form nitrogen-fixing root nodules on legume host plants, the idea ... Database searches with the deduced amino acid .... 4B, lane 5) in cell-free enzyme assays.
Molecular Microbiology (2001) 39(5), 1186±1198
Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant Alexander C. Minder,1 Karel E. E. de Rudder,2 Franz Narberhaus,1 Hans-Martin Fischer,1 Hauke Hennecke1 and Otto Geiger3* 1 Institut fuÈr Mikrobiologie, EidgenoÈssische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 ZuÈrich, Switzerland. 2 Institute of Biotechnology, Technical University Berlin, Seestrasse 13, D-13353 Berlin, Germany. 3 Centro de InvestigacioÂn sobre FijacioÂn de NitroÂgeno, Universidad Nacional AutoÂnoma de MeÂxico, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, MeÂxico. Summary Phosphatidycholine (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. PmtAdeficient 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 Accepted 15 December, 2000. *For correspondence. E-mail otto@cifn. unam.mx; Tel. (152) 73 131 697; Fax (152) 73 175 581. Q 2001 Blackwell Science Ltd
to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant. Introduction Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes, and it is essential for their survival. Eukaryotic organisms usually possess two alternative pathways for PC biosynthesis, the CDP-choline pathway and the methylation pathway (reviewed by Kent, 1995). In the CDP-choline pathway, choline is activated to choline phosphate and, subsequently, to CDP-choline, which condenses with diacylglycerol to obtain PC. In the methylation pathway, phosphatidylethanolamine (PE) is N-methylated three times by phospholipid N-methyltransferase (Pmt) using S-adenosylmethionine as the methyl donor in order to yield PC. In higher eukaryotes, such as humans or rats (Ridgway and Vance, 1987), only one phospholipid N-methyltransferase seems to be present performing all three methylation steps. Lower eukaryotes, such as yeast (Kodaki and Yamashita, 1987) or Neurospora (Crocken and Nyc, 1964), possess two different phospholipid N-methyltransferases with distinct substrate specificities. One phospholipid N-methyltransferase predominantly performs the first methylation step, converting PE to monomethylphosphatidylethanolamine (MMPE) and is therefore sometimes called phosphatidylethanolamine N-methyltransferase. A second phospholipid Nmethyltransferase predominantly performs the second and third methylation converting MMPE to dimethylphosphatidylethanolamine (DMPE) and further to PC. In bacteria, PC was thought to occur in only a few species, and it was speculated that, in such highly specialized bacteria, PC might fulfil a special function. In the case of members of the Rhizobiaceae that are able to form nitrogen-fixing root nodules on legume host plants, the idea was propagated that rhizobial PC might be of importance for a successful interaction with the eukaryotic host (Goldfine, 1982; Miller et al., 1990; Geiger, 1998). As no enzymatic activities of the CDP-choline pathway have been detected in Agrobacterium tumefaciens (Sherr and Law, 1965), it was generally believed that bacteria possess only the methylation pathway of PC biosynthesis (Vance and Ridgway, 1988). In fact, mutants of Rhodobacter sphaeroides and Zymomonas mobilis (bacteria
Phosphatidylcholine biosynthesis in Bradyrhizobium japonicum 1187 that normally contain PC as a membrane phospholipid) have been isolated that are deficient in Pmt and also lack PC (Arondel et al., 1993; Tahara et al., 1994). Surprisingly, PC-deficient mutants of Rhodobacter or Zymomonas are fully functional in their vegetative functions. In Sinorhizobium meliloti, a single gene coding for Pmt activity constitutes the methylation pathway (de Rudder et al., 2000). Apart from the methylation pathway, a second bacterial pathway for PC biosynthesis exists in this organism (de Rudder et al., 1997) involving the novel enzymatic activity, phosphatidylcholine synthase (Pcs), which forms PC directly from choline and CDP-diacylglycerol (de Rudder et al., 1999; Sohlenkamp et al., 2000). We have also shown that PC is required for normal growth of S. meliloti (de Rudder et al., 2000). Here, we describe the isolation of a gene from Bradyrhizobium japonicum that encodes a phospholipid N-methyltransferase predominantly performing the conversion from PE to MMPE. Inactivation of this gene led to significantly decreased PC synthesis in B. japonicum. Although the growth of such mutants is largely unaffected, we show here that, if normal PC biosynthesis is impaired, the symbiosis between Bradyrhizobium and its soybean host plant is inefficient.
