Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

Abstract

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.

Pathways for phosphatidylcholine biosynthesis in bacteria

“…A similar conclusion was drawn from recent studies performed by Wilderman et al (2002). Earlier work on the methylation pathway of PC biosynthesis in Bradyrhizobium japonicum (Minder et al, 2001) suggested that besides a phospholipid N-methyltransferase (PmtA) catalysing efficiently the first methylation, and therefore the formation of MMPE, another phospholipid methyltransferase (PmtX) activity must exist in this organism which efficiently performs the second and third methylation required for PC formation via the methylation pathway. In the Bradyrhizobium japonicum genome sequence (Kaneko et al, 2002), two homologues (Bjap ORF1 and Bjap ORF2) of the rhodobacterial PmtA exist that could qualify as candidates for PmtX.…”

“…Studies with Sinorhizobium meliloti demonstrated that PC can be synthesized via two different routes, the PMT (de Rudder et al, 2000) pathway, represented by phospholipid Nmethyltransferase (PmtA) enzymes, or the PCS (de Rudder et al, 1999;Sohlenkamp et al, 2000) pathway. In recent years, numerous homologues of PmtA or Pcs have been described, but only a few have been evaluated experimentally (Hanada et al, 2001;Minder et al, 2001;Wilderman et al, 2002).…”

“…Expression of the ORF with the higher similarity to the sinorhizobial PmtA (mll4753), present in pFM20, caused the formation of MMPE, DMPE and PC (lane 5); therefore, this ORF encodes a Pmt (Mlot PmtA). Expression of the ORF with the lower similarity to the sinorhizobial PmtA (mlr5374), cloned in pFM18, might cause the formation of MMPE and possibly DMPE (lane 6) and, like the PmtA of Bradyrhizobium japonicum (Minder et al, 2001), the expressed ORF mlr5374 seems unable to form PC (lane 6). Expression of an ORF from L. pneumophila from pFM14 which encodes a homologue to the rhodobacterial PmtA leads to the formation of MMPE, DMPE and PC (lane 7), demonstrating that this ORF encodes a functional Pmt.…”

“…Recently, the gene encoding Pmt in Acetobacter aceti has been identified (Hanada et al, 2001) and its product belongs to the rhodobacterial PmtA family. A Pmt protein (PmtA) from Bradyrhizobium japonicum resembles the sinorhizobial PmtA but surprisingly the bradyrhizobial PmtA shows a peculiar substrate specificity as it seems to perform only the first methylation step efficiently, converting PE to MMPE (Minder et al, 2001).…”

Bradyrhizobium daqingense sp. nov., isolated from soybean nodules

“…Analysis of respiratory quinones and polar lipids have been suggested for characterization for prokaryotes (Tindall et al, 2010), but there are few publications on taxonomy of rhizobia (Choma & Komaniecka, 2003;Miller et al, 1990;Minder et al, 2001;Orgambide et al, 1993). The respiratory quinones for CCBAU 15774 T , B. liaoningense USDA 3622 T , B. yuanmingense CCBAU 10071 T , B. betae LMG 21987 T and B. japonicum USDA 6 T were analysed by HPLC as described by Lee et al (2001).…”