RELOCATION OF RNA POLYMERASE CORE GENES IMPACTS Vibrio cholerae’s GROWTH AND FITNESS

LETICIA LAROTONDA; BELEN BORDIGNON; ELISA OJEDA; DIEGO COMERCI; ALFONSO SOLER BISTUE

Chapadmalal

Congreso; SAMIGE 2023; 2023

SAMIGE

Growth rate is a key parameter of bacterial physiology that varieswidely among microorganisms. However, its genetic basis remains unclear. Geneorder along the chromosomes could play a role: in fast-growing bacteria, thegenes encoding ribosomal proteins (RP) and RNA polymerase (RNAP) are locatednear the origin of replication (oriC).In optimal growth conditions, fast-growing bacteria overlap replication rounds,a process called multi-fork replication (MFR). Hence, genes close to the oriC benefit from a higher dosage duringexponential growth with respect to those in the terminal region(ter)increasingtheir global expression. The positional bias of RNAP and RP genes maximizetheir expression. To experimentally test these bioinformatic correllations  we employed recombineeringtechniques to manipulate the genomic location of the rplKAJL-rpoBC locus, which encodes the catalytic core of the soleRNAP of Vibrio cholerae (Vc). After relocatingit to increasing distances from oriC,we measured the growth rate and fitness of this strain set at maximum  and slow growth conditions when MFR does notoccur. Relocation of the locus closeto its original location shows no phenotype indicating that neither thetransposition process nor the exact genetic context impacts rplKAJL-rpoBC function. In optimal growth conditions,rplKAJL-rpoBC relocation to themiddle of the chromosome, or to ter1and ter2 regions led to a cell sizedecrease, a generation time (GT) increment and a fitness loss closelycorrelating to oriC distance. In slow growing conditions, no phenotype wasobserved. These results suggest that the relocation of rplKAJL-rpoBC far from oriCaffects the GT due to a decrease in its gene dose in exponential phase underoptimal growth conditions. To demonstrate this we generated merodiploid strainswith two copies of the locus: bothnear the oriC, one copy in theoriginal site and other in ter1, andwith copies in the terminal regions of both chromosomes. We observed arestoration of growth rate when a second copy of rplKAJL-rpoBC was added in those strains where it was relocated farfrom the oriC. On the other hand, theexcess of rplKAJL-rpoBC dosage, didnot alter cell physiology. Finally, to test if our observations were due to rpoBC genes and not to the other genesin the locus we deleted the RNAP coregenes at the original position in merodiploid strains. In optimal growthconditions, we observed statistically lower increment in GT and fitness losswhen only one copy of rpoBC genesremain at ter1 region. Thus, RNAPgenes genomic location are partially responsible for the observed phenotypes.Overall, this study shows that the chromosomal gene order has been selectedalong evolution to maximize growth when nutrients are abundant and efficientlyoccupy the niche. [U1]  [U1]Frase final que le de una contenido más amplioy ponga tu investigación en contexto.