CONICET | Buscador de Institutos y Recursos Humanos

Background: Ribosomalprotein (RP) genes locate near the replication origin (oriC) in fast-growing bacteria. Thus, higher RP dosage duringexponential phase due to overlapping replication rounds would optimize translationlevels. We built a set of strains where S10-spc-αlocus (S10), coding most of RP, was relocated to alternative genomic positions.S10 relative distance to the oriC tightly correlates toreduction of its dosage and growth rate. Objectives: Uncovermechanisms linking S10 dosage to bacterial physiology. Methods: We studiedstrains were only S10 location was altered. We estimated protein production. Wedeep sequences their DNA and RNA. Flourescence Recovery After Photobleaching (FRAP) allowedcytoplasm fluidity assessment. Results: S10 locationdid not affect protein production at the population level. At the single-celllevel, there was less reporter synthesys when S10 was far from oriC. RNA and DNA deep-sequencing revealedgenome-wide alterations transcription and replication dynamics. The number of differentially expressed genesand replication dynamics varied as a function of S10-oriC distance. Transcriptionally altered genes were implicated in amino acidmetabolism, transport and protein folding. Since RP constitute a significantpropotion of cell mass we hypothesized that lower S10 expression could reducethe cytoplasm macromolecular crowding, globally impairing cell physiology. Wegathered evidence supporting this idea. FRAP experiments showed higher cytoplasmfluidity when S10 dosage is low. Differences in growth and replication dynamicsin the most affected mutans were alleviated in a hyperosