1 Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan
2 Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka 4, Toyohira-Ku, Sapporo 062-0045, Japan
3 Akita Research Institute of Food and Brewing, Sanuki 4-26, Araya-Machi, Akita 010-1623, Japan
Correspondence
Yoshifumi Itoh
yosifumi{at}arif.pref.akita.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Present address: Japan International Research Center for Agricultural Science, Ohwashi 1-1, Tsukuba, Ibaraki 305-8686, Japan.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Some strains of B. subtilis and Bacillus anthracis produce capsular poly(-glutamic acid) (
PGA; Thorne, 1993
). In both B. subtilis and B. anthracis, the membrane
PGA synthetic proteins encoded by the capBCA (also referred to ywsCywtAB or pgsBCA in B. subtilis) operon catalyse synthesis of the capsule polypeptide (Ashiuchi et al., 1999
; Makino et al., 1989
; Urushibata et al., 2002
). However, the stereochemistry of these bacterial
PGAs depends upon the structure of the
-glutamyl linkage, and the synthesis of each
PGA is regulated differently. B. anthracis
PGA consists of D-glutamate only, and it is produced in the presence of serum or under high atmospheric CO2 concentrations, circumstances that mimic host environments where the capsule functions as a protective barrier against phagocytosis by macrophages (Makino et al., 1988
, 1989
, 2002
). On the other hand, B. subtilis produces capsule
PGA consisting of both D- and L-glutamate specifically during the early stationary phase. This growth-phase-dependent synthesis of the capsule is mediated through the ComQXPA quorum-sensing mechanism, which also controls the expression of other stationary-phase-specific traits (Dubnau, 1999
; Lazazzera et al., 1999
; Tran et al., 2000
).
Both B. anthracis and B. subtilis degrade their capsule PGAs, but probably via different enzymes. B. anthracis degrades the capsule polypeptide into 214 kDa fragments via a depolymerase encoded by capD, which lies immediately downstream of the cap operon (Makino et al., 2002
). The resultant polypeptide fragments appear to be required for the pathogen to flourish in hosts (Makino et al., 2002
). The B. subtilis capsule is degraded during late stationary phase. This bacterium has the ywtD gene encoding
-DL-glutamyl hydrolase at a locus corresponding to capD (Suzuki & Tahara, 2003
). The ywtD product, however, has no amino acid sequence similarity to the CapD depolymerase, and it cleaves
PGA in vitro into fragments of 490 and 11 kDa (Suzuki & Tahara, 2003
). B. subtilis GGT appears to be capable of generating D- and L-glutamate in vitro from
PGA (Abe et al., 1997
). However, the precise hydrolytic mechanism of this enzyme has not been defined, and whether YwtD and GGT participate in the in vivo degradation process remains unknown.
B. subtilis can utilize both D- and L-glutamate as nitrogen sources (Kimura et al., 2004). D-glutamate catabolism by this bacterium proceeds after conversion to the L-form by glutamate racemases (the racE and yrpC products). Mutants of racE or yrpC accumulate D-glutamate in late-stationary-phase cultures (Kimura et al., 2004
), indicating that B. subtilis cells degrade capsule
PGA into its constituent glutamates outside the cells, and utilize them as nitrogen sources during late stationary phase.
We report here that B. subtilis GGT has powerful exo--glutamyl hydrolase activity towards
PGA, and generates both the amino-terminal D- and L-glutamate of the polypeptide. Experiments with a mutant lacking GGT activity demonstrated that this enzyme is involved in
PGA degradation in vivo to yield the constituent amino acids, and that B. subtilis has, in addition to YwtD, a second endo-
PGA hydrolase that degrades the capsule polypeptide into 1x105 Da fragments. Furthermore, we showed that when the nitrogen supply is limited, mutant cells lacking GGT sporulate more frequently than the wild-type strain, suggesting that capsule glutamates serve B. subtilis as nitrogen sources during the stationary phase.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of mutants.
