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Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis.

ABSTRACT: The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of Mycobacterium smegmatis mc(2)155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The mariner transposon insertion mapped to a GPL biosynthesis gene (atf1) which encodes a putative acetyltranferase involved in the transfer of acetyl groups to the glycopeptide core. Physical characterization of the GPLs from the atf1 mutant demonstrated that they were not acetylated.

SUBMITTER: Recht J

PROVIDER: S-EPMC95464 | biostudies-literature | 2001 Oct

REPOSITORIES: biostudies-literature

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Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis.

Recht J J Kolter R R

Journal of bacteriology 20011001 19

The absence of glycopeptidolipids (GPLs) abolishes the ability of mycobacteria both to slide over the surface of motility plates and to form biofilms on polyvinyl chloride. In a screen for biofilm-defective mutants of Mycobacterium smegmatis mc(2)155, a new mutant was obtained that resulted in partial inhibition of both processes and also showed an intermediate rough colony morphology. The mariner transposon insertion mapped to a GPL biosynthesis gene (atf1) which encodes a putative acetyltranfe ...[more]

PMID: 11544235

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Project description:Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG_5700 (rpfA), MSMEG_5439 (rpfB), MSMEG_4640 (rpfE2), and MSMEG_4643 (rpfE). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG_4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG_4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the ΔrpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules.IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis, a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis, encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance.

Dataset Information

Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis.

Publications

Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis.

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