NORMAL GENOMIC

• Sidote DJ, Barbieri CM, Wu T, Stock AM
• Structure of the Staphylococcus aureus AgrA LytTR domain bound to DNAreveals a beta fold with an unusual mode of binding.
• Structure. 2008; 16: 727-35
• Display abstract

• The LytTR domain is a DNA-binding motif found within the AlgR/AgrA/LytRfamily of transcription factors that regulate virulence factor and toxingene expression in pathogenic bacteria. This previously uncharacterizeddomain lacks sequence similarity with proteins of known structure. Thecrystal structure of the DNA-binding domain of Staphylococcus aureus AgrAcomplexed with a DNA pentadecamer duplex has been determined at 1.6 Aresolution. The structure establishes a 10-stranded beta fold for theLytTR domain and reveals its mode of interaction with DNA. Residues withinloop regions of AgrA contact two successive major grooves and theintervening minor groove on one face of the oligonucleotide duplex,inducing a substantial bend in the DNA. Loss of DNA binding uponsubstitution of key interacting residues in AgrA supports the observedbinding mode. This mode of protein-DNA interaction provides a potentialtarget for future antimicrobial drug design.

• Molina-Henares AJ, Krell T, Eugenia Guazzaroni M, Segura A, Ramos JL
• Members of the IclR family of bacterial transcriptional regulatorsfunction as activators and/or repressors.
• FEMS Microbiol Rev. 2006; 30: 157-86
• Display abstract

• Members of the IclR family of regulators are proteins with around 250residues. The IclR family is best defined by a profile covering theeffector binding domain. This is supported by structural data and by anumber of mutants showing that effector specificity lies within a pocketin the C-terminal domain. These regulators have a helix-turn-helix DNAbinding motif in the N-terminal domain and bind target promoters as dimersor as a dimer of dimers. This family comprises regulators acting asrepressors, activators and proteins with a dual role. Members of the IclRfamily control genes whose products are involved in the glyoxylate shuntin Enterobacteriaceae, multidrug resistance, degradation of aromatics,inactivation of quorum-sensing signals, determinants of plantpathogenicity and sporulation. No clear consensus exists on thearchitecture of DNA binding sites for IclR activators: the MhpR bindingsite is formed by a 15-bp palindrome, but the binding sites of PcaU andPobR are three perfect 10-bp sequence repetitions forming an inverted anda direct repeat. IclR-type positive regulators bind their promoter DNA inthe absence of effector. The mechanism of repression differs amongIclR-type regulators. In most of them the binding sites of RNA polymeraseand the repressor overlap, so that the repressor occludes RNA polymerasebinding. In other cases the repressor binding site is distal to the RNApolymerase, so that the repressor destabilizes the open complex.

• McGowan S, Lucet IS, Cheung JK, Awad MM, Whisstock JC, Rood JI
• The FxRxHrS motif: a conserved region essential for DNA binding of theVirR response regulator from Clostridium perfringens.
• J Mol Biol. 2002; 322: 997-1011
• Display abstract

• The VirSR two-component signal transduction pathway regulates virulenceand toxin production in Clostridium perfringens, the causative agent ofgas gangrene. The response regulator, VirR, binds to repeat sequenceslocated upstream of the promoter and is directly responsible for thetranscriptional activation of pfoA, the structural gene for thecholesterol-dependent cytolysin, perfringolysin O. Comparative sequenceanalysis of the 236 amino acid residue VirR protein revealed a two-domainstructure: a typical N-terminal response regulator domain and anuncharacterised C-terminal domain. Database searching revealed that over40 other proteins, many of which appeared to be response regulators ortranscriptional activators, had homology with the VirR C-terminal domain(VirRc). Multiple sequence alignment of this VirRc family revealed ahighly conserved region that was designated the FxRxHrS motif. By deletionanalysis this motif was shown to be essential for the functional integrityof the VirR protein. Alanine scanning mutagenesis and subsequentphenotypic analysis indicated that conserved residues located within themotif were required for activity. These residues extended from L179 toN194. More detailed site-directed mutagenesis showed that amino acidresidues R186, H188 and S190 were essential for activity since evenconservative substitutions in these positions resulted in non-functionalproteins. Three of the mutant proteins, R186K, S190A and S190C, werepurified and shown by in vitro gel shift analysis to be unable to bind tothe specific target DNA with the same efficiency as the wild-type protein.These data reveal for the first time that VirRc functions as a DNA bindingdomain in which the highly conserved FxRxHrS motif has a functional role.These studies have important implications for this new family oftranscriptional factors since they imply that the conserved FxRxHrS motifmay be involved in DNA binding in all of these proteins, irrespective oftheir biological role.

