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Journal of Bacteriology, January 2008, p. 571-580, Vol. 190, No. 2
0021-9193/08/$08.00+0     doi:10.1128/JB.01423-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Multiple Phospholipid N-Methyltransferases with Distinct Substrate Specificities Are Encoded in Bradyrhizobium japonicum{triangledown}

Stephanie Hacker,1,{dagger} Christian Sohlenkamp,2,{dagger} Meriyem Aktas,1 Otto Geiger,2 and Franz Narberhaus1*

Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, Bochum, Germany,1 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México2

Received 3 September 2007/ Accepted 26 October 2007


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ABSTRACT
 
Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.


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INTRODUCTION
 
Phosphatidylcholine (PC) or lecithin is one of the major structural constituents of eukaryotic membranes (10). In contrast, only ca. 10% of all bacterial species are predicted to produce PC (39). Eukaryotes synthesize PC by two alternative pathways: the CDP-choline pathway or the methylation pathway (22). In the CDP-choline pathway, also known as the Kennedy pathway, free choline is converted to PC via choline-phosphate and CDP-choline as intermediates (21). In the methylation pathway, PC is formed by three successive methylations of phosphatidylethanolamine (PE) via the intermediates monomethylphosphatidylethanolamine (MMPE) and dimethylphosphatidylethanolamine (DMPE) using the methyl donor S-adenosyl-L-methionine (SAM). In higher eukaryotes, such as human or rats, a single phospholipid N-methyltransferase (Pmt) performs all three methylation reactions (36). Lower eukaryotes, such as yeast or Neurospora, possess two different Pmts with distinct substrate specificities (6, 24). One Pmt predominantly catalyzes the first methylation step, converting PE to MMPE, and is therefore referred to as phosphatidylethanolamine N-methyltransferase. A second Pmt mainly catalyzes the second and third methylation steps from MMPE via DMPE to PC.

Prokaryotes were originally believed to use only the methylation pathway for PC biosynthesis (43). Indeed, mutants of Rhodobacter sphaeroides and Zymomonas mobilis deficient in Pmt were unable to form PC (1a, 41). However, an alternative pathway in which choline is directly condensed with CDP-diacylglycerol (CDP-DAG) by the PC synthase (Pcs) was later discovered in the legume symbiont Sinorhizobium meliloti (38). Similar activities have been demonstrated in Pseudomonas aeruginosa, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti, Legionella pneumophila, Agrobacterium tumefaciens, and Brucella abortus (4, 26, 45, 46). Most intriguingly, evidence for a eukaryotic-like CDP-choline pathway was found in Treponema denticola (23), indicating that prokaryotic PC biosynthesis is very diverse.

The lipid composition of biological membranes defines their stability and integrity (34). It undergoes adaptational changes in response to environmental conditions, such as oxygen tension, temperature, salinity, or medium composition (27, 42). Recently, a fundamental role of PC during infection of eukaryotic hosts has been recognized. A B. japonicum mutant with decreased PC levels was unable to establish an efficient symbiosis with its soybean host, although vegetative growth was largely unaffected (30). An S. meliloti mutant unable to form PC failed to establish a proper symbiosis with its host (8, 39). PC is also required for virulence of the plant pathogen A. tumefaciens (45). Important human pathogens such as P. aeruginosa, L. pneumophila, Borrelia burgdorferi, and Brucella melitensis are also known to possess PC (26, 44, 46). Interestingly, it has been reported recently that PC is necessary for the full virulence of Brucella abortus (4, 5). This clearly supports the earlier hypothesis that PC might play an important role in many host-microbe interactions (14, 39).

