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Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada
Correspondence
Ivan J. Oresnik
oresniki{at}cc.umanitoba.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) relatively little is known of the determinants that are necessary for the bacterium to switch from a saprophytic to a symbiotic existence.
The rhizosphere has been defined as the volume of soil surrounding the root influenced by the exudation of plant-derived compounds (Bowen & Rovira, 1976). To enter into an endosymbiotic relationship, a bacterium must first be able to survive in the rhizosphere and to colonize the roots of the host. In addition to the exudation of compounds into the rhizosphere, the plant also influences the rhizosphere environment by actively removing nutrients such as water and other trace elements that are essential for its own survival, and excluding others such as sodium which are deleterious to the plant's survival. It has been predicted that this exclusion may salinize the rhizosphere and lead to an osmolality within the rhizosphere that is higher than that found in bulk soil (Miller & Wood, 1996).
S. meliloti is more halotolerant than many other rhizobia (Botsford, 1984; Breedveld et al., 1990). A number of studies have shown that osmoadaptation in Rhizobium appears to be atypical compared to that found in many enteric bacteria (Miller & Wood, 1996). Whereas enteric bacteria often synthesize glycine betaines as osmoprotectants, S. meliloti responds by accumulating glutamate, the novel dipeptide N-acetylglutaminyl glutamine amide (NAGGN) and potassium (Botsford & Lewis, 1990; Smith & Smith, 1989). In response to extreme osmotic shock, trehalose accumulation occurs (Smith et al., 1994). In addition, a number of compounds, including disaccharides, are capable of acting as non-accumulated osmoprotectants by S. meliloti (Gouffi et al., 1999, 2000; Le Rudulier & Bernard, 1986).
A number of studies have shown that S. meliloti polysaccharide determinants are altered with changes to either media or salt concentrations (Breedveld et al., 1990; Lloret et al., 1995, 1998). S. meliloti has the ability to synthesize two different exopolysaccharides (EPS), succinoglycan (EPS I), which when bound to Calcofluor causes it to fluoresce under long-wave UV light, and galactoglucan (EPS II), which confers a mucoid colony phenotype when it is synthesized (Glazebrook & Walker, 1989; Leigh et al., 1985). S. meliloti strain Rm1021 normally only produces EPS I (Zhan et al., 1991). Inability to produce EPS I has been correlated with an inability to establish a functional symbiotic association (Keller et al., 1988; Leigh et al., 1985). The necessity for EPS I in symbiosis can be replaced by the presence of low-molecular-mass EPS II or a symbiotically active form of a capsular polysaccharide termed K antigen or KPS (González et al., 1996a; Pellock et al., 2000; Reuhs et al., 1995).
Regulation of EPS II is complex and is in part dependent upon the presence of the expR+ allele (Glazebrook & Walker, 1989; Pellock et al., 2002). The wild-type, strain Rm1021, contains an insertion element (ISRm2011-1) within the expR open reading frame and only synthesizes EPS II under low-phosphate conditions (Mendrygal & González, 2000; Rüberg et al., 1999; Zhan et al., 1991). In the halotolerant S. meliloti isolate EFB1 (growth unaffected by the addition of 300 mM NaCl), EPS II production is decreased in response to NaCl (Lloret et al., 1998). The mechanism by which this occurs has not been investigated. Microarray analysis of expR+-dependent gene expression in Rm1021 has shown that a large number of genes influencing many cellular processes are affected, including some involved in low-molecular-mass EPS I biosynthesis (Hoang et al., 2004).