Results Cloning of the B. japonicum pmtA gene Database searches with the deduced amino acid sequence of the recently found S. meliloti phospholipid N-methyltransferase gene pmtA (de Rudder et al., 2000) revealed a significant similarity (38% identical amino
acids) to the B. japonicum orf.65 product encoded 87 bp downstream of dnaJ (Minder et al., 1997; de Rudder et al., 2000). On the basis of this finding, the complete DNA sequence of the presumed B. japonicum pmtA gene was expected to be located on plasmid pRJ8162, which had been constructed during the characterization of the dnaKJ gene region (Fig. 1). DNA sequence analysis of a 1.1 kb EcoRI subfragment (insert of plasmid pRJ5565) revealed an open reading frame (ORF) of 600 nucleotides (position 838±1437 in Fig. 1). The corresponding gene product consists of 199 amino acids (Mr 21 955) and indeed shows significant similarity to the S. meliloti PmtA protein (34.2% identical amino acids). Based on this similarity and the enzymatic activity described below, the predicted B. japonicum ORF was designated pmtA. To address the question of whether there was more than one copy of the pmtA gene in the B. japonicum chromosome, as is the case for groESL (Fischer et al., 1993) and rpoH (Narberhaus et al., 1996), a bradyrhizobial pmtA probe was hybridized under low-stringency conditions (5 SSC, 578C) against B. japonicum chromosomal DNA digested with several different restriction enzymes. No evidence for additional pmtA-like genes was obtained. Nucleotide sequence of the pmtA gene region In order to get an overview of the genetic structure of the B. japonicum pmtA gene region, the DNA sequence of the 3.3 kb XhoI insert of plasmid pRJ8162 was established, continuing the previously described sequence of the dnaKJ gene region (Fig. 1; Minder et al., 1997). Three additional ORFs, all oriented as pmtA, were identified. Fig. 1. Physical map of the B. japonicum gene region containing the pmtA gene and the heat shock genes hrcA, grpE, dnaK and dnaJ. The blow-up below the physical map shows the insert of plasmids pRJ8162 and pRJ5565. Numbers indicate start and stop codon positions of ORFs, the transcription start site (horizontal arrow) and recognition sites of the following restriction enzymes: B, BamHI; E, EcoRI; K, KpnI; N, NotI; S, SalI; X, XhoI. The strategy for constructing the B. japonicum pmtA deletion strains 5569 and 5570 is indicated.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1186±1198
1188 A. C. Minder et al. The first ORF (orf192) starts 58 bp downstream of pmtA and codes for a putative protein of 192 amino acids (Mr 20 669), which shows significant similarity to the hypothetical 20.4 kDa YieF protein of E. coli (33.9% identical amino acids; accession number P31465). Only 7 bp downstream of orf192, a second ORF starts (orf237). Its gene product consists of 237 amino acids (Mr 24 673), and it exhibits a striking sequence similarity to orotidine5 0 -monophosphate decarboxylases (e.g. 42.3% identical amino acids compared with the pyrF gene product of Pseudomonas aeruginosa; Strych et al., 1994). The gene product of the third B. japonicum ORF (orf103; Mr 11 350) showed no significant similarity to any known protein sequence in the database. Interestingly, a similar chromosomal organization of pmtA with regard to an adjacent orf of unknown function (orf162) and a pyrF-homologous gene was also found in S. meliloti (de Rudder et al., 2000). However, the deduced gene products of B. japonicum orf192 and S. meliloti orf162 display no significant similarity.
