scholarly journals SwsB and SafA Are Required for CwlJ-Dependent Spore Germination in Bacillus subtilis

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Jeremy D. Amon ◽  
Akhilesh K. Yadav ◽  
Fernando H. Ramirez-Guadiana ◽  
Alexander J. Meeske ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Developmental Process ◽  
Protective Layer ◽  
Uncharacterized Protein ◽  
Content Type ◽  
Essential Step ◽  
Dormant Spore

ABSTRACT When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore’s proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro. While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities. IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.

scholarly journals Role of PhoP∼P in Transcriptional Regulation of Genes Involved in Cell Wall Anionic Polymer Biosynthesis in Bacillus subtilis

1998 ◽  
Vol 180 (15) ◽  
pp. 4007-4010 ◽  
Author(s):  
Ying Qi ◽  
F. Marion Hulett
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Acid Synthesis ◽  
In Vitro Transcription ◽  
Anionic Polymer ◽  
Transcription System ◽  
Teichuronic Acid

ABSTRACT tagA, tagD, and tuaA operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in Bacillus subtilis. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation oftuaA and the repression of tagA andtagD. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the tagA and tagD promoters and also to activate the transcription of the tuaA promoter.

scholarly journals An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Richard E. Wiemels ◽  
Stephanie M. Cech ◽  
Nikki M. Meyer ◽  
Caleb A. Burke ◽  
Andy Weiss ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Active Form ◽  
Ppiase Activity ◽  
Content Type ◽  
Associated Proteins

ABSTRACT Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus. IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

scholarly journals Bifunctional Malic/Malolactic Enzyme Provides a Novel Mechanism for NADPH-Balancing in Bacillus subtilis

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Manuel Hörl ◽  
Tobias Fuhrer ◽  
Nicola Zamboni
Keyword(s):  
Bacillus Subtilis ◽  
Metabolic Flux ◽  
Central Carbon Metabolism ◽  
Flux Analysis ◽  
Content Type ◽  
Malolactic Enzyme ◽  
First Time

ABSTRACT The redox cofactor NADPH is required as a reducing equivalent in about 100 anabolic reactions throughout metabolism. To ensure fitness under all conditions, the demand is fulfilled by a few dehydrogenases in central carbon metabolism that reduce NADP+ with electrons derived from the catabolism of nutrients. In the case of Bacillus subtilis growing on glucose, quantitative flux analyses indicate that NADPH production largely exceeds biosynthetic needs, suggesting a hitherto unknown mechanism for NADPH balancing. We investigated the role of the four malic enzymes present in B. subtilis that could bring about a metabolic cycle for transhydrogenation of NADPH into NADH. Using quantitative 13C metabolic flux analysis, we found that isoform YtsJ alone contributes to NADPH balancing in vivo and demonstrated relevant NADPH-oxidizing activity by YtsJ in vitro. To our surprise, we discovered that depending on NADPH, YtsJ switches activity from a pyruvate-producing malic enzyme to a lactate-generating malolactic enzyme. This switch in activity allows YtsJ to adaptively compensate for cellular NADPH over- and underproduction upon demand. Finally, NADPH-dependent bifunctional activity was also detected in the YtsJ homolog in Escherichia coli MaeB. Overall, our study extends the known redox cofactor balancing mechanisms by providing first-time evidence that the type of catalyzed reaction by an enzyme depends on metabolite abundance. IMPORTANCE A new mechanism for NADPH balancing was discovered in Bacillus subtilis. It pivots on the bifunctional enzyme YtsJ, which is known to catalyze NADP-dependent malate decarboxylation. We found that in the presence of excessive NADPH, the same enzyme switches to malolactic activity and creates a transhydrogenation cycle that ultimately converts NADPH to NADH. This provides a regulated mechanism to immediately adjust NADPH/NADP+ in response to instantaneous needs.

scholarly journals Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4276-4291 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Shonna M. McBride
Keyword(s):  
Clostridium Difficile ◽  
Hamster Model ◽  
Content Type ◽  
Gastrointestinal Pathogen ◽  
Dormant Spore ◽  
Sporulation Initiation ◽  
Spore Forming Bacteria

