Genetic basis of the activation of the cryptic dct genes in Mesorhizobium loti
Previous studies showed that, although non-symbiotic mesorhizobia carry a copy of the dctABD genes responsible for C4-dicarboxylate transport, most strains isolated from soil were unable to utilise succinate as a carbon source, indicating that the genes were cryptic. Following prolonged incubation on solid media containing succinate as a sole carbon source, two strains (CJ1 and N18) gave rise to succinate-utilising colonies at a frequency much greater than expected by random mutation alone. Sequence analysis revealed that point mutations had occurred within the dctB or dctD genes in these strains. A cosmid clone pJW5 that conferred the mutator phenotype on other Dct- non-symbiotic strains was isolated from a CJ1 genomic DNA library. Two gene clusters similar to the toxin-antitoxin module hipAB, designated hipB1A1 and hipA2B2, found on pJW5 were implicated in the mutator phenotype. pJW5 was mutagenised with transposon Tn5 and two insertions both within hipB2 were the only insertions found that abolished the mutator phenotype. Furthermore, it was reported that subclones of pJW5 containing either or both hipAB loci conferred the mutator phenotype. These findings led to the hypothesis that the hipAB loci enable the cells to mutate to succinate utilisation by allowing them to undergo adaptive mutation through a dormant state known as persistence (Weaver, 2003 and personal communication). In this study subcloning was employed in an effort to confirm that hipAB act as a toxin-antitoxin module and further define the contribution of the hipAB loci to the mutator phenotype. Attempts to express hipA1 or hipA2 from an inducible promoter failed to show that HipA acted as a toxin. Further subcloning studies suggested that neither individual hip genes, hipB1A1, hipA2B2, or both hipAB clusters alone could promote mutation to succinate metabolism, indicating that other gene(s) in addition to hipB2 present on pJW5 are required. Subsequent restriction digests of pJW5 demarcated a region to the left end of pJW5 as most likely being involved in the mutator phenotype. The underlying mechanism of mutation to succinate utilisation, although still ambiguous, seems to harbour some functions of stress-induced mutagenesis (adaptive mutation) and cryptic activation of silent genes. Involvement of other genes in the phenotype was postulated and the Bacterial Transcription Repair Coupling Factor (TRCF or Mfd) gene was identified as a possible candidate. Mfd facilitates recruitment of repair proteins to DNA lesions present on the coding DNA strand of actively transcribed genes. An R7ANS mfd mutant was constructed and analysis of the mutant suggested that the mfd gene plays a role in the mutator phenotype as the onset of mutant colonies was delayed in the mutant and their numbers were reduced.
Advisor: Ronson, Clive; Sullivan, John
Degree Name: Master of Science
Degree Discipline: Department of Microbiology and Immunology
Publisher: University of Otago
Keywords: Cryptic genes; Mutation; Bacteria; Genetics
Research Type: Thesis