. Additionally, the csrA mutant poorly expressed the genes of the mal operon, required for transport and use of maltose and maltodextrins, and had reduced amounts of maltoporin, normally a dominant protein of the outer membrane. These results show that csrA controls a number of gene classes in addition to those required for invasion, some of them unique to Salmonella, and suggests a co-ordinated bacterial response to conditions that exist at the site of bacterial invasion, the intestinal tract of a host animal.

Pei J, Sadreyev R, Grishin NV
PCMA: fast and accurate multiple sequence alignment based on profile consistency.
Bioinformatics. 2003; 19: 427-8
Display abstract
PCMA (profile consistency multiple sequence alignment) is a progressive multiple sequence alignment program that combines two different alignment strategies. Highly similar sequences are aligned in a fast way as in ClustalW, forming pre-aligned groups. The T-Coffee strategy is applied to align the relatively divergent groups based on profile-profile comparison and consistency. The scoring function for local alignments of pre-aligned groups is based on a novel profile-profile comparison method that is a generalization of the PSI-BLAST approach to profile-sequence comparison. PCMA balances speed and accuracy in a flexible way and is suitable for aligning large numbers of sequences. AVAILABILITY: PCMA is freely available for non-commercial use. Pre-compiled versions for several platforms can be downloaded from ftp://iole.swmed.edu/pub/PCMA/.

Landraud L, Gibert M, Popoff MR, Boquet P, Gauthier M
Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein.
Mol Microbiol. 2003; 47: 1653-67
Display abstract
Examination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD-cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co-regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti-Shine-Dalgarno sequence present in the cnf1 coding sequence [fold-back inhibition (cnf1 fbi)]. The cnf1 fbi was not regulated by elements present in the igs. An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100-fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co-regulation.

Kiupakis AK, Reitzer L
ArgR-independent induction and ArgR-dependent superinduction of the astCADBE operon in Escherichia coli.
J Bacteriol. 2002; 184: 2940-50
Display abstract
For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by sigma(54) and nitrogen regulator I (NR(I), also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a sigma(54)-dependent promoter utilized in exponential phase during nitrogen limitation and a sigma(S)-dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NR(I), and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the sigma(54)-dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NR(I)-dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression.

Cano DA, Dominguez-Bernal G, Tierrez A, Garcia-Del Portillo F, Casadesus J
Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA.
Genetics. 2002; 162: 1513-23
Display abstract
Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.

Schnetz K
Silencing of the Escherichia coli bgl operon by RpoS requires Crl.
Microbiology. 2002; 148: 2573-8
Display abstract
Silencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS. Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter. However, the expression level of activated bgl operon derivatives varies with the strain background. Here it is shown that the repression of the bgl operon by RpoS requires Crl. Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes. In a Crl-negative strain RpoS had no effect on the bgl operon. The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E. coli strains. Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.

Sperandio V, Torres AG, Kaper JB
Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli.
Mol Microbiol. 2002; 43: 809-21
Display abstract
Quorum sensing is a cell-to-cell signalling mechanism in which bacteria secrete hormone-like compounds called autoinducers. When these auto-inducers reach a certain threshold concentration, they interact with bacterial transcriptional regulators, thereby regulating gene expression. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 as well as E. coli K-12 produces the autoinducer-2 (AI-2), which is synthesized by the product of the luxS gene, and previous work from our laboratory has shown that genes encoding the EHEC type III secretion system were activated by quorum sensing. Recently, by hybridizing an E. coli K-12 gene array with cDNA synthesized from RNA extracted from EHEC strain 86-24 and its isogenic luxS mutant, we observed that other potential virulence-associated factors, such as genes encoding the expression and assembly of flagella, motility and chemotaxis, were also activated by quorum sensing. The array data also indicated that several genes encoding putative E. coli regulators were controlled by quorum sensing. In this report, we describe a two-component system regulated by quorum sensing that shares homology with Salmonella typhimurium PmrAB, which we have named quorum sensing E. coli regulator B and C (QseBC). The qseBC genes, previously identified only as open reading frames b3025 and b3026, are organized in an operon in the E. coli chromosome, with qseB encoding the response regulator and qseC the sensor kinase. We confirmed the regulation of qseBC by quorum sensing using qseB::lacZ transcriptional fusions and characterized the phenotypes of an isogenic qseC mutation in EHEC. This mutant expressed less flagellin and had reduced motility compared with the wild-type and complemented strains. Transcription of flhD, fliA, motA and fliC::lacZ fusions was decreased in the qseC mutant, suggesting that qseBC is a transcriptional regulator of flagella genes. A qseC mutant was also generated in E. coli K-12 strain MC1000 that showed the same phenotypes as the EHEC mutant, indicating that qseBC regulates flagella and motility by quorum sensing in both EHEC and K-12. QseBC activates transcription of flhDC, which is the master regulator for the flagella and motility genes and, in the absence of flhD, QseBC failed to activate the transcription of fliA. Motility of a luxS, but not of a qseC, mutant can be restored by providing AI-2 exogenously as preconditioned media, suggesting that the qseC mutant is unable to respond to AI-2. However, QseC has no effect on the expression of other quorum sensing-controlled genes such as those encoding for the type III secretion system. These data indicate that QseBC is one component of the quorum-sensing regulatory cascade in both EHEC and K-12 that is involved in the regulation of flagella and motility genes, but that additional regulators in this cascade remain to be characterized.

Puchkova LI, Ushakova TA, Mikhailova VK, Serov GD, Krivopalova GN, Repin VE
[Testing and isolation of high-purity restriction endonucleases].
Prikl Biokhim Mikrobiol. 2002; 38: 20-4
Display abstract
A new method of testing restriction nucleases is proposed. This method is based on high-temperature treatment of crude cell extracts. Disrupted cells were heated at 50-60 degrees C, centrifuged, and assayed for restrictases. This method provides the opportunity for screening new enzymes in microbial strains enriched with nonspecific restrictases. High-temperature treatment of cell extracts of certain producers reduces the number of steps of the procedure used for isolating high-purity restrictases; the resulting preparations are capable of maintaining high enzymatic activity during long-term storage. It was shown that high-temperature treatment can be applied not only to thermophilic but also to mesophilic strains of microorganisms of different taxa.

Tamulaitis G, Solonin AS, Siksnys V
Alternative arrangements of catalytic residues at the active sites of restriction enzymes.
FEBS Lett. 2002; 518: 17-22
Display abstract
A catalytic sequence motif PDX10-30(E/D)XK is found in many restriction enzymes. On the basis of sequence similarities and mapping of the conserved residues to the crystal structure of NgoMIV we suggest that residues D160, K182, R186, R188 and E195 contribute to the catalytic/DNA binding site of the Ecl18kI restriction endonuclease. Mutational analysis confirms the functional significance of the conserved residues of Ecl18kI. Therefore, we conclude that the active site motif 159VDX21KX12E of Ecl18kI differs from the canonical PDX10-30(E/D)XK motif characteristic for most of the restriction enzymes. Moreover, we propose that two subfamilies of endonucleases Ecl18kI/PspGI/EcoRII and Cfr10I/Bse634I/NgoMIV, specific, respectively, for CCNGG/CCWGG and RCCGGY/GCCGGC sites, share conserved active site architecture and DNA binding elements.

Brown PK, Dozois CM, Nickerson CA, Zuppardo A, Terlonge J, Curtiss R 3rd
MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium.
Mol Microbiol. 2001; 41: 349-63
Display abstract
Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coli chi7122 conferred mannose-resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR-like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain chi7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA-lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild-type S. typhimurium SL1344 or SR-11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low-copy-number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature- and RpoS-independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS-dependent curli and extracellular matrix production by E. coli and S. typhimurium.

Bujnicki JM, Rychlewski L
Identification of a PD-(D/E)XK-like domain with a novel configuration of the endonuclease active site in the methyl-directed restriction enzyme Mrr and its homologs.
Gene. 2001; 267: 183-91
Display abstract
The Escherichia coli K-12 restriction enzyme Mrr recognizes and cleaves N6-methyladenine- and 5-methylcytosine-containing DNA. Its amino acid sequence has been subjected to structure prediction and comparison with other sequences from publicly available sources. The results obtained suggest that Mrr and related putative endonucleases possess a cleavage domain typical for all the so far structurally characterized type II restriction enzymes, however with an unusual glutamine residue at the central position of the (D/E)-(D/E)XK hallmark of the active site. The "missing" acidic side chain was instead found anchored in a different, unusual position, suggesting that Mrr represents a third topological variant of the endonuclease active site in addition to the two alternatives determined previously (Skirgaila et al., 1998. J. Mol. Biol. 279, 473-481). One of the newly identified putative endonucleases from the Mrr family is composed of two diverged cleavage domains, which possess both the "typical" D-EXK and the "Mrr-like" D-QXK variants of the active site. Among the Mrr homologs there are also proteins from yeast, in which restriction phenotype has not been observed, suggesting that the free-standing Eukaryotic PD-(D/E)XK superfamily members might be implicated in other cellular processes involving enzymatic DNA cleavage.

Interthal H, Pouliot JJ, Champoux JJ
The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily.
Proc Natl Acad Sci U S A. 2001; 98: 12009-14
Display abstract
The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.

Zou X, Caufield PW, Li Y, Qi F
Complete nucleotide sequence and characterization of pUA140, a cryptic plasmid from Streptococcus mutans.
Plasmid. 2001; 46: 77-85
Display abstract
Approximately 5% of strains of Streptococcus mutans contain plasmid DNA. Strain UA140 harbors a 5.6-kb cryptic plasmid, pUA140, with an overall G+C content of 32.7%. Five open reading frames (ORF), encoding peptides of larger than 100 amino acid residues, were initially designated as ORF1 to ORF5. These five ORFs were located on the same strand of pUA140. ORF1 (258 amino acids) resembled a replication protein, Rep. Upstream of the putative Rep gene, a double-stranded origin for plasmid replication that showed strong similarity to those of a number of plasmids in the pT181 family was identified. Further upstream was a region constituting the single-stranded origin of replication. A single-stranded DNA intermediate was detected during plasmid replication. Taken together, these results suggest that pUA140 replicated by the rolling circle replication mechanism but exhibited several characteristics that differ from those of other members of the pT181 plasmid family.

Gulati A, Mahadevan S
Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti-terminator BglG, CRP-cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiation.
Genes Cells. 2000; 5: 239-50
Display abstract
BACKGROUND: Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic beta-glucosides, is extremely sensitive to the presence of glucose in the growth medium. We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression. RESULTS: Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose. Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho-independent terminator present within the leader sequence. Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose-mediated repression is the anti-terminator protein, BglG. Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression. Studies using crp, cya and crr strains show that both CRP-cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation. Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator. Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose-grown cultures of a crp+ strain. Glucose repression is lost in the crr mutant strain. CONCLUSIONS: The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti-termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF. The CRP-cAMP complex is also involved in this regulation. The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes.