Regulation of pmtA gene expression Recent findings indicate that membrane lipids may assist in protein folding and might therefore have chaperone function (Bogdanov and Dowhan, 1999). The localization of the B. japonicum pmtA gene just downstream of the heat shock-controlled dnaKJ chaperone operon prompted us to investigate the potential heat shock regulation of pmtA. Primer extension analysis with oligonucleotides complementary to the 5 0 end of pmtA revealed a single transcription start site 46 nucleotides upstream of the proposed translational start site of pmtA (Fig. 2A). The amount of pmtA transcript was reduced under heat shock conditions (3.7-fold decrease in reverse transcripts; Fig. 2A), and the putative pmtA promoter region shows significant similarity to the constitutive B. japonicum 235/ 210-type rrn promoter (Fig. 2B; Beck et al., 1997). We conclude that pmtA belongs to the class of housekeeping genes. Phospholipid N-methyltransferase activity measurements, based on the transfer of radiolabelled methyl groups to phospholipids, support the notion that PmtA is not a heat shock protein. In cell-free extracts of normally grown B. japonicum, a specific activity of 5.2 pmol mg21 protein min21 was determined, whereas only 1.4 pmol mg21 protein min21 was found when cellfree extracts were prepared from cells after heat shock. Part of this reduction in enzyme activity after heat shock might be explained by the reduced amount of mRNA formed. In addition, the bradyrhizobial phospholipid N-methyltransferase activity is not stable when incubated at 438C (data not shown), which is another
factor contributing to the much reduced phospholipid Nmethyltransferase activity after heat shock. Expression of the bradyrhizobial pmtA gene in E. coli In order to perform a functional analysis of the pmtA gene of B. japonicum, the gene was amplified by polymerase chain reaction (PCR), cloned into a pET3a expression vector to obtain plasmid pTB2117 and expressed in E. coli BL21 (DE3) by induction with IPTG as described in Experimental procedures. Analysis of protein extracts by SDS±PAGE (Fig. 3A) shows that, when the bradyrhizobial pmtA is expressed from pTB2117, a protein is formed that migrates according to a molecular mass of 22 kDa (Fig. 3A, lane 4). This value is in close agreement with the calculated mass of 21 955 Da for the bradyrhizobial pmtA gene product. Such a protein is not formed in E. coli BL21 (DE3) that does not contain pTB2117 (Fig. 3A, lane 1). From analogous constructs (de Rudder et al., 2000), the PmtA protein of S. meliloti (Fig. 3A, lane 2) or the PmtA protein of Rhodobacter sphaeroides (Fig. 3A, lane 3) can be readily expressed in E. coli BL21 (DE3). The lipid compositions of the respective E. coli cells were determined after in vivo labelling with [1-14C]-acetate in complex LB medium after 4 h of induction. Lipid extracts were separated by one-dimensional thin-layer chromatography (TLC) (Fig. 3B). E. coli BL21 (DE3) forms only PG, CL and PE as membrane phospholipids (Fig. 3B, lane 1). When the sinorhizobial pmtA was expressed in E. coli BL21 (DE3) pTB2084, three additional lipids (MMPE, DMPE and PC) were formed (Fig. 3B, lane 2), whereas only PC was produced as an additional lipid when the rhodobacterial pmtA gene was expressed in E. coli BL21 (DE3) pPMT-F. The `methylated intermediates' (MMPE or DMPE) of the methylation pathway of PC biosynthesis were not found in this background (Fig. 3B, lane 3). Surprisingly, when the bradyrhizobial pmtA was expressed in E. coli BL21 (DE3) pTB2117 (Fig. 3B, lane 4), the predominant lipid was MMPE. Some DMPE was produced, but only very minor amounts of PC. The fact that hardly any PE was detected in E. coli BL21 (DE3) pTB2117 suggests that the bradyrhizobial PmtA enzyme very efficiently performs the methylation reaction from PE to MMPE. Apparently, the subsequent methylations leading to DMPE and to PC are catalysed very inefficiently by this enzyme. Evidence for a second pathway of phospholipid Nmethyltransfer in B. japonicum An initial analysis of the membrane lipid composition of B. japonicum demonstrates that, in the strain 110spc4 (wild type), PC is by far the most prominent methylated PE derivative (Fig. 4A, lane 1). MMPE as well as DMPE Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1186±1198
Phosphatidylcholine biosynthesis in Bradyrhizobium japonicum 1189 Fig. 2. A. Determination of the transcription start sites of pmtA by primer extension mapping. Total RNA was isolated fromB. japonicum 110spc4 cells harvested before and 30 min after a heat shock from 308C to 438C. The extension and sequencing reactions (TCGA) were performed with the primer DnaK36. The transcription start site is marked with an arrow. B. Comparison of the deduced B. japonicum pmtA promoter sequence with the promoter of B. japonicum rrn (KuÈndig et al., 1995). Nucleotides matching the E. coli s70 consensus promoter (Lisser and Margalit, 1993) and transcriptional start sites are emphasized in bold letters.