A DNA region corresponding to the mature part of GGT was amplified using KOD DNA polymerase (Toyobo Biochemicals), primers [5'-GATGAGTCAAAACAAGTAGATGTTGGA-3', nt 106132 relative to the translation initiation codon (1) of ggt (DDBJ/EMBL/GenBank accession number AB095984) and 5'-TATTTACGTTTTAAATTAATGCCGATCGC-3', complementary to nt 17341762 of ggt], and the chromosomal DNA of B. subtilis NAFM5 (Kimura & Itoh, 2003) as the template. The amplified DNA region was then cloned into the HincII site on plasmid pUC118 (Vieira & Messing, 1987
) to verify the nucleotides by sequencing. A spectinomycin (Spc)-resistance cassette, isolated from plasmid pDG1726 (Guérout-Fleury et al., 1995
) as an EcoRVHincII fragment, was then inserted into the StuI site of ggt on the resultant plasmid. After linearization at the unique ScaI site on the vector sequence, the plasmid DNA was used to knock out the ggt of strain NAFM5 by double-crossover recombination, generating strain NAFM90 (ggt : : Spc). The SspISphI fragment carrying the entire ggt gene was integrated into the amyE locus of strain NAFM90 via plasmid pDG1661 (Guérout-Fleury et al., 1996
) by homologous recombination to create strain NAFM96 (ggt : : Spc amyE : : ggt). Replacing comP in strain NAFM5 with comP : : Spc using a pUC118 derivative carrying a 4·4 kb HindIII fragment containing comP (Tran et al., 2000
), which had been inactivated by insertion of the EcoRVHincII Spc-resistance cassette (see above) at the ClaI site, resulted in strain NAFM65 (comP : : Spc). Southern blotting (Nakada & Itoh, 2002
) confirmed that the Spc-resistance cassette and ggt at the target loci were correctly inserted.
Preparation of PGA and
-glutamyltetrapeptides.
We purified PGA from B. subtilis NAFM90 (ggt : : Spc) cultures incubated for either 2 or 7 days on E9 agar (without glutamate) containing 0·5 µg biotin ml1, as described by Nagai et al. (1997)
. We determined the molecular masses of the polypeptides by gel-permeation HPLC using an Asahipak GFA-7M column (Asahi Chemical Industry) (Nagai et al., 1997
). The content of D- and L-glutamate in the polypeptides was determined after hydrolysis with 1 M HCl for 3 h, and by using CrownPack CR (+) and CrownPack CR () chiral columns (Daicel Chemical Industry) (Nagai et al., 1997
). The molecular masses of the polypeptides isolated from the 2 and 7 day cultures were 2x106 and 1x105 Da, respectively, and they both comprised 54 % D-glutamate.
PGA of 1x105 Da, with a high D-glutamate content (76 %), was also prepared from strain NAFM90 cultures incubated on GSP agar containing 0·1 mM MnCl2 (Nagai et al., 1997
) for 7 days. The synthetic glutamyltetrapeptides
-D-glu-(
-L-Glu)3, (
-L-glu)3-
-D-Glu,
-L-glu-(
-L-glu)3 and (
-L-glu)3-
-L-Glu were obtained from Hokkaido System Science (Sapporo, Japan). The oligopeptides were constructed using a Pioneer Dual Column Peptide Synthesizer (Applied Biosystems). We confirmed the molecular mass and purity (99 %) of the synthetic peptides by mass spectrometry (Apex II 70e, Bruker Daltonics) and gel-permeation HPLC, respectively (Nagai et al., 1997
).
Enzyme assays.
We measured GGT activity using -glutamyl-p-nitroanilide (
GNA) as the substrate in the presence of the acceptor glycylglycine, according to Suzuki et al. (1986)
. One unit was defined as the amount of enzyme that was required to produce 1 µmol p-nitroaniline (
410 8800 M1 cm1) per min. We assayed the hydrolytic activity of GGT towards
PGA and
-glutamyltetrapeptide in a reaction mixture (400 µl) containing 2 mg ml1 1x105 Da
PGA or 0·5 mM synthetic
-glutamyltetrapeptide, 20 mM sodium phosphate buffer (pH 6·9), 150 mM NaCl (omitted from the reaction with the tetrapeptide) and GGT (0·4 µg), at 37 °C. Portions (60 µl) of the reaction mixtures were withdrawn at 0, 5, 10, 20, 30 and 45 min, and then boiled for 10 min to terminate the reaction. The D- and L-glutamate reaction products were separated using HPLC chiral columns (see above), and quantified using a Shimadzu RF-10AXL fluorescent detector (excitation at 345 nm, emission at 455 nm) after coupling with o-phthalaldehyde.
