Role of uricase in the triggering of germination of Bacillus fastidiosus spores.
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ABSTRACT:
SUBMITTER: Salas JA
PROVIDER: S-EPMC1145173 | biostudies-other | 1985 Jul
REPOSITORIES: biostudies-other
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Project description:The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ?sleB and ?ypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup.
| S-EPMC4187665 | biostudies-literature
Project description:BACKGROUND: The genome of Bacillus licheniformis DSM 13 harbours three neighbouring open reading frames showing protein sequence similarities to the proteins encoded from the Bacillus subtilis subsp. subtilis 168 gerA operon, GerAA, GerAB and GerAC. In B. subtilis, these proteins are assumed to form a germinant receptor involved in spore germination induced by the amino acid L-alanine. RESULTS: In this study we show that disruption of the gerAA gene in B. licheniformis MW3 hamper L-alanine and casein hydrolysate-triggered spore germination, measured by absorbance at 600 nm and confirmed by phase contrast microscopy. This ability was restored by complementation with a plasmid-borne copy of the gerA locus. Addition of D-alanine in the casein hydrolysate germination assay abolished germination of both B. licheniformis MW3 and the complementation mutant. Germination of both B. licheniformis MW3 and the gerA disruption mutant was induced by the non-nutrient germinant Ca2+-Dipicolinic acid. CONCLUSIONS: These results demonstrate that the B. licheniformis MW3 gerA locus is involved in germination induced by L-alanine and potentially other components present in casein hydrolysate.
| S-EPMC3359204 | biostudies-literature
Project description:Spore germination is the first step to Bacillus anthracis pathogenicity. Previous work has shown that B. anthracis spores use germination (Ger) receptors to recognize amino acids and nucleosides as germinants. Genetic analysis has putatively paired each individual Ger receptor with a specific germinant. However, Ger receptors seem to be able to partially compensate for each other and recognize alternative germinants. Using kinetic analysis of B. anthracis spores germinated with inosine and L-alanine, we previously determined kinetic parameters for this germination process and showed binding synergy between the cogerminants. In this work, we expanded our kinetic analysis to determine kinetic parameters and binding order for every B. anthracis spore germinant pair. Our results show that germinant binding can exhibit positive, neutral, or negative cooperativity. Furthermore, different germinants can bind spores by either a random or an ordered mechanism. Finally, simultaneous triggering of multiple germination pathways shows that germinants can either cooperate or interfere with each other during the spore germination process. We postulate that the complexity of germination responses may allow B. anthracis spores to respond to different environments by activating different germination pathways.
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Project description:Rates of commitment to germinate and germination of Bacillus subtilis spores with mixtures of low concentrations of germinants acting on different germinant receptors (GRs) were much higher than the sums of the rates of commitment and germination with individual germinants alone. This synergism with mixtures of nutrient germinants was not seen with spores lacking GRs responsible for recognizing one or several components of the germinant mixtures and was not eliminated by either a gerD mutation or overexpression of one of the GRs involved in this synergism. This synergism was also not seen between the germinant L-valine, which acts via a GR, and the germinant dodecylamine, which does not act via any GR. These results indicate that spores not only integrate but can also amplify signals from multiple germinants and multiple GRs in determining rates of commitment and overall spore germination. This amplification can be explained by a simple mechanism in which a single signal integrator triggers germination above an accumulation threshold. Direct cooperative action between GRs may further add to the synergism seen in germination triggered by multiple GRs. Further experiments and modeling are required to determine the relative contributions of these different mechanisms.
| S-EPMC3165675 | biostudies-literature
Project description:Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.
| S-EPMC3994143 | biostudies-other
Project description:Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.
| S-EPMC2687284 | biostudies-literature
Project description:Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed.
2010-11-01 | GSE19186 | GEO
Project description:Insertional inactivation of the yrbA gene of Bacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with L-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in various sig mutants indicated that yrbA was transcribed by RNA polymerase with sigma(E) beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the -35 (TCATAAC) and -10 (CATATGT) regions were similar to the consensus sequences of genes recognized by sigma(E). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination of yrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.
| S-EPMC93988 | biostudies-literature
Project description:BackgroundBacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis.ResultsConserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization.ConclusionsThe YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.
| S-EPMC6660665 | biostudies-literature
Project description:The effect of high NaCl concentrations on nutrient and nonnutrient germination of Bacillus subtilis spores was systematically investigated. Under all conditions, increasing NaCl concentrations caused increasing, albeit reversible, inhibition of germination. High salinity delayed and increased the heterogeneity of germination initiation, slowed the germination kinetics of individual spores and the whole spore population, and decreased the overall germination efficiency, as observed by a variety of different analytical techniques. Germination triggered by nutrients which interact with different germinant receptors (GRs) was affected differently by NaCl, suggesting that GRs are targets of NaCl inhibition. However, NaCl also inhibited GR-independent germination, suggesting that there is at least one additional target for NaCl inhibition. Strikingly, a portion of the spore population could initiate germination with l-alanine even at NaCl concentrations near saturation (∼5.4 M), suggesting that spores lack a salt-sensing system preventing them from germinating in a hostile high-salinity environment. Spores that initiated germination at very high NaCl concentrations excreted their large depot of Ca(2+)-pyridine-2,6-dicarboxylic acid and lost their heat resistance, but they remained in a phase-gray state in the phase-contrast microscope, suggesting that there was incomplete germination. However, some metabolic activity could be detected at up to 4.8 M NaCl. Overall, high salinity seems to exert complex effects on spore germination and outgrowth whose detailed elucidation in future investigations could give valuable insights on these processes in general.
| S-EPMC3911066 | biostudies-literature