(D) Western blot analyses of IID and IIP in 630Δ erm, Δ 0A, and associated sporulation sigma factor mutants. Data were analyzed using a one-way ANOVA and Tukey’s test. Data represents the average of three independent biological replicates. The normalized transcript levels for each strain was compared to the normalized transcript level of spo0A –. Transcript levels were normalized to the housekeeping gene, rpoB, using the standard curve method. (C) qRT-PCR analyses of spoIID, spoIIP, and spoIIM transcription in JIR8094, spo0A:: ermB ( 0A –), and sporulation sigma factor mutants. Promoters are represented as bent arrows, and the sigma factor binding sites identified by Saujet et al. (B) Schematic of spoIID ( CD0126), spoIIP ( CD2469), and spoIIM ( CD1221) transcriptional units identified by RNA-Seq analyses (Fig. IID has lytic transglycoslase activity (pink triangles). IIP has both endopeptidase and amidase activity (scissors), both of which are shown in the schematic. Yellow hexagons (NAG) represent N-acetylglucosamine blue hexagons (NAM) represent N-acetylmuramic acid, purple circles represent L-alanine, pink circles represent D-glutamic acid, grey circles represent m-2,6-diaminopimelic acid (DAP) green circles represent D-alanine and the black line indicates cross-linking between DAP and D-ala residues. (A) Schematic of IID and IIP activities on peptidoglycan. © 2018 The Authors Molecular Microbiology Published by John Wiley & Sons Ltd. Collectively, these findings advance our understanding of several stages during infectious C. Lastly, engulfment mutants mislocalized polymerized coat even though the basement layer coat proteins, SpoIVA and SipL, remained associated with the forespore. Catalytic mutant complementation analyses revealed that these phenotypes depend on the enzymatic activities of SpoIIP and SpoIID, respectively. Loss of SpoIID and SpoIIQ also led to unique morphological phenotypes: asymmetric engulfment and forespore distortions, respectively. Interestingly, a small percentage of ∆spoIID and ∆spoIIQ cells generated heat-resistant spores through the actions of SpoIIQ and SpoIID, respectively. While SpoIID, SpoIIP and SpoIIQ were critical for engulfment, loss of SpoIIM minimally impacted C. Using genetic analyses, we determined the functions of these engulfment-related proteins along with the putative endopeptidase, SpoIIQ, during C. In Bacillus subtilis, engulfment is mediated by a peptidoglycan degradation complex consisting of SpoIID, SpoIIP and SpoIIM, which are all individually required for spore formation. Completion of this process depends on the mother cell engulfing the developing forespore, but little is known about how engulfment occurs in C. Spore formation is essential for the bacterial pathogen and obligate anaerobe, Clostridioides (Clostridium) difficile, to transmit disease.
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