To determine if mutants unable to tolerate high salt concentrations are less efficient in competition for nodule occupancy, S. meliloti Rm1021 was mutagenized, and the mutants screened for salt sensitivity and their ability to compete for nodule occupancy. The results show some of the strategies used by S. meliloti in dealing with high solute regimes.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Complex (LB) and defined (VMM) media were as previously described (Miller, 1972; Vincent, 1970). Antibiotics were used as necessary at the following concentrations: streptomycin (Sm), 200 µg ml–1; neomycin (Nm), 200 µg ml–1; tetracycline (Tc), 5 µg ml–1; chloramphenicol (Cm), 20 µg ml–1; gentamicin (Gm), 20 µg ml–1; spectinomycin, 100 µg ml–1; rifampicin, 100 µg ml–1. To assay for ion sensitivity, LB base medium containing 10 g tryptone and 5 g yeast extract per litre was amended with the appropriate concentration of salt (NaCl, MgCl2, KCl, CaCl2, LiCl) as indicated.
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). Phage transductions using 
M12 were carried out as previously described (Finan et al., 1984). Standard procedures were used for plasmid isolation, restriction endonuclease digestions, ligations, transformations, gel electrophoresis and Southern transfers (Sambrook et al., 1989).
The point of insertion for Tn5 mutants was determined by a modification of an arbitrary PCR protocol (Caetano-Anollés, 1993; Raffa & Raivio, 2002). Strains containing Tn5, or Tn5-233, inserts were purified and the genomic DNA was used as a template. A low-stringency PCR amplification (45 °C) using the primers IS50(1) (5'-CACGATGAAGAGCAGAAG-3') and DGEN(1) (5'-GGCCACGCGTCGACTAGTCAGNNNNNNNNNNACGCC-3') was carried out. The products of this reaction were subsequently amplified with a higher-stringency PCR amplification (60 °C) using the primers IS50(2) (5'-TAGGAGGTCACATGGAAGTCAGAT-3') and DGEN(2) (5'-GGCCACGCGTCGACTAGTCAG-3'). The primers IS50(2) and IS50(1) are found within the IS50 elements of Tn5 such that the IS50(2) primer is nested between primer IS50(1) and the sequence flanking the transposon. The DGEN(2) primer was designed to be complementary to the 3' end of the amplification product produced by using the IS50(1) primer [i.e. identical to the 5' end of DGEN(1)]. The products of the second PCR were gel isolated and sequenced using the IS50(2) primer as previously described (Oresnik et al., 1998). Sequencing was performed at the University of Calgary Core DNA facilities. Sequence data were trimmed of IS50 sequences and database searches were done with BLASTX (Altschul et al., 1997) against the S. meliloti database at http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/ (Barnett et al., 2001; Finan et al., 2001; Galibert et al., 2001).
Plant assays.
Plant symbiotic assays were carried out as previously described (Oresnik et al., 1994). Alfalfa (Medicago sativa cv. Rangelander) seeds were surface sterilized by treating the seeds for 1 min with 70 % ethanol followed by 10 min in a 1/5 dilution of bleach (approx. 1 % hypochlorite). The seeds were then extensively washed with sterile distilled water and germinated on 1.5 % water agar plates. Seedlings were transferred after about 2–3 days to sterile Leonard jar assemblies. Plants were grown in a quartz sand : vermiculite mixture, and nitrogen-free Jensen's nutrient solution and water were added as required (Vincent, 1970). Plants were inoculated by growing S. meliloti cultures overnight in LB, diluting the cultures 1/100 in sterile distilled water and inoculating each Leonard jar assembly with 10 ml. Plants were harvested between 28 and 35 days after inoculation. The plants were unearthed and the shoots (above the cotyledon) were dried to determine plant dry weight. The nodules on the root systems were counted and representative nodules were crushed and the bacteria isolated. Bacteria that were isolated from nodules were streaked onto LB agar plates and single colonies were checked for relevant phenotypes. Plant competition assays were carried out as previously described (Oresnik et al., 1998). Data from the competition experiments were evaluated for statistical significance using both binomial probability and a Fisher's least significance difference (LSD) assuming that a P value of less than 0.05 indicated a difference in strain competitiveness.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). LB generally contains about 86 mM NaCl. A brief survey of the S. meliloti strains showed that strains Rm1021, Rm2011, AK631, Rm102F34, Rm102F21, RCR2012 and CC2013 were able to grow on LB whereas strain 104A14 could not (data not shown). To identify mutants unable to tolerate elevated concentrations of NaCl we first determined what concentration of NaCl could be tolerated by strain Rm1021 when grown on LB medium. Initial experiments determined that Rm1021 could grow on LB containing a final concentration of 300 mM NaCl, whereas growth was very poor on 400 mM and sparse on 500 mM (data not shown). Transposon-induced mutants were therefore screened for the ability to grow on LB agar containing 100 mM NaCl and an inability to grow on LB containing either 300 or 350 mM NaCl. Approximately 5000 Tn5 mutants were screened. To ensure that the transposon insert was the cause of the salt-sensitive phenotype, the insert was transduced into Rm1021 and screened for cotransduction of the antibiotic-resistance marker associated with the transposon and the salt-sensitive phenotype. In all cases the insertion and the phenotype were 100 % linked by transduction (typically 50–100 colonies screened), strongly suggesting that the transposon insertion was the cause of the salt sensitivity. From these screens, approximately 20 putative mutants were isolated and further tested.
