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.

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 Virus Evolution toward Limited Dependence on Nonessential Functions of the Host: the Case of Bacteriophage SPP1

2014 ◽  
Vol 89 (5) ◽  
pp. 2875-2883 ◽  
Author(s):  
Virginija Cvirkaite-Krupovic ◽  
Rut Carballido-López ◽  
Paulo Tavares
Keyword(s):  
Bacillus Subtilis ◽  
Host Cell ◽  
Exponential Growth ◽  
Receptor Protein ◽  
Exponential Growth Phase ◽  
Exact Nature ◽  
Lytic Bacteriophage ◽  
Content Type ◽  
Cell Functions ◽  
Bacteriophage Infection

ABSTRACTAll viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if nonessentialBacillus subtilisgenes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2,514 single-gene knockouts of nonessentialB. subtilisgenes yielded only a few genes necessary for efficient SPP1 propagation. Among these were genes belonging to theyukoperon, which codes for the Esat-6-like secretion system, including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication was negatively affected by the absence of two other genes,putBandefp. The geneefpencodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline-containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection in the post-exponential growth phase ofB. subtilis, when the bacterium undergoes a complex genetic reprogramming. TheputBknockout shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment, whereB. subtilisrarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved toward limited dependence on nonessential host functions.IMPORTANCEA successful viral infection largely depends on the ability of the virus to hijack cellular machineries and to redirect the flow of building blocks and energy resources toward viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host cross talk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of nonessential genes of its host,Bacillus subtilis, with only a few cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant “evolutionary arms race” and might be much more widespread than currently thought.

scholarly journals Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Marina Borisova ◽  
Rosmarie Gaupp ◽  
Amanda Duckworth ◽  
Alexander Schneider ◽  
Désirée Dalügge ◽  
...  
Keyword(s):  
Cell Wall ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Model Organisms ◽  
Exponential Growth Phase ◽  
Rich Medium ◽  
Late Stationary Phase ◽  
Gram Positive ◽  
Term Survival ◽  
Gram Positive Bacteria

ABSTRACTPeptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms,Staphylococcus aureus,Bacillus subtilis, andStreptomyces coelicolor, all recycle the sugarN-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ inE. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P toN-acetylglucosamine-6-phosphate andd-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQcells ofS. aureusandB. subtilisrevealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQcultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQmutants ofS. aureusandB. subtilisshowed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival.IMPORTANCEThe peptidoglycan of the bacterial cell wall is turned over steadily during growth. As peptidoglycan fragments were found in large amounts in spent medium of exponentially growing Gram-positive bacteria, their ability to recycle these fragments has been questioned. We conclusively showed recycling of the peptidoglycan component MurNAc in different Gram-positive model organisms and revealed that a MurNAc-6P etherase (MurQ or MurQ ortholog) enzyme is required in this process. We further demonstrated that recycling occurs predominantly during the transition to stationary phase inS. aureusandB. subtilis, explaining why peptidoglycan fragments are found in the medium during exponential growth. We quantified the intracellular accumulation of recycling products in MurNAc-6P etherase gene mutants, revealing that about 5% and 10% of the MurNAc of the cell wall per generation is recycled inS. aureusandB. subtilis, respectively. Importantly, we showed that MurNAc recycling and salvaging does not sustain growth in these bacteria but is used to enhance survival during late stationary phase.

scholarly journals The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σVin Response to Lysozyme

2016 ◽  
Vol 84 (6) ◽  
pp. 1902-1916 ◽  
Author(s):  
Emily C. Woods ◽  
Kathryn L. Nawrocki ◽  
Jose M. Suárez ◽  
Shonna M. McBride
Keyword(s):  
Cell Wall ◽  
Clostridium Difficile ◽  
Sigma Factor ◽  
Intestinal Tract ◽  
Dependent Manner ◽  
Hamster Model ◽  
Content Type ◽  
Rapid Amplification ◽  
Extracytoplasmic Function Sigma Factor

Clostridium difficile(also known asPeptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary forC. difficileto cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yetC. difficileis able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition ofd-alanine to teichoic acids, is important forC. difficileresistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that adltnull mutant is severely attenuated for growth in lysozyme and that expression of thedltDABCoperon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σVdoes not inducedltexpression in response to lysozyme, indicating that σVis required for regulation of lysozyme-dependentd-alanylation of the cell wall. Using reporter gene fusions and 5′ RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independentdltexpression. In addition, we observed that both asigVmutant and adltmutant are more virulent in a hamster model of infection. These findings demonstrate that cell walld-alanylation inC. difficileis induced by lysozyme in a σV-dependent manner and that this pathway impacts virulencein vivo.

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.

Identification of transposon-inserted mutations including rnpB::Tn that abolished glucose induction of sigX encoding extracytoplasmic function-sigma factor in Bacillus subtilis

2021 ◽  
Author(s):  
Mitsuo Ogura
Keyword(s):  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Stringent Response ◽  
Rnase P ◽  
Glucose Induction ◽  
Extracytoplasmic Function Sigma Factor

Abstract We investigated the regulators of the glucose induction (GI) of the ECF-sigma genes sigX/M. During further screening of transposon-inserted mutants, we identified several regulators including an RNA component of RNase P (rnpB), which is required for tRNA maturation. A depletion of rnpB is known to trigger the stringent response. We showed evidence that the stringent response inhibited GI of sigX/M.

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 Regulation of the Bacillus subtilis Extracytoplasmic Function Protein σY and Its Target Promoters

2003 ◽  
Vol 185 (16) ◽  
pp. 4883-4890 ◽  
Author(s):  
Min Cao ◽  
Letal Salzberg ◽  
Ching Sung Tsai ◽  
Thorsten Mascher ◽  
Carla Bonilla ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Negative Regulation ◽  
Wild Type ◽  
Induced Mutations ◽  
Mutant Strains ◽  
Elevated Expression ◽  
Direct Target ◽  
Extracytoplasmic Function Sigma Factor ◽  
Target Promoters

ABSTRACT The Bacillus subtilis extracytoplasmic function sigma factor σY is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, PY. We selected for transposon-induced mutations that upregulate PY transcription in an attempt to identify genes involved in σY regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of PY. Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of σY activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for σY. This was supported by analysis of direct targets for σY transcription with whole-genome runoff transcription followed by macroarray analysis.

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.

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