Identification of the regulatory genes required for the acid activation of the low pH inducible gene lpiA in Sinorhizobium medicae

The study of low pH responsive gene expression in Sinorhizobium medicae has identified many genes that are regulated in response to acidic conditions. One of these, the lpiA (low pH inducible gene A) gene, is acid-activated at least twenty fold by low pH and not by any other stress. The acid-activated expression of lpiA has been shown to require functional FsrR (fused sensor-regulator). However, even in the absence of FsrR, there is still a 6-fold acid-induction of lpiA revealing that other proteins are required for the acid-activation process. Such regulators could be encoded by tcsA (two component sensor gene A) and tcrA (two component regulator gene A) which are located upstream of fsrR in the lpiA gene region. Furthermore, the identification of a potential sigma factor binding motif upstream of lpiA suggested the requirement for RpoN (RNA polymerase sigma factor for nitrogen metabolism) for the transcription process. These regulatory proteins may also be required for the transcription of the acvB (acid virulence induced gene B) since the DNA sequence contains a lpiA stop codon that is coupled to the acvB start codon. This would infer that these two genes constitute an operon. It was the aim of this work to therefore investigate the additional regulatory aspects critical for the acid activation of lpiA and to identify if the lpiA and acvB genes are co-transcribed in response to acid. Mutations were constructed by inserting the CAS-1116 cassette into the tcsA, acvB and lpiA genes of S. medicae WSM419 to create the mutants MUR2121, MUR2124 and MUR2127, respectively. Each mutant was constructed by single crossover insertional inactivation to disrupt the targeted gene and to enable expression to be monitored in each background. In addition, mutants were obtained from previous studies carrying single crossover mutations in tcrA, rpoN and fsrR. Phenotypic characterization of tcsA, tcrA, fsrR, acvB, lpiA and rpoN mutants revealed that these genes are not required for resistance to ZnCl2 (up to 0.7 mM), CdCl2 (up to 0.175 mM), CuSO4 (up to 1.75 mM), NaN3 (up to 0.175 ml) or ethanol (7%) in comparison to the wild-type WSM419. These genes were also found not to be essential for cell growth in moderately acidic conditions (pH 5.7). However, it was found that a mutation in lpiA or rpoN slightly decreased the growth rate of the mutants when they were exposed to pH 5.7. Furthermore, in comparison to the wild-type S. medicae WSM419, the two mutants were more sensitive to the antibiotic polymyxin B, and were only marginally more sensitive to the antibiotics vancomycin, bacitracin and spectinomycin at pH 5.7. In addition, it was also discovered that the mutation in rpoN, but not lpiA, caused a symbiotic nitrogen fixation defect for the hosts Medicago sativa, Medicago murex, Medicago polymorpha and Medicago truncatula. All of the above-mentioned mutants described were constructed by the insertion of a CAS-1116 cassette into the targeted gene. The promoterless gusA reporter was used to describe enable transcription to be monitored at the point of insertion. s-glucuronidase (GUS) determinations at pH 7.0 and pH 5.7 revealed that all of the CAS-1116 induced fusions, including that to lpiA, were constitutively expressed in the conditions tested. This finding indicated the unexpected existence of a strong unidentified promoter in the cassette sequence upstream of gusA. Expression of each gene was therefore measured in cells cultured at pH 7.0 and pH 5.7 using the qRT-PCR technique. This technique confirmed the acid-induction of lpiA and revealed that acvB was also acid-activated to the same extent. This result reinforced the concept that acvB was co-transcribed with lpiA in S. medicae. In contrast, tcsA, tcrA, fsrR and rpoN were shown to be constitutively transcribed with respect to pH. To investigate the expression of lpiA in each mutant, gusA negative backgrounds were constructed. The CAS-1116 cassette (flanked by loxP sites) was excised from MUR2121 (tcsA:CAS-1116), MUR2124 (acvB:CAS-1116), MUR2090 (tcrA:CAS-1116), MUR2127 (lpiA:CAS-1116) and MUR2088 (rpoN:CAS-1116) by Cre-mediated recombination. The resulting S. medicae mutants MUR2122, MUR2125, MUR2092, MUR2128, and MUR2093 contained ΔCAS-1116 loxP mutations in tcsA, acvB, tcrA, lpiA and rpoN, respectively. To monitor the expression of lpiA in these backgrounds, a plasmid borne lpiA-gusA fusion located on pWR220-101H was mobilized into the deletion mutant backgrounds MUR2122, MUR2125, MUR2092, MUR2128 and MUR2093 to create the strains MUR2123, MUR2126, MUR2094, MUR2129 and MUR2095, respectively. The expression of the plasmid borne lpiA-gusA fusion was examined in each mutant cultured at low and neutral pH. The fusion was partially induced at pH 5.7 in MUR2092 (~6-fold) and MUR2125 (~10-fold) revealing that TcrA and AcvB both act positively to acid activate lpiA transcription. The expression of the lpiA-gusA fusion in MUR2122 or MUR2092 was totally abolished revealing that both TcsA and RpoN are essential for lpiA expression. This result confirmed a role for RpoN and revealed for the first time a specific sigma factor that is required for pH regulated gene expression in S. medicae. Furthermore, the finding provided credence to the suggestion that the putative RpoN binding motif located upstream of lpiA may in fact be the lpiA promoter. To further reinforce this concept, the start site of acid–induced transcription was determined using the 5′ Rapid Amplification of cDNA ends (RACE) technique. The transcription start site for both lpiA and acvB was found to occur 206 bp upstream of the lpiA start codon. The rpoN binding site (5′-TGGCACG-N4-TTGCW-3′) was located adjacent to this identified transcription start site but with the bold G and C in the motif positioned -27 and -14 bases upstream of the transcription start site rather than the expected positioning at -24 and -12. RpoN is known to have a requirement for an enhancer binding protein (EBP) to form the open form complex required for transcriptional activation. A bioinformatics search against the WSM419 genome for proteins carrying a sigma54_interaction domain (Pfam accession number: pfam00158) revealed that the gene product of Smed_5956, located upstream of tcsA, is a putative EBP. Future work could investigate whether it is this EBP, or another, that is required for the expression of the lpiA/acvB operon. The results presented reveal that the expression of the low pH induced lpiA/acvB operon is regulated in a multifaceted way. Interestingly, weak induction of lpiA occurs in the absence of TcrA or FsrR. Since induction was totally abolished in a TcsA mutant, this could infer that TcsA is the cognate sensor for each of these two-component regulatory proteins. In addition, full expression of lpiA was shown to occur only if the RpoN sigma factor was present. This is the first report that has detailed a role for the alternative sigma factor RpoN to regulate the transcription of acid-induced genes. The findings of this thesis have been used to construct a model for the lpiA acid-induced regulatory circuit and the implications of this model are discussed.