Background: Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated.
Results: AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC.
Conclusions: In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.
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http://dx.doi.org/10.1186/s12866-024-03231-6 | DOI Listing |
Elife
November 2024
Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Phage-derived peptidoglycan hydrolases (i.e. lysins) are considered promising alternatives to conventional antibiotics due to their direct peptidoglycan degradation activity and low risk of resistance development.
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October 2024
Faculty of Biotechnology and Genetic Engineering, Sylhet Agricultural University, Sylhet 3100, Bangladesh.
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November 2024
Department of Biological and Environmental Science, Nanoscience Center, University of Jyvaskyla, Jyväskylä, Finland.
Orchestrated action of peptidoglycan (PG) synthetases and hydrolases is vital for bacterial growth and viability. Although the function of several PG synthetases and hydrolases is well understood, the function, regulation, and mechanism of action of PG hydrolases characterised as lysostaphin-like endopeptidases have remained elusive. Many of these M23 family members can hydrolyse glycyl-glycine peptide bonds and show lytic activity against whose PG contains a pentaglycine bridge, but their exact substrate specificity and hydrolysed bonds are still vaguely determined.
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October 2024
INRAE, Université Paris-Saclay, AgroParisTech, Micalis Institute, Jouy-en-Josas, France.
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October 2024
Inserm, BRM (Bacterial RNAs and Medicine)-UMR_S 1230, Université de Rennes, 35000 Rennes, France
Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. In , SprC is an antivirulent -acting sRNA known to base-pair with the major autolysin mRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing, we looked for its sRNA-RNA interactome and identified 14 novel mRNA targets.
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