Based on their growth characteristics on various media, eight of the putative mutants appeared to be salt sensitive without any other notable growth defects. One of these, strain SRmA259, was subsequently identified as carrying a pgm (phosphoglucomutase) mutation (see below). Since this mutation is pleiotropic, SRmA259 was characterized in a limited manner. Growth rates were determined on the remaining mutants to ensure that they could grow in LB broth containing 100 mM NaCl at rates comparable to the wild-type and to show that they were compromised in their ability to grow in LB broth containing elevated salt (Table 2).
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). The growth rate of SRmA251 and SRmA267 correlated well with their growth on agar (some growth could be seen) whereas when SRmA258 was streaked on LB agar containing 350 mM NaCl it was not uncommon to see some isolated colonies. A number of these were shown to have acquired second-site mutations that were transductionally unlinked to the original mutation (M. Miller-Williams & I. J. Oresnik, unpublished).
Identification of mutations
The point of insertion of the transposon in each mutant was identified and the annotated name or number as well as a brief description of the gene are presented in Table 3. The mutations break down into three broad classes: mutations that directly affect the cell structure (fabG, exoF1, pgm, Smc02909), uptake of metabolite(s) (Smb20056, Smb20057, Smb20058) as well as a number of mutations that are more likely to have an indirect or pleiotropic effect (ftsEX, tig, Smb20912).
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). For example, in strain SRmA258, the insertion in exoF1 affects other genes in succinoglycan production; this has been shown by others experimentally (González et al., 1996b). The Tn5 insertions in strains SRmA284 and SRmA239 are likely to have polar effects on downstream genes since the inserts are in the first genes of operons and there appears not to be enough space for a downstream promoter. The insertion in strain SRmA282 is within an ATP-binding cassette protein with similarity to ftsE and is likely polar on a gene that is related to ftsX (Smc00716) since the two genes have a 4 bp overlap. It is unclear whether the insertion could have a polar effect on Smc00715 and Smc00714 since the spacing between these genes is 109 and 105 bp, respectively, yet they are transcribed in the same orientation as Smc00717. Whereas Smc00715 is predicted to be a gene that encodes a hypothetical protein, Smc00714 is predicted to encode a putative 1-acyl-sn-glycerol-3-phosphate acyltransferase. It is also worth pointing out that ftsEX mutations in Escherichia coli also have an ion-related phenotype (de Leeuw et al., 1999; Schmidt et al., 2004).