Nieto JM et al.
Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS.
Mol Gen Genet. 2000; 263: 349-58
Display abstract
The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Deibert M, Grazulis S, Sasnauskas G, Siksnys V, Huber R
Structure of the tetrameric restriction endonuclease NgoMIV in complex with cleaved DNA.
Nat Struct Biol. 2000; 7: 792-9
Display abstract
The crystal structure of the NgoMIV restriction endonuclease in complex with cleaved DNA has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a protein tetramer and two DNA molecules cleaved at their recognition sites. This is the first structure of a tetrameric restriction enzyme-DNA complex. In the tetramer, two primary dimers are arranged back to back with two oligonucleotides bound in clefts on opposite sides of the tetramer. The DNA molecules retain a B-type conformation and have an enclosed angle between their helical axes of 60 degrees. Sequence-specific interactions occur in both the major and minor grooves. Two Mg2+ ions are located close to the cleaved phosphate at the active site of NgoMIV. Biochemical experiments show that interactions between the recognition sites within the tetramer greatly increase DNA cleavage efficiency.

Bilcock DT, Daniels LE, Bath AJ, Halford SE
Reactions of type II restriction endonucleases with 8-base pair recognition sites.
J Biol Chem. 1999; 274: 36379-86
Display abstract
Type II restriction endonucleases usually recognize 4-6-base pair (bp) sites on DNA and cleave each site in a separate reaction. A few type II endonucleases have 8-bp recognition sites, but these seem unsuited for restriction, since their sites are rare on most DNA. Moreover, only one endonuclease that recognizes a target containing 8 bp has been examined to date, and this enzyme, SfiI, needs two copies of this site for its DNA cleavage reaction. In this study, several endonucleases with 8-bp sites were tested on plasmids that have either one or two copies of the relevant sequence to determine if they also need two sites. SgfI, SrfI, FseI, PacI, PmeI, Sse8781I, and SdaI all acted through equal and independent reactions at each site. AscI cleaved the DNA with one site at the same rate as that with two sites but acted processively on the latter. In contrast, SgrAI showed a marked preference for the plasmid with two sites and cleaved both sites on this DNA in a concerted manner, like SfiI. Endonucleases that require two copies of an 8-bp sequence may be widespread in nature, where, despite this seemingly inappropriate requirement, they may function in DNA restriction.

Yanagihara S, Iyoda S, Ohnishi K, Iino T, Kutsukake K
Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium.
Genes Genet Syst. 1999; 74: 105-11
Display abstract
The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium. Here we report the molecular structure and in vivo and in vitro expression of the flhD operon. Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site. Primer extension analysis demonstrated six possible transcription start sites for this operon. They include CRP-dependent and CRP-repressible transcription start sites. The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the sigma 70-dependent promoter. The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site. The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site. These two start sites were confirmed by in vitro transcription experiments using sigma 70-RNA polymerase with or without cAMP-CRP.

Burova E et al.
Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein.
Mol Microbiol. 1999; 31: 1783-93
Display abstract
The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the lambda nutR site in vivo. We show that it is also essential for Nun termination at lambda nutL. Three recessive mis-sense nusG mutations have been isolated that inhibit termination by Nun at lambda nutR. The mutations are ineffective in a lambda pL nutL fusion, even when lambda nutR replaces lambda nutL. The mutant strains support lambda growth, indicating that lambda N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both lambda nutR and lambda nutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild-type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho-dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.

Dahai T, Ando S, Takasaki Y, Tadano J
Site-directed mutagenesis of restriction endonuclease HindIII.
Biosci Biotechnol Biochem. 1999; 63: 1703-7
Display abstract
Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467-473 (1998)). Seven kinds of mutants were obtained by the PCR, and their enzymatic and biochemical properties were examined. Three mutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108-110, are responsible for the enzymic reaction of HindIII.

Li H, Trotta CR, Abelson J
Crystal structure and evolution of a transfer RNA splicing enzyme.
Science. 1998; 280: 279-84
Display abstract
The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

Zhang Y, Cronan JE Jr
Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster.
J Bacteriol. 1998; 180: 3295-303
Display abstract
The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome. A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y. Zhang and J. E. Cronan, Jr., J. Bacteriol. 178:3614-3620, 1996) is that several of these genes are essential for the growth of E. coli. We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E. coli, to provide functions necessary for growth. The S. typhimurium fab cluster was isolated by complementation of an E. coli fabD mutant and was found to encode proteins with > 94% homology to those of E. coli. However, the S. typhimurium sequences cannot recombine with the E. coli sequences required to direct polar allele duplication via homologous recombination. Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth. We have also found that the fabG gene is obligatorily cotranscribed with upstream genes. Insertion of a transcription terminator cassette (omega-Cm cassette) between the fabD and fabG genes of the E. coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene. Insertion of the omega-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s).

Webb E, Claas K, Downs D
thiBPQ encodes an ABC transporter required for transport of thiamine and thiamine pyrophosphate in Salmonella typhimurium.
J Biol Chem. 1998; 273: 8946-50
Display abstract
In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli.

