Downloaded from www.microbiologyresearch.org by IP: 23.22.250.46 On: Tue, 23 Feb 2016 12:31:30 Osmotic stress adaptation in Lactobacillus casei BL23 leads to structural changes in the cell wall polymer lipoteichoic acid Maria Mercedes Palomino, 1,2 Mariana C. Allievi, 1 Angelika Gru ¨ ndling, 2 Carmen Sanchez-Rivas 1 and Sandra M. Ruzal 1 Correspondence Sandra M. Ruzal sandra@qb.fcen.uba.ar Received 17 June 2013 Accepted 4 September 2013 1 Departamento de Quı ´mica Biolo ´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, IQUIBICEN-CONICET, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina 2 Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, South Kensington Campus, London, UK The probiotic Gram-positive bacterium Lactobacillus casei BL23 is naturally confronted with salt- stress habitats. It has been previously reported that growth in high-salt medium, containing 0.8 M NaCl, leads to modifications in the cell envelope of this bacterium. In this study, we report that L. casei BL23 has an increased ability to form biofilms and to bind cations in high-salt conditions. This behaviour correlated with modifications of surface properties involving teichoic acids, which are important cell wall components. We also showed that, in these high-salt conditions, L. casei BL23 produces less of the cell wall polymer lipoteichoic acid (LTA), and that this anionic polymer has a shorter mean chain length and a lower level of D-alanyl-substitution. Analysis of the transcript levels of the dltABCD operon, encoding the enzymes required for the incorporation of D-alanine into anionic polymers, showed a 16-fold reduction in mRNA levels, which is consistent with a decrease in D-alanine substitutions on LTA. Furthermore, a 13-fold reduction in the transcript levels was observed for the gene LCABL_09330 coding for a putative LTA synthase. To provide further experimental evidence that LCABL_09330 is a true LTA synthase (LtaS) in L. casei BL23, the enzymic domain was cloned and expressed in E. coli. The purified protein was able to hydrolyse the membrane lipid phosphatidylglycerol as expected for an LTA synthase enzyme, and hence LCABL_09330 was renamed LtaS. The purified enzyme showed Mn 2+ -ion dependent activity, and its activity was modulated by differences in NaCl concentration. The decrease in both ltaS transcript levels and enzyme activity observed in high-salt conditions might influence the length of the LTA backbone chain. A putative function of the modified LTA structure is discussed that is compatible with the growth under salt-stress conditions and with the overall envelope modifications taking place during this stress condition. INTRODUCTION Lactobacilli are normal inhabitants of the human oral cavity and digestive tract where some strains play beneficial roles. They also are used in food fermentation processes and as probiotics in the dairy industry. In most of these habitats, lactobacilli are confronted with salt stress. We have previously reported that Lactobacillus casei BL23 shows alterations in the cell wall, when it is subjected to high-salt growth conditions (Piuri et al., 2005). We have observed that growth in the presence of 0.8 M NaCl increases its sensitivity to mutanolysin and lysozyme and antibiotics that target cell wall synthesis pathways, and also leads to increased sensitivity to positively charged anti- microbial peptides, such a nisin (Piuri et al., 2005). Some of these effects are likely to be a consequence of a decrease in peptidoglycan (PEPG) cross-linkage caused by the altered expression and activity of high and low molecular mass penicillin-binding proteins (PBPs) (Piuri et al., 2005; Palomino et al., 2009). In addition, osmotic stress induced by a high salt concentration has been reported to increase the autolytic activity and survival following lyophilization in Lactobacillus delbrueckii subsp. lactis (Koch et al., 2007). We have used these properties to develop a new electroporation protocol involving growth in high-salt medium as a cell- wall-weakening agent (Palomino et al., 2010). Besides PEPG, lactobacilli cell walls contain secondary cell wall polymers named teichoic acids (Debabov et al., 1996; Abbreviations: CW, total cell wall fraction; Gro-P, glycerolphosphate; LTA, lipoteichoic acid; LtaS, LTA synthase; PBP, penicillin-binding protein; PG, phosphatidylglycerol; PEPG, peptidoglycan; WTA, wall teichoic acid. Microbiology (2013), 159, 2416–2426 DOI 10.1099/mic.0.070607-0 2416 070607 G 2013 SGM Printed in Great Britain