Mimicking the last step of gene elongation: hints from the bacterial hisF gene

Gene elongation consists in an in-tandem duplication of a gene and divergence and fusion of the two copies, resulting in a gene constituted by two divergent paralogous modules. Many present-day proteins show internal repeats of amino acid sequences, generated by gene elongation events; however, gene elongation is still a poorly studied evolutionary molecular mechanism. The most documented case is that of the histidine biosynthetic genes hisA and hisF, which derive from the gene elongation of an ancestral gene half the size of the extant ones. The aim of this work was to experimentally simulate the possible last step of the gene elongation event occurred during hisF gene evolution under selective pressure conditions. Azospirillum brasilense hisF gene, carrying a single nucleotide mutation that generates a stop codon between the two halves of the gene, was used to transform the histidine-auxotrophic Escherichia coli strain FB182 (hisF892). The transformed strain was subjected to selective pressure (i.e., low concentration/absence of histidine in the growth medium) and the obtained mutants were characterized. The restoration of prototrophy was strongly dependent on the time of incubation and on the strength of the selective pressure. The mutations involved the introduced stop codon with a single base substitution and none of the mutants restored the wild-type codon. Possible correlations between the different mutations and i) E. coli codon usage, ii) three-dimensional structures of the mutated HisF proteins, and iii) growth ability of the mutants were investigated. On the contrary, when the experiment was repeated by mutating a more conserved codon, only a synonymous substitution was obtained. Thus, experiments performed in this study allowed to mimic a possible gene elongation event occurred during the evolution of hisF gene, evidencing the ability of bacterial cells to modify their genome in short times under selective conditions.