• Kenney LJ
• Structure/function relationships in OmpR and other winged-helixtranscription factors.
• Curr Opin Microbiol. 2002; 5: 135-41
• Display abstract

• Response regulators are the output component of two-component regulatorysystems, the predominant form of signal transduction systems utilized byprokaryotes. The majority of response regulators function as transcriptionfactors, yet detailed descriptions of their mechanisms of DNA binding andits consequences are lacking. Versatility in the modes of DNA binding isevident with winged helix-turn-helix proteins, raising doubts thatmechanisms of DNA binding will be generalizable among members of thefamily. The current focus of some of the research efforts aimed atunderstanding activation and DNA binding by response regulators ishighlighted in this review.

• Vannini A et al.
• The crystal structure of the quorum sensing protein TraR bound to itsautoinducer and target DNA.
• EMBO J. 2002; 21: 4393-401
• Display abstract

• The quorum sensing system allows bacteria to sense their cell density andinitiate an altered pattern of gene expression after a sufficient quorumof cells has accumulated. In Agrobacterium tumefaciens, quorum sensingcontrols conjugal transfer of the tumour- inducing plasmid, responsiblefor plant crown gall disease. The core components of this system are thetranscriptional regulator TraR and its inducing ligandN-(3-oxo-octanoyl)-L-homoserine lactone. This complex binds DNA andactivates gene expression. We have determined the crystal structure ofTraR in complex with its autoinducer and target DNA (PDB code 1h0m). Theprotein is dimeric, with each monomer composed of an N-terminal domain,which binds the ligand in an enclosed cavity far from the dimerizationregion, and a C-terminal domain, which binds DNA via a helix-turn-helixmotif. The structure reveals an asymmetric homodimer, with one monomerlonger than the other. The N-terminal domain resembles GAF/PAS domains,normally fused to catalytic signalling domains. In TraR, the gene fusionis between a GAF/PAS domain and a DNA-binding domain, resulting in aspecific transcriptional regulator involved in quorum sensing.

• Ducros VM et al.
• Crystal structure of GerE, the ultimate transcriptional regulator of sporeformation in Bacillus subtilis.
• J Mol Biol. 2001; 306: 759-71
• Display abstract

• The small, DNA-binding protein GerE regulates gene transcription in theterminally differentiated mother-cell compartment during late stages ofsporulation in Bacillus subtilis. This versatile transcription factorshares sequence homology with the LuxR/FixJ/UhpA family of activators andmodulates the expression of a number of genes, in particular thoseencoding the components of the coat that surrounds the mature spore. GerEorchestrates the final stages of coat deposition and maturation that leadto a spore with remarkable resistance properties but that must beresponsive to low levels of germination signals. As this germinationprocess is largely passive and can occur in the absence of de novo proteinsynthesis, the correct assembly of germination machinery, includinggerminant receptors and energy storage compounds, is crucial to thesurvival of the cell. The crystal structure of GerE has been solved at2.05 A resolution using multi-wavelength anomalous dispersion techniquesand reveals the nature of the GerE dimer. Each monomer comprises fouralpha-helices, of which the central pair forms a helix-turn-helixDNA-binding motif. Implications for DNA-binding and the structuralorganisation of the LuxR/FixJ/UhpA family of transcription activatordomains are discussed.

