Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Troiano, Anthony J.; Zhang, Jingqiao; Cowan, Ann E.; Ji, Yu; Setlow, Peter

doi:10.1128/jb.02274-14

Cited by 19 publications

(25 citation statements)

References 31 publications

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“…Our results support the first model whereby CspB, CspA, and CspC are in a germinosome complex – similar to the germinosome complex found in B. subtilis [53]. Recent studies on GerS have shown that GerS likely does not form a complex with other germination proteins [52, 54].…”

Section: Discussionsupporting

confidence: 86%

The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

Shrestha

Sorg

2018

Preprint

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13Clostridium difficile spore germination is critical for the transmission of disease. C. difficile 14 spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino 15 acids, such as glycine or alanine. Although the bile acid germinant receptor is known, the amino 16 acid germinant receptor has remained elusive. Here, we used EMS mutagenesis to generate 17 mutants with altered requirements for the amino acid co-germinant, similar to the strategy used 18 previously to identify the bile acid receptor, CspC. Surprisingly, we identified strains that do not 19 require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. 20 Upon sequencing these mutants, we identified different mutations in yabG. In C. difficile, yabG 21 expression is required for the processing of CspBA to CspB and CspA and preproSleC to 22 proSleC during spore formation. A defined yabG mutant exacerbated the EMS mutant 23 phenotype. Moreover, we found that various mutations in cspA caused spores to germinate in the 24 presence of TA alone without the requirement of an amino acid. Thus, our study provides 25 evidence that apart from regulating the CspC levels in the spore, CspA is important for 26 recognition of amino acids as co-germinants during C. difficile spore germination and that two 27 pseudoproteases (CspC and CspA) function as the C. difficile germinant receptors.28 86 inhibitory pro-peptide from proSleC, a spore cortex hydrolase that degrades the cortex 87 peptidoglycan, thereby activating the protein [38][39][40][41]. In C. difficile, CspB and CspA are encoded 88 by one ORF and the resulting protein is post-translationally processed into CspB and CspA and 89 then further processed by the sporulation specific protease, YabG [14,[42][43][44]. CspC is encoded 90 downstream of cspBA and is part of the same transcriptional unit. Interestingly, the catalytic 91 residues in CspA and CspC are lost, suggesting that only the CspB protein can process proSleC 92 to its active, cortex degrading form [37, 44]. Although present in the spore, and essential for C. 93 difficile spore germination, the CspA pseudoprotease has only been shown to regulate the 94 incorporation of CspC into the spore [42, 43]. 95In our working model for spore germination, activation of CspC by TA leads to the 96 activation of the CspB protein which cleaves proSleC into its active form. Activated SleC 97 degrades cortex and the core releases CaDPA in exchange for water by a mechanosensing 98 mechanism [45]. Because the receptor with which amino acids interact is unknown, we sought to 99 screen for chemically-generated mutants that have altered amino acid requirements during spore 100 germination [similar to the strategy used to identify the bile acid germinant receptor [37]]. Here, 101 we report that a mutation in yabG gene results in strains whose spores no longer respond to or 102 require amino acid co-germinants but respond to TA alone as a spore germinant. We hypothesize 103 that the misprocessing o...

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Section: Discussionsupporting

confidence: 86%

The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

Shrestha

Sorg

2018

Preprint

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“…2 ), an extension of this model is that CspC, CspB and CspA form a complex in dormant spores analogous to the B. subtilis germinosome signaling complex [72]. The CspB substrate SleC may also associate with this complex in mature spores, since CspC and CspB levels are reduced in a sleC mutant ([39], Fig.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain

2016

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Clostridium difficile is a spore-forming obligate anaerobe that is a leading cause of healthcare-associated infections. C. difficile infections begin when its metabolically dormant spores germinate in the gut of susceptible individuals. Binding of bile salt germinants to the Csp family pseudoprotease CspC triggers a proteolytic signaling cascade consisting of the Csp family protease CspB and the cortex hydrolase SleC. Conserved across the Clostridia, Csp proteases are subtilisin-like serine proteases that activate pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the protective cortex layer, allowing spores to resume metabolism and growth. This signaling pathway, however, is differentially regulated in C. difficile, since CspC functions both as a germinant receptor and regulator of CspB activity. CspB is also produced as a fusion to a catalytically inactive CspA domain that subsequently undergoes interdomain processing during spore formation. In this study, we investigated the role of the CspA pseudoprotease domain in regulating C. difficile spore germination. Mutational analyses revealed that the CspA domain controls CspC germinant receptor levels in mature spores and is required for optimal spore germination, particularly when CspA is fused to the CspB protease. During spore formation, the YabG protease separates these domains, although YabG itself is dispensable for germination. Bioinformatic analyses of Csp family members suggest that the CspC-regulated signaling pathway characterized in C. difficile is conserved in related Peptostreptococcaceae family members but not in the Clostridiaceae or Lachnospiraceae. Our results indicate that pseudoproteases play critical roles in regulating C. difficile spore germination and highlight that diverse mechanisms control spore germination in the Clostridia.

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“…Individual knockout of all ger operons in B. cereus ATCC 14579 failed to identify a role in germination for three (GerK, GerL and GerS) out of the seven Ger receptors, although all operons were transcribed during sporulation 16 . Clarification of the structure-function relationship of Ger receptors has also been hindered by the difficulty to isolate functional receptors, because they are membrane complexes composed of three subunits and associated with other proteins in a so-called germinosome complex 17 , 18 .…”

Section: Introductionmentioning

confidence: 99%

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

Clauwers

Lood

Bergh

et al. 2017

Sci Rep

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Clostridium botulinum is an anaerobic sporeforming bacterium that is notorious for producing a potent neurotoxin. Spores of C. botulinum can survive mild food processing treatments and subsequently germinate, multiply, produce toxin and cause botulism. Control of spore germination and outgrowth is therefore essential for the safety of mildly processed foods. However, little is known about the process of spore germination in group II C. botulinum (gIICb), which are a major concern in chilled foods because they are psychrotrophic. The classical model of spore germination states that germination is triggered by the binding of a germinant molecule to a cognate germinant receptor. Remarkably, unlike many other sporeformers, gIICb has only one predicted canonical germinant receptor although it responds to multiple germinants. Therefore, we deleted the gerBAC locus that encodes this germinant receptor to determine its role in germination. Surprisingly, the deletion did not affect germination by any of the nutrient germinants, nor by the non-nutrient dodecylamine. We conclude that one or more other, so far unidentified, germinant receptors must be responsible for nutrient induced germination in gIICb. Furthermore, the gerBAC locus was strongly conserved with intact open reading frames in 159 gIICb genomes, suggesting that it has nevertheless an important function.

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Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Cited by 19 publications

References 31 publications

The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

The requirement for the amino acid co-germinant duringC. difficilespore germination is influenced by mutations inyabGandcspA

Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain

Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219

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