Since a B. japonicum pmtA mutant was still able to produce reduced amounts of MMPE, DMPE, and PC, at least one additional Pmt (PmtX) was predicted in this organism (30). In the present study, we set out to determine the complete PC biosynthesis inventory of B. japonicum. We describe a Pcs and multiple Pmt enzymes, that have distinct substrate specificities.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were routinely grown at 37°C in Luria-Bertani (LB) medium (29) supplemented with ampicillin (200 µg ml–1), kanamycin (50 µg ml–1), chloramphenicol (50 µg ml–1), or tetracycline (10 µg ml–1) if required. B. japonicum strains were propagated aerobically at 30°C in PSY complex medium (35) supplemented with 0.1% (wt/vol) L-arabinose. Anaerobic cultures were grown in yeast extract-mannitol (YEM) medium supplemented with 10 mM KNO3 (7) under nitrogen atmosphere. If appropriate, antibiotics were added at the following concentrations) to B. japonicum cultures: chloramphenicol, 20 µg ml–1 (for counterselection against E. coli donor strains); kanamycin, 100 µg ml–1 (solid media) and 50 µg ml–1 (liquid media); spectinomycin, 100 µg ml–1; streptomycin, 50 µg ml–1; and tetracycline, 50 µg ml–1 (solid media) and 30 µg ml–1 (liquid media).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Routine DNA work and sequence analysis. Recombinant DNA work and Southern blotting were performed according to standard protocols (37). Probes for Southern blot hybridizations were generated by PCR and labeled with digoxigenin according to manufacturer's instructions (Roche, Mannheim, Germany). The isolation of chromosomal DNA from B. japonicum was carried out as described previously (15).

Homology searches were performed by using the National Center for Biotechnology Information's BLAST network server (http://www.ncbi.nlm.nih.gov/BLAST/).

Construction of pmt and pcs mutants. Construction of the pmtA deletion mutant has been described (30). In order to construct a pmtX1 mutant by a gene disruption strategy, an internal fragment comprising nucleotides 38 to 477 was PCR amplified using the oligonucleotides Sig238 and Sig239 (for oligonucleotide sequences, see Table 2). The resulting 440-bp StuI-Ecl136II fragment was directly cloned into the suicide vector pSUP202pol4 linearized with SmaI to yield plasmid pRJ5295 (see Fig. 3B).


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  		TABLE 2. Oligonucleotides used in this study


Figure 3
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  		FIG. 3. Physical and genetic maps of the B. japonicum pmtA (A), pmtX1-4 (B to E), and pcs (F) gene regions. Physical maps are given for SalI, StuI, Ecl136II, and PstI (indicated by S, St, E, and P, respectively). Black arrows indicate the position of chromosomally integrated translational lacZ fusions and point to the corresponding strain designation. Below each map, the insertion sites for antibiotic resistance cassettes (Km in the case of pmtA and pmtX2-4; {Omega}Sm/Sp in the case of pcs) or the plasmid integration site are depicted. Antibiotic resistance cassettes are not drawn to scale. Km, kanamycin; Sm, streptomycin; Sp, spectinomycin; dnaJ, chaperone protein DnaJ; pmtA, Pmt; blr0682, putative oxidoreductase; pyrF, orotidine-5'-monophosphat-decarboxylase; blr0684, blr6996, bll6993, bll6633, blr8167, bll4801, and bll4584, hypothetical ORFs; trmU, tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase; pmtX1, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17); blr6636, ATP synthase subunit; pmtX2, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17); bll6635, probable glycosyl transferase; bll8168, Na+/H+ antiporter; pmtX3, Pmt; bll8165, putative alkaline phosphatase; blr4802, two-component response regulator; blr4803, two-component hybrid sensor and regulator; pmtX4, Pmt; pyrG, CTP synthetase; blr4586, putative oxidoreductase protein; pcs, Pcs; qor, quinone oxidoreductase.

For construction of the pmtX2 deletion mutant, the pmtX2 up- and downstream regions (645 and 593 bp, respectively) were amplified by using the oligonucleotide pairs O45-O46 and O53-O54, respectively. After cloning both fragments into pBCSK(+), the 1.2-kb PstI fragment of pBSL86 containing the neomycin phosphotransferase II cassette (Kmr) was placed between them in both possible orientations. The 2.4-kb EcoRI-NotI inserts of the resulting plasmids were ligated into the 6.7-kb EcoRI-NotI vector fragment of pSUP202pol4, yielding plasmids pBO230 and pBO231.

In order to delete pmtX3, a 1.8-kb DNA fragment carrying the gene was PCR amplified by using primer pair O1-O2. A 1.7-kb EcoRI-NotI fragment from the amplification product was cloned into vector plasmid pBCSK(+). Subsequently, a 249-bp PstI fragment of the pmtX3 coding region was replaced by a 1.2-kb PstI kanamycin cartridge from pBSL86 (see Fig. 3D). The 2.6-kb EcoRI-NotI inserts of the resulting plasmids were ligated into the 6.7-kb EcoRI-NotI vector fragment of pSUP202pol4, yielding plasmids pBO203 and pBO204.

