S10-spec-α Repositioning in Vibrio cholerae Provides Insight into Genome Organization Rules of Fast-Growers

ALFONSO SOLER BISTUE; JUAN MONDOTTE; VAL ME; BLAND MJ

Boston

Congreso; ASM General Meeting; 2014

American Society for Microbiology

Comparative genomics revealed a conserved trend ingenome organization of fast-growing bacteria: position of ribosomal and RNApolymerase genes are biased towards origin of replication (oriC). It has been speculated that this bias would allow increasedgene dosage during early exponential phase. Experimental evidence is scarcethough. In bacteria, a single locus S10-spc-α(S10) encodes half of ribosomal protein genes. Vibrio cholerae, a bichromosomal fast-growing pathogen, was tested forprecise relocation of a highly conserved locus. S10repositioning was achieved using lamboid phage recombinases through transientexcision followed by targeted reintegration. A set of isogenic mutants whereS10 was moved next to its original position, to the middle of the replichore,to the terminal region of Chromosome 1 and to the secondary chromosome wasobtained. Analysis of growth kinetics and time-lapse microscopy experiments showedthat, in fast-growing conditions, the further away S10 was moved the lowergrowth rate (GR) was obtained. Interestingly, this phenotype correlated with areduction of S10-dosage and S10 mRNA abundance. Importantly, upon the returningthe locus to its original position GR is recovered.Although S10 could be easily moved to several positions within the genome, infectiontests on the model organism Drosophilamelanogaster showed that mutants are highly impaired in host invasion. Thisfact suggests that, in nature, strong selective pressure drove this locus near oriC. This work provides direct genetic experimentalevidence of how genome is organized and will certainly contribute to SyntheticBiology for future efforts to rationally reprogram bacterial generation time.