Purification of GGT.
B. subtilis GGTs have been purified from strains NR-1 and 168, and amino acid sequencing of the large and small subunits has confirmed that they are the products of ggt (Minami et al., 2003; Ogawa et al., 1991
, 1997
; Kunst et al., 1997
). We purified GGT from stationary-phase cultures (2 l) of B. subtilis NAFM5 in E9 medium. The culture supernatant was dialysed against 25 mM Tris/HCl buffer (pH 7·5) containing 0·5 mM DTT, and eluted through a Hiprep 16/10 DEAE column (Amersham Biosciences) using a linear gradient of NaCl (00·4 M). After dialysis against 10 mM sodium phosphate buffer (pH 6·8) containing 0·5 mM DTT, fractions containing the enzyme were applied to a hydroxyapatite column (CHT5-I; Bio-Rad), and the enzyme was eluted with a gradient (0·010·5 M) of sodium phosphate, pH 6·8. Active fractions were dialysed against 10 mM sodium phosphate (pH 6·8) containing 0·5 mM DTT, and then eluted through a MonoQ column (HR 5/5; Amersham Biosciences) using a linear NaCl gradient (00·35 M) in the same buffer. Combined active fractions were concentrated using Centriprep-10 (Millipore), and finally gel filtered through a Superose12 column (Amersham Biosciences) using 10 mM sodium phosphate (pH 6·8) containing 0·15 M NaCl as the running buffer. These procedures resulted in a 2·4 % yield of GGT that was purified 122-fold. The purified GGT (56 µg) was apparently homogeneous, and consisted of 44 and 23 kDa subunits as shown by SDS-PAGE. We partially purified E. coli GGT from exponentially proliferating cultures (5 l) of E. coli W3110 in LB medium, according to Suzuki et al. (1986)
. Bovine kidney GGT was purchased from Wako Pure Chemicals. The protein concentration was determined using a Protein Assay kit (Bio-Rad) with bovine serum albumin as the standard. We performed SDS-PAGE using Mini PROTEAN II electrophoresis apparatus and 12·5 % (w/v) polyacrylamide gels (Bio-Rad).
Two-dimensional immunoelectrophoresis.
PGA was extracted from portions (1 ml) of B. subtilis strains NAFM5 (wild-type) and NAFM90 (ggt : : Spc) incubated in medium E9 (100 ml) as described by Nagai et al. (1997)
. After dissolution in 100 µl 20 mM sodium phosphate buffer (pH 6·9), 8 µl portions of the samples were resolved by electrophoresis through 1·2 % (w/v) agarose gels containing 0·1 M Tris/HCl (pH 8·5) at 2 mA cm1 for 6 h. Second-dimension electrophoresis proceeded on 1·2 % (w/v) agarose gels containing 0·1 M Tris/HCl (pH 8·5) and 10 % (v/v) anti-
PGA serum (Uchida et al., 1993
) at 2 mA cm1 for 18 h. After electrophoresis, the gels were soaked in PBS (25 mM sodium phosphate pH 7·0, 150 mM NaCl) to remove free antiserum, and then
PGAantibody complexes were stained with Amido black (Uchida et al., 1993
).
Primer extension and Northern blotting.
