1887

Abstract

Mutants of serovar Typhimurium lacking DNA adenine (Dam) methylase show reduced secretion of invasion effectors encoded in the -pathogenicity island 1 (SPI-1). Concomitant with this alteration, a high number and quantity of extracellular proteins are detected in cultures of Dam mutants. This study shows by subcellular fractionation analysis that the presence of numerous extracellular proteins in cultures of Dam mutants is linked to an exacerbated release of membrane particulate material. The membrane ‘leaky’ phenotype and the impaired functionality of type III secretion systems were, however, unrelated since exacerbated release of proteins to the medium was evident in Dam strains carrying mutations in either SPI-1 (, ) or flagellar () genes. This result supports the view that Dam methylation controls a plethora of cellular processes. Electron microscopy analysis demonstrated that the accumulation of membrane particulate material occurs preferentially as vesicles in stationary cultures of Dam strains. In addition, a reduction in the relative amount of peptidoglycan-associated lipoprotein (PAL), TolB, OmpA and murein lipoprotein (Lpp) bound to peptidoglycan was observed in actively growing Dam mutants. The existence of an envelope defect was further confirmed by the increased sensitivity to deoxycholate exhibited by Dam mutants, mostly during exponential growth. Unexpectedly, lack of Dam methylation neither increased envelope instability nor impaired the association of PAL-Tol-Lpp proteins to the peptidoglycan in . Accordingly, Dam mutants did not show sensitivity to deoxycholate. Altogether, these results indicate that, besides its role in modulating the secretion of effectors by the SPI-1-encoded type III apparatus, Dam methylation controls cell envelope integrity in

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2002-04-01
2020-01-19
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