The inserts in strains SRmA262 and SRmA267 do not have polar effects since the next annotated genes are transcribed in the opposite orientation to the genes interrupted by the Tn5 insertion. The insert in strain SRmA251 is within fabG. Although the next gene downstream is transcribed in the same orientation, there is a gap of 471 bp between these genes, making it unlikely that they are cotranscribed. The insert in strain SRmA259 is within pgm. The space between pgm and the next gene, glgX1, which is transcribed in the same orientation is 233 bp and it is unclear whether the Tn5 has a polar effect. However, Rhizobium tropici contains an identical arrangement of genes and glgX has been shown to transcribed independently of pgm (Marroquí et al., 2001)
Ion phenotypes of salt-sensitive mutants
PEG has been used experimentally to generate high osmotic environments because it is excluded from cells, non-metabolizable and osmotically active. The mutants we had isolated were streaked onto LB agar plates containing 5 % PEG 8000 and 100 mM NaCl. The results showed that the mutants were not sensitive to high osmolarity under these conditions (data not shown).
To determine if the mutants were specifically sensitive to Na+, their ability to tolerate high concentrations of other cations was determined. Since strains SRmA258 and SRmA259 were shown to carry mutations that affect EPS synthesis (Leigh et al., 1985), the other mutants were also screened for their ability to fluoresce on Calcofluor (Table 4). Of the eight mutants analysed, SRmA251 (fabG : : Tn5) was the only one that appeared to be sensitive only to NaCl. It is noteworthy to point out that of all the mutants isolated with a salt-sensitivity phenotype, SRmA251 appeared to be the least sensitive (Table 2, Table 4). The rest of the mutants tended to show some degree of sensitivity to the other ions that were tested.
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). Consistent with this, neither strain was fluorescent on Calcofluor (Table 4). However, unlike SRmA258, which was sensitive to Mg2+, SRmA259 was not sensitive to Mg2+, but it was very sensitive to Ca2+. The addition of as little as 3.5 mM CaCl2 to an LB base medium containing 100 mM NaCl gave an observable salt-sensitive phenotype (data not shown).
EPS I mutations and salt sensitivity
The original genetic screens yielded two mutants that were first reported as exo mutations: pgm (exoC) and exoF (Leigh et al., 1985). Mutations in pgm are pleiotropic, and affect the synthesis of both succinoglycan and LPS. To our knowledge, the inability to synthesize succinoglycan has not previously been shown to be related to a salt-sensitive phenotype in S. meliloti. We note that cultures of strain SRmA258 that were grown from inocula taken from older plates had a tendency to lose their salt-sensitivity phenotype, and cultures of SRmA258 also generated a high number of isolated colonies if they were left on LB base medium with 50 mM MgCl2.
Strain Rm7055, which carries an exoF (Leigh et al., 1985) mutation, in our hands was not sensitive to salt and did not show any of the phenotypes that were associated with either strain SRmA259 (pgm) or strain SRmA305 (exoF). Since we had seen a number of second-site mutations arise in SRmA305, we hypothesized that this may have also occurred in Rm7055. The transposons from both Rm7055 and SRmA305 were transduced into strain Rm1021. Transductants from both crosses were 100 % linked with their respective salt phenotypes, suggesting that either both phenotypes were correct, or a second-site mutation had arisen that was tightly linked with one of the exoF alleles. The position of the Tn5 insertion in Rm7055 was determined by generating an arbitrary PCR fragment from the genomic DNA flanking the Tn5. Nucleotide sequencing of the PCR fragment clearly showed that the exoF55 allele in Rm7055 was not within the exoF gene as previously reported but within the exoY gene, which is predicted to be responsible for the transfer of the initial galactose onto a carrier as the first step in succinoglycan biosynthesis (Reuber & Walker, 1993).
It is a common practice to grow strain Rm1021 on LB that contains Mg2+ and Ca2+ (Glazebrook & Walker, 1991). Since we had found a difference in the salt sensitivity of strains SRmA305 (exoF1) and Rm7055 (exoY), and we had seen that 3.5 mM CaCl2 could have an effect on a strain's sensitivity to salt, we wanted to know if the difference between the strains could be attributed to the genetic screens that had been employed or the media on which they were purified.