Wah DA, Bitinaite J, Schildkraut I, Aggarwal AK
Structure of FokI has implications for DNA cleavage.
Proc Natl Acad Sci U S A. 1998; 95: 10564-9
Display abstract
FokI is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. FokI consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain. The bipartite nature of FokI has led to the development of artificial enzymes with novel specificities. We have solved the structure of FokI to 2.3 A resolution. The structure reveals a dimer, in which the dimerization interface is mediated by the cleavage domain. Each monomer has an overall conformation similar to that found in the FokI-DNA complex, with the cleavage domain packing alongside the DNA recognition domain. In corroboration with the cleavage data presented in the accompanying paper in this issue of Proceedings, we propose a model for FokI DNA cleavage that requires the dimerization of FokI on DNA to cleave both DNA strands.

Norris V, Alexandre J, Barray S, Mouchard L, Quillet L
A mechanical approach to the distribution and orientation of genes on genetic maps.
Mol Microbiol. 1998; 27: 236-7

Kholti A, Charlier D, Gigot D, Huysveld N, Roovers M, Glansdorff N
pyrH-encoded UMP-kinase directly participates in pyrimidine-specific modulation of promoter activity in Escherichia coli.
J Mol Biol. 1998; 280: 571-82
Display abstract
The carAB operon of the enterics Escherichia coli K-12 and Salmonella typhimurium LT2, encoding the sole carbamoylphosphate synthetase (CPSase) of these organisms, is transcribed from two promoters in tandem, carP1 upstream and carP2 downstream, repressed respectively by pyrimidines and arginine. We present evidence that the pyrH gene product (the hexameric UMP-kinase) directly participates in the pyrimidine-specific control of carP1 activity. Indeed, we have isolated in E. coli a particular type of pyrH mutation (pyrH41) that retains a quasi-normal UMP-kinase activity, but yet is impaired in the pyrimidine-specific repression of the P1 promoter of the carAB operon of E. coli and of S. typhimurium. Moreover, the pyrimidine-dependent inhibition of in vivo Dam methylase modification of adenine -106 upstream of the carP1 promoter is altered in this pyrH mutant. The recessive pyrH41 allele bears a single C-G to A-T transversion that converts alanine 94 into glutamic acid (A94E). Although overexpression of pyrH41 results in UMP-kinase levels far above that of a wild-type strain, pyrimidine-specific repression of the carAB operon is not restored under these conditions. Similarly, overexpression of the UMP-CMP-kinase gene of Dictyostelium discoideum in the pyrH41 mutant does not restore pyrimidine-mediated control of carP1 promoter activity, in spite of the elevated UMP-kinase activity measured in such transformants. These results indicate that besides its catalytic function in the de novo pyrimidine biosynthesis, E. coli UMP-kinase fulfils an additional, but previously unrecognized role in the regulation of the carAB operon. UMP-kinase might function as the real sensor of the internal pyrimidine nucleotide pool and act in concert with the integration host factor (IHF) and aminopeptidase A (PepA alias CarP and XerB) in the elaboration of the complex nucleoprotein structure required for pyrimidine-specific repression of carP1 promoter activity.

Xu H et al.
Plant homologue of human excision repair gene ERCC1 points to conservation of DNA repair mechanisms.
Plant J. 1998; 13: 823-9
Display abstract
Nucleotide excision repair (NER), a highly versatile DNA repair mechanism, is capable of removing various types of DNA damage including those induced by UV radiation and chemical mutagens. NER has been well characterized in yeast and mammalian systems but its presence in plants has not been reported. Here it is reported that a plant gene isolated from male germline cells of lily (Lilium longiflorum) shows a striking amino acid sequence similarity to the DNA excision repair proteins human ERCC1 and yeast RAD10. Homologous genes are also shown to be present in a number of taxonomically diverse plant genera tested, suggesting that this gene may have a conserved function in plants. The protein encoded by this gene is able to correct significantly the sensitivity to the cross-linking agent mitomycin C in ERCC1-deficient Chinese hamster ovary (CHO) cells. These findings suggest that the NER mechanism is conserved in yeast, animals and higher plants.

Frymier JS, Reed TD, Fletcher SA, Csonka LN
Characterization of transcriptional regulation of the kdp operon of Salmonella typhimurium.
J Bacteriol. 1997; 179: 3061-3
Display abstract
The transcriptional control of the kdpFABC (K+ transport) operon of Salmonella typhimurium was characterized with a lacZ fusion. The kdpFABC operon of this organism was induced by K+ limitation and high osmolality, and osmotic induction was antagonized by a high concentration of K+. In the trkA (sapG) kdp+ mutant background, high concentrations of K+ inhibited growth, along with repressing the kdp operon. This result, which has not been reported for Escherichia coli, is inconsistent with the model in which the signal for the induction of the kdp operon is turgor loss.

Korsa I, Bock A
Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis.
J Bacteriol. 1997; 179: 41-5
Display abstract
The FhlA protein belongs to the NtrC family of transcriptional regulators. It induces transcription from the -12/-24 promoters of the genes of the formate regulon by sigma54 RNA polymerase. FhlA is activated by binding of the ligand formate and does not require phosphorylation. A mutational analysis of the fhLA gene portion coding for the A and C domains was conducted with the aim of gaining information on the interaction between formate binding and ATP hydrolysis plus transcription activation. Four mutations were identified, all located in the A domain; one of them rendered transcription completely independent from the presence of formate, and the others conferred a semiconstitutive phenotype. The FhlA protein of one of the semiconstitutive variants was purified. Catalytic efficiency of ATP hydrolysis of the mutant FhlA was increased in the absence of formate in the same manner as formate influences the activity of wild-type FhlA. Moreover, in vitro transcription occurred at much lower threshold concentrations of the mutant protein and of nucleoside triphosphates than with the wild-type FhlA.