Cells cultivated in the media specified in Results (100 ml) were incubated in 15 ml of 20 % (w/v) sucrose containing 6 mg egg-white lysozyme per ml, 50 mM Tris/HCl (pH 7·5) and 50 mM EDTA, at 37 °C for 3 min. The resultant protoplasts were quickly sedimented by centrifugation, and suspended in 10 ml acetate/EDTA buffer (pH 4·8) containing 30 mM sodium acetate, 1 mM EDTA and 10 mM Tris. Thereafter, total RNA was extracted with hot phenol (Nakada & Itoh, 2002). For primer extension analysis, RNA samples (20 µg) were annealed with an oligonucleotide (5'-AGCGACTAACAGAACACTAAGCAGAGC-3', complementary to nt 3158 of ggt) labelled with 32P at the 5' end by using [
-32P]ATP (220 TBq mmol1; Amersham Biosciences) and T4 polynucleotide kinase (Toyobo Biochemicals). Complementary strands were synthesized using AMV reverse transcriptase XL (Toyobo Biochemicals), and resolved on a denatured 6 % (w/v) polyacrylamide gel. Sequence ladders were generated using a BcaBest sequencing kit (Takara Shuzo; http://www.takara-bio.co.jp) with the oligonucleotide as the primer, and plasmid pNAG201 carrying an SspIBglII ggt fragment as the template. Total RNA (10 µg) was resolved for Northern blotting on 1·2 % (w/v) agarose gels, and blotted onto nylon membranes (Hybond-N+; Amersham Biosciences). A ggt DNA fragment amplified by PCR, as described above, was labelled using a random-prime labelling kit (Nippon Gene) and [
-32P]dCTP (220 TBq mmol1; Amersham Biosciences), and hybridized with membrane ggt mRNA. Hybridized probes were visualized on X-ray films.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
To test whether GGT synthesis is under the control of the quorum-sensing system and L-glutamate, we measured GGT activities in NAFM5 (wild-type) cells cultured with or without L-glutamate, and in comP mutant NAFM65 (comP : : Spc) cells incubated without L-glutamate. In the absence of L-glutamate, the wild-type strain initiated GGT synthesis immediately after the culture entered the stationary phase, and this continued for another 3 days, producing maximal levels of the enzyme after 5 days (Fig. 3). Exogenous L-glutamate reduced GGT synthesis to marginal levels (2·1x103 units ml1). The comP mutant produced negligible amounts (0·08x103 units ml1) of the enzyme during the entire stationary phase (up to 6 days; Fig. 3
), showing that the quorum-sensing system positively controls GGT synthesis and that L-glutamate antagonizes this positive control.
Regulation of ggt transcription
The ggt gene shares a 184 bp intergenic promoter region with the upstream divergent yoeD gene (Kunst et al., 1997). Primer extension experiments with total RNA from the stationary-phase wild-type cells incubated without L-glutamate identified the 5' end of the ggt transcript at position 34 relative to the translation initiation codon (Fig. 6
, lane 1). The 35 (5'-TTGTCA-3') and 10 (5'-TTTTAC-3') sequences proceed at the corresponding sites relative to the inferred transcription initiation point. In contrast, ggt cDNA was not detected using total RNA from comP mutant cells cultured under the same conditions (Fig. 6
, lane 2). Northern blots (not shown) showed that ggt was scarcely transcribed during the exponential phase (i.e. 1 day culture), but became actively transcribed after the culture entered the stationary phase (2 days incubation). The amounts of the ggt transcripts reached maximal levels after 3 days, and these were maintained for at least 1 day. Very small amounts of ggt mRNA were detected in stationary-phase cells incubated with L-glutamate, indicating that exogenous L-glutamate inhibits GGT synthesis at the level of transcription.