The pmtX4 deletion constructs were cloned as described for pmtX2. The up- and downstream regions (418 and 520 bp, respectively) of pmtX4 were amplified with the primer pairs O63/O64 and O82/O83.

To delete the pcs gene, up- and downstream regions (704 and 662 bp, respectively) were PCR amplified with the primer pairs Sig241-Sig242 and Sig243-Sig244. After PCR products were cloned in pUC18, the 2-kb BamHI fragment of pHP45::{Omega} containing a Sm/Sp resistance cassette was inserted in the same orientation as the pcs gene. Finally, the 3.4-kb EcoRI-PstI insert was cloned into pSUP202pol4, yielding pRJ5702.

Finally, the suicide plasmids were mobilized from E. coli S17-1 into B. japonicum 110spc4 for marker replacement mutagenesis as described previously (16). The correct genomic structure of all mutant strains was confirmed by Southern blot analysis of genomic DNA digested with appropriate restriction enzymes.

Construction of chromosomally integrated, translational 'lacZ fusions. For the construction of 'lacZ fusion strains, suitable translational lacZ fusions present on pSUP202pol4 derivates were integrated into the chromosome of wild-type B. japonicum via homologous recombination (see Fig. 3). The correct genomic structure of all mutant strains was confirmed by Southern blot hybridization with appropriate digoxigenin-11-dUTP-labeled DNA probes.

β-Galactosidase assays. The β-galactosidase activity of B. japonicum cells grown aerobically in PSY complex medium or anaerobically in YEM medium was measured according to standard protocols (29).

In vivo labeling of B. japonicum with [14C]acetate and quantitative analysis of lipid extracts. The lipid compositions of B. japonicum wild type and mutant strains were determined after labeling with [1-14C]acetate. Cultures (1 ml) in PSY were inoculated from precultures grown in the same medium. After the addition of 0.5 µCi of [1-14C]acetate (56.0 mCi/mmol) to each culture, the cultures were incubated for 48 h. Cells were harvested by centrifugation, washed with 500 µl of water, and resuspended in 100 µl of water. Lipid extraction was done according to the method of Bligh and Dyer (2). The chloroform phase was used for lipid analysis on HPTLC silica gel 60 plates (Merck, Darmstadt, Germany). After one-dimensional separation with n-propanol-propionic acid-chloroform-water (3:2:2:1) as the running solvent the individual lipids were quantified by using a Bio-Imager (Fujifilm Europe GmbH, Düsseldorf, Germany) and the Aida Image Analyzer v. 4.03 software (Raytest GmbH, Straubenhardt, Germany).

Cloning and expression of the putative pmt genes of B. japonicum in E. coli. Candidate open reading frames (ORFs) pmtX1, pmtX2, and pmtX3 were PCR amplified by using oligonucleotide primers that introduced NdeI/BamHI restriction sites (Table 2) and were cloned into the expression plasmid pET9a (40), resulting in plasmids pCCS20, pCCS36, and pCCS37, respectively. Candidate ORF pmtX4 was PCR amplified by using oligonucleotide primers that introduced NdeI/HindIII restriction sites and cloned into pET24b (40), resulting in plasmid pBO234. Expression plasmids were transformed into the E. coli expression strain BL21(DE3)/pLysS. Cultures were grown in LB medium containing the appropriate antibiotics. At a density of 5 x 108 cells/ml, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 200 µM. Aliquots of 1 ml were transferred to sterile culture tubes and after 1 h of induction 0.5 µCi of [1-14C]acetate (56.0 mCi/mmol; Amersham) or [14C-methyl]methionine (40 to 60 mCi/mmol; Amersham) were added to each culture. One hour later the cells were harvested and extracted as described above.

To coexpress pmtA from B. japonicum with the candidate ORFs in E. coli, the expression cassette containing the T7 promoter and the pmtA gene was subcloned from pTB2117 (30) as a BamHI/BglII fragment into the broad-host-range plasmid pRK404 (9), resulting in plasmid pCCS27. E. coli BL21(DE3)/pLysS was transformed with pCCS27 and one of the plasmids pCCS20, pCCS36, pCCS37, or pBO234. Growth of the cells, induction, labeling, and extraction of the lipids was done as described above.