Parekh BS, Hatfield GW
Growth rate-related regulation of the ilvGMEDA operon of Escherichia coli K-12 is a consequence of the polar frameshift mutation in the ilvG gene of this strain.
J Bacteriol. 1997; 179: 2086-8
Display abstract
In Escherichia coli K-12 the intracellular levels of threonine deaminase and transaminase B, products of ilvA and ilvE, respectively, in the ilvGMEDA operon, increase with increasing growth rates (S. Pedersen, P. L. Bloch, S. Reeh, and F. C. Neidhardt, Cell 14:179-190, 1978). However, the transcriptional activities of the upstream ilvpG and the internal ilvpE promoters do not increase. Therefore, the growth rate-related expression of this operon is not regulated at the level of transcription initiation. Unlike other wild-type E. coli strains, E. coli K-12 contains a polar frameshift mutation in the ilvG gene (R. P. Lawther, D. H. Calhoun, C. W. Adams, C. A. Hauser, J. Gray, and G. W. Hatfield, Proc. Natl. Acad. Sci. USA 78:922-925, 1981). In an E. coli K-12 (IlvG+) derivative strain, where the reading frame of the ilvG gene is restored, no growth rate-related expression of the ilvGMEDA operon is observed. Thus, the growth rate-related expression of the ilvGMEDA operon in E. coli K-12 is the fortuitous consequence of the polar frameshift mutation in the ilvG gene of this strain.

Altschul SF et al.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 1997; 25: 3389-402
Display abstract
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

Prival MJ
Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli.
J Bacteriol. 1996; 178: 2989-90
Display abstract
Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.

Savic DJ
Adaptive mutations: an additional view.
Mol Microbiol. 1996; 21: 429-30

Nakagawa Y, Oda K, Nakada S
The PB1 subunit alone can catalyze cRNA synthesis, and the PA subunit in addition to the PB1 subunit is required for viral RNA synthesis in replication of the influenza virus genome.
J Virol. 1996; 70: 6390-4
Display abstract
We indicated that the PB1 and PA subunits of RNA polymerase and nucleoprotein (NP) can support replication of the influenza virus genome as well as transcription to yield uncapped poly(A)(+)-RNA (Y. Nakagawa, N. Kimura, T. Toyoda, K. Mizumoto, A. Ishihama, K. Oda, and S. Nakada, J. Virol. 69:728-733, 1995). To analyze the functions of the PB1 and PA subunits in replication and transcription, YP1N clones in which the PB1 and NP genes can be expressed in response to dexamethasone were established. cRNA was transcribed from model viral RNA (vRNA), but vRNA synthesis from model cRNA was not detected in YP1N clones. Furthermore, poly(A)(+)-RNA directed from model vRNA was synthesized in YP1N clones. These results indicated that PB1 and NP can support the syntheses of cRNA and poly(A)(+)-RNA and that the PA subunit, in addition to that of PB1 and to NP, is required for vRNA synthesis. In summary, the PB1 subunit is involved in the catalytic activities of nucleotide elongation, and the PA subunit may act as an allosteric modulator and cause a conformational change from a cRNA-to a vRNA-synthesizing form of the PB1 subunit.

Eberl L, Christiansen G, Molin S, Givskov M
Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon.
J Bacteriol. 1996; 178: 554-9
Display abstract
The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.

Merkel WK, Nichols BP
Characterization and sequence of the Escherichia coli panBCD gene cluster.
FEMS Microbiol Lett. 1996; 143: 247-52
Display abstract
A 4589 bp DNA segment containing the Escherichia coli panBCD gene cluster was sequenced, and found to contain 6 complete open reading frames. panB, panC, and panD were identified by subcloning and insertional mutagenesis. The orientation of panD was also confirmed by orientation-specific expression of asparate-1-decarboxylase. panB and panC lie adjacent to one another, but are separated from panD by orf3, which is oriented in the opposite direction. Interruptions in the remaining open reading frames did not affect growth on glucose-minimal medium. No significant similarity to sequences in databases was found for orf1 and orf2. Orf3 contained extensive similarity to reading frames defined by E. coli yjiP, yjiQ, yhgA, and yafD. The function of these amino acid sequences is as yet undefined.

Stein DC, Gunn JS, Radlinska M, Piekarowicz A
Restriction and modification systems of Neisseria gonorrhoeae.
Gene. 1995; 157: 19-22
Display abstract
An individual strain of Neisseria gonorrhoeae may produce up to 16 different DNA methytransferases (MTases). We have used a novel cloning system that is able to detect MTase clones in the absence of direct selection [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] to identify 14 different MTase clones. Initial characterization of these clones indicates that at least seven of these MTases are linked to restriction endonuclease (ENase) systems. Six of these systems have been characterized by DNA sequence analysis, and the open reading frames encoding each of these systems have been identified. The recognition sequences for the cloned systems have the following specificities: S.NgoI, RGCGCY; S.NgoII, GGCC; S.NgoIV, GCCGCC; S.NgoV, GGNNCC; S.NgoVII, GCSGC; S.NgoVIIIA, GGTGA; and S.NgoVIIIC, TCACC. Of those systems that have been cloned, NgoI-NgoVII are typical type II R-M systems, with each encoding a DNA MTase that methylates cytosine in position 5. NgoVIII is a type IIS system, containing an ENase and two different MTases. One of these is a cytosine MTase (NgoVIIIC) and the other is an adenine MTase (NgoVIIIA). Although most of our clones encodes both the ENase and the MTase, none of the six R-M systems are genetically linked on the chromosome.