|
B. subtilis cells develop spores during the nutrient-poor stationary phase (Phillips & Strauch, 2002). We assumed that in the absence of any other nitrogen source, ggt mutant cells unable to utilize capsule glutamate as a nitrogen source would sporulate more frequently than wild-type cells. To test this hypothesis, we counted spores in wild-type (strain NAFM5) and ggt mutant (NAFM90) cultures during the stationary phase. In the presence of excess NH4Cl (e.g. 100 mM), both wild-type and mutant culture spores constituted less than 2 % of the total cells, even after 7 days of incubation (data not shown). At 10 mM NH4Cl, the wild-type cells initiated sporulation after 5 days, and spores accounted for 25 % of the total cells after 7 days (Fig. 7
). In contrast, the mutant culture began to develop spores after 2 days, and 40 % cells sporulated after 4 days, when most wild-type cells remained in a vegetative stage (Fig. 7
). Limiting the carbon source did not significantly change sporulation frequencies between these strains.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The involvement of GGT in the utilization of glutathione or other -glutamylpeptides has been demonstrated in B. subtilis, E. coli, Saccharomyces cerevisiae and animal cells (Hanigan & Ricketts, 1993
; Lieberman et al., 1996
; Mehdi & Penninckx, 1997
; Minami et al., 2004
; Suzuki et al., 1993
). B. subtilis GGT has high activity towards
PGA, which seems to be absent in the E. coli and bovine kidney counterparts. Among 49 independent B. subtilis strains, 23 (47 %) were found to produce
PGA (unpublished results). The wide distribution of
PGA-producing strains and the results described herein (Figs 4 and 5
) favour the view that
PGA is a natural substrate of B. subtilis GGT. In minimal medium, B. subtilis strains produce about 1 mg
PGA ml1. However, because of the high molecular mass of
PGA (2x106 Da), molar concentrations of the polypeptide in the culture are around 0·5 µM. This concentration is far below the Km value (9·0 µM). To degrade
PGA by GGT, B. subtilis must fragment the polypeptide to increase the molar concentrations of substrate to nearer the Km value. The endo-type
PGA hydrolase inferred by this study appears to perform such fragmentation (Figs 3 and 4
). Degradation of
PGA by the endo-type enzyme yields 1x105 Da fragments at a concentration around 10 µM, which is appropriate for hydrolysis by GGT. When
PGA is completely hydrolysed by the combined action of the two hydrolytic enzymes, the total concentrations of D- and L-glutamate in the growth medium would reach about 8 mM. This concentration would represent a significant nitrogen source for B. subtilis in the stationary phase (Fig. 7
). During in vivo capsule degradation, GGT levels would be properly modulated through the negative control of ggt transcription by the product L-glutamate (Fig. 3
). This feedback regulation should prevent overdegradation of
PGA, and steadily supply B. subtilis cells with the required amounts of glutamate.
The ComQXPA quorum-sensing system plays a pivotal role in the mechanism through which B. subtilis adapts to nutrient starvation during the stationary phase (Lazazzera et al., 1999). This system monitors increasing cell population, and expresses an array of cellular processes, including exoenzyme production and flagellation, through which the cells cope with the nutrient shortage imposed by a dense cell population (Lazazzera et al., 1999
; Phillips & Strauch, 2002
). Exoenzymes enable the cells to utilize energetically less favourable polysaccharides, proteins or lipids, whereas flagella allow the cells to translocate to nutritionally favourable sites. Integration of capsule
PGA and GGT synthesis by B. subtilis into the regulatory circuit of the quorum-sensing system (Figs 3 and 6
; Tran et al., 2000
) enables them to fulfil their respective roles as an extracellular glutamate reserve and as a cognate degradation enzyme. Thus, B. subtilis can adapt to starvation during the stationary phase, not only by utilizing polymer nutrients in the environment or moving to other sites, but also by preserving nutrients as capsule
PGA.
The response regulator ComA of the quorum-sensing system either directly or indirectly expresses a set of genes that determine the stationary-phase-specific phenotypes (Lazazzera et al., 1999). This regulatory protein stimulates expression of the relevant genes through binding to specific sites having the consensus sequence 5'-TTGCGGNNNNCCGCAA-3' in the promoters (Lazazzera et al., 1999
). Neither the capBCD operon nor ggt has a ComA-binding site in its promoter, implying that the quorum-sensing system indirectly regulates the
PGA synthetic and degradation systems. Identification of the cascade pathways that transduce the quorum-sensing signal to the regulatory machineries of the
PGA synthetic and degradation enzyme genes would provide further insight into the regulatory mechanisms of capsule
PGA, a unique extracellular reserve of glutamate.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ashiuchi, M., Soda, K. & Misono, H. (1999). A poly--glutamate synthetic system of Bacillus subtilis IFO 3336: gene cloning and biochemical analysis of poly-
-glutamate produced by Escherichia coli clone cells. Biochem Biophys Res Commun 263, 612.[CrossRef][Medline]
Birrer, G. A., Cromwick, A.-M. & Gross, R. A. (1994). -Poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies. Int J Biol Macromol 16, 265275.[CrossRef][Medline]
Del Bello, B., Paolicchi, A., Comporti, M., Pompella, A. & Maellaro, E. (1999). Hydrogen peroxide produced during -glutamyl transpeptidase activity is involved in prevention of apoptosis and maintenance of proliferation in U937 cells. FASEB J 13, 6979.
