Lipoteichoic acid synthesis and modifications during osmotic stress in Lactobacillus casei

PALOMINO MARIA MERCEDES; GRUNDLIG ANGELIKA; SANCHEZ -RIVAS, C; RUZAL SANDRA MONICA

Mar del Plata

Congreso; VIII SAMIGE; 2012

Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria. LTA consists of a glycerolphosphate (GroP) chain retained by a glycolipid anchor in the bacterial membrane, the GroP backbone chain is modified with D-alanine. In previous work we have described several modifications in cell wall of Lactobacillus casei when cells are exposed to an osmotic stress condition In the present work we focused on studying the structural alterations in LTA under high salt conditions (NaCl = 0. 8 M). Polymers extracted from high salt condition cultures migrate on SDS-PAGE faster than those from control condition. To gain insight into structural alterations, LTA architecture was analyzed by Nuclear Magnetic Resonance (NMR). For that purpose, it was first purified using a 1-butanol extraction method and further by hydrophobic interaction chromatography using an Octyl-Sepharose column. Based on the NMR analysis, we found a significative difference of the GroP chain length and the percentage of D-Alanine substitution in LTA from the high salt condition. NMR analysis from three independently isolated LTA samples showed an average of D-Ala substitution of 64?}4 and 27?}9 for the control and N condition respectively and an average of glycerolphosphate chain length of 42?}3 and 31?}4 for these same conditions. The differences in D-Ala substitution and in chain length are considered to be statically significant, with P value p=0,0039 and p= 0,0128 respectively. Gene expression studies of genes for D-Alanilation of LTA (dltA and dltC) showed a decrease in the transcriptional activity during growth in high salt condition, supporting the biochemichal results. Furthermore, yfnI gene present in this Lactobacillus strain, was in fact an ltaS orthologo responsible for polyglycerolphosphate LTA synthesis and we showed a decreased in its transcriptional activity during growth in high salt condition. Also, we determined that Lactobacillus casei LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. LtaS activity is Mn2+ ion dependent. Addition of Zn2+ and EDTA inhibited the enzyme activity even in the presence of Mn2+. The highest enzyme activity was seen at 100 mM NaCl, higher concentration had an inhibitory effect. In sum, these results show a modification in the LTA architecture when Lactobacillus casei cells are grown in high salt condition. We also demonstrated that the putative Lactobacillus casei YfnI protein is enzymatically active and is involved in LTA synthesis. ?@. For that purpose, it was first purified using a 1-butanol extraction method and further by hydrophobic interaction chromatography using an Octyl-Sepharose column. Based on the NMR analysis, we found a significative difference of the GroP chain length and the percentage of D-Alanine substitution in LTA from the high salt condition. NMR analysis from three independently isolated LTA samples showed an average of D-Ala substitution of 64?}4 and 27?}9 for the control and N condition respectively and an average of glycerolphosphate chain length of 42?}3 and 31?}4 for these same conditions. The differences in D-Ala substitution and in chain length are considered to be statically significant, with P value p=0,0039 and p= 0,0128 respectively. Gene expression studies of genes for D-Alanilation of LTA (dltA and dltC) showed a decrease in the transcriptional activity during growth in high salt condition, supporting the biochemichal results. Furthermore, yfnI gene present in this Lactobacillus strain, was in fact an ltaS orthologo responsible for polyglycerolphosphate LTA synthesis and we showed a decreased in its transcriptional activity during growth in high salt condition. Also, we determined that Lactobacillus casei LtaS hydrolyzes the glycerolphosphate head group of the membrane lipid phosphatidylglycerol (PG) and catalyzes the formation of the polyglycerolphosphate LTA backbone chain. LtaS activity is Mn2+ ion dependent. Addition of Zn2+ and EDTA inhibited the enzyme activity even in the presence of Mn2+. The highest enzyme activity was seen at 100 mM NaCl, higher concentration had an inhibitory effect. In sum, these results show a modification in the LTA architecture when Lactobacillus casei cells are grown in high salt condition. We also demonstrated that the putative Lactobacillus casei YfnI protein is enzymatically active and is involved in LTA synthesis. ?@ ?@