Role of SpoIVA ATPase Motifs during Clostridioides difficile Sporulation

The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins SpoIVA and SipL regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in the C. difficile SpoIVA ATPase motifs result in relatively minor defects in spore formation, in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in the SipL C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

; green arrows highlight immature phase-dark forespores; orange arrows highlight phase-gray sporelets, which look swollen and are surrounded by a phase-dark ring; purple arrows highlight phase-bright sporelets, which are swollen and surrounded by a phase-dark ring; yellow arrows mark mature phase-bright forespores (phase-brightness reflects cortex formation (7,8)); blue arrows highlight phase-bright free spores. Heat resistance efficiencies are based on 20-24 hr sporulating cultures and represent the mean and standard deviation for a given strain relative to wild type based on a minimum of three biological replicates. Statistical significance for all assays was determined relative to wild type using a one-way ANOVA and Tukey's test. Scale bar represents 5 µm. The limit of detection of the assay is 10 -6 . (B) Western blot analyses of SpoIVA and SipL. SpoIVA levels were quantified based on analyses of three biological replicates using (9). Statistical significance for all assays was determined relative to wild type using a one-way ANOVA and Tukey's test. No statistically significant differences were detected by western blotting using a one-way ANOVA and Tukey's test. Figure S3. Spore purifications of SpoIVA ATPase motif mutants. The "Input" sample was taken after cells were scraped off sporulation media and resuspended in ice-cold water. The "Purified" sample was taken after spores were washed repeatedly and purified on a density gradient. Pink arrows mark regions of probable coat attachments or "appendages" (https://www.biorxiv.org/content/10.1101/468637v1); orange arrows highlight phase-gray sporelets, which look swollen and are surrounded by a phase-dark ring; purple arrows highlight phase-bright sporelets, which are swollen and surrounded by a phase-dark ring.     (5,6); green arrows highlight immature phase-dark forespores; orange arrows highlight phase-gray sporelets, which look swollen and are surrounded by a phase-dark ring; purple arrows highlight phase-bright sporelets, which are swollen and surrounded by a phase-dark ring; yellow arrows mark mature phase-bright forespores (phase-brightness reflects cortex formation (7,8)); blue arrows highlight phase-bright free spores. Heat resistance efficiencies are based on 20-24 hr sporulating cultures and represent the mean and standard deviation for a given strain relative to wild type based on a minimum of three biological replicates. The limit of detection of the assay is 10 -6 . Statistical significance was determined relative to wild type using a one-way ANOVA and Tukey's test.
Supplementary Text S1 -E. coli strain construction pMTL-YN1C-spoIVA ATPase motif mutations. To clone the K35A spoIVA pyrE locus complementation construct, primer pair #2036 and 1483 was used to amplify the region 117 bp upstream of spoIVA up to the region around the K35 codon off C. difficile genomic DNA. Primer pair #1482 and 2037 was used to amplify the spoIVA gene starting from the region around the K35 codon through to the stop codon off C. difficile genomic DNA. Primers 1482 and 1483 encode the K35A mutation. The resulting PCR products were cloned into pMTL-YN1C digested with NotI and XhoI using Gibson assembly, although a SOE PCR step was sometimes employed to join the two spoIVA fragments together (10). The same procedure was used to clone the K35E, T75A, and D102A mutations except primer pairs #711 and 712; #1215 and 1216; and #1217 and 1218, respectively were used to introduce the indicated mutations.
pMTL-YN3-spoIVA ATPase motif mutations. Primer pair #1927 and 1483 were used to amplify the region 1043 bp upstream of the spoIVA gene through to the K35 codon off C. difficile genomic DNA. Primer pair #1482 and 1928 were used to amplify the region downstream of the K35 codon through to 975 bp downstream of the spoIVA gene off C. difficile genomic DNA, although a SOE PCR step was sometimes employed to join the two spoIVA fragments together (10). The PCR products resulting PCR products were cloned into pMTL-YN3 digested with AscI and SbfI using Gibson assembly.

pMTL-YN1C-mCherry-spoIVA ATPase motif mutations.
To generate a construct encoding mCherry fusions to SpoIVA ATPase motif mutants, primer pair #2036 and 1483 was used to amplify the promoter region of spoIVA, a codon-optimized mCherry gene up to the region around the K35 codon using pMTL-YN1C mCherry-spoIVA as the template (6). Primer pair #1482 and 2037 was used to amplify the spoIVA gene starting from the region around the K35 codon through to the stop codon off C. difficile genomic DNA. The resulting PCR products were gel purified and used in a PCR SOE reaction (10) to generate the mCherry-spoIVA ATPase motif mutant constructs, which were assembled into pMTL-YN1C using Gibson assembly. The same procedure was used to clone the K35E, T75A, and D102A mutations except primer pairs #711 and 712; #1215 and 1216; and #1217 and 1218, respectively were used to introduce the indicated mutations.
pKNT25-sipL. Primer pair #1453 and 1454 were used to amplify the sipL gene (without the stop codon) with BamHI and KpnI sites flanking the 5' and 3' ends of the gene. The resulting PCR product was digested with BamHI and KpnI then ligated into pKNT25 digested with the same enzymes (11). The ligation was transformed into DH5α. The resulting plasmid encodes sipL as an N-terminal fusion to the T25 fragment of adenylate cyclase. pUT18C-spoIVA. Primer pair #1452 and 1337 were used to amplify the spoIVA gene including the stop codon with BamHI and KpnI sites flanking the 5' and 3' ends of the gene. The resulting PCR product was digested with BamHI and KpnI then ligated into pUT18C digested with the same enzymes (11). The ligation was transformed into DH5α. The resulting plasmid encodes spoIVA as a C-terminal fusion to the T18 fragment of adenylate cyclase. pUT18C-spoIVA ATPase motif mutations. Primer pair #3064 and #3065 was used to amplify spoIVA encoding ATPase motif mutations off pMTL-YN1C spoIVA K35A, K35E, T75A, and D102A, respectively. The resulting PCR products were cloned into pUT18C digested with BamHI and KpnI.