Because of the simplicity of the screen, an additional 2000 Tn5 mutants were visually screened on LB that contained Calcofluor (exo mutants show a dark phenotype on LB Calcofluor plates). Two dark mutants were isolated, purified and designated SRmA337and SRmA339. Both mutants were sensitive to 350 mM salt, and arbitrary PCR analysis of these mutants showed that Tn5 in SRmA337 was located in pgm whereas the Tn5 insertion in SRmA339 was in exoA. Consistent with the previous data, the strain carrying the pgm mutation was sensitive to Ca2+, and the strain carrying the exoA mutation was sensitive to Mg2+. The isolation of these mutations further supports the hypothesis that EPS I plays a role in protecting the cell against high ion concentrations.
The Mg2+ sensitivity phenotype of exoF is suppressed by expR+
The ability of expR+ to suppress EPS-associated symbiotic phenotypes has been previously reported (Glazebrook & Walker, 1989). To determine if expR+ also affects the level of sensitivity of exoF mutants to NaCl and MgCl2, the exoF allele was transduced from strain SRmA305 into RmG182. Because RmG182 also contains the insert 
317 : : Tn5-233 marker linked in transduction to the expR+ allele, it was also transduced from RmG182 into Rm1021 to make strain SRmA287 so that all possible combinations of markers could be tested. The presence of the expR+ allele did not affect the salt sensitivity associated with the exoF allele but it did alleviate the associated sensitivity to Mg2+ (Table 5). Surprisingly, RmG182 was found to be sensitive to NaCl (Table 5), but SRmA287 was not (Table 5).
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317 : : Tn5-233 in SRmA287 might be helpful. The insertion was located 23 aa from the C terminus of RpoH2, placing the insert approximately 25 kb from expR+, which correlates well with the 65 % cotransduction frequency (data not shown). rpoH2 is known to affect EPS synthesis in Rhizobium sp. strain TAL1145 (Kaufusi et al., 2004). Expression of rpoH2 in a TAL1145 rpoH2 background resulted in functional complementation (Kaufusi et al., 2004).
It was deemed prudent to construct an expR+ strain that was completely devoid of transposons. A strategy that employed a Tn5 insert that was unable to grow on defined media and was linked in transduction was needed. The pckA gene is located at approximately 140 kb from the expR+ allele, whereas the rpoH2 : : Tn5-233 is about 165 kb from pckA. Thus these two markers flank the expR+ gene. Utilizing strain Rm5439, which contains pckA : : TnV [unable to use succinate as a sole carbon source (Suc–)] (Finan et al., 1988), a lysate was grown on this strain and used to transduce strain RmG182 to NmR. The resultant transductants were Muc+, GmRSpR and Suc–. One colony was purified and designated strain SRmA309. A lysate was grown on SRmA309 and the pckA : : TnV marker from this strain was transduced into strain Rm1021. All of these transductants were GmSSpS and 1 % of the transductants were both NmR (unable to use succinate) and mucoid, suggesting that the expR+ allele and the pckA allele were cotransduced into Rm1021. One of these was purified and designated strain SRmA316. To eliminate the pckA : : TnV mutation, a wild-type lysate was used to transduce SRmA316 to prototrophy. The resultant strain was purified and designated SRmA363. Utilizing the published primer sequences that flank expR+ (Pellock et al., 2002) a 0.9 kb PCR fragment was generated by using SRmA363 genomic DNA as template. Subsequent gel isolation and nucleotide sequencing of both strands of the PCR fragment showed that SRmA363 contained the expR+ gene (data not shown).
The resultant strain, SRmA363, was used to construct strains carrying either exoA, expR+ or exoF, expR+. These strains were designated SRmA458 and SRmA457, respectively, and tested for NaCl and MgCl2 sensitivity (Table 5). The results showed that there is a strong correlation between the presence of expR+ and salt sensitivity. Moreover, expR+ was clearly capable of reversing the sensitivity to MgCl2 associated with exoF1, but not exoA, and the addition of 100 mM MgCl2 appeared to reverse the mucoid phenotype associated with the expR+ allele, suggesting that the presence of mucoidy (and presumably EPS II) was not the determining factor in reversing the MgCl2 phenotype associated with exoF1.
