Abstract
One contentious method by which bacteria deal with a maladapted situation is what has been coined ‘adaptive’, ‘directed’ or ‘stress induced-mutation’. This involves mutants that compensate for some form of sub-lethal stress appearing in large numbers from an apparently non-dividing cell population after a protracted period of time. In Mesorhizobium loti this was first observed in the non-symbiotic strains CJ1 and N18. The stress-induced mutation involved the activation of a cryptic chromosomal dctABD locus which allowed these strains to utilize succinate as a sole carbon source (Dct+). In the current study the symbiotic strain R7A, which containsan integrative and conjugative element ICEMlSymR7A, was also shown to produce Dct+ mutants when under stress, when its active dct locus on ICEMlSymR7A was deleted. However a derivative of R7A cured of ICEMlSymR7A (strain R7ANS), did not form mutant colonies strongly suggesting that the causative locus was located on ICEMlSymR7A. In this work, the causative locus was identified as the nadQABC locus, which consists of genes encoding enzymes for the first three steps of the NAD de novo biosynthetic pathway (nadABC) and an upstream hypothetical ORF encoding a putative nudix hydrolase-like repressor (nadQ). Addition of this locus conferred the mutator phenotype on R7ANS. Furthermore, addition of increased concentrations of nicotinic acid, a precursor to NAD biosynthesis via a separate salvage pathway, also conferred the mutator phenotype on R7ANS, whereas increased nicotinic acid led to partial repression of the mutator phenotype in strains that contained the nadQABC locus. Mutants of R7A in the nadA, nadB and nadC genes were both nicotinic acid auxotrophs and unable to form Dct+ colonies without high concentrations of nicotinic acid. Expression data for this locus showed that all three de novo genes had significantly higher expression during prolonged incubation when compared to exponential growth, while nadQ appeared to be constitutive. Addition of nicotinic acid repressed expression of all three de novo genes in a concentration-dependent manner provided that nadQwas intact. Mutants in recA and mfd, genes essential to double-strand-break repair and transcription-coupled repair respectively, exhibited the mutator phenotype in the presence of nadQABC or high levels of nicotinic acid. The addition of exogenous catalase did not prevent the mutator phenotype, though it did lead to a small but consistent decrease in Dct+ colony formation. In conclusion, increased production of NAD was responsible for the mutator phenotype in M. loti, through a mechanism independent of either double-strand-break or transcription-coupled repair. This can occur through either de novo synthesis or salvage of NAD precursors, though nicotinic acid acted with NadQ as a co-repressor of de novo synthesis, leading to a decrease in mutator phenotype function in NAD prototrophic strains. A model is proposed in which the increase in the concentration of pro-oxidant NAD(H) leads to the formation of reactive oxygen species which in turn elevate the mutation rate due to genotoxic stress.