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

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.

Probing for pH-regulated genes in Sinorhizobium medicae using transcriptional analysis.

The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis. Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021. Acid activated expression was detected for fixNO, kdpC, lpiA, and phrR and for genes encoding a putative lipoprotein, two ABC-transporter components, a putative DNA ligase and a MPA1-family protein. These findings implicate cytochrome synthesis, potassium ion cycling, lipid biosynthesis and transport processes as key components of pH response in S. medicae.

Probing for pH-regulated proteins in Sinorhizobium medicae using proteomic analysis.

To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media. Fifty pH-regulated proteins were identified; N-terminal sequences for 15 of these were obtained using the Edman degradation. Transient acid exposure downregulated GlnA and GlnK and upregulated a hypothetical protein. Continuing acid exposure downregulated ClpP, an ABC transporter, a hypothetical protein, a lipoprotein, the Trp-like repressor WrbA1 and upregulated DegP, fructose bisphosphate aldolase, GroES, malate dehydrogenase and two hypothetical proteins. These findings implicate proteolytic, chaperone and transport processes as key components of pH response in S. medicae.

Sinorhizobium medicae genes whose regulation involves the ActS and/or ActR signal transduction proteins.

ActS-ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global regulation in the alpha-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae. To do this, random gusA fusions were created in S. medicae to follow gene transcription in an actS chromosomal knockout mutant containing plasmid-borne actS. Plasmid borne actS was cured from the mutants and beta-glucuronidase (GUS) activity compared between the different genetic backgrounds. We detected actS-dependent regulation of the genes gst1 (detoxification), hyuA (hydantoin utilization) and fixN2 (microaerobic respiration). We show that ActR is involved in regulating cbbS (CO2 fixation), narB (nitrate assimilation) and required for low pH and microaerobic induction of the nitrogen fixation regulators fixK and nifA. In particular, we demonstrate that the transcriptional activation of fixN2 is regulated by ActR through FixK.

ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH-induced copper toxicity.

Two 'calcium-irreparable' acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily. Here, we show that both actP-knockout mutants show sensitivity to copper; omission of this heavy metal from low pH-buffered media restores acid tolerance to these strains. Furthermore, complementation of the mutant phenotype requires only the actPgene. An actP-gusA fusion in R. leguminosarum was transcriptionally regulated by copper in a pH-dependent manner.Downstream to actP in both organisms is the hmrR gene that encodes a heavy metal-responsive regulator (HmrR) that belongs to the merR class of regulatory genes. Insertional Inactivation of hmrR abolished transcriptional activation of actP by copper ions and increased the basal level of its expression in their absence. These observations suggest that HmrR can regulate actP transcription positively and negatively. We show that copper homeostasis is an essential mechanism for the acid tolerance of these root nodule bacteria since it prevents this heavy metal from becoming overtly toxic in acidic conditions.

Survival and exopolysaccharide production in Sinorhizobium meliloti WSM419 are affected by calcium and low pH.

Cells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown at pH 5.8 instead of pH 7.0. Increasing concentrations of calcium in the exposure medium significantly decreased the death rate of WSM419 cells under conditions of acid stress (pH 4.0). The effect of calcium on survival at pH 4.0 however, appears unconnected to exopolysaccharide (EPS), since a strain with a mutation in exoY (Rm0540) responded to calcium in the exposure medium in the same way as its wild-type parent (Rm2011). The concentration of calcium in the growth medium also affected subsequent survival at pH 4.0, and the effect varied with pH. In cells grown at pH 5.8, higher calcium concentrations also markedly increased the rate of synthesis of EPS; this was not seen in cells grown at pH 7.0. 1H NMR spectra for isolated EPS from WSM419 cultures grown at pH 5.8 and pH 7.0 showed that low pH markedly lowered the degree of substitution with acetyl and pyruvyl groups, but not the degree of substitution with succinyl groups; calcium concentration did not affect the pattern of substitution at either pH. For EPS to be involved in the effect of calcium concentration in the growth medium on survival would imply a deleterious effect of the EPS produced at low pH.

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses.

