Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity

Abstract

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of <i>Bacillus subtilis</i> cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (<i>ywiE</i>, <i>ywnE</i> and <i>ywjE</i>) were obtained. Only the <i>ywnE</i> mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true <i>clsA</i> gene. The osmotolerance of a <i>clsA</i> mutant was impaired: although at NaCl concentrations lower than 1.2 M the growth curves were similar to those of its wild-type control, at 1 .5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the <i>clsA</i> mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the <i>clsA</i> mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and <i>clsA</i> mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the <i>clsA</i> mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and <i>clsA</i> mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled <i>pgsA</i> mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the <i>clsA</i> and <i>pgsA</i> genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of <i>B. subtilis</i> in media containing elevated NaCl concentrations.