To coexpress pmtA from B. japonicum with two or three candidate ORFs in E. coli, new constructs had to be made: the pmtX3 gene was subcloned as a BamHI/BglII fragment from pCCS37 into the BamHI site of pCCS27, resulting in plasmid pCCS67, containing pmtA and pmtX3 both under control of the T7 promoter. The pmtX1 gene was subcloned as a BamHI/SphI fragment from pCCS20 into the BglII/SphI-digested plasmid pBO234, resulting in plasmid pCCS115, containing pmtX1 and pmtX4 under the control of the T7 promoter. Growth of the cells, induction, labeling, and lipid extraction were done as described above.

Plant infection tests. Surface sterilization of soybean seeds (G. max L.Merr cv. Williams 82), inoculation, plant cultivation, and acetylene reduction assays were done as described previously (15, 16). For determination of specific acetylene reduction activity (expressed as µmol of ethylene formed/h/g [dry weight] of nodule) all nodules from each plant were collected, dried overnight at 80°C, and weighed.


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RESULTS
 
Multiple genes coding for putative Pmts in the genome of B. japonicum. Earlier work had suggested that at least two Pmts are present in B. japonicum (30). However, previous attempts to identify a pmtX gene in the B. japonicum genome via low-stringency Southern blot hybridization had failed (30). More recently, the complete genome sequence of B. japonicum USDA110 (19) made it possible to search for pmtX candidates using bioinformatics tools. Two different families of prokaryotic Pmts have been described (1a, 8). We used as a query the sequences of S. meliloti PmtA and R. sphaeroides PmtA, which were the first members to be described in each family (25) to identify candidate genes coding for additional Pmts. Using the amino acid sequence of PmtA from S. meliloti as a query revealed three close homologs. As described earlier, B. japonicum PmtA shows homology to PmtA from S. meliloti (30) (Fig. 1). Both proteins share 35% identical and 56% similar amino acids (30, 39). Two additional candidates were identified that we named PmtX3 (Bll8166; identity on amino acid level, 33%; similarity, 53%) and PmtX4 (Blr4804; identity, 32%; similarity, 50%).


Figure 1
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  		FIG. 1. Unrooted phylogenetic tree of selected bacterial Pmt enzymes. The sequences used for the construction of the tree include the B. japonicum Pmt proteins (Bjap_PmtA, Blr0681; Bjap_PmtX1, Bll6994; Bjap_PmtX2, Bll6634; Bjap_PmtX3, Bll8166; Bjap_PmtX4, Blr4804) (19), the PmtA proteins from S. meliloti (Smel_PmtA; accession number AF201699) and R. sphaeroides (Rsph_PmtA, L07247), PmtA from A. tumefaciens (Atum PmtA, AE009001), a Pmt-like ORF from the genome of Rhodobacter capsulatus (Rcaps RRC03911; Ergo-light database; http://www.ergo-light.com/), and one from the genome of M. loti (Mlot Mlr5374, BA000012). The bradyrhizobial Pmt enzymes are highlighted by surrounding black boxes. The dashed line indicates the separation of the different types of Pmt enzymes. The tree was constructed by using the program CLUSTAL X (ftp://ftp-igbmc.u-strasbg.fr/pub/). Distances between sequences are expressed as 0.1 changes per amino acid residue.

Using the amino acid sequence of PmtA from R. sphaeroides as a query, two close homologs showed up, which we named PmtX1 (Bll6994; identity, 39%; similarity, 59%) and PmtX2 (Bll6634; identity, 41%; similarity, 61%). All predicted Pmt-like proteins contain the consensus motif for S-adenosylmethionine (SAM)-utilizing methyltransferases (17) thought to be involved in SAM binding (18) (data not shown).

To summarize, our bioinformatic analysis revealed that in addition to the already-known pmtA gene the B. japonicum genome contains four ORFs coding for putative Pmts. To make PC biosynthesis in this organism even more complex, its genome also contains a pcs gene encoding a Pcs (26).