The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.g. phosphate limitation, elevated temperature, high concentrations of sucrose or iron). A DNA fragment containing the wild-type phrR gene could not be cloned and inverse PCR was therefore used to amplify a 3.5 kb BamHI fragment containing phrR from the mutant S. meliloti TG2-6 (actA::Tn5). DNA fragments from a BamHI/SalI digest of the amplified product were cloned into pUK21 and sequenced. The phrR open reading frame contiguous to actA appears to code for a 15.2 kDa protein showing significant identity with the proteins encoded by y4wC and y4aM in Rhizobium sp. NGR234. All three proteins resemble transcriptional regulators in containing a DNA-binding helix-turn-helix motif similar to that reported for URF4 in Rhodospirillum rubrum and repressors in coliphage.

Rhizobium leguminosarum bv. viciae produces a novel cyclic trihydroxamate siderophore, vicibactin.

Trihydroxamate siderophores were isolated from iron-deficient cultures of three strains of Rhizobium leguminosarum biovar viciae, two from Japan (WSM709, WSM710) and one from the Mediterranean (WU235), and from a Tn5-induced mutant of WSM710 (MNF7101). The first three all produced the same compound (vicibactin), which was uncharged and could be purified by solvent extraction into benzyl alcohol. The gallium and ferric complexes of vicibactin were extractable into benzyl alcohol at pH 5.0, while metal-free vicibactin could be extracted with good yield at pH 8.0. The trihydroxamate from MNF7101 (vicibactin 7101) could not be extracted into benzyl alcohol, but its cationic nature permitted purification by chromatography on Sephadex CM-25 (NH+ 4 form). Relative molecular masses and empirical formulae were obtained from fast-atom-bombardment MS. The structures were derived from one- and two-dimensional 1H and 13C NMR spectroscopy, using DQF-COSY, NOESY, HMQC and HMBC techniques on the compounds dissolved in methanol-d 4 and DMSO-d 6. Vicibactin proves to be a cyclic molecule containing three residues each of (R)-2,5-diamino-N 2-acetyl-N 5-hydroxypentanoic acid (N 2-acetyl-N 5-hydroxy-D-ornithine) and (R)-3-hydroxybutanoic acid, arranged alternately, with alternating ester and peptide bonds. Vicibactin 7101 differed only in lacking the acetyl substitution on the N2 of the N 5-hydroxyornithine, resulting in net positive charge; it was still functional as a siderophore and promoted 55Fe uptake by iron-starved cells of WSM710 in the presence of an excess of phosphate. The rate of vicibactin biosynthesis by iron-deficient cells of WSM710 was essentially constant between pH 5.5 and 7.0, but much decreased at pH 5.0. When iron-starved cultures were supplemented with potential precursors for vicibactin, the rates of its synthesis were consistent with both ß-hydroxybutyrate and ornithine being precursors. At least three genes seem likely to be involved in synthesis of vicibactin from ornithine and ß-hydroxybutyrate: a hydroxylase adding the -OH group to the N5 of ornithine, an acetylase adding the acetyl group to the N2 of ornithine, and a peptide synthetase system.

Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR.

A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71.3% identity over 892 bp). At the protein level there was 70% identity and 93.3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.

An essential role for actA in acid tolerance of Rhizobium meliloti.

The actA gene, which is disrupted by Tn5 in the acid-sensitive mutant of Rhizobium meliloti TG2-6, was cloned and sequenced. It encodes a protein of 541 amino acids with a calculated molecular mass of 57,963 Da and an estimated pl of 9.0. The ActA protein sequence has 30% identity, and much higher similarity (69%), with the CutE protein of Escherichia coli. Like the cutE mutant of E. coli, TG2-6 is sensitive to copper. The reconstructed wild-type actA gene complemented the low pH- and copper-sensitive phenotype of TG2-6. Studies with an actA-lacZ gene fusion showed that actA is constitutively expressed at pH 5.8 and 7.0. The actA gene appears to be chromosomal and is present in all seven strains of R. meliloti tested.

Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti.

The development and function of the Rhizobium meliloti-Medicago sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable. The intracellular pH of R. meliloti was measured using either radioactively labeled weak acids (5,5-dimethyloxazolidine-2,4-dione and butyric acid) or pH-sensitive fluorescent compounds; both methods gave similar values. Six acid-tolerant strains (WSM419, WSM533, WSM539, WSM540, WSM852, and WSM870) maintained an alkaline intracellular pH when the external pH was between 5.6 and 7.2. In contrast, two Australian commercial inoculant strains (CC169 and U45) and four acid-sensitive strains from alkaline soils in Iraq (WSM244, WSM301, WSM365, and WSM367) maintained an alkaline intracellular pH when the external pH was >/=6.5, but had intracellular pH values of