NORMAL GENOMIC

• Candela T, Mock M, Fouet A
• CapE, a 47-amino-acid peptide, is necessary for Bacillus anthracispolyglutamate capsule synthesis.
• J Bacteriol. 2005; 187: 7765-72
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• Polyglutamate is found in various bacteria, but displays differentfunctions depending on the species and their environment. Here, wedescribe a minimal polyglutamate synthesis system in Bacillus anthracis.In addition to the three genes previously described as sufficient forpolyglutamate synthesis, this system includes a small open reading frame,capE, belonging to the cap operon. The polyglutamate system's requirementfor the five cap genes, for capsulation and anchoring, was assayed innonpolar mutants. The capA, capB, capC, and capE genes are all necessaryand are sufficient for polyglutamate synthesis by B. anthracis. capD isrequired for polyglutamate anchoring to the peptidoglycan. The47-amino-acid peptide encoded by capE is localized in the B. anthracismembrane. It is not a regulator and it is required for polyglutamatesynthesis, suggesting that it has a structural role in polyglutamatesynthesis. CapE appears to interact with CapA. Bacillus subtilis ywtC issimilar to capE and we named it pgsE. Genes similar to capE or pgsE werefound in B. subtilis natto, Bacillus licheniformis, and Staphylococcusepidermidis, species that produce polyglutamate. All the bacterialpolyglutamate synthesis systems analyzed show a similar geneticorganization and, we suggest, the same protein requirements.

• Candela T, Fouet A
• Bacillus anthracis CapD, belonging to the gamma-glutamyltranspeptidasefamily, is required for the covalent anchoring of capsule topeptidoglycan.
• Mol Microbiol. 2005; 57: 717-26
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• Several examples of bacterial surface-structure anchoring have beendescribed, but they do not include polyglutamate capsule. Bacillusanthracis capsule, which is composed only of poly-gamma- d-glutamate, isone of the two major virulence factors of the bacterium. We analysed itsanchoring. We report that the polyglutamate is anchored directly to thepeptidoglycan and that the bond is covalent. We constructed a capD mutantstrain, capD being the fourth gene of the capsule biosynthetic operon. Themutant bacilli are surrounded by polyglutamate material that is notcovalently anchored. Thus, CapD is required for the covalent anchoring ofpolyglutamate to the peptidoglycan. Sequence similarities suggest thatCapD is a gamma-glutamyltranspeptidase. Furthermore, CapD is cleaved atthe gamma-glutamyltranspeptidase consensus cleavage site, and the twosubunits remain associated, as necessary for gamma-glutamyltranspeptidaseactivity. Other Gram-positive gamma-glutamyltranspeptidases are secreted,but CapD is located at the Bacillus surface, associated both with themembrane and the peptidoglycan. Polyglutamate is hydrolysed by CapDindicating that it is a CapD substrate. We suggest that CapD catalyses thecapsule anchoring reaction. Interestingly, the CapD(-) strain is far lessvirulent than the parental strain.

• Kada S, Nanamiya H, Kawamura F, Horinouchi S
• Glr, a glutamate racemase, supplies D-glutamate to both peptidoglycansynthesis and poly-gamma-glutamate production in gamma-PGA-producingBacillus subtilis.
• FEMS Microbiol Lett. 2004; 236: 13-20
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• Poly-gamma-glutamate (gamma-PGA)-producing Bacillus subtilis contains twoglutamate racemase genes, glr and yrpC, as does gamma-PGA-nonproducing B.subtilis strain 168. glr and yrpC on the chromosome of gamma-PGA-producingstrain r22 were separately disrupted by means of gene replacement with anerythromycin resistance determinant. yrpC-disruption caused no effects ongrowth or gamma-PGA-production, whereas glr was disrupted only when anexogenous glr copy was present on a plasmid. In addition, the D-glutamatecontent of gamma-PGA produced by the yrpC-disruptant was the same as thatproduced by the parental strain r22. Glr in strain r22 is thereforeresponsible for the supply of D-glutamate to the synthesis of bothpeptidoglycan and gamma-PGA. Consistent with this idea, glr wastranscribed actively during the exponential growth phase for peptidoglycansynthesis and continuously at a low, but distinct, level during thestationary phase for gamma-PGA production, whereas yrpC was transcribed ata very low level throughout growth. Phylogenetic analysis of glutamateracemases from eubacteria showed that YrpC is distinct from otherglutamate racemases.