Expression of the individual pmt genes in E. coli. Analysis of B. japonicum mutants deficient in the pmtA gene and heterologous expression of the B. japonicum pmtA in E. coli indicated that PmtA appears to be responsible mainly for the first methylation step in PC biosynthesis (30). Additional Pmts should therefore carry out the subsequent methylation steps converting MMPE into DMPE and PC. To detect this activity, we expressed PmtA, PmtX1, PmtX2, PmtX3, and PmtX4 in E. coli BL21(DE3)/pLysS. Lipids were radiolabeled by [14C]acetate or [methyl-14C]methionine, and the lipid composition of the strains was analyzed by using thin-layer chromatography (TLC) (Fig. 2). The amount of [14C]methionine-labeled lipids was quantified (Table 3). E. coli does not have Pmts and therefore only produces the membrane lipids PE, phosphatidylglycerol (PG), and cardiolipin. Expression of pmtA from B. japonicum led to the formation of mainly MMPE and minor amounts of DMPE (Fig. 2A and Table 3) as described earlier (30). If at all, only minute amounts of methylated PE derivatives were formed when PmtX1 or PmtX2 were expressed in E. coli. When PmtX3 was expressed, MMPE and DMPE were produced in almost equal amounts. Expression of PmtX4 led to the formation of large quantities of MMPE and only small amounts of DMPE.


Figure 2
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  		FIG. 2. Lipid formation after heterologous expression of pmtA from B. japonicum and four genes encoding putative Pmt enzymes from B. japonicum in E. coli. E. coli BL21(DE3)/pLysS cells expressing the indicated enzymes were labeled with [14C]acetate or [methyl-14C]methionine during growth on LB medium. Panel A shows the resulting phospholipid profile after single expression of (putative) Pmts in E. coli: panels B and C show the resulting phospholipid profile after coexpression of the phospholipid methyltransferase PmtA from B. japonicum with ORFs encoding putative Pmts in E. coli. After extraction, lipids were separated by one-dimensional TLC. Lane M, marker (S. meliloti lipids labeled with [14C]acetate); lane C, control with BL21(DE3)/pLysS/pET9a (+pRK404 for panels B and C); lane A, pmtA; lane X1, pmtX1; lane X2, pmtX2; lane X3, pmtX3; and lane X4, pmtX4 (genes encoded in the indicated vector backbone [see also Materials and Methods and Table 1]); lane Bj, B. japonicum lipids labeled with [14C]acetate. The lipids PG, cardiolipin (CL), PE, MMPE, DMPE, and PC are indicated.


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  		TABLE 3. Formation of methylated derivatives of PE upon expression of genes encoding putative Pmts from B. japonicum in E. colia

Coexpression of multiple pmt genes in E. coli. Since PmtA catalyzes very efficiently the formation of MMPE, one of the pmtX genes might encode a Pmt responsible for the second and/or third methylation step of PC biosynthesis. We therefore expressed PmtA together with each of the candidate ORFs in E. coli. Lipids were radiolabeled by [14C]acetate or [methyl-14C]methionine, and the lipid composition of the strains was analyzed by using TLC (Fig. 2B). The amount of [14C]methionine incorporated into each lipid was quantified (Table 3). As described above, mainly MMPE was detected when only PmtA was expressed. Coexpression of PmtA with PmtX1 led to the formation of significant amounts of DMPE and PC. When PmtA and PmtX2 were expressed together, mainly MMPE and possibly minor amounts of DMPE and PC were detected, indicating that, if at all, PmtX2 is an inefficient Pmt enzyme under these conditions. Coexpression of PmtA and PmtX3 caused a lipid spectrum similar to that caused by the expression of PmtX3 alone, with slightly increased levels of DMPE. Since both PmtA and PmtX4 preferentially catalyze the first methylation step, their coexpression also led mainly to the formation of MMPE.

Wild-type B. japonicum grown in complex PSY medium produces mainly PC and only small amounts of MMPE and DMPE (30). In an attempt to simulate this lipid composition heterologously, we coexpressed two, three, or four different Pmts from B. japonicum in E. coli. However, the expression of PmtA and PmtX1 caused the maximal formation of DMPE and PC methylation products in E. coli hosts. The relative amounts of DMPE and PC could not be increased by expressing additionally PmtX3 or PmtX3 and PmtX4 (Fig. 2C), indicating that PmtX3 and PmtX4 have no important role for overall PC formation.

Expression of pmt genes and the pcs gene in B. japonicum. In order to understand the complexity of PC biosynthesis in B. japonicum, we studied the expression of all potential PC biosynthesis enzymes using the corresponding reporter gene fusions to these and some flanking genes (Fig. 3). We first measured β-galactosidase activity during aerobic growth in complex medium. The reporter strains harboring chromosomally integrated, translational lacZ fusions were assayed 24, 48, and 72 h after inoculation to an optical density at 600 nm (OD600) of 0.02, which corresponds to early-exponential (OD600 = 0.3), late-exponential (OD600 = 1.0), and stationary-phase (OD600 = 1.2) cultures, respectively. B. japonicum wild type, which does not encode an endogenous β-galactosidase, served as a negative control.

