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1.
Microbiology (Reading) ; 164(3): 277-286, 2018 03.
Article in English | MEDLINE | ID: mdl-29458655

ABSTRACT

Biopolymers on the cell surface are very important for protecting microorganisms from environmental stresses, as well as storing nutrients and minerals. Synthesis of biopolymers is well studied, while studies on the modification and degradation processes of biopolymers are limited. One of these biopolymers, poly-γ-glutamic acid (γ-PGA), is produced by Bacillus species. Bacillus subtilis PgdS, possessing three NlpC/P60 domains, hydrolyses γ-PGA. Here, we have demonstrated that several dl-endopeptidases with an NlpC/P60 domain (LytE, LytF, CwlS, CwlO, and CwlT) in B. subtilis digest not only an amide bond of d-γ-glutamyl-diaminopimelic acid in peptidoglycans but also linkages of γ-PGA produced by B. subtilis. The hydrolase activity of dl-endopeptidases towards γ-PGA was inhibited by IseA, which also inhibits their hydrolase activity towards peptidoglycans, while the hydrolysis of PgdS towards γ-PGA was not inhibited. PgdS hydrolysed only the d-/l-Glu‒d-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3) and l-Glu-rich γ-PGA (d-Glu:l-Glu=1 : 9), indicating that PgdS can hydrolyse only restricted substrates. On the other hand, the dl-endopeptidases in B. subtilis cleaved d-/l-Glu‒d-/l-Glu linkages of d-Glu-rich γ-PGA (d-Glu:l-Glu=7 : 3), indicating that these enzymes show different substrate specificities. Thus, the dl-endopeptidases digest γ-PGA more flexibly than PgdS, even though they are annotated as "dl-endopeptidase, digesting the d-γ-glutamyl-diaminopimelic acid linkage (d‒l amino acid bond)".


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Cell Wall/enzymology , Endopeptidases/metabolism , Hydrolases/metabolism , Polyglutamic Acid/analogs & derivatives , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Biopolymers/metabolism , Catalytic Domain , Cell Wall/metabolism , Endopeptidases/chemistry , Hydrolysis , Peptidoglycan/metabolism , Polyglutamic Acid/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Substrate Specificity
2.
Microbiology (Reading) ; 164(3): 299-307, 2018 03.
Article in English | MEDLINE | ID: mdl-29458657

ABSTRACT

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.


Subject(s)
Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Endopeptidases/metabolism , Peptidoglycan/metabolism , Bacillus subtilis/cytology , Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Catalytic Domain/genetics , Cell Division , Cell Wall/metabolism , Endopeptidases/chemistry , Endopeptidases/genetics , Peptidoglycan/biosynthesis , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/metabolism , Synthetic Lethal Mutations
3.
Methods Mol Biol ; 1440: 87-98, 2016.
Article in English | MEDLINE | ID: mdl-27311666

ABSTRACT

Zymography of cell wall hydrolases is a simple technique to specifically detect cell wall or peptidoglycan hydrolytic activity. The zymographic method can be used for assessing the hydrolytic activities of purified target proteins, cell surface proteins, and proteins secreted to culture. Here, methods of cell wall and peptidoglycan purification, extraction of cell surface proteins containing cell wall hydrolases, and zymographic analysis are described. The purified or extracted proteins are separated by electrophoresis using an SDS gel containing cell wall or peptidoglycan material and then the proteins are renatured in the gel. The renatured cell wall hydrolases in the gel hydrolyze the material around the proteins. The cell wall or peptidoglycan in the gel is stained by methylene blue and the hydrolyzed material cannot be stained, resulting in the detection of cell wall hydrolytic activities of the enzymes on the gel.


Subject(s)
Bacillus subtilis/enzymology , Peptidoglycan/metabolism , Bacterial Proteins/metabolism , Cell Wall/enzymology , Hydrolases/isolation & purification , Hydrolases/metabolism , Hydrolysis , Peptidoglycan/isolation & purification
4.
Microbiology (Reading) ; 160(Pt 12): 2639-2649, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25288647

ABSTRACT

Bacillus subtilis LytF plays a principal role in cell separation through its localization at the septa and poles on the vegetative cell surface. In this study, we found that a mutation in a major lipoteichoic acid (LTA) synthase gene--ltaS--results in a considerable reduction in the σ(D)-dependent transcription of lytF. The lytF transcription was also reduced in mutants that affected glycolipid anchor biosynthesis. Immunofluorescence microscopy revealed that both the numbers of cells expressing LytF and the LytF foci in these mutants were decreased. In addition, the transcriptional activity of lytF was almost abolished in the double (ltaS yfnI), triple (ltaS yfnI yqgS), and quadruple (ltaS yfnI yqgS yvgJ) mutants during vegetative growth. Cell separation defects in these mutants were partially restored with artificial expression of LytF. Interestingly, when lytF transcription was induced in the ltaS single or multiple mutants, LytF was localized not only at the septum, but also along the sidewall. The amounts of LytF bound to cell wall in the single (ltaS) and double (ltaS yfnI) mutants gradually increased as compared with that in the WT strain, and those in the triple (ltaS yfnI yqgS) and quadruple mutants were almost similar to that in the double mutant. Moreover, reduction of the lytF transcription and chained cell morphology in the ltaS mutant were completely restored with artificial induction of the yqgS gene. These results strongly suggest that LTA influences the temporal, σ(D)-dependent transcription of lytF and is an additional inhibitory component to the vegetative cell separation enzyme LytF.


Subject(s)
Bacillus subtilis/chemistry , Bacillus subtilis/enzymology , Bacterial Proteins/analysis , Endopeptidases/analysis , Gene Expression Regulation, Bacterial , Bacillus subtilis/genetics , Bacillus subtilis/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Division , Endopeptidases/genetics , Microscopy, Fluorescence , Sigma Factor/metabolism , Transcription, Genetic
5.
Biosci Biotechnol Biochem ; 78(8): 1428-34, 2014.
Article in English | MEDLINE | ID: mdl-25130749

ABSTRACT

The Gram-positive bacterium Bacillus subtilis has a thick cell wall. The cell wall contains various proteins, both for secretion and for peptidoglycan (PG) maintenance. Penicillin-binding proteins for PG synthesis, PG hydrolases (autolysins), and regulator proteins for the autolysins are the known components of the PG maintenance system. YqgA was identified as an abundant protein attached to the cell wall of B. subtilis through a proteomics analysis. The YqgA protein was localized at cell division sites during the transition period between the exponential and the stationary phases. YqgA localization was affected by mutations in the dl-endopeptidases (DLEPases), which are the autolysins involved in cell morphogenesis. Furthermore, yqgA mutations on a background of defective DLEPases led to delays in cell growth and cell morphological changes. These results demonstrate that yqgA is genetically related to the genes encoding DLEPases involved in cell morphogenesis.


Subject(s)
Bacillus subtilis/cytology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Cell Wall/metabolism , Endopeptidases/genetics , Sequence Homology, Nucleic Acid , Bacillus subtilis/enzymology , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Cell Proliferation , Gene Expression Regulation, Bacterial , Intracellular Space/metabolism , Mutation , Protein Transport
6.
J Biol Chem ; 287(53): 44736-48, 2012 Dec 28.
Article in English | MEDLINE | ID: mdl-23091053

ABSTRACT

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3(10)-helix, and eight ß-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between ß4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the ß4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the ß4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.


Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Protease Inhibitors/chemistry , Amino Acid Sequence , Bacillus subtilis/chemistry , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Molecular Sequence Data , Protease Inhibitors/metabolism , Protein Folding , Protein Structure, Secondary , Sequence Homology, Amino Acid
7.
J Biol Chem ; 287(13): 9765-9776, 2012 Mar 23.
Article in English | MEDLINE | ID: mdl-22277649

ABSTRACT

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Esterases/chemistry , Peptidoglycan/chemistry , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Esterases/genetics , Esterases/metabolism , Gene Deletion , Kinetics , Peptidoglycan/genetics , Peptidoglycan/metabolism , Sequence Homology, Amino Acid , Streptococcus pneumoniae/enzymology , Streptococcus pneumoniae/genetics
8.
J Bacteriol ; 194(4): 796-803, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22139507

ABSTRACT

Bacterial peptidoglycan acts as an exoskeleton to protect the bacterial cell. Although peptidoglycan biosynthesis by penicillin-binding proteins is well studied, few studies have described peptidoglycan disassembly, which is necessary for a dynamic structure that allows cell growth. In Bacillus subtilis, more than 35 genes encoding cell wall lytic enzymes have been identified; however, only two D,L-endopeptidases (lytE and cwlO) are involved in cell proliferation. In this study, we demonstrated that the D,L-endopeptidase activity at the lateral cell wall is essential for cell proliferation. Inactivation of LytE and CwlO by point mutation of the catalytic residues caused cell growth defects. However, the forced expression of LytF or CwlS, which are paralogs of LytE, did not suppress lytE cwlO synthetic lethality. Subcellular localization studies of these D,L-endopeptidases showed LytF and CwlS at the septa and poles, CwlO at the cylindrical part of the cell, and LytE at the septa and poles as well as the cylindrical part. Furthermore, construction of N-terminal and C-terminal domain-swapped enzymes of LytE, LytF, CwlS, and CwlO revealed that localization was dependent on the N-terminal domains. Only the chimeric proteins that were enzymatically active and localized to the sidewall were able to suppress the synthetic lethality, suggesting that the lack of D,L-endopeptidase activity at the cylindrical part of the cell leads to a growth defect. The functions of LytE and CwlO in cell morphogenesis were discussed.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Cell Wall/enzymology , Endopeptidases/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Bacillus subtilis/growth & development , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacteriolysis , Cell Wall/chemistry , Genes, Bacterial , Genotype , N-Acetylmuramoyl-L-alanine Amidase/biosynthesis , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/genetics , Peptidoglycan/metabolism , Point Mutation
9.
Biosci Biotechnol Biochem ; 75(6): 1119-28, 2011.
Article in English | MEDLINE | ID: mdl-21670523

ABSTRACT

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.


Subject(s)
Bacillus subtilis , Bacterial Proteins/metabolism , DNA, Intergenic/genetics , Gene Expression Regulation, Bacterial , Recombinant Fusion Proteins/metabolism , Spores, Bacterial , Amino Acid Sequence , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Blotting, Western , DNA, Intergenic/chemistry , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Molecular Sequence Data , Mutation , Plasmids , Polymerase Chain Reaction , Promoter Regions, Genetic , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/genetics , Spores, Bacterial/chemistry , Spores, Bacterial/genetics , Spores, Bacterial/metabolism , Transcription, Genetic , Transformation, Bacterial
10.
J Gen Appl Microbiol ; 57(1): 35-43, 2011.
Article in English | MEDLINE | ID: mdl-21478646

ABSTRACT

Poly-γ-glutamic acid (γPGA), a polymer of glutamic acid, is a component of the viscosity substance of natto, a traditional Japanese food made from soybeans fermented with Bacillus subtilis (natto). Here we investigate the effects of the cell wall lytic enzymes belonging to the D,L-endopeptidases (LytE, LytF, CwlO and CwlS) on γPGA production by B. subtilis (natto). γPGA levels in a cwlO disruptant were about twofold higher than that of the wild-type strain, whereas disruption of the lytE, lytF and cwlS genes had little effect on γPGA production. The molecular size of γPGA in the cwlO disruptant was larger than that of the wild-type strain. A complementary strain was constructed by insertion of the entire cwlO gene into the amyE locus of the CwlO mutant genome, and γPGA production was restored to wild-type levels in this complementary strain. These results indicated that the peptidoglycan degradation enzyme, CwlO, plays an important role in γPGA production and affects the molecular size of γPGA.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/metabolism , Cell Wall/metabolism , Glutamic Acid/metabolism , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Wall/enzymology , Cell Wall/genetics , Endopeptidases/genetics , Endopeptidases/metabolism , Fermentation/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Weight , Mutagenesis, Insertional , Mutation , N-Acetylmuramoyl-L-alanine Amidase/genetics , Peptidoglycan/metabolism , Polymers , Glycine max/chemistry , Glycine max/metabolism , Glycine max/microbiology , Time Factors
11.
J Biol Chem ; 285(53): 41232-43, 2010 Dec 31.
Article in English | MEDLINE | ID: mdl-20980266

ABSTRACT

For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-ß prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.


Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Muramidase/physiology , Peptidoglycan/chemistry , Cations , Chromatography, High Pressure Liquid/methods , Endopeptidases/chemistry , Mass Spectrometry/methods , Muramidase/chemistry , Muramidase/genetics , Plasmids/metabolism , Polymers/chemistry , Polysaccharides/chemistry , Prophages , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization/methods , Zinc/chemistry
12.
Biochem Biophys Res Commun ; 398(3): 606-12, 2010 Jul 30.
Article in English | MEDLINE | ID: mdl-20609359

ABSTRACT

CwlQ (previous YjbJ) is one of the putative cell wall hydrolases in Bacillus subtilis. Its domain has an amino acid sequence similar to the soluble-lytic transglycosylase (SLT) of Escherichia coli Slt70 and also goose lysozyme (muramidase). To characterize the enzyme, the domain of CwlQ was cloned and expressed in E. coli. The purified CwlQ protein exhibited cell wall hydrolytic activity. Surprisingly, RP-HPLC, mass spectrometry (MS), and MS/MS analyses showed that CwlQ produces two products, 1,6-anhydro-N-acetylmuramic acid and N-acetylmuramic acid, thus indicating that CwlQ is a bifunctional enzyme. The site-directed mutagenesis revealed that glutamic acid 85 (Glu-85) is an amino acid residue essential to both activities.


Subject(s)
Bacillus subtilis/enzymology , Glycosyltransferases/chemistry , Muramidase/chemistry , Catalytic Domain/genetics , Cloning, Molecular , Escherichia coli/genetics , Glutamic Acid/chemistry , Glutamic Acid/genetics , Glycosyltransferases/genetics , Muramidase/genetics , Mutagenesis, Site-Directed , Protein Structure, Tertiary
13.
Microbiology (Reading) ; 155(Pt 7): 2137-2147, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19389778

ABSTRACT

Bacillus subtilis Mrp is a unique Na+/H+ antiporter with a multicomponent structure consisting of the mrpABCDEFG gene products. We have previously reported that the conserved and putative membrane-embedded Glu-113, Glu-657, Asp-743 and Glu-747 of MrpA (ShaA) are essential for the transport function. In this study, we further investigated the functional involvement of the equivalent conserved acidic residues of other Mrp proteins in heterologous Escherichia coli and natural B. subtilis backgrounds. Asp-121 of MrpB and Glu-137 of MrpD were additionally identified to be essential for the transport function in both systems. Glu-137 of MrpD and Glu-113 of MrpA were found to be conserved in the homologous MrpD/MrpA proteins as well as in the homologous subunits of H+-translocating primary active transporters such as Nuo and Mbh, suggesting their critical role in ion binding. The remaining essential acidic residues clustered in the C-terminal domain of MrpA (Glu-657, Asp-743 and Glu-747) and MrpB (Asp-121); these subunits are fused in some Gram-negative species. It is possible that the MrpA, MrpB and MrpD subunits, which contain essential transmembrane acidic residues, form the ion translocation site(s) of the Mrp antiporter complex.


Subject(s)
Amino Acids, Acidic/chemistry , Bacillus subtilis/chemistry , Cell Membrane/chemistry , Sodium-Hydrogen Exchangers/chemistry , Amino Acid Sequence , Bacillus subtilis/metabolism , Bacillus subtilis/ultrastructure , Bacterial Proteins/chemistry , Conserved Sequence , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Genes, Bacterial , Molecular Sequence Data , Mutagenesis, Site-Directed , Sodium/metabolism , Sodium-Hydrogen Exchangers/metabolism
14.
Appl Microbiol Biotechnol ; 82(4): 741-8, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19205688

ABSTRACT

The effects of cortex-lysis related genes with the pdaA, sleB, and cwlD mutations of Bacillus subtilis (natto) NAFM5 on sporulation and germination were investigated. Single or double mutations did not prevent normal sporulation, but did affect germination. Germination was severely inhibited by the double mutation of sleB and cwlD. The quality of natto made with the sleB cwlD double mutant was tested, and the amounts of glutamic acid and ammonia were very similar to those in the wild type. The possibility of industrial development of natto containing a reduced number of viable spores is presented.


Subject(s)
Bacillus subtilis/physiology , Mutation , Soy Foods/microbiology , Spores, Bacterial/physiology , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Fermentation , Soy Foods/analysis , Spores, Bacterial/genetics
15.
Biosci Biotechnol Biochem ; 72(12): 3091-9, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19060408

ABSTRACT

Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 at 80 degrees C for Chi-A and Chi-C, and at pH 1.0 and 70 degrees C for Chi-B. Toward N-acetylchitooligosaccharide (GlcNAc(n)) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc(5) and GlcNAc(6) and produced GlcNAc(n) (n=2 to 4). GlcNAc(n) (n=3, 4) with the reducing end-side of beta anomer was detected in the hydrolysis products. These results indicate that the reactions of Chi-A, B, and C for GlcNAc(n) were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen beta-chitin more than crab shell or shrimp shell alpha-chitin.


Subject(s)
Chitinases/isolation & purification , Chitinases/metabolism , Chondrus/enzymology , Amino Acid Sequence , Chitinases/chemistry , Electrophoresis , Enzyme Stability , Hydrogen-Ion Concentration , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Solubility , Substrate Specificity , Temperature
16.
Mol Microbiol ; 70(1): 168-82, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18761694

ABSTRACT

Three D,L-endopeptidases, LytE, LytF and CwlS, are involved in the vegetative cell separation in Bacillus subtilis. A novel cell surface protein, IseA, inhibits the cell wall lytic activities of these d,l-endopeptidases in vitro, and IseA negatively regulates the cell separation enzymes at the post-translational level. Immunofluorescence microscopy indicated that the IseA-3xFLAG fusion protein was specifically localized at cell separation sites and poles on the vegetative cell surface in a similar manner of the d,l-endopeptidases. Furthermore, pull-down assay showed that IseA binds to the catalytic domain of LytF, indicating that IseA is localized on the cell surface through the catalytic domain of LytF. Overexpression of IseA caused a long-chained cell morphology in the exponential growth phase, indicating that IseA inhibits the cell separation D,L-endopeptidases in vivo. Besides, overexpression of IseA in a cwlO disruptant affected cell growth, implying that IseA is also involved in the cell elongation event. However, although IseA inhibits the activities of LytE, LytF, CwlS and CwlO in vitro, it is unlikely to inhibit CwlS and CwlO in vivo. This is the first demonstration that the cell separation event is post-translationally controlled through a direct interaction between cell separation enzymes and a specific novel inhibitor in bacteria.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Cell Wall/enzymology , Endopeptidases/metabolism , Protein Processing, Post-Translational , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Catalytic Domain , DNA, Bacterial/genetics , Enzyme Inhibitors/metabolism , Genes, Bacterial , Membrane Proteins/metabolism , Microscopy, Fluorescence , Plasmids
17.
Mol Microbiol ; 70(2): 297-310, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18761696

ABSTRACT

Cell separation in Bacillus subtilis depends on specific activities of DL-endopeptidases CwlS, LytF and LytE. Immunofluorescence microscopy (IFM) indicated that the localization of LytF depended on its N-terminal LysM domain. In addition, we revealed that the LysM domain efficiently binds to peptidoglycan (PG) prepared by chemically removing wall teichoic acids (WTAs) from the B. subtilis cell wall. Moreover, increasing amounts of the LysM domain bound to TagB- or TagO-depleted cell walls. These results strongly suggested that the LysM domain specifically binds to PG, and that the binding may be prevented by WTAs. IFM with TagB-, TagF- or TagO-reduced cells indicated that LytF-6xFLAG was observed not only at cell separation site and poles but also as a helical pattern along the sidewall. Moreover, we found that LytF was localizable on the whole cell surface in TagB-, TagF- or TagO-depleted cells. These results strongly suggest that WTAs inhibit the sidewall localization of LytF. Furthermore, the helical LytF localization was observed on the lateral cell surface in MreB-depleted cells, suggesting that cell wall modification by WTAs along the sidewall might be governed by an actin-like cytoskeleton homologue, MreB.


Subject(s)
Bacillus subtilis/enzymology , Cell Wall/chemistry , Cell Wall/enzymology , Endopeptidases/metabolism , Teichoic Acids/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Endopeptidases/genetics , Microscopy, Fluorescence , Microscopy, Immunoelectron , Peptidoglycan/metabolism , Protein Binding , Protein Structure, Tertiary , Teichoic Acids/genetics
18.
J Biol Chem ; 283(17): 11117-25, 2008 Apr 25.
Article in English | MEDLINE | ID: mdl-18305117

ABSTRACT

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to dl-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a dl-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."


Subject(s)
Bacillus subtilis/enzymology , Cell Wall/enzymology , Endopeptidases/physiology , Glycoside Hydrolases/physiology , Hydrolases/physiology , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/physiology , Amino Acid Sequence , Aspartic Acid/chemistry , Binding Sites , Endopeptidases/chemistry , Glutamic Acid/chemistry , Glycoside Hydrolases/chemistry , Hydrolases/chemistry , Mass Spectrometry/methods , Models, Biological , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid
19.
J Biosci Bioeng ; 104(2): 135-43, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17884659

ABSTRACT

In Bacillus subtilis, extracellular protease-deficient mutants have been used in attempts to increase the productivity of heterologous proteins. We detected protease activity of AprX using protease zymography in the culture medium at the late stationary growth phase. An alpha-amylase-A522-PreS2 hybrid protein, in which the PreS2 antigen of human hepatitis B virus (HBV) is fused with the N-terminal 522-amino-acid polypeptide of B. subtilis alpha-amylase, has been produced in multiple-protease-deficient mutants. The B. subtilis KA8AX strain, which is deficient in eight extracellular proteases and AprX, did not show the proteolysis of alpha-amylase-A522-PreS2 in the late stationary growth phase. Moreover, the production of alpha-amylase-A522-PreS2 was about 80 mg/l, which was eight times higher than that by the KA8AX strain previously reported. In addition, we showed the degradation of the heterologous protein by AprX that leaked to the culture medium (probably caused by cell lysis) during the late stationary growth phase.


Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/growth & development , Bacterial Proteins/metabolism , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolism , Bacillus subtilis/classification , Bacillus subtilis/genetics , Biodegradation, Environmental , Species Specificity
20.
J Bacteriol ; 189(20): 7511-4, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17693497

ABSTRACT

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na(+)/H(+) antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.


Subject(s)
Bacillus subtilis/metabolism , Membrane Transport Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Centrifugation , Electrophoretic Mobility Shift Assay , Membrane Transport Proteins/chemistry , Protein Binding , Protein Subunits/analysis , Sodium-Hydrogen Exchangers/chemistry
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