• Ashiuchi M et al.
• Enzymatic synthesis of high-molecular-mass poly-gamma-glutamate andregulation of its stereochemistry.
• Appl Environ Microbiol. 2004; 70: 4249-55
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• For the first time, we succeeded in synthesizing in vitropoly-gamma-glutamate (PGA) with high molecular masses (>1,000 kDa) by theuse of enzyme-associated cell membranes from Bacillus subtilis subsp.chungkookjang. The activity for PGA synthesis, however, was readily lostin the presence of critical concentrations of detergents tested inmicelles. The optimum pH for the reaction was found to be approximately7.0. We examined the effects of some divalent cations on PGA synthesis andfound that Mg(2+) was essential in catalysis and that Zn(2+) additionallyboosted the activity. In contrast, Fe(2+) and Ca(2+) acted as inhibitors.Mn(2+) did not apparently influence the in vitro formation of PGA.DL-Glutamate (D isomer content, 60 to 80%) apparently served as the bestsubstrate; d-Glutamate was preferable to the L isomer as a substrate. WhenD- and L-glutamate were used for the reaction, the elongated chains ofPGAs were composed of the D- and L-isomers, respectively. Our resultssuggest that the stereochemical properties of enzymatically synthesizedPGAs substantially depend on the stereochemistry (DL ratio) of glutamateas the substrate. Furthermore, genetic analysis indicated that all thepgsB, -C, and -A gene products, which are responsible for PGA productionby B. subtilis cells, were also indispensable for enzymatic PGA synthesis.

• Ashiuchi M, Misono H
• Biochemistry and molecular genetics of poly-gamma-glutamate synthesis.
• Appl Microbiol Biotechnol. 2002; 59: 9-14
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• Current research into poly-gamma-glutamate (PGA) and its biosynthesis isreviewed. In PGA-producing Bacillus subtilis, glutamate racemase suppliesabundant DL-glutamate, the substrate for PGA synthesis. The pgsBCA genesof PGA-producing B. subtilis, which encode the membrane-associated PGAsynthetase complex PgsBCA, were characterized and the enzyme complex wassuggested to be an atypical amide ligase based on its structure andfunction. A novel reaction mechanism of PGA synthesis is proposed.

• Kambourova M, Tangney M, Priest FG
• Regulation of polyglutamic acid synthesis by glutamate in Bacilluslicheniformis and Bacillus subtilis.
• Appl Environ Microbiol. 2001; 67: 1004-7
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• The synthesis of polyglutamic acid (PGA) was repressed by exogenousglutamate in strains of Bacillus licheniformis but not in strains ofBacillus subtilis, indicating a clear difference in the regulation ofsynthesis of capsular slime in these two species. Although extracellulargamma-glutamyltranspeptidase (GGT) activity was always present inPGA-producing cultures of B. licheniformis under various growthconditions, there was no correlation between the quantity of PGA andenzyme activity. Moreover, the synthesis of PGA in the absence ofdetectable GGT activity in B. subtilis S317 indicated that this enzyme wasnot involved in PGA biosynthesis in this bacterium. Glutamate repressionof PGA biosynthesis may offer a simple means of preventing unwanted slimeproduction in industrial fermentations using B. licheniformis.

• Ashiuchi M et al.
• Physiological and biochemical characteristics of poly gamma-glutamatesynthetase complex of Bacillus subtilis.
• Eur J Biochem. 2001; 268: 5321-8
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• An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillussubtilis, the PgsBCA system, was investigated. The gene-disruptionexperiment showed that the enzymatic system was the sole machinery of PGAsynthesis in B. subtilis. We succeeded in achieving the enzymaticsynthesis of elongated PGAs with the cell membrane of the Escherichia coliclone producing PgsBCA in the presence of ATP and D-glutamate. The enzymepreparation solubilized from the membrane with 8 mM Chaps catalyzedADP-forming ATP hydrolysis only in the presence of glutamate; theD-enantiomer was the best cosubstrate, followed by the L-enantiomer. Eachcomponent of the system, PgsB, PgsC, and PgsA, was translated in vitro andthe glutamate-dependent ATPase reaction was kinetically analyzed. The PGAsynthetase complex, PgsBCA, was suggested to be an atypical amide ligase.

• King EC, Blacker AJ, Bugg TD
• Enzymatic breakdown of poly-gamma-D-glutamic acid in Bacilluslicheniformis: identification of a polyglutamyl gamma-hydrolase enzyme.
• Biomacromolecules. 2000; 1: 75-83
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• A polyglutamyl gamma-hydrolase enzyme has been identified which catalysesthe hydrolytic breakdown of poly-gamma-D-glutamic acid (PGA) from Bacilluslicheniformis 9945a. The enzyme was found to be physically associated withthe polymer and was activated by Zn2+ or Ca2+ salts. The enzyme can besolubilized from the polymer by treatment with 0.5% SDS and 1 mM ZnCl2 andcan then be renatured onto exogenous PGA upon dilution below the detergentcritical micellar concentration. The enzyme was partially purified byaffinity chromatography, using immobilized PGA. Peptide thioesterscontaining one and two gamma-glutamyl units were synthesized as potentialchromogenic substrates but showed no activity with the solubilized enzyme.Examination of 14C-labeled reaction products indicated that the enzyme isan endo-type hydrolase.

