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The molecular basis of acid-tolerance in Rhizobium

Reeve, Wayne (1995) The molecular basis of acid-tolerance in Rhizobium. PhD thesis, Murdoch University.

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The aim of this study was to investigate the molecular basis of acidtolerance in Rhizobium. Transposon Tn5-induced mutants were used to identify genes required for acid-tolerance in the Sardinian strain Rhizobium meliloti WSM419 and the Japanese strainR. leguminosarum bv. viceae WSM710. Plant inoculation tests showed that nodulation by these acid-sensitive mutants on their respective hosts was comparable to that of the wild-type.

Calcium affected the growth of both wild-type Rhizobium strains at acidic pH. Cells of R. meliloti WSM419 grew faster at acidic pH and could grow at a progressively lower pH as the calcium concentration was increased. R.
leguminosarum WSM710 was able to grow below pH 4.9 if the concentration of calcium was increased in the medium. The acid-sensitive mutants could be divided into two groups on the basis of their response at acidic pH to the external calcium concentration. The first group (R. meliloti strains TG 1-6, TG 1-11, TG2-6, TGS-46 and R. leguminosarum WR6-35) grew at low pH if extra calcium was supplied in the medium. In the second group were those mutants (R. meliloti RT3-27 and R. leguminosarum WRl-14) that were unable to grow at low pH even if a high concentration of calcium was supplied.

Southern hybridisation studies using Tn5 as a probe demonstrated that each mutant contained only a single copy of Tn5. The re-insertion of Tn5 back into the wild-type using a suitable site-directed homologous recombination strategy (with an appropriate suicide vehicle for R. meliloti or phage RL38 mediated transduction for R. leguminosarum) recreated the acid-sensitive phenotype which verified that Tn5 was the causative agent of the disruption of a gene required for acid-tolerance.

The rhizobial DNA flanking Tn5 was cloned from the mutants TG2-6, TGS-46, RT3-27, WR6-35, and WRl-14 and the DNA was sequenced and analysed for protein encoding regions. DNA or protein sequences were then used to search for similarity in the GenBank, EMBL, or GenPeptide databases. The genes interrupted by Tn5 in the mutants were designated as act genes (acidtolerance genes) and numbered according to the name of the mutant; for example, the gene disrupted by Tn5 in TG2-6 was labelled as act206.

The predicted protein (Act206) encoded by act206 in WSM419 is 541 amino acids in length and has an estimated molecular weight of 57, 963 D and a pl of 9.0. An incomplete open reading frame contiguous to act206 appears to code for a DNA binding protein homologous to URF4 of Rhodospirillum rubrum and n~pressors in coliphage. The Act206 protein has a small degree of identity (30 % over 465 amino acids) but higher similarity (69 % over 465 amino acids) to CutE from Escherichia coli. Disruption of the latter protein caused a copper sensitive phenotype in cells of E. coli. S. typhimurium also contains an allele of cutE which, if mutated, causes a temperature-sensitive and copper-sensitive phenotype and a reduction in the activity of the lipid metabolising enzyme apolipoprotein Nacy ltransferase. The act206 mRNA was found in cells grown under both acidic (pH 5.8) and neutral (pH 7.0) conditions. The act206 gene appears to be chromosomal and was found in all seven strains of R. meliloti examined. At this stage the role of Act206 in acid-tolerance remains unclear.

The gene inactivated in WR6-35 has a strong similarity to the exoR sequence of R. meliloti Rm1021 (71.3 % over 892 bp). The protein (Act635) encoded by act635 is predicted to be 267 amino acids in length with a molecular weight of 28, 920 D and a calculated pl of 5.5. The Act635 protein has 93.3 % similarity and 70 % identity over 267 amino acids with R. meliloti Rm1021 ExoR. Strain WR6-35 produced approximately twice as much EPS as the wild-type WSM710 under the conditions used. NMR spectra of EPS produced by the wildtype and WR6-35 were indistinguishable. Disruption of exoR in R. leguminosarum caused a mildly acid-sensitive phenotype; one possible explanation is that a perturbation of the cytoplasmic membrane has resulted through an overproduction of EPS which subsequently caused an increased susceptibility to proton influx. The gene disrupted by Tn5 in R. meliloti TG5-46 (actR; previously called act546) encodes a protein (ActR) of 193 amino acids with a predicted molecular weight of 21, 463 D and a pl of 8.3. This protein has a similar amino-terminal region to regulatory proteins of the histidine protein kinase/regulator protein family involved in signal transduction in bacteria. ActR has a high degree of similarity over the entire protein sequence of PrrA from Rhodobacter capsulatus (94.9 % similarity, 69.3 % identity over 176 amino acids). It also had a high degree of similarity with RegA from Rhodobacter capsulatus (92.6 % similarity, 70.5 % identity over 176 amino acids) or R. sphaeroides (92.6 % similarity, 67.0 % identity over 176 amino acids). Neither, PrrA or RegA belong to any recognised subclass ofregulators and hence have been classified in a new subgroup. It is postulated that ActR falls into this new subgroup based on protein similarity. The predicted protein encoded by the gene upstream to actR showed similarity with sensor proteins, such as PhoR (67.2 % similarity and 24.6 % identity over 244 amino acids) from Escherichia coli and FixL (69.5 % similarity and 25.4 % identity over 256 amino acids) from Bradyrhizobium japonicum, which are members of the histidine protein kinase/regulator family. It is speculated that this protein, consequently labelled as ActS, may play a role in the detection of the hydrogen ion concentration and transfer this signal to ActR which in turn regulates one or more structural genes.

The genes inactivated in R. meliloti RT3-27 (act327) or R. leguminosarum WRl-14 (actl 14) showed similarity with genes which encode P-type ATPases in a variety of eukaryotes and prokaryotes. These genes may encode proteins which have a direct role in proton translocation or an indirect role through ion transport in the cell.

The possible implications of these findings are discussed and a tentative model for the genetic basis of acid-tolerance in Rhizobium is presented.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Glenn, Andrew and Dilworth, Michael
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