Neither pmtX2, pmtX3, and pmtX4 nor the genes potentially forming an operon with pmtX2 and pmtX3 (bll6635 and bll8165, respectively) were expressed in B. japonicum (Fig. 4). This finding was confirmed by primer extension analysis (data not shown) and by recently deposited microarray data in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE8478). Consistent with our previous primer extension analysis (30), significant expression between 200 and 300 MU was detected for the pmtA'-'lacZ fusion. The only other expressed Pmt gene was pmtX1, with approximately 10 MU throughout the growth curve. The trmU gene, a potential tRNA methyltransferase that is located upstream of pmtX1 (Fig. 3B) shows about 50 MU (Fig. 4). The pcs gene is only weakly (3 to 4 MU) expressed.


Figure 4
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  		FIG. 4. Expression of B. japonicum PC biosynthesis genes. The expression of chromosomally integrated, translational lacZ reporter gene fusions (see Fig. 3) was measured as described previously (see Materials and Methods). Samples were collected after 24, 48, and 72 h. The results of a typical experiment, in which two parallel cultures of each strain were grown and assayed in duplicate, are shown. Four independent experiments were performed with individual strains.

The pmtA gene is symbiotically important (30), and a main physiological signal during nodulation is a reduction in the cellular oxygen level (11, 12). Hence, we tested whether the expression of PC biosynthesis genes was altered under anaerobic conditions. In order to control the establishment of anaerobic conditions, a nifH'-'lacZ fusion was measured in parallel. Again, pmtA, trmU, and pmtX1 were the only significantly expressed enzymes. An overall reduced expression of the pmtA'-'lacZ fusion under anaerobic growth (aerobically, 200 to 350 MU; anaerobically, 100 to 150 MU) might be due to some differences in the medium for aerobic and anaerobic cultivation (see Materials and Methods). Aerobic cultivation in YEM medium normally used for anaerobic cultivation resulted in similar reduction of β-galactosidase activity (data not shown). Similar to aerobic conditions, pmtX2 and pmtX3 were not expressed during oxygen deprivation (data not shown). The pmtX4 and pcs fusions were not tested. At the time these fusions were obtained, recent microarray studies had shown that expression of these genes did not change under microaerobic conditions (NCBI GEO database; http://www.ncbi.nlm.nih.gov/geo/; accession number GSE8478).

Construction and phospholipid profiling of B. japonicum PC biosynthesis mutants. To examine the phenotypic importance of each putative PC biosynthesis gene, we constructed a series of individual deletion mutants (Fig. 3). Despite numerous attempts, the pmtX1 gene could not be inactivated by a simple plasmid insertion strategy.

The lipid composition of B. japonicum wild type and all obtained PC biosynthesis mutants was examined after in vivo labeling with [1-14C]acetate. The wild-type strain possesses PE (52.2% of total phospholipids) and PC (42.4%) as major phospholipids and contains only small amounts of MMPE (2.3%) and DMPE (1.8%) in the membrane (Fig. 5). Except for the pmtA mutant, all other strains showed a wild-type-like membrane composition, as one might expect from the expression data described above. The pmtA mutant, on the other hand, showed a marked decrease of PC and a concomitant increase of PE in the membrane (30.4 and 63.7% of total phospholipids, respectively), The MMPE and DMPE levels remained unchanged (2.5 and 1.8%).


Figure 5
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  		FIG. 5. Phospholipid profile of PC biosynthesis mutants after in vivo labeling with [14C]acetic acid. B. japonicum strains were labeled with [14C]acetate during growth in PSY medium. After extraction, lipids were separated by one-dimensional TLC. The lipids PC, DMPE, MMPE, and PE are indicated. The relative PC content (as the percentage of total phospholipids) is given below each PC spot.

Since the pmtA mutant still produced significant amounts of PC, we tested whether the alternative Pcs pathway or another Pmt enzyme might be upregulated in this strain. Since expression of pmtX4 was strongly induced (Fig. 6), it appears that PmtX4 partially compensates for the loss of pmtA under these conditions.


Figure 6
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  		FIG. 6. Expression of the PC biosynthesis genes in the pmtA mutant compared to the wild type. The expression of chromosomally integrated, translational lacZ reporter gene fusions (see Fig. 3) was measured as described previously (see Materials and Methods). Samples were collected after 24, 48, and 72 h. The results of a typical experiment, in which two parallel cultures of each strain were grown and assayed in duplicate, are shown. Four independent experiments were performed with individual strains.

Symbiotic performance of B. japonicum pmt and pcs mutants. Since disruption of the pmtA gene resulted in a severe nitrogen fixation defect (30), we tested whether any of the other mutants also showed reduced symbiotic properties. Plants infected with B. japonicum wild type showed nitrogen fixation activity of about 93.7 µmol of C2H4/h/g (dry weight) of nodule, whereas plants infected with a nifH mutant showed only 5% of the wild-type level (Fig. 7). All plants infected with B. japonicum pmtX2, pmtX3, pmtX4, or pcs mutants developed normal nodules with the typical red color indicative of the presence of leghemoglobin (data not shown) and showed wild-type levels of nitrogen fixation activity. Consistent with previous results, the pmtA mutant elicited defective nodules capable of only reduced nitrogen fixation activity. This indicates that the PmtA activity and/or the presence of sufficient PC are of importance for the symbiotic performance of B. japonicum.


Figure 7
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  		FIG. 7. Symbiotic phenotype of B. japonicum pmt and pcs mutants. The nitrogen fixation activity was measured as the amount of C2H2 reduced per hour per gram (dry weight) of nodule. A 100% wild-type (WT) activity corresponds to 93.7 µmol of C2H2/h/g. Mean values and standard deviations derive from at least eight individual plants, except for the pcs mutant with six individual plants.


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DISCUSSION
 
All PC-synthesizing prokaryotes with an active methylation pathway that have been characterized thus far, such as S. meliloti, R. sphaeroides, or A. tumefaciens, possess only one Pmt enzyme capable of performing all three methylation steps (1a, 8, 45). Here, we report the presence of four functional Pmt enzymes with different substrate specificities in B. japonicum. The presence of more than one Pmt might be rare but is probably not exclusive to B. japonicum. In a bioinformatic survey, we identified four good Pmt candidates in the genome of Rhodopseudomonas palustris, which is closely related to Bradyrhizobium. Three putative Pmt-encoding genes are present in the genomes of Rhizobium etli, R. leguminosarum, and Nitrobacter winogradsky. Two putative Pmt enzymes are encoded in Granulibacter bethesdensis, M. loti MAFF303099, Fulvimarina pelagi, and Aurantimonas sp. (data not shown; genome sequences were accessed via the TIGR database; http://www.tigr.org/). The presence of two Pmts seems to be typical in lower eukaryotes, such as Saccharomyces cerevisiae or Neurospora crassa, which possess two different Pmt enzymes with distinct substrate specificities (6, 24). The presence of multiple pmt genes possibly reflects a need to adjust the membrane composition in response to environmental changes.

Why are there so many pmt genes in some organisms but not in others? We studied the complexity of prokaryotic PC biosynthesis using B. japonicum as a model organism. Apart from PmtA, at least one additional enzyme is required since PmtA alone produces predominantly MMPE. PmtX1 was shown to be responsible for converting MMPE via the DMPE intermediate to PC. Only pmtA and pmtX1 genes were significantly expressed in B. japonicum under all conditions tested. However, expression experiments with pmtX2, pmtX3, or pmtX4 in E. coli show that at least PmtX3 and PmtX4 function as Pmt enzymes. The fact that B. japonicum retains these Pmts intact suggests that there is some physiological demand for them. Otherwise, cryptic genes tend to accumulate missense mutations, as was shown for a member of the small heat shock gene family in Bradyrhizobium sp. (Parasponia) (33). B. japonicum is notorious for encoding multigene families such as the sigma factors RpoH1 to RpoH3 (32), the groESL-like chaperonin family (13), small heat shock proteins (31), and the transcription activators FixK1 and FixK2 (28). Members of these families often have overlapping functions but differ in the way they are regulated.

