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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 IpiA, 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 IpiA required the fused sensor--regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. melilotistrain 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.

A pH-regulated (phrR) gene was discovered in the Sinorhizobium meliloti WSM491, formerly known as Rhizobium meliloti, located downstream from the lipid acyl transferase (actA) gene. The organism was observed to be more tolerant against acid soils than other forms of the strains. It was indicated that the actA gene of the organism was required for acid tolerance and growth at low pH. The phrR gene encoded a putative repressor protein, PhrR, induced by low pH and other forms of environmental stresses.

A number of Tn5-induced acid sensitive mutants of Rhizoium meliloti and rhizobium leguminosarum bv. viciae were isolated to determine the basis of acid tolerance in Rhizobium. Results indicat the discovery of a mutant of R. leguminosarum bv. visiae WSM710 (WR6-35) which showed a more mucoid phenotype on minimal medium than the parent. Furthermore, it was found that Tn5 disrupted a gene encoding regulatory protein (ExoR) which negatively modulates the biosynthesis of EPS in R. leguminosarum bv. viciae WSM710.

The TG5-46 mutant, arising from Tn5 mutagenesis of Rhizobium meliloti, shows acid sensitivity due to inactivation of actR or actS genes in R. meliloti WSM419. DNA sequence analysis reveals ORF1 (actR) and ORF2 (actS), open reading frames flanking Tn5 in TG5-46, as sensor-regulator system controlling expression of genes involved in resistance to pH lower than 6.0. Acid sensitivity is directly related to the presence of Tn5 in TG5-46.

The DNA sequence of the actA gene, predicted nature of the ActA protein, its similarity to the cutE gene of Escherichia coli, its distribution in a range of bacteria and its expression at acidic and neutral pH are presented. The rhizobial gene is required to isolate and sequence an acid-tolerant phenotype. The membrane-pound, lipid-metabolizing protein can be compared to the Cut/Lnt protein of enteric bacteria, which, when inactivated, becomes prone to environmental stress.

A study was conducted to analyze alternative minitransposons that may be utilized to complement Tn5 derivatives. The number of restriction sites in the mTn5s was significantly minimize to increase the range of endonucleases which can be used to cut mutated target DNA in preparation for cloning. Results indicated novel constructs that provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies.