| Summary: | Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of <i>Lactobacillus paracasei</i>, GCRL 46, we observed temporal up-regulation of proteins in the 40–45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the <i>L. casei</i> group (<i>L. casei</i>, <i>L. paracasei</i> and <i>L. rhamnosus</i>) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan <span style="font-variant: small-caps;">d</span>,<span style="font-variant: small-caps;">l</span>-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of <i>L. rhamnosus</i> LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in <i>Enterococcus</i>, <i>Streptococcus</i> and <i>Bifidobacterium</i> species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the <i>L. casei</i> group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in <i>L. casei</i> is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.
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