RELOCALIZATION OF CATALYTIC CORE OF RNAP AFFECTS THE GROWTH RATE IN V.CHOLERAE THROUGH GENE DOSAGE DURING MULTIFORK REPLICATION

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

Virtual

Congreso; SAIB SAMIGE 2021; 2021

SAIB y SAMIGE

Growth rate (GR) is a key parameter of bacterial physiology that varies widely among microorganisms. However, itsgenetic basis has not yet been clarified. Gene order in the bacterial chromosomes could play a role: in fast-growingbacteria, the genes encoding for ribosomal proteins (PR) and RNA polymerase (RNAP) are located near the origin ofreplication (oriC). Under optimal conditions, fast-growing bacteria overlap replication rounds, a process called multi-forkreplication (MFR). Hence, genes close to the oriC benefit from a higher dose during exponential growth with respect tothose in the terminal region (ter). Such positional bias may be a strategy to maximize the expression of the transcriptionand translation genes. As most of these observations come from bioinformatic studies, our goal is to experimentally testthese correlations. Our model is Vibrio cholera (Vc), an easy to handle, fast growing (GT: 16min) pathogen and also oneof the best-studied models of bacteria with compound genomes since it has a main chromosome and a secondary one.Using recombination techniques based on lambdoid phage recombinases, we altered the genomic location of the rplKAJL-rpoBC locus, which encodes the catalytic core of the sole RNAP in Vc, by placing it at increasing distances from the oriC.All the strains were checked by PCR and Southern Blot. First, we tested the growth of this strain set. Relocation of thelocus in close proximity of its original location shows no phenotype indicating that neither the transposition process northe exact genetic context impacts rpoBC function. At maximum growth condition (BHI, 37°C) relocation of the rpoBClocus far from oriC caused an increase of 12 % in GT. No differences between strains were observed when they weregrown in minimal medium at 30°C, a condition in which MFR does not occur (GT: 60 min). These results suggest that therelocation of rpoBC away from oriC affects the GT due to a decrease in its gene dose in the exponential phase underoptimal growth conditions. However, by relocating rpoBC we simultaneously modify its dose and its subcellular location.To discriminate between these possibilities, we generated merodiploid strains with two copies of the locus: both near theoriC, one copy in the original site and other in ter1, and also with copies in the terminal regions of both chromosomes. Weobserved a restoration of growth speed when a second copy of rpoBC was added in those strains where it was relocatedfar from the oriC. On the other hand, the excess of rpoBC dosage, didn't show an increase in the growth speed. In sum,our results suggest that the relocation of the core of RNAP away from the oriC is detrimental to cell physiology due toreduction on rpoBC locus dosage. In future experiments we intend to elucidate which genes within the locus are thoseresponsible for the observed phenotypes.