Johanson U, Hughes D
Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium.
Gene. 1994; 143: 55-9
Display abstract
We have sequenced fusA, the gene coding for elongation factor G (EF-G), in 18 different mutants of Salmonella typhimurium selected as fusidic acid resistant (FuR). In addition, we have sequenced two previously described FuR mutants from Escherichia coli. In all cases, the resistance is due to a mutation in one of three separate regions in fusA. The three clusters of mutant sites superimpose on regions that are well conserved, suggesting that they are of a more general functional importance. To further classify the mutants, we have measured the minimal inhibitory concentration (MIC) for Fu and for two other antibiotics which interfere with translocation on the ribosome, kanamycin (Km) and spectinomycin (Sp). The levels of resistance to Fu for each of the mutants are significantly higher than in the wild type (wt), and vary by about one order of magnitude between the highest and the lowest. Most of the mutants are also more resistant to Km than the wt, although the level of resistance is low and the variation small. In contrast, about half of the mutants are more sensitive to Sp than the wt, with only one being more resistant. Only three of the twenty mutants behave like the wt with respect to the non-selected phenotypes, KmR and SpR.

Blanc-Potard AB, Bossi L
Phenotypic suppression of DNA gyrase deficiencies by a deletion lowering the gene dosage of a major tRNA in Salmonella typhimurium.
J Bacteriol. 1994; 176: 2216-26
Display abstract
One of the pleiotropic phenotypes of mutations affecting DNA gyrase activity in Salmonella typhimurium is the constitutive deattenuation of the histidine operon. In the present work, we isolated and characterized a suppressor mutation which restores his attenuation in the presence of a defective gyrase. Such a suppressor, initially named sgdA1 (for suppressor gyrase deficiency), was found to correct additional phenotypes associated with defective gyrase function. These include the aberrant nucleoid partitioning of a gyrB mutant and the conditional lethality of a gyrA mutation. Furthermore, the sgdA1 mutation was found to confer low-level resistance to nalidixic acid. The last phenotype permitted isolation of a number of additional sgdA mutants. Genetic analysis established the recessive character of these alleles as well as the position of the sgdA locus at 57 U on the Salmonella genetic map. All of the sgdA mutants result from the same molecular event: a deletion removing three of the four tandemly repeated copies of argV, the gene which specifies tRNA(2Arg), the major arginine isoacceptor tRNA. These findings, combined with the observation of some Sgd-like phenotypes in a tRNA modification mutant (hisT mutant), lead us to propose that protein synthesis contributes, directly or indirectly, to the pathology of gyrase alterations in growing bacteria. We discuss plausible mechanisms which may be responsible for these effects.

Old IG, Saint Girons I, Richaud C
Physical mapping of the scattered methionine genes on the Escherichia coli chromosome.
J Bacteriol. 1993; 175: 3689-91
Display abstract
Methionine is an important amino acid which acts not only as a substrate for protein elongation but also as the initiator of protein synthesis. The genes of the met regulon, which consists of 10 biosynthetic genes (metA, metB, metC, metE, metF, metH, metK, metL, metQ, and metX), two regulatory genes (metJ and metR), and the methionyl tRNA synthetase gene (metG), are scattered throughout the chromosome. The only linked genes are metK and metX at 63.6 min, metE and metR at 86.3 min, and the metJBLF gene cluster at 89 min. metBL form the only met operon.

Johanson U, Hughes D
Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli.
Gene. 1992; 120: 93-8
Display abstract
The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level. The order of conservation at the nt and aa level is rpsL greater than tufA greater than rpsG greater than f usA. A striking difference is that the rpsG-encoded ribosomal protein, S7, in E. coli K-12 is 23 aa longer than in S. typhimurium. The very low (0.18) codon adaptation index of this part of the E. coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension. A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S. typhimurium is noted. In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events. An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.

Brazas R, Davie E, Farewell A, Rothfield LI
Transcriptional organization of the rfaGBIJ locus of Salmonella typhimurium.
J Bacteriol. 1991; 173: 6168-73
Display abstract
The transcriptional organization of the rfaGBIJ gene cluster of Salmonella typhimurium was studied by using lacZ and cat transcriptional probes. The results indicated that the leftward end of the gene cluster (rfaG-rfaB-rfaI) is an operon that is transcribed from one or more promoters that lie upstream of rfaG. The results further indicated that the product of the rfaH (sfrB) gene acts as a positive regulator of transcription of the entire rfaGBIJ cluster. At least one site required for the RfaH-mediated transcriptional regulation lies within or very close to the upstream promoter.

Farewell A, Brazas R, Davie E, Mason J, Rothfield LI
Suppression of the abnormal phenotype of Salmonella typhimurium rfaH mutants by mutations in the gene for transcription termination factor Rho.
J Bacteriol. 1991; 173: 5188-93
Display abstract
Mutations in the rfaH gene have previously been shown to cause premature termination of transcription of the traYZ operon of the F factor and also to prevent expression of the rfaGBIJ gene cluster of Salmonella typhimurium. In the present study, mutants were selected for their ability to restore the normal pattern of rfaGBIJ function. On the basis of this initial section, several classes of extragenic suppressor mutants were isolated that completely or partially corrected the Tra- and Rfa- phenotypes of the prototype rfaH mutant. The suppressor mutations included mutations in rho and mutations that mapped in or close to rpoBC. Other suppressor mutations were located elsewhere on the chromosome, presumably identifying other genes that play a role in the RfaH-mediated transcriptional regulation.

Ekena K, Maloy S
Regulation of proline utilization in Salmonella typhimurium: how do cells avoid a futile cycle?
Mol Gen Genet. 1990; 220: 492-4
Display abstract
Salmonella typhimurium can degrade proline for use as a carbon, nitrogen, or energy source. To determine whether a futile cycle occurs which degrades the proline accumulated by proline biosynthesis, we studied the expression and enzymatic activity of the proline utilization (put) pathway under conditions which increase the concentration of the intracellular proline pools: catabolism of the dipeptide glycyl-proline, overproduction of proline due to a mutation which prevents feedback inhibition of proline biosynthesis, and accumulation of proline due to osmotic stress. The results indicate that: (i) internal proline induces the put genes, but only when accumulated to concentrations greater than the normal proline biosynthetic pool; and (ii) degradation of proline pools accumulated under high osmotic pressure is limited because proline oxidase is directly inhibited under these conditions.

Paton EB, Vudmaska MI, Zolotukhin SB, Zhivolun AN, Krupskaia IV
[Highly conserved primary structure of the L10 protein from Salmonella typhimurium in comparison to Escherichia coli assures the possibility of their regulation of expression of genes in the rplJL operon of E. coli].
Bioorg Khim. 1990; 16: 1280-2

Van Dyk TK, Gatenby AA, LaRossa RA
Demonstration by genetic suppression of interaction of GroE products with many proteins.
Nature. 1989; 342: 451-3
Display abstract
The way in which proteins attain and maintain their final form is of fundamental importance. Recent work has focused on the role of a set of ubiquitous proteins, termed chaperonins, in the assembly of phage and multisubunit proteins. The range of chaperonin action is unknown; they could interact with most cellular polypeptides or have a limited subset of protein partners. Included in the chaperonin family is the essential heat-shock regulated Escherichia coli groEL gene product. Over-expression of the groE operon in E. coli causes enhanced assembly of heterologously expressed ribulose bisphosphate carboxylase subunits and suppresses the heat-sensitive mutant phenotype of several dnaA alleles. It has been inferred that suppression of heat-sensitive mutations is confined to dnaA alleles and that this confinement could reflect an interaction between the groE operon products and a dnaA protein aggregate at the replication origin. We now report that multiple copies of the groE operon suppress mutations in genes encoding several diverse proteins. Our data indicate a general role for the groE operon products, the GroEL and GroES proteins, in the folding-assembly pathways of many proteins.

Bjork GR
E. coli ribosomal protein operons: the case of the misplaced genes.
Cell. 1985; 42: 7-8

Creeger ES, Schulte T, Rothfield LI
Regulation of membrane glycosyltransferases by the sfrB and rfaH genes of Escherichia coli and Salmonella typhimurium.
J Biol Chem. 1984; 259: 3064-9
Display abstract
The role of sfrB and rfaH genes in the regulation of expression of membrane glycosyltransferases was studied in Escherichia coli and Salmonella typhimurium. The transferase enzymes form part of a multienzyme system involved in biosynthesis of the polysaccharide core of Gram-negative bacterial lipopolysaccharides. Several sfrB mutants of E. coli showed reductions of 90-98% in the activities of two of the glycosyltransferases (UDP-galactose:(glucosyl)lipopolysaccharide 1,6-galactosyltransferase and UDP-glucose: (glucosyl)lipopolysaccharide 1,3-glucosyltransferase). Introduction of a recombinant ColE1 plasmid restored the transferase levels to normal and simultaneously corrected the F-factor defects that also characterize sfrB mutants; recombinant plasmids containing other regions of the E. coli chromosome were ineffective. An amber mutation of the S. typhimurium rfaH gene (thought to be the homologue of the E. coli sfrB gene) resulted in 97% loss of activity of the Salmonella UDP-galactose:(glucosyl)lipopolysaccharide galactosyltransferase. Antibody precipitation studies showed that the loss of enzyme activity in the amber mutant was associated with a corresponding decrease in amount, but not in size, of the transferase protein, indicating that the gene is not the structural gene for the S. typhimurium galactosyltransferase. Taken together, the results indicate that the sfrB(rfaH) gene acts as a positive regulatory element in expression of multiple glycosyltransferases in E. coli and S. typhimurium.

Hogarth BG, Higgins CF
Genetic organization of the oligopeptide permease (opp) locus of Salmonella typhimurium and Escherichia coli.
J Bacteriol. 1983; 153: 1548-51
Display abstract
The opp locus of Salmonella typhimurium and Escherichia coli encodes the oligopeptide permease. In this study we showed by complementation analysis that the locus in both species consists of four separate genes, which we named oppA, oppB, oppC, and oppD. These genes are organized as a single operon. The direction of transcription was determined.