EPS II is affected by ions and not osmolarity
The basis for the original isolation of expR+ was the exhibition of a mucoid phenotype (Glazebrook & Walker, 1989). Because MgCl2 appears to reverse the mucoid phenotype, and expR+ appears to confer sensitivity to NaCl, the effects of other ions and osmolarity were investigated. Streaking of either strain SRmA363 or strain RmG182 onto LB containing different ions revealed that either 50 mM Mg2+ or 350 mM K+ in LB could reverse the mucoid phenotype conferred by the presence of expR+ (Fig. 1). The strains did not appear to grow slower than those streaked onto LB not containing the ions. To ensure that the ions did not delay the onset of mucoidy, plates were incubated for extended periods. Delayed mucoidy was not observed. The mucoid colony phenotype was not reversed by Li+, Ca2+, or the presence of high osmolarity induced by the presence of 5 % PEG 8000.
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; Leigh et al., 1985). The six remaining strains were symbiotically proficient and not different from the wild-type with respect to nitrogen fixation, nodule number and morphology (data not shown).
Each strain's ability to compete for nodule occupancy was tested by inoculating approximately equal amounts of wild-type and mutant onto alfalfa seedlings. Nodules were harvested and the bacteria were reisolated and tested for phenotype. The results showed that of the six strains tested, two, SRmA239 and SRmA284, occupied a percentage of nodules not significantly different from the proportion of the strain found in the inoculum (Table 6). The remaining four strains, SRmA262, SRmA282, SRmA251 and SRmA267, were all found to occupy a significantly lower proportion of nodules than would be expected from the proportion of each strain found in the inoculum, suggesting that these strains were less competitive for nodule occupancy than the wild-type (Table 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), or previous genetic studies that focused on salt effects and Rhizobium (Barra et al., 2003; Nogales et al., 2002). The mutations isolated appear to be diverse, suggesting that many loci affect the ability of S. meliloti to grow at elevated salt concentrations. Although it is unlikely that the concentration of salt in the rhizosphere is equivalent to that which was used to screen for mutants, it is pertinent to consider that elevated concentrations of salt also increase the osmotic stress encountered by the bacteria. It has been suggested that bacteria may need to adapt to changes in osmolarity during symbiosis (Gore & Miller, 1993; Spaink, 2000; Vedam et al., 2003). Therefore, the use of this screen, followed by assaying the mutants for a subtle phenotype (competition for nodule occupancy), has the potential to unveil not only genes that affect salt sensitivity but also those that affect growth in the rhizosphere or during symbiosome development.
Genes Smb20192 and Smb20056 (represented by strains SRmA239 and SRmA284, respectively) do not appear to play pivotal roles in competition within the rhizosphere. The nodulation competition data suggest that mutants fabG (strain SRmA251), Smc02909 (strain SRmA262), tig (strain SRmA267) and Smc00717 (strain SRmA282) are significantly affected in competing against the wild-type for nodule occupancy (Table 6). Proteomic studies have identified trigger factor (Tig) as a protein that is increased in nodule bacteria or during stress (Djordjevic et al., 2003). In E. coli, trigger factor is responsible for the correct targeting of some secreted proteins to either the cytoplasmic membrane or the periplasm (Beck et al., 2000). Our data showing that SRmA267 (tig : : Tn5) has a reduced ability to grow in LB with elevated salt as well as being unable to compete against the wild-type for nodule occupancy corroborate this finding (Table 2, Table 6). Strain SRmA267 can, however, form effective nodules; therefore tig is dispensable for normal nodule formation and nitrogen fixation.