• Ma S, Selvaraj U, Ohman DE, Quarless R, Hassett DJ, Wozniak DJ
• Phosphorylation-independent activity of the response regulators AlgB andAlgR in promoting alginate biosynthesis in mucoid Pseudomonas aeruginosa.
• J Bacteriol. 1998; 180: 956-68
• Display abstract

• Overproduction of the capsular polysaccharide alginate appears to confer aselective advantage for Pseudomonas aeruginosa in the lungs of cysticfibrosis patients. The regulators AlgB and AlgR, which are both requiredas positive activators in alginate overproduction, have homology with theregulator class of two-component environmental responsive proteins whichcoordinate gene expression through signal transduction mechanisms. Signaltransduction in this class of proteins generally occurs viaautophosphorylation of the sensor kinase protein and phosphotransfer fromthe sensor to a conserved aspartate residue, which is present in the aminoterminus of the response regulator. Recently, kinB was identifieddownstream of algB and was shown to encode the cognate histidine proteinkinase that efficiently phosphorylates AlgB. However, we show here that anull mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosaFRD1, did not block alginate production. The role of the conservedaspartate residue in the phosphorylation of AlgB was examined. Thepredicted phosphorylation site of AlgB (D59) was mutated to asparagine(N), and a derivative of an AlgB lacking the entire amino-terminalphosphorylation domain (AlgB delta1-145) was constructed. A hexahistidinetag was included at the amino terminus of the wild-type (H-AlgB), H-AlgBdelta1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives werepurified by Ni2+ affinity chromatography and examined for in vitrophosphorylation by the purified sensor kinase protein, KinB. The resultsindicated that while KinB efficiently phosphorylated H-AlgB, nophosphorylation of H-AlgB delta1-145 or H-AlgB.D59N was apparent. Anallelic exchange system was developed to transfer mutant algB alleles ontothe chromosome of a P. aeruginosa algB mutant to examine the effect onalginate production. Despite the defect in AlgB phosphorylation, P.aeruginosa strains expressing AlgB.D59N or H-AlgB delta1-145 remainedmucoid. The roles of the conserved aspartate residues in thephosphorylation of AlgR were also examined. As seen with AlgB, mutationsin the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N)did not affect alginate production. These results indicate that in vivophosphorylation of AlgB and AlgR are not required for their roles inalginate production. Thus, the mechanism by which these responseregulators activate alginate genes in mucoid P. aeruginosa appears not tobe mediated by conventional phosphorylation-dependent signal transduction.

• Yu H, Mudd M, Boucher JC, Schurr MJ, Deretic V
• Identification of the algZ gene upstream of the response regulator algRand its participation in control of alginate production in Pseudomonasaeruginosa.
• J Bacteriol. 1997; 179: 187-93
• Display abstract

• Alginate production in mucoid Pseudomonas aeruginosa isolates from cysticfibrosis patients is under direct control by AlgU, the P. aeruginosaequivalent of the extreme heat shock sigma factor sigma(E) ingram-negative bacteria, and AlgR, a response regulator from thesuperfamily of two-component signal transduction systems. In this report,we describe the identification of the algZ gene, located immediatelyupstream of algR, which is involved in the control of alginate production.The predicted product of the algZ gene showed similarity to a subset ofsensory components from the superfamily of signal transduction systems butlacked several of the highly conserved motifs typical of histidine proteinkinases. Inactivation of algZ in the wild-type standard genetic strainPAO1 did not affect its nonmucoid morphology. However, inactivation ofalgZ in a mucoid mutant P. aeruginosa strain, which had AlgU freed fromcontrol by the anti-sigma factor MucA, resulted in increased alginateproduction under growth conditions which did not permit expression ofmucoidy in the parental algZ+ strain. The observed effects were abrogatedwhen algR was inactivated in the algZ::Tc(r) background. These findingsindicate that algZ plays a regulatory role in alginate production,possibly interacting with AlgR, and that it may have negative effects onexpression of the mucoid phenotype under the conditions tested. Thepresented results suggest that elements of negative regulation exist atthe levels of both the alternative sigma factor AlgU and thetranscriptional activator AlgR which, once relieved from that suppression,cooperate to bring about the expression of the alginate system.