Anton DN, de Micheli AT, Palermo AM
Isolation of round-cell mutants of Salmonella typhimurium.
Can J Microbiol. 1983; 29: 170-3

Merrick MJ
A new model for nitrogen control.
Nature. 1982; 297: 362-3

Zengel JM, Lindahl L
Oversynthesis of elongation factors G and Tu in Escherichia coli.
J Bacteriol. 1982; 149: 793-7
Display abstract
We induced the oversynthesis of elongation factors Tu and G by using multicopy plasmids carrying the structural genes for these proteins under the control of the lac operator-promoter. We found no evidence that accumulation of excess elongation factor Tu or G affects the expression of genes for ribosomal proteins or elongation factors.

Alkhimova RA, Mironov AS, Sukhodolets VV
[Regulation of the activity of the Escherichia coli K-12 uridine phosphorylase gene. I. Mapping of the structural gene mutations and a determination of the direction of transcription].
Genetika. 1981; 17: 1730-6
Display abstract
A fine genetic mapping of some point and deletion mutations for uridine phosphorylase (udp) gene, including deletions covering simultaneously the udp and the adjacent metE gene was carried out. Deletions of the latter type do not recombine with the udp promoter mutations isolated previously (Mironov, Sukhodolets 1979) thus suggesting that the udp promoter is situated at the site adjoining metE.

Bendiak DS, Friesen JD
Organization of genes in the four minute region of the Escherichia coli chromosome: evidence that rpsB and tsf are co-transcribed.
Mol Gen Genet. 1981; 181: 356-62
Display abstract
Restriction endonuclease-generated DNA fragments of lambda polC-9 (Friesen et al. 1976) have been cloned on plasmid vehicles. Expression of genes carried by these plasmids was determined either by genetic complementation of the appropriate mutants, or in ultraviolet-irradiated cells. On the basis of these experiments we have inferred the following gene order in the four minute region of the Escherichia coli chromosome: tonA-dapD4-dapD2-rpsB-tsf-22 kilodalton protein - fir 27,000-firA-dnaE. We suggest that rpsB and tsf are in one transcriptional unit, with rpsB being promoter-proximal. We also suggest the possible position of the promoter for dnaE.

Sanderson KE, Stocker BA
Gene rfaH, which affects lipopolysaccharide core structure in Salmonella typhimurium, is required also for expression of F-factor functions.
J Bacteriol. 1981; 146: 535-41
Display abstract
Mutations in gene rfaH of Salmonella typhimurium at 84 units on the linkage map make lipopolysaccharide of chemotypes Ra, Rb2, Rb3, and Rc (A. A. Lindberg and C. G. Hellerqvist, J. Gen. Microbiol, 116:25--32, 1980). F-factor expression in RfaH- strains was reduced in the following properties when compared with RfaH+ strains: transfer of Flac, number of phage f2 infective centers, lysis by and propagation of phages f2 and M13, proportion of cells with visible F-pili, and formation of mating aggregates with F- cells. Inhibition of multiplication of Br60, a female-specific phage, was not reduced in RfaH- Flac strains. Plasmid transfer from RfaH- strains was reduced for Inc groups FI, FII, and T, unaffected for Inc groups beta, I alpha, L, N, P, and W. and increased for Inc group M when compared with plasmid transfer from RfaH+ strains. Reduced F-factor function in RfaH- strains was not due to defective lipopolysaccharide since strains with mutations in other rfa genes were unaffected in plasmid transfer. Gene rfaH appears to be homologous with gene sfrB in Escherichia coli K-12, which maps at the same location, influences F-factor function, and affects synthesis of lipopolysaccharide. The gene product of sfrB has been proposed to be a transcription antiterminator.

Snyder MA, Koshland DE Jr
Identification of the esterase peptide and its interaction with the cheZ peptide in bacterial sensing.
Biochimie. 1981; 63: 113-7
Display abstract
Previous genetic evidence has indicated that the methylesterase activity of the chemotaxis system in Escherichia coli and Salmonella typhimurium resides with the cheB gene product, and that this protein may interact with the cheZ protein. In this article we report biochemical evidence for association of the cheB protein with esterase activity. We also show that while the cheZ gene product is not needed for catalysis, certain mutations in it can eliminate esterase activity in vitro, suggesting association of cheZ and cheB in an oligomeric protein complex.

Kutsukake K, Iino T, Komeda Y, Yamaguchi S
Functional homology of fla genes between Salmonella typhimurium and Escherichia coli.
Mol Gen Genet. 1980; 178: 59-67

Creeger ES, Chen JF, Rothfield LI
Cloning of genes for bacterial glycosyltransferases. II. Selection of a hybrid plasmid carrying the rfah gene.
J Biol Chem. 1979; 254: 811-5
Display abstract
A hybrid ColE1 plasmid containing DNA from Escherichia coli K12 were identified which was capable of correcting the defect in UDP-galactose:lipopolysaccharide alpha1,3-galactosyltransferase in an rfaH mutant of Salmonella typhimurium. Expression of the gene for this enzyme was also demonstrated in several strains of E. coli by direct assay. The E. coli and S. typhimurium enzymes are similar in catalytic properties and immunologic specificity. The finding of the galactosyltransferase activity in E. coli extracts is surprising since the alpha1,3-galactosylglucose disaccharide which is the product of the enzyme-catalyzed reaction does not appear to be present in the E. coli lipopolysaccharide.

Rudbach JA
Some requisites in systems leading to hybrid formation between bacterial endotoxins.
J Infect Dis. 1970; 122: 139-45

Demerec M
Gene differentiation.
Natl Cancer Inst Monogr. 1965; 18: 15-20