Within the genome there are eight genes annotated as transmembrane glycosylases, one of which, Smc02909, is mutated in strain SRmA262. A conserved-domain search recognizes this gene to encode a lytic transmembrane transglycosylase that belongs to COG 2951 and is 90 % aligned with MltB (membrane-bound lytic murein transglycosylase B), an enzyme that is capable of both hydrolysing and synthesizing 
-1,4-glycosidic linkages in peptidoglycan. The most straightforward interpretation of the salt and competition phenotype associated with this mutation is that growth and/or peptidoglycan remodelling occurs during both these processes. Although this suggests that the mutation could be pleiotropic, we note that the ability of this strain to grow on standard laboratory media does not seem compromised and it does appear normal with respect to its ability to nodulate and fix nitrogen.
The insert in fabG is intriguing because FabG reduces the -ketoester from the condensation of malonyl-CoA with a growing acyl chain (Cronan & Rock, 1996). In E. coli and Salmonella enterica this gene has been shown to be essential for viability, with only temperature-sensitive mutants having been isolated (Lai & Cronan, 2004; Zhang & Cronan, 1998). In strain Rm1021 nodG appears to be a duplication of fabG (López-Lara & Geiger, 2001) and expression of nodG is regulated by NodD (Maillet et al., 1990), suggesting that nodG should not be transcribed under free-living conditions. The fact that the mutation in strain SRmA251 is not lethal suggests that either there is expression of nodG or there is a third copy of a gene encoding a FabG-like protein. The latter possibility is unlikely since the entire genome has been sequenced. It has been shown that expression of nodG does increase in the presence of nodD3 in the absence of inducer, but this effect is not seen with nodE, nodH, nodA or nodD3 fusions (Maillet et al., 1990). These data suggest that it may be possible to have some nodG expressed under free-living conditions and this may allow a fabG strain to be viable in S. meliloti.
The reason for the inability of strain SRmA251 to compete for nodule occupancy is unknown but may involve a change in membrane composition in response to high ion concentrations. Fatty acid synthesis is involved in providing the acyl portion of acylated homoserine lactones in Pseudomonas aeruginosa (Hoang et al., 2002) and in its potential interaction with the synthesis of acylated lipochito-oligosaccharides in rhizobia (Spaink, 2000). Therefore, mechanisms involving either of these processes in compromising a strain carrying a fabG mutation from effectively competing may be proposed, but further work is necessary.
Strain SRmA282 carries a mutation in ftsE and appears to be less competitive for nodule occupancy than strain Rm1021 (Table 6). These results should be viewed with caution. The results presented represent a single dataset. Although the strain was assayed multiple times, and consistently showed a decreased ratio of nodules recovered containing the mutant when compared to the inoculation ratio, we noted that upon dilution of SRmA282, we would continually attain inoculation ratios far below the anticipated ratios (data not presented). The results we present may represent a pleiotropic effect reflecting the fitness of the strain rather than an inability to compete against the wild-type.
The finding that EPS mutants were sensitive to NaCl should not have been unexpected. A number of reports have shown that either polysaccharide expression or composition are altered with the addition of NaCl (Breedveld et al., 1990; Lloret et al., 1998; Rüberg et al., 2003). We have isolated two strains unable to synthesize EPS I using two different genetic screens and both had similar ion sensitivities (Table 4). Testing of a previously characterized strain, Rm7055, was not consistent with our findings.
The site of the mutation in strain Rm7055 is in exoY and not within exoF as has been previously reported (Leigh et al., 1985). It is postulated that ExoF and ExoY are necessary for the initial addition of galactose during EPS I biosynthesis (Reuber & Walker, 1993). The representative exoF allele used in the study was the exoF55 allele from Rm7055 (Reuber & Walker, 1993). Effectively this means that ExoF more than likely does not play a role in the initiation of EPS I synthesis, and based on the annotation within the S. meliloti database, it is likely to be an outer-membrane protein involved in polysaccharide export (http://bioinfo.genopole-toulouse.prd.fr/annotation/iANT/bacteria/rhime/). Thus we would predict that a strain carrying an exoF1 mutation should produce a polysaccharide equivalent to that of a strain carrying an exoQ mutation. exoQ mutants have been shown to produce complete succinoglycan subunits and are believed to be unable to make high-molecular-mass polysaccharide (Reuber & Walker, 1993). It is therefore possible that the difference in the sensitivity to NaCl between Rm7055 and the mutations we isolated may be due to the ability to initiate EPS I synthesis. Further work will be necessary to resolve these differences.
The sensitivity to Mg2+ exhibited by a strain carrying an exoF1 mutation was suppressed by expR+, but exoA was unaffected by expR+ (Table 5). The suppression of the exoF phenotype was not due to the production of EPS II because both strain SRmA321 (exoF expR+ rpoH2) and strain SRmA457 (exoF expR+) were non-mucoid on media containing Mg2+. This suggests that expR+ does not suppress the exoF-associated phenotype by the production of EPS II. It also shows that EPS II production is somehow regulated by the presence of Mg2+. Recent microarray studies demonstrate that ExpR affects many genes (Hoang et al., 2004). It is not clear which of the ExpR target gene(s) may be responsible for the suppression of Mg2+ sensitivity.
The reversal of the mucoid phenotype associated with expR+ prompted us to try different ions. We found that both Mg2+ and K+ had the same effect. Presumably the lack of mucoidy is due to a lack of EPS II biosynthesis. Although EPS II is not normally synthesized by strain Rm1021, it can be synthesized under low-phosphate conditions (Mendrygal & González, 2000; Rüberg et al., 1999; Zhan et al., 1991). Mutations affecting phosphate uptake show an osmolarity-dependent EPS II synthesis that is reversed by the addition of NaCl, MgCl2 or KCl (Oresnik et al., 1994). Taken together, the data suggest that in addition to being regulated by ExpR and phosphate, EPS II synthesis may also be regulated in an ion-dependent manner independent of ExpR.
EPS II is regulated by NaCl in a S. meliloti strain that was isolated as being halotolerant (Lloret et al., 1998). Since the presence of ExpR in strain Rm1021 is correlated with increased salt sensitivity (Table 5), the effect of NaCl on the mucoid phenotype could not be determined directly.
The finding that strain Rm1021 derivatives carrying expR+ were sensitive to salt was unexpected. It appears that in addition to regulating EPS II biosynthesis, ExpR must also regulate genes that play a role in either osmotic adaptation or salt tolerance. Using published microarray data, it is clear that ExpR affects the expression of EPS I biosynthesis genes, as well as ndvA regulation (Hoang et al., 2004). Our data show that strains carrying some EPS I biosynthesis mutations are sensitive on media containing salt (Table 3, Table 4). Previous work has also shown that strains carrying ndvA mutations are compromised under hypo-osmotic conditions (Dylan et al., 1990). Interestingly, ndv osmotic pseudorevertants were shown to be suppressed by the presence of loci corresponding to EPS I biosynthesis (Nagpal et al., 1992). Taking these findings together, it might not be unexpected that changes in either salt or osmotic sensitivities may be seen in an expR+ background.
In summary, this work has investigated the hypothesis that S. meliloti mutants that are affected in their ability to survive at elevated NaCl concentrations will also be unable to compete against the wild-type for nodulation in the rhizosphere. The analysis has shown that of the six salt-sensitive mutants that were capable of a symbiotic association, four were compromised in their ability to compete against the wild-type for nodule occupancy. In addition, a role for EPS I contributing to the tolerance of S. meliloti to high NaCl or MgCl2 concentrations has been shown. The sensitivity of an exoF mutation to Mg2+ is altered in an expR+-dependent, EPS II-independent manner. Moreover, we have observed that expR+-dependent mucoidy is regulated by the addition of K+ or Mg2+. To our knowledge, the role ions play in gene regulation has not been previously addressed in S. meliloti. It seems unlikely that only EPS determinant genes are affected by ions. In light of this, our work is currently focused on pursuing both how ions are perceived and what determinants are affected by changes in ion concentration.
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REFERENCES |
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Received 20 February 2006;
revised 17 March 2006;
accepted 21 March 2006.
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