• Brown DP, Ganova-Raeva L, Green BD, Wilkinson SR, Young M, Youngman P
• Characterization of spo0A homologues in diverse Bacillus and Clostridiumspecies identifies a probable DNA-binding domain.
• Mol Microbiol. 1994; 14: 411-26
• Display abstract

• Spo0A is a phosphorylation-activated transcription factor of Bacillussubtilis. It is a member of the response regulator superfamily ofbacterial signal transduction proteins and controls many of the changes ingene expression that occur during the transition into stationary phase andduring the initiation of sporulation. To identify the domains of Spo0Amost critical for determining its structural and functional features,presumptive homologues of the spo0A gene were characterized in acollection of eight Bacillus species and six Clostridium speciesrepresenting phylogenetically diverse members of these genera. Analignment of the partial or complete DNA sequences of these homologuesrevealed three regions of especially high conservation in the effectordomain. We speculate that the most highly conserved of these correspondsto the recognition helix of a putative helix-turn-helix motif, and,therefore, represents the actual DNA-containing surface of the protein. Inthe case of homologues identified in Bacillus anthracis and Clostridiumacetobutylicum and retrieved by polymerase chain reaction amplification,we confirmed by gene-disruption analysis that the homologue actually isrequired for initiation of sporulation. Apparent homologues of the B.subtilis spoIVB gene were also discovered immediately upstream from thespo0A homologues in all Bacillus and Clostridium species examined. Thediscovery of homologues of B. subtilis sporulation genes in these diversespecies implies that the gene products required for specifying pathways ofsporulation-specific gene activation and for determining key morphogeneticchanges may be highly conserved and suggests that an approach similar tothat undertaken here might be used as a general strategy to retrieve andcompare their gene sequences. Exhaustive efforts to detect a spo0A-likegene in non-endospore formers, including close relatives of Bacillus suchas Listeria and Staphylococcus, were uniformly unsuccessful, suggestingthat regulation of gene activity during the transition into stationaryphase mediated by Spo0A-like proteins may be exclusive to theendospore-forming bacteria.

• Wozniak DJ, Ohman DE
• Pseudomonas aeruginosa AlgB, a two-component response regulator of theNtrC family, is required for algD transcription.
• J Bacteriol. 1991; 173: 1406-13
• Display abstract

• Most strains of Pseudomonas aeruginosa isolated from the respiratorytracts of cystic fibrosis patients have a mucoid colony morphology due tothe synthesis of an expolysaccharide called alginate. The algB geneproduct (AlgB) is necessary for the high-level production of alginate inmucoid P. aeruginosa. In this study, AlgB was shown to be involved in thetranscription of algD, a gene previously demonstrated to be activated inmucoid P. aeruginosa. In vitro and in vivo expression studies reveal thatalgB encodes a protein with a molecular size of 49 kDa. The DNA sequenceof a 2.2-kb P. aeruginosa fragment containing algB was also determined.The amino-terminal domain of AlgB was found to be conserved with theamino-terminal domains of the response regulator class of two-componentregulatory proteins. The central domain of AlgB has sequences highlyconserved with those in the NtrC subfamily of transcriptional activators(NtrC, NifA, HydG, DctD, FlbD, TyrR, and PgtA). The central domain of AlgBalso contains a potential nucleotide binding site. AlgB is the first NtrChomolog described from P. aeruginosa. At the carboxy terminus of AlgB, ahelix-turn-helix motif was observed, suggesting that AlgB is a DNA-bindingprotein. The strongly conserved NtrC-like central domain of AlgB is notpresent in AlgR, another alginate response regulator. This study thereforeidentifies and characterizes the second of at least two unique responseregulators used by P. aeruginosa to control alginate gene expression.