Genomic position of rpoBC impacts the physiology of Vibrio cholerae

JULIETA VIGLINO ; DIEGO COMERCI; BELEN BORDIGNON; ALFONSO SOLER BISTUE

Congreso; LIV Congreso Anual de SAIB; 2018

SAIB

Growth rate (GR) is a key parameter ofbacterial physiology widely varying among microorganisms. The genetic basis ofGR is not well understood. In fast-growing bacteria, the genes encoding RNApolymerase (RNAP) are close to the origin of replication (oriC). During exponential growth, fast growers perform multi-forkreplication. Thus, genes near to oriCbenefit of higher dosage. Therefore, such positional bias can be a strategy tomaximize RNAP expression. To experimentally test this, we used Vibriocholerae, a fast-growing human pathogen. Its genome can be widely modifiedby natural transformation coupled to recombineering techniques based on lambdaphage recombination sites. We aimed at modifying the genomic location of the rpoBC locus, encoding the sole bacterialRNAP. For this we flanked it with lambda phage attR and attL sites allowing itsexcision or inversion. We inserted attB? sites enabling site-specific re-integrationin heterologous locations. Growth curves show that the insertion of att sites donot affect bacterial physiology. Next, Int and Xis recombinases were expressedtransiently. Recombination reaction reconstitutes a marker that enables mutantselection. Two strains were built: one with an inverted rpoBC and a second where the locus was relocated far from the oriC.  Locus inversion does not present any evidentphenotype. Relocation of rpoBC producedcolonies of smaller size and slower GR. These physiological alterations areprobably due to the difference in gene dosage that occurs during the rapidgrowth phase by multi-fork replication.