ABSTRACTThe anaerobic gastrointestinal pathogenClostridium difficilemust form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by whichC. difficileinitiates and controls the early stages of sporulation inC. difficileare not highly conserved in otherClostridiumorBacillusspecies. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation inC. difficile. These permeases are known to positively affect sporulation inBacillusspecies through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases inC. difficileresulted in the earlier expression of early sporulation genes and increased sporulationin vitro. Furthermore, disruption ofoppandappresulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model ofC. difficileinfection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence inC. difficilethan inBacillus subtilisand suggest that nutrient availability plays a significant role in pathogenesis and sporulationin vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation inC. difficile.

scholarly journals Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

2019 ◽  
Vol 201 (10) ◽  
Author(s):  
Benjamin D. Labbe ◽  
Cherisse L. Hall ◽  
Stephanie L. Kellogg ◽  
Yao Chen ◽  
Olivia Koehn ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Kinase Activity ◽  
Cell Envelope ◽  
Activation Loop ◽  
Intrinsic Resistance ◽  
Content Type

ABSTRACTTransmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP]andSer/Thr-associated) domains are ubiquitous amongActinobacteriaandFirmicutesspecies. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylationin vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood.Enterococcus faecalisis a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. InE. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreKin vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their functionin vivoandin vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activityin vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreKin vivoupon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activityin vivoto promote adaptation to cell wall stresses.IMPORTANCETransmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous amongActinobacteriaandFirmicutesspecies and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase ofEnterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistancein vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

scholarly journals Aag Hypoxanthine-DNA Glycosylase Is Synthesized in the Forespore Compartment and Involved in Counteracting the Genotoxic and Mutagenic Effects of Hypoxanthine and Alkylated Bases in DNA during Bacillus subtilis Sporulation

2016 ◽  
Vol 198 (24) ◽  
pp. 3345-3354 ◽  
Author(s):  
Víctor M. Ayala-García ◽  
Luz I. Valenzuela-García ◽  
Peter Setlow ◽  
Mario Pedraza-Reyes
Keyword(s):  
Bacillus Subtilis ◽  
Developmental Process ◽  
Specific Pattern ◽  
Dna Glycosylase ◽  
Regulatory Sequences ◽  
Regulation Of Expression ◽  
Content Type ◽  
Mutagenic Effects

ABSTRACTAag fromBacillus subtilishas been implicated inin vitroremoval of hypoxanthine and alkylated bases from DNA. The regulation of expression ofaaginB. subtilisand the resistance to genotoxic agents and mutagenic properties of an Aag-deficient strain were studied here. A strain with a transcriptionalaag-lacZfusion expressed low levels of β-galactosidase during growth and early sporulation but exhibited increased transcription during late stages of this developmental process. Notably,aag-lacZexpression was higher inside the forespore than in the mother cell compartment, and this expression was abolished in asigG-deficient background, suggesting a forespore-specific mechanism ofaagtranscription. Two additional findings supported this suggestion: (i) expression of anaag-yfpfusion was observed in the forespore, and (ii)in vivomapping of theaagtranscription start site revealed the existence of upstream regulatory sequences possessing homology to σG-dependent promoters. In comparison with the wild-type strain, disruption ofaagsignificantly reduced survival of sporulatingB. subtiliscells following nitrous acid or methyl methanesulfonate treatments, and the Rifrmutation frequency was significantly increased in anaagstrain. These results suggest that Aag protects the genome of developingB. subtilissporangia from the cytotoxic and genotoxic effects of base deamination and alkylation.IMPORTANCEIn this study, evidence is presented revealing thataag, encoding a DNA glycosylase implicated in processing of hypoxanthine and alkylated DNA bases, exhibits a forespore-specific pattern of gene expression duringB. subtilissporulation. Consistent with this spatiotemporal mode of expression, Aag was found to protect the sporulating cells of this microorganism from the noxious and mutagenic effects of base deamination and alkylation.

Role of kallikrein-kinin system in pathogenesis of bacterial cell wall-induced inflammation

1991 ◽  
Vol 260 (2) ◽  
pp. G213-G219 ◽  
Author(s):  
R. A. DeLa Cadena ◽  
K. J. Laskin ◽  
R. A. Pixley ◽  
R. B. Sartor ◽  
J. H. Schwab ◽  
...  
Keyword(s):  
Cell Wall ◽  
Acute Phase ◽  
Bacterial Cell ◽  
Chronic Phase ◽  
Bacterial Cell Wall ◽  
Kinin System ◽  
Kallikrein Kinin System ◽  
Bacterial Products

The plasma kallikrein-kinin system is activated in Gram-negative sepsis and typhoid fever, two diseases in which bacterial products have been shown to initiate inflammation. Because a single intraperitoneal injection of bacterial cell wall peptidoglycan-polysaccharide polymers from group A steptococci (PG-APS) into a Lewis rat produces a syndrome of relapsing polyarthritis and anemia, we investigated changes in the role of the kallikrein-kinin system in this model of inflammation. Coagulation studies after injection of PG-APS revealed an immediate and persistent decrease in prekallikrein levels. High-molecular-weight kininogen levels decreased significantly during the acute phase and correlated with the severity of arthritis. Factor XI levels were decreased only during the acute phase. Antithrombin III levels remained unchanged, indicating that neither decreased hepatic synthesis nor disseminated intravascular coagulation caused the decreased plasma contact factors. Plasma T-kininogen (an acute phase protein) was significantly elevated during the chronic phase. PG-APS failed to activate the contact system in vitro. Thus the kallikrein-kinin system plays an important role in this experimental model of inflammation, suggesting that activation of this system may play a role in the pathogenesis of inflammatory bowel disease and rheumatoid arthritis in which bacterial products might be etiologically important.

scholarly journals Promising Role of Calcium Hypochlorite as a Disinfectant: An in vitro Evaluation Regarding its Effect on Type V Dental Stone

2012 ◽  
Vol 13 (6) ◽  
pp. 856-866
Author(s):  
Prasanta Kumar Swain ◽  
Sharanbasappa C Nagaral ◽  
Pawan Kumar Kamalapurker ◽  
Ravishankar Damineni
Keyword(s):  
Tensile Strength ◽  
Bacillus Subtilis ◽  
Deleterious Effect ◽  
Strength Testing ◽  
Calcium Hypochlorite ◽  
Resistant Species ◽  
Type V ◽  
Mixing Water

ABSTRACT Aim The current study has been chosen to evaluate the efficacy of calcium hypochlorite as a disinfecting additive for the gypsum products and its effect on compressive and tensile strength of the set material. It is hypothesized that, the addition of calcium hypochlorite to type V dental stone in sufficient quantity to disinfect the material would have no deleterious effect on compressive or tensile strength. Materials and methods Total of 160 samples made up of type V dental stone were divided broadly into two groups of 80 samples each for the sake of compressive and tensile strength testing in dry and wet conditions: Out of each group, 10 samples without addition of any disinfectant (0% calcium hypochlorite) was compared with other group of 30 samples after adding disinfectant, i.e. each subgroup containing 10 samples each (0.5, 1.0 and 1.5% calcium hypochlorite). Conclusion Within limitations of this in vitro study it is assumed to prepare type V dental stone that contains a disinfectant, has adequate compressive strength and tensile strength, and can significantly act against a resistant species like Bacillus subtilis. Clinical significance When calcium hypochlorite was added to dental stone, extra mixing water was required to produce a material of nearly same pouring consistency. The samples, which were put to microbiological tests, showed effective action of disinfectant on Bacillus subtilis. No deleterious effect on compressive or tensile strength could be found after putting the selected samples with calcium hypochlorite. How to cite this article Swain PK, Nagaral SC, Kamalapurker PK, Damineni R. Promising Role of Calcium Hypochlorite as a Disinfectant: An in vitro Evaluation Regarding its Effect on Type V Dental Stone. J Contemp Dent Pract 2012;13(6):856-866.

scholarly journals Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Jonathan L. Portman ◽  
Qiongying Huang ◽  
Michelle L. Reniere ◽  
Anthony T. Iavarone ◽  
Daniel A. Portnoy
Keyword(s):  
Protein Function ◽  
Inhibitory Effect ◽  
Cysteine Residue ◽  
Infection Model ◽  
Listeriolysin O ◽  
Content Type ◽  
Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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