Dubnau, D. (1999). DNA uptake in bacteria. Annu Rev Microbiol 53, 217244.[CrossRef][Medline]
Elsenhans, B., Ahmad, O. & Rosenberg, I. H. (1984). Isolation and characterization of pteroylglutamate hydrolase from rat intestinal mucosa. J Biol Chem 259, 63646368.
Guérout-Fleury, A.-M., Shazand, K., Frandsen, N. & Stragier, P. (1995). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335336.[CrossRef][Medline]
Guérout-Fleury, A.-M., Frandsen, N. & Stragier, P. (1996). Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 5761.[CrossRef][Medline]
Hanigan, M. H. & Ricketts, W. A. (1993). Extracellular glutathione is a source of cysteine for cells that express -glutamyl transpeptidase. Biochemistry 32, 63026306.[Medline]
Karp, D. R., Shimooku, K. & Lipsky, P. E. (2001). Expression of -glutamyl transpeptidase protects Ramos B cells from oxidation-induced cell death. J Biol Chem 276, 37983804.
Kimura, K. & Itoh, Y. (2003). Characterization of poly--glutamate hydrolase encoded by a bacteriophage genome: possible role in phage infection of Bacillus subtilis encapsulated with poly-
-glutamate. Appl Environ Microbiol 69, 24912497.
Kimura, K., Tran, L.-S. P. & Itoh, Y. (2004). Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis. Microbiology 150, 29112920.[CrossRef][Medline]
Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G. & 146 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[CrossRef][Medline]
Lazazzera, B. A., Palmer, T., Quisel, J. & Grossman, A. D. (1999). Cell density control of gene expression and development in Bacillus subtilis. In CellCell Signalling in Bacteria, pp. 2746. Edited by G. M. Dunny & S. C. Winans. Washington, DC: American Society for Microbiology.
Lieberman, M. W., Wiseman, A. L., Shi, Z. Z. & 10 other authors (1996). Growth retardation and cysteine deficiency in -glutamyl-transpeptidase-deficient mice. Proc Natl Acad Sci U S A 93, 79237926.
Makino, S., Sasakawa, C., Uchida, I., Terakado, N. & Yoshikawa, M. (1988). Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis. Mol Microbiol 2, 371376.[Medline]
Makino, S., Uchida, I., Terakado, N., Sasakawa, C. & Yoshikawa, M. (1989). Molecular characterization and protein analysis of the cap region, which is essential for encapsulation in Bacillus anthracis. J Bacteriol 171, 722730.[Medline]
Makino, S., Watarai, M., Cheun, H.-I., Shirahata, T. & Uchida, I. (2002). Effect of the low molecular capsule released from the cell surface of Bacillus anthracis on the pathogenesis of anthrax. J Infect Dis 186, 227233.[CrossRef][Medline]
Mehdi, K. & Penninckx, M. J. (1997). An important role for glutathione and -glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast Saccharomyces cerevisiae. Microbiology 143, 18851889.[Medline]
Meijer, W. J. J., de Boer, A. J., van Tongeren, S., Venema, G. & Bron, S. (1995). Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a novel type of replicon. Nucleic Acids Res 23, 32143223.[Abstract]
Meijer, W. J. J., Wisman, G. B. A., Terpstra, P., Thorsted, P. B., Thomas, C. M., Holsappel, S., Venema, G. & Bron, S. (1998). Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analysis of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparison with related plasmids from Gram-positive bacteria. FEMS Microbiol Rev 21, 337368.[CrossRef][Medline]
Minami, H., Suzuki, H. & Kumagai, H. (2003). Salt-tolerant -glutamyltranspeptidase from Bacillus subtilis 168 with glutaminase activity. Enzyme Microb Technol 32, 431438.[CrossRef]
Minami, H., Suzuki, H. & Kumagai, H. (2004). -Glutamyltranspeptidase, but not YwrD, is important in utilization of extracellular glutathione as a sulfur source in Bacillus subtilis. J Bacteriol 186, 12131214.
Nagai, T., Koguchi, K. & Itoh, Y. (1997). Chemical analysis of poly--glutamic acid produced by plasmid-free Bacillus subtilis (natto): evidence that plasmids are not involved in poly-
-glutamic acid production. J Gen Appl Microbiol 43, 139143.[Medline]
Nakada, Y. & Itoh, Y. (2002). Characterization and regulation of the gbuA gene, encoding guanidinobutyrase in the arginine dehydrogenase pathway of Pseudomonas aeruginosa PAO1. J Bacteriol 184, 33773384.
Ogawa, Y., Hosoyama, H., Hamano, M. & Motai, H. (1991). Purification and properties of -glutamyltranspeptidase from Bacillus subtilis (natto). Agric Biol Chem 55, 29712977.[Medline]
Ogawa, Y., Sugiura, D., Motai, H., Yuasa, K. & Tahara, Y. (1997). DNA sequencing of Bacillus subtilis (natto) NR-1 -glutamyltranspeptidase gene, ggt. Biosci Biotechnol Biochem 61, 15961600.[Medline]
Phillips, Z. E. & Strauch, M. A. (2002). Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol Life Sci 59, 392402.[CrossRef][Medline]
Rosenberg, I. & Saini, P. K. (1980). Folylpolyglutamate endopeptidase from chicken intestine: isolation with the aid of affinity chromatography. Methods Enzymol 66, 667670.[Medline]
Suzuki, T. & Tahara, Y. (2003). Characterization of the Bacillus subtilis ywtD gene, whose product is involved in -polyglutamic acid degradation. J Bacteriol 185, 23792382.
Suzuki, H., Kumagai, H. & Tochikura, T. (1986). -Glutamyltranspeptidase from Escherichia coli K-12: purification and properties. J Bacteriol 168, 13251331.[Medline]
Suzuki, H., Hashimoto, W. & Kumagai, H. (1993). Escherichia coli K-12 can utilize an exogenous -glutamyl peptide as an amino acid source, for which
-glutamyltranspeptidase is essential. J Bacteriol 175, 60386040.[Abstract]
Tanaka, T., Hiruta, O., Futamura, T., Uotani, K., Satoh, A., Taniguchi, M. & Oi, S. (1993). Purification and characterization of poly(-glutamic acid) hydrolase from a filamentous fungus, Myrothecium sp. TM-4222. Biosci Biotechnol Biochem 57, 21482153.
Tate, S. S. & Meister, A. (1981). -Glutamyl transpeptidase: catalytic, structural and functional aspects. Mol Cell Biochem 39, 357368.[Medline]
Tate, S. S. & Meister, A. (1985). -Glutamyl transpeptidase from kidney. Methods Enzymol 113, 400419.[Medline]
Thorne, C. B. (1993). Bacillus anthracis. In Bacillus subtilis and Other Gram-Positive Bacteria, pp. 113124. Edited by A. L. Sonenshein, J. A. Hock & R. Losick. Washington, DC: American Society for Microbiology.
Tran, L.-S. P., Nagai, T. & Itoh, Y. (2000). Divergent structure of the comQXPA quorum-sensing components: molecular basis of strain-specific communication mechanism in Bacillus subtilis. Mol Microbiol 37, 11591171.[CrossRef][Medline]
Uchida, I., Makino, S., Sasakawa, C., Yoshikawa, M., Sugimoto, C. & Terakado, N. (1993). Identification of a novel gene, dep, associated with depolymerization of the capsule polymer in Bacillus anthracis. Mol Microbiol 9, 487496.[Medline]
Urushibata, Y., Tokuyama, S. & Tahara, Y. (2002). Characterization of the Bacillus subtilis ywsC gene, involved in -polyglutamic acid production. J Bacteriol 184, 337343.
Vieira, J. & Messing, J. (1987). Production of single-stranded plasmid DNA. Methods Enzymol 153, 311.[Medline]
Xu, K. & Strauch, M. A. (1996). Identification, sequence, and expression of the gene encoding -glutamyltranspeptidase in Bacillus subtilis. J Bacteriol 178, 43194322.[Abstract]
Received 7 July 2004;
revised 17 August 2004;
accepted 16 September 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |