scholarly journals Localization of the Vegetative Cell Wall Hydrolases LytC, LytE, and LytF on the Bacillus subtilis Cell Surface and Stability of These Enzymes to Cell Wall-Bound or Extracellular Proteases

2003 ◽  
Vol 185 (22) ◽  
pp. 6666-6677 ◽  
Author(s):  
Hiroki Yamamoto ◽  
Shin-ichirou Kurosawa ◽  
Junichi Sekiguchi
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Surface ◽  
Fusion Protein ◽  
Growth Phase ◽  
Vegetative Growth ◽  
Cell Separation ◽  
Vegetative Cell ◽  
Exponential Growth Phase ◽  
Cell Wall Hydrolases

ABSTRACT LytF, LytE, and LytC are vegetative cell wall hydrolases in Bacillus subtilis. Immunofluorescence microscopy showed that an epitope-tagged LytF fusion protein (LytF-3xFLAG) in the wild-type background strain was localized at cell separation sites and one of the cell poles of rod-shaped cells during vegetative growth. However, in a mutant lacking both the cell surface protease WprA and the extracellular protease Epr, the fusion protein was observed at both cell poles in addition to cell separation sites. This suggests that LytF is potentially localized at cell separation sites and both cell poles during vegetative growth and that WprA and Epr are involved in LytF degradation. The localization pattern of LytE-3xFLAG was very similar to that of LytF-3xFLAG during vegetative growth. However, especially in the early vegetative growth phase, there was a remarkable difference between the shape of cells expressing LytE-3xFLAG and the shape of cells expressing LytF-3xFLAG. In the case of LytF-3xFLAG, it seemed that the signals in normal rod-shaped cells were stronger than those in long-chain cells. In contrast, the reverse was found in the case of LytE-3xFLAG. This difference may reflect the dependence on different sigma factors for gene expression. The results support and extend the previous finding that LytF and LytE are cell-separating enzymes. On the other hand, we observed that cells producing LytC-3xFLAG are uniformly coated with the fusion protein after the middle of the exponential growth phase, which supports the suggestion that LytC is a major autolysin that is not associated with cell separation.

Induction of autolysis in Bacillus subtilis by ochratoxin A

1978 ◽  
Vol 24 (5) ◽  
pp. 563-568 ◽  
Author(s):  
U. Singer ◽  
R. Röschenthaler
Keyword(s):  
Cell Wall ◽  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Ochratoxin A ◽  
Optical Density ◽  
Growth Phase ◽  
Exponential Growth ◽  
Rna Synthesis ◽  
Exponential Growth Phase ◽  
Cell Wall Synthesis

Ochratoxin A (OTA) added during the exponential growth phase at a concentration higher than 12 μg/ml caused autolysis of Bacillus subtilis. Optical density of cultures decreased, and at higher concentrations the cultures became sterile. Optimum OTA-induced lysis was about pH 5. At concentrations below 10 μg/ml, protein synthesis was inhibited more strongly than RNA synthesis. Cell wall synthesis was also strongly inhibited. A fraction extracted from the lysates had the property of a lysis inhibitor. The relevance of this fraction in respect to autolysis is discussed.

scholarly journals Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

2000 ◽  
Vol 66 (8) ◽  
pp. 3174-3179 ◽  
Author(s):  
M. Carmen Martínez-Cuesta ◽  
Jan Kok ◽  
Elisabet Herranz ◽  
Carmen Peláez ◽  
Teresa Requena ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sodium Dodecyl Sulfate ◽  
Growth Phase ◽  
Cell Walls ◽  
Cell Damage ◽  
Sodium Dodecyl ◽  
Exponential Growth Phase ◽  
Cell Wall Degradation ◽  
Intracellular Proteins ◽  
Autolytic Activity

ABSTRACT The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus andLactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co2+ and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAΔ1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactisMG1363, or of L. lactis MG1363acmAΔ1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA.

scholarly journals Mutations in the lrpE Gene of Ralstonia solanacearum Affects Hrp Pili Production and Virulence

2006 ◽  
Vol 19 (8) ◽  
pp. 884-895 ◽  
Author(s):  
Yukio Murata ◽  
Naoyuki Tamura ◽  
Kazuhiro Nakaho ◽  
Takafumi Mukaihara
Keyword(s):  
Cell Surface ◽  
Growth Phase ◽  
Ralstonia Solanacearum ◽  
Vascular System ◽  
Exponential Growth Phase ◽  
Bacterial Cells ◽  
Wild Type ◽  
Bacterial Surface ◽  
Bacterial Morphology ◽  
In The Wild

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the ΔlrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the ΔlrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the ΔlrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.

scholarly journals Localization and expression of the Bacillus subtilis dl-endopeptidase LytF are influenced by mutations in LTA synthases and glycolipid anchor synthetic enzymes

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2639-2649 ◽  
Author(s):  
Yuuka Kiriyama ◽  
Kazuya Yazawa ◽  
Tatsuhito Tanaka ◽  
Ritsuko Yoshikawa ◽  
Hisaya Yamane ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Separation ◽  
Vegetative Cell ◽  
Transcriptional Activity ◽  
Immunofluorescence Microscopy ◽  
Lipoteichoic Acid ◽  
Synthetic Enzymes ◽  
Principal Role ◽  
Artificial Induction ◽  
Glycolipid Anchor

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 σD-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, σD-dependent transcription of lytF and is an additional inhibitory component to the vegetative cell separation enzyme LytF.

scholarly journals Regulation of a New Cell Wall Hydrolase Gene,cwlF, Which Affects Cell Separation in Bacillus subtilis

1998 ◽  
Vol 180 (9) ◽  
pp. 2549-2555 ◽  
Author(s):  
Shu Ishikawa ◽  
Yoshiko Hara ◽  
Ryo Ohnishi ◽  
Junichi Sekiguchi
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exponential Growth Phase ◽  
High Sequence Similarity ◽  
Cell Wall Hydrolase ◽  
Mutant Cells

ABSTRACT Bacillus subtilis produces a 35-kDa cell wall hydrolase, CwlF, during vegetative growth. The CwlF protein was extracted from B. subtilis cwlB sigD mutant cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequencing revealed that its sequence is completely identical to that of the internal region of thepapQ gene product. Disruption of the papQ gene in the B. subtilis chromosome led to the complete loss of CwlF, indicating that papQ is identical tocwlF. CwlF exhibits high sequence similarity to the p60 proteins of Listeria species, NlpC proteins ofEscherichia coli and Haemophilus influenzae, and Enp2 protein of Bacillus sphaericus. The β-galactosidase activity of the cwlF-lacZ transcriptional fusion and Northern blot analysis of the cwlF gene indicated that the gene is expressed as a monocistronic operon during the exponential growth phase, and primer extension analysis suggested that the cwlF gene is transcribed mainly by EςA RNA polymerase and weakly by EςH RNA polymerase. While the cells of the cwlF-deficient mutant were about twice as long as those of the wild-type strain, the cwlF sigD double mutant cells exhibited extraordinary microfiber formation, in contrast to the filamentation of the sigD mutant. The CwlF production was not affected by the pleiotropic mutationsflaD1 and degU32(Hy), which endow cells with the ability of extensive filamentation.

scholarly journals CcpA-Independent Regulation of Expression of the Mg2+-Citrate Transporter Gene citM by Arginine Metabolism in Bacillus subtilis

2003 ◽  
Vol 185 (3) ◽  
pp. 854-859 ◽  
Author(s):  
Jessica B. Warner ◽  
Christian Magni ◽  
Juke S. Lolkema
Keyword(s):  
Bacillus Subtilis ◽  
Growth Phase ◽  
Exponential Growth ◽  
Stationary Growth Phase ◽  
Luria Bertani ◽  
Transition Phase ◽  
Exponential Growth Phase ◽  
Uptake System ◽  
Regulation Of Expression ◽  
Citrate Transporter

ABSTRACT Transcriptional regulation of the Mg2+-citrate transporter, CitM, the main citrate uptake system of Bacillus subtilis, was studied during growth in rich medium. Citrate in the growth medium was required for induction under all growth conditions. In Luria-Bertani medium containing citrate, citM expression was completely repressed during the exponential growth phase, marginally expressed in the transition phase, and highly expressed in the stationary growth phase. The repression was relieved when the cells were grown in spent Luria-Bertani medium. The addition of a mixture of 18 amino acids restored repression. l-Arginine in the mixture appeared to be solely responsible for the repression, and ornithine appeared to be an equally potent repressor of citM expression. Studies of mutant strains deficient in RocR and SigL, proteins required for the expression of the enzymes of the arginase pathway, confirmed that uptake into the cell and, most likely, conversion of arginine to ornithine were required for repression. Arginine-mediated repression was independent of a functional CcpA, the global regulator protein in carbon catabolite repression (CCR). Nevertheless, CCR-mediated repression was the major mechanism controlling the expression during exponential growth, while the newly described, CcpA-independent arginine-mediated repression was specifically apparent during the transition phase of growth.

scholarly journals A New d,l-Endopeptidase Gene Product, YojL (Renamed CwlS), Plays a Role in Cell Separation with LytE and LytF in Bacillus subtilis

2006 ◽  
Vol 188 (15) ◽  
pp. 5541-5550 ◽  
Author(s):  
Tatsuya Fukushima ◽  
Anahita Afkham ◽  
Shin-ichirou Kurosawa ◽  
Taichi Tanabe ◽  
Hiroki Yamamoto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Separation ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Diaminopimelic Acid ◽  
Amino Acid Sequence Similarity ◽  
Northern Hybridization ◽  
Triple Mutant ◽  
High Amino Acid

ABSTRACT A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of d-γ-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. β-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.

scholarly journals An Exhaustive Multiple Knockout Approach to Understanding Cell Wall Hydrolase Function in Bacillus subtilis

2021 ◽  
Author(s):  
Sean Wilson ◽  
Ethan Garner
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Growth ◽  
Bond Cleavage ◽  
Normal Growth ◽  
Bacterial Cells ◽  
Single Strain ◽  
Cell Wall Hydrolase ◽  
Cell Wall Hydrolases ◽  
Synthetically Lethal

ABSTRACTMost bacteria are surrounded by their cell wall, a highly crosslinked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate cell wall hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “Δ40” strain shows a normal growth rate, indicating that none of the 40 hydrolases are necessary for cell growth. The Δ40 strain does not shed old cell wall, demonstrating that turnover is not essential for growth.The remaining two hydrolases in the Δ40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be knocked out in Δ40, indicating that either hydrolase alone is sufficient for cell growth. Environmental screening and zymography revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results demonstrate that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone is sufficient for this function. These experiments introduce the Δ40 strain as a tool to study hydrolase activity and regulation in B. subtilis.IMPORTANCEIn order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall – cell wall hydrolases – has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the Δ40 strain is a research tool to characterize hydrolases, using it to identify 3 ‘helper’ hydrolases that act in certain stress conditions.

scholarly journals SigM, an Extracytoplasmic Function Sigma Factor of Bacillus subtilis, Is Activated in Response to Cell Wall Antibiotics, Ethanol, Heat, Acid, and Superoxide Stress

2003 ◽  
Vol 185 (12) ◽  
pp. 3491-3498 ◽  
Author(s):  
Penny D. Thackray ◽  
Anne Moir
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Exponential Growth ◽  
Sigma Factor ◽  
Promoter Region ◽  
Normal Cell ◽  
Tween 20 ◽  
Exponential Growth Phase ◽  
Extracytoplasmic Function Sigma Factor ◽  
Triton X

ABSTRACT The extracytoplasmic function sigma M of Bacillus subtilis is required for normal cell growth under salt stress. It is expressed maximally during exponential growth and is further induced by the addition of 0.7 M NaCl. The promoter region of the sigM operon contains two promoters; one (PA) is sigma A dependent, and the other (PM) is sigma M dependent. These have been placed separately at the amy locus, directing expression of a lacZ reporter gene. Only the PM fusion responded to salt induction. This promoter, which was responsive to the level of active sigma M in the cell, was also induced by 5% ethanol, by vancomycin, bacitracin, or phosphomycin (inhibitors of cell wall biosynthesis; 2 μg per ml), and by heat shock of 50°C for 10 min. It was very strongly induced by acid (pH 4.3) and 80 μM paraquat, but after a 15- to 30-min delay. There was no induction by alkali (pH 9), 5 mM H2O2, the detergents 0.1% Triton X-100 and 0.1% Tween 20, or 50 μM monensin. In addition to their reduced tolerance to salt, null mutants of sigM were unable to grow at pH 4.3 and lysed after exposure to 5% ethanol. Genes regulated by SigM were also tested for their response to pH 4.3, 5% ethanol, and 2 μg of vancomycin per ml. Expression of the genes may have been activated by increased levels of sigma M, but at least some were also subject to additional controls, as they responded to one type of stress but not another. Expression of yrhJ, which encodes a cytochrome P450/NADPH reductase, was induced in response to acid and vancomycin. yraA expression was acid, ethanol, and vancomycin induced, whereas yjbD showed only ethanol induction. YraA protein was extremely important to acid survival—a mutation in yraA, like a sigM mutation, resulted in the failure of B. subtilis to grow at pH 4.3. Sigma M is therefore involved in maintaining membrane and cell wall integrity in response to several different stresses in exponential growth phase and is activated by such stresses.

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