Cell wall and autolytic properties of a cold-sensitive Listeria monocytogenes mutant deficient in branched-chain fatty acid biosynthesis.

摘要

Cellular membranes modulate several critical functions of bacterial physiology, including solute diffusion and nutrient transport. Biophysical properties of phospholipids, as well as environmental pressures such as temperature, can have a profound effect on these membrane functions and the kinetics of associated enzymes. Listeria monocytogenes is a facultative intracellular pathogen capable of robust low temperature growth, a characteristic thought to be facilitated by a membrane highly enriched in branched-chain fatty acids (BCFA). Mutants deficient in the biosynthesis of these fatty acids exhibit severe growth defects under low temperature and extreme pH conditions. Morphological investigations have also revealed these mutants to undergo aberrant cell division and separation, suggesting appropriate membrane fluidity may be integral to these processes. Proper cell division and bacterial morphology requires the coordinated activity of many membrane- and surface-associated proteins. In particular, cell wall structure, biosynthesis, and hydrolysis are integral components of this process, and the relationship between these molecular mechanisms and membrane fatty acid composition is poorly understood. This study seeks to elucidate potential determinants of aberrant cell division in the BCFA biosynthesis-deficient mutant, MOR401. Properties of interest include autolytic rates, susceptibility to lytic enzymes, peptidoglycan hydrolase profiles, and surface-associated protein profiles. No statistically significant alterations to autolytic rate were observed for whole cells exposed to Triton X-100, mutanolysin, or lysozyme, while EDTA-treated MOR401 was observed to undergo decreased autolysis relative to the wild-type. In contrast, crude cell walls of MOR401 were observed to lyse more rapidly than wild-type. Zymographic analyses reveal decreased extracellular concentrations of certain higher molecular weight enzymes in the MOR401 strain, as well as the absence of a ca. 32 kDa enzyme known to hydrolyze L. monocytogenes and Bacillus subtilis, but not Micrococcus luteus, cell walls. Non-covalent surface protein profiles show several bands of increased intensity in the MOR401 strain, while the covalently-attached surface proteins display a markedly distinct profile compared to the wild-type. Some of these altered properties may suggest alterations to cell wall chemistry, peptidoglycan hydrolase profiles, or reduced access of proteins to the extracellular space. To support future investigations, methods for the purification of cell wall and the extraction of teichoic acid and peptidoglycan have been refined. These procedures will facilitate the analysis of cell wall chemistry, structure, and macromolecular constituents.