Little is known about regulation of PC biosynthesis in prokaryotes. It is unclear under which conditions, if at all, pmtX2, pmtX3, and pmtX4 are expressed in B. japonicum. In contrast to the pmtA mutant, deletion of these genes did not confer a symbiotic defect, indicating that they do not play an essential role in plant-microbe interaction under the conditions tested. Accordingly, anaerobic conditions did not induce expression of the pmtX genes. Expression of pmtA was affected by the medium composition rather than the oxygen supply. This seems to contradict results from a previous study showing a decrease in PC levels under anaerobic conditions and suggesting a possible oxygen-dependent regulation of PC biosynthesis gene expression (42). However, both the medium ingredients and the cultivation conditions were different in both studies. There is more evidence that external conditions and the nutrient supply influence PC biosynthesis in B. japonicum. The amount of pmtA transcripts was reduced three- to fourfold under heat shock conditions (30). Recent experiments with Bradyrhizobium SEMIA 6144 showed that PC amounts were decreased by heat stress and increased with salinity (27). Finally, a microarray analysis of an irr mutant lacking the iron-responsive regulator Irr showed that pmtX1 (and its upstream trmU gene) was downregulated approximately threefold under iron-limited conditions compared to the parental strain (47). This result was reproduced with our pmtX1'-'lacZ fusion (data not shown). Interestingly, transcription of the gene for the phosphatidylglycerophosphate synthase (pgsA), catalyzing the first reaction from CDP-DAG to PG, was decreased 2.6-fold under the same conditions (47). These findings suggest a possible regulatory connection between iron availability and phospholipid biosynthesis in B. japonicum.

Although it has been reported that B. japonicum is unable to take up choline (3), it expresses a functional Pcs, which requires choline as precursor. A previous study already showed Pcs activity in cell extracts of B. japonicum (26). Expression of the pcs gene was low but significant under all conditions tested. As speculated for A. tumefaciens and B. abortus (5, 20), B. japonicum might be able to synthesize choline.

On the basis of our analysis we propose an updated model for PC biosynthesis in B. japonicum (Fig. 8). The previously postulated Pmt activity catalyzing the second and third methylation step was shown to derive from PmtX1. PC biosynthesis is achieved by the concerted action of PmtA and PmtX1. Although PmtX4 and PmtX3 are able to perform the initial one or two methylation reactions when expressed in E. coli, their role in B. japonicum is not clear. The conditions under which they come into play remain to be elucidated. In an alternative route, PC can be synthesized in a one-step reaction by the Pcs pathway. One interesting avenue for future studies will be to study the mechanistic differences between the various Pmts biochemically, especially in cases in which closely related sequences produce different product spectra.


Figure 8
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  		FIG. 8. Model of PC biosynthesis in B. japonicum. The model is based on the phospholipid profiles after expression of each enzyme in E. coli and integrates the corresponding gene expression data from B. japonicum grown aerobically in complex culture medium. Thick arrows and boldface letters indicate the predominant reaction(s) performed by each enzyme. Enzymes in brackets are not expressed in B. japonicum wild type but are functional when expressed in E. coli. SAH, S-adenosylhomocysteine.


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ACKNOWLEDGMENTS
 
We are grateful to Hans-Martin Fischer (Zürich, Switzerland) for providing B. japonicum H1 and 110-48 and soybean seeds for plant infection tests. We thank Christiane Fritz and Jan Gleichenhagen (Bochum, Germany) for technical assistance and Sylvia Balsiger (Zürich, Switzerland) for the construction of some plasmids.

A fellowship from the Allgemeines Promotionskolleg der Ruhr-Universität Bochum to M.A. and funding by the German Research Foundation (DFG SFB 480) to F.N. are gratefully acknowledged. This research was supported by grants from CONACyT-Mexico (46020-N and 42578-Q), DGAPA/UNAM (IN217907) and the Howard Hughes Medical Institute (HHMI 55003675).


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FOOTNOTES
 
* Corresponding author. Mailing address: Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, NDEF 06/783, D-44780 Bochum, Germany. Phone: 49 (234) 32 23100. Fax: 49 (234) 32 14620. E-mail: franz.narberhaus{at}rub.de Back

{triangledown} Published ahead of print on 9 November 2007. Back

{dagger} S.H. and C.S. contributed equally to this study. Back


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Journal of Bacteriology, January 2008, p. 571-580, Vol. 190, No. 2
0021-9193/08/$08.00+0     doi:10.1128/JB.01423-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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