Abstract
Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.
Highlights
The,75-kDa (LGG_00324 or p75) and,40-kDa (LGG_00031 or p40) Major secreted proteins are the two most abundant proteins found in the spent culture supernatant of probiotic Lactobacillus rhamnosus GG (LGG)
The Msp1 and Msp2 proteins both show homology to cell wall hydrolases [1], the physiological function associated with such an enzymatic activity has not yet been elucidated in LGG
The cell wall of Gram-positive bacteria is mainly composed of a thick layer of peptidoglycan (PG), an amino-sugar polymer crosslinked with peptide bridges
Summary
The ,75-kDa (LGG_00324 or p75) and ,40-kDa (LGG_00031 or p40) Major secreted proteins are the two most abundant proteins found in the spent culture supernatant of probiotic Lactobacillus rhamnosus GG (LGG). The cell wall of Gram-positive bacteria is mainly composed of a thick layer of peptidoglycan (PG), an amino-sugar polymer crosslinked with peptide bridges. Cell wall hydrolases catalyze the cleavage of PG sugar or peptide chains Based on their cleavage specificities, cell wall hydrolases can be divided in N-acetyl muramoyl-L-alanine amidases, carboxypeptidases, endopeptidases, N-acetylglucosaminidases and N-acetylmuramidases [5]. These cell wall hydrolases play important roles in the regulation of cell wall growth, turnover and maintenance, and in the separation of daughter cells [6]. Besides their hydrolytic specificity, cell wall hydrolases differ by their site of action (e.g. at septum or whole cell wall) and timing of action (e.g. during cell division or during growth for turnover and maintenance) [6]
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