5570 (Fig. 4B, lane 3) are also able to form all three methylated PE derivatives, again indicating that, in pmtAdeficient mutants, a second phospholipid N-methyltransferase activity is functional. However, the amounts of MMPE, DMPE and PC formed by extracts of 5569 (Fig. 4B, lane 2) or 5570 (Fig. 4B, lane 3) were reduced compared with the wild type (Fig. 4B, lane 1). The calculated specific phospholipid N-methyltransferase activities of 5569 were are about one-third of the activities found in the wild type (data not shown). If mutant 5569 was complemented with a broad-host-range plasmid containing the B. japonicum pmtA gene (pRJ5575), wildtype amounts of MMPE, DMPE and PC were formed (Fig. 4B, lane 5) in cell-free enzyme assays. As pmtA deletion mutants 5569 and 5570 were still able to form all three methylated derivatives of PE (MMPE, DMPE and PC), a detailed quantitative analysis of the lipid composition was performed after two-dimensional separation of the individual lipid extracts (Table 1). Whereas the relative amounts of anionic phospholipids (PG and CL) remained relatively constant in all B. japonicum strains, the relative amounts of the zwitterionic phospholipids (PE 1 MMPE versus PC) varied drastically.
are present only in trace amounts. Therefore, the lipid patterns of an E. coli strain producing bradyrhizobial PmtA (Fig. 3B) and of a B. japonicum wild type are strikingly different with regard to the relative amounts of the individual methylated PE derivatives. This obvious discrepancy can only be explained by postulating the presence of a second methylating enzyme or pathway involved in PC biosynthesis in B. japonicum. When lipids of bradyrhizobial pmtA deletion mutants 5569 (Fig. 4A, lane 2) and 5570 (Fig. 4A, lane 3) were analysed, all three methylated PE derivatives (MMPE, DMPE and PC) were still formed, although at much lower relative amounts than in the wild type. This also suggests that pmtA-deficient mutants possess an alternative methylation pathway for PC biosynthesis. Mutants 5569 and 5570 also formed MMPE, DMPE and PC after growth on HM minimal medium (data not shown) and therefore in the absence of choline. Assaying cell-free extracts for phospholipid N-methyltransferase activity demonstrates that all three methylated PE derivatives (MMPE, DMPE and PC) are formed by B. japonicum wild-type extracts (Fig. 4B, lane 1). Extracts of the pmtA-deficient mutants 5569 (Fig. 4B, lane 2) or
Table 1. Membrane lipid composition of B. japonicum pmtA mutants. Composition (% of total
14
C)
Lipid
Wild type 110spc4
5569
5570
5569 pRK290X
5569 pRJ5575
5569 pRJ5576
PG CL PE 1 MMPE DMPE PC
11.0 3.7 32.8 0.3 52.0
10.1 2.9 81.0 ± 5.6
11.3 2.3 79.3 ± 6.9
12.1 3.4 75.3 ± 6.6
10.9 4.2 36.0 0.2 48.8
12.0 5.6 36.1 0.5 44.9
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1186±1198
1190 A. C. Minder et al. Whereas in B. japonicum wild type, PC comprised 52% of all the membrane lipids, pmtA-deficient mutants showed much reduced relative amounts of PC (5.6% for 5569 and 6.9% for 5570). The relative amount of PC could be restored to wild-type levels (48.8%) by the introduction of the bradyrhizobial pmtA gene on a broad-host-range plasmid (Bj5569 pRJ5575) and close to wild-type levels (44.9%) by the introduction of the sinorhizobial pmtA gene on the same replicon (Bj5569 pRM5576). No complementation (6.6% PC) was achieved by the introduction of
the empty broad-host-range vector (pRK290X). These data clearly demonstrate that a deletion in the bradyrhizobial pmtA gene causes a greatly reduced rate of PC biosynthesis. Growth phenotype of B. japonicum pmtA deletion mutants In order to initially characterize the phenotype of pmtAdeficient B. japonicum mutants, their growth behaviour was monitored. Under aerobic growth conditions in rich medium (PSY medium), the generation time of the pmtA mutant strains 5569 and 5570 (about 8.7 h) seemed slightly elevated compared with the wild type (about 7.5 h). The generation times of mutants and wild type were indistinguishable (