• Cromwick AM, Gross RA
• Effects of manganese (II) on Bacillus licheniformis ATCC 9945A physiologyand gamma-poly(glutamic acid) formation.
• Int J Biol Macromol. 1995; 17: 259-67
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• Bacillus licheniformis ATCC 9945A was cultivated in shake flasks usingcitrate (12 gl-1), glutamate (20 gl-1) and glycerol (80 gl-1) as carbonsources for cell growth and gamma-poly(glutamic acid) (gamma-PGA)production. The effect of the MnSO4 concentration in the medium over arange from 0.0 to 615 microns was studied. The number of viable cellsincreased for all concentrations of MnSO4 from approximately 10(5) to10(9) colony-forming units (cfu) ml-1 by the early stationary phase (24h). However, after 50 h, the cell viability decreased rapidly forrelatively lower MnSO4 concentrations (0.615 and 0 microns). Theutilization of carbon sources by B. licheniformis was greater for culturescontaining 33.8 and 615 microns MnSO4 relative to cultures with no addedMnSO4. For example, cultures with 615 microns MnSO4 utilized 37, 54 and93% and cultures with no added MnSO4 utilized 19, 10 and 17% of glutamate,glycerol and citrate, respectively. The gamma-PGA volumetric yieldincreased from approximately 5 to 17 gl-1 for corresponding increases inMnSO4 concentration from 0 to 33.8 microns and then decreased at higherMnSO4 concentrations. The stereochemical content of gamma-PGA was found tovary inversely with MnSO4 concentration, and ranged from 59 to 10%L-glutamate units for MnSO4 concentrations of 0 and 615 microns,respectively. For all of the MnSO4 concentrations investigated, thegamma-PGA molecular weights decreased rapidly as the gamma-PGA volumetricyield simultaneously increased for cultivation times from 24 toapproximately 50 h. Mw and Mn values after approximately 50 h cultivationtimes, determined by gel permeation chromatography (GPC), were 1.3 to 1.6and 0.5 to 0.8 million g mol-1, respectively. A complex gamma-PGAmolecular weight distribution that appeared bimodal by GPC analysis due tothe presence of a low-molecular-weight product fraction was observed incultures containing 33.8 and 61.5 microns MnSO4 at extended cultivationtimes. A high-molecular-weight fraction and the unfractionated gamma-PGAsample from the 33.8 microns MnSO4 culture contained 13 +/- 4 and 30 +/-1% L-repeat units, respectively. A relationship between the productmolecular weight and its stereochemical composition was thus established.

• Birrer GA, Cromwick AM, Gross RA
• Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a:physiological and biochemical studies.
• Int J Biol Macromol. 1994; 16: 265-75
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• Cryogenically frozen vegetative cells of Bacillus licheniformis 9945aderived from young mucoid colonies were used to inoculategamma-poly(glutamate) (gamma-PGA) production media containing L-glutamate,citrate and glycerol as carbon sources. A gel permeation chromatography(GPC) method was developed to determine gamma-PGA volumetric yield andmolecular weight directly using culture filtrates. For GPC volumetricyield measurements, a calibration curve was generated using purifiedgamma-PGA to relate the gamma-PGA GPC peak area and polymer weight.Purified gamma-PGA was characterized by elemental analysis, 1H- and13C-NMR spectroscopy. Cultures of B. licheniformis using all three carbonsources showed the following characteristics: cell growth mainly duringthe first 24 h; largest gamma-PGA volumetric productivity (approximately0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by96 h; reduction (utilization) of glycerol, glutamate and citrate during a96 h cultivation time from 80 to 45 g l-1, 18 to 10 g l-1 and 12 toapproximately 1 g l-1, respectively; a decrease in pH from 7.4 toapproximately 5.5 by 42 h cultivation; acetic acid secretion into themedium at a maximum level of approximately 4.5 g l-1 and detection of themetabolite 2,3-butanediol (as acetoin) as a fermentation by-product atapproximately 42 h and through a 96 h cultivation period. The presence of2,3-butanediol indicated that the level of oxygen in the medium no longersupported a fully aerobic mode of metabolism. When the medium formulationwas altered by removal of either citrate, L-glutamate or glycerol in shakeflask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1,respectively, of gamma-PGA were formed. Variation of the medium ionicstrength by the addition of up to 4% (w/v) NaCl led to the formation ofgamma-PGA of relatively higher molecular weight but lower volumetricyield. Studies carried out on 5-day-old B. licheniformis culturessuggested that gamma-PGA depolymerase is intracellularly located orcell-bound. Culture filtrates showed no significant gamma-PGA depolymeraseactivity.