The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

Abstract

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5?7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4?5) conditions.

The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

“…When we initiated this work, it was not known whether the gene products encoded by mprF homologs of gram-negative bacteria would catalyze the formation of LPG. The lpiA gene from R. tropici was cloned and expressed in the same way as the mprF homolog from B. subtilis, and mprF from S. aureus in a heterologous E. coli system (Oku et al 2004;Reeve et al 2006). Our data show that the expression of lpiA from R. tropici in E. coli leads to the formation of LPG.…”

“…E. coli does not possess genes homologous to lpiA/mprF and, therefore, can potentially be used for studying the functionality of lpiA/mprF gene products. Expression of S. aureus mprF in E. coli DH5α (Oku et al 2004) or of the mprF homolog of B. subtilis (Reeve et al 2006) in E. coli BL21(DE3)/pLysS leads to the formation of LPG (Fig. 1) (Fig.…”

“…When expressing R. tropici lpiA in E. coli, formation of LPG was observed, indicating that LpiA is able to catalyze formation of LPG, similarly to what has been described for MprF from S. aureus. The lpiA/atvA operon is induced upon acid shock in Sinorhizobium medicae (Reeve et al 1999(Reeve et al , 2006 and R. tropici CIAT899 (Vinuesa et al 2003), and it has been reported that the relative amounts of LPG increase in S. aureus and B. subtilis when grown in acidic conditions (Houtsmuller and van Deenen 1964;Op den Kamp et al 1969). Therefore, R. tropici CIAT899 wild type was grown in TY medium adjusted to a pH of 4.5, 5.5, 6.8, or 8.0, and lipids were labeled with [ 14 C]acetate.…”

“…This finding implies that LPG might play a role in interactions between bacteria and eukaryotic hosts. For some of the bacteria in which lpiA/mprF homologs are present, e.g., Lactobacillus plantarum, the formation of LPG has been described, whereas for others, such as Agrobacterium tumefaciens, Sinorhizobium meliloti , Sinorhizobium medicae (Reeve et al 2006), or Rhizobium tropici (Rojas-Jiménez et al 2005), LPG has not been described to form part of the membrane.…”

Rhizobial plasmids — replication, structure and biological role

“…The strain is of great agricultural importance because of its saprophytic competence in the acidic, infertile soils and its high effectiveness in nitrogen fixation with a broad range of annual medics of Mediterranean origin. It was shown that enhanced viability of cells exposed to lethal acid conditions depended on the pSmed02 located, acid-activated lpiA gene transcription, which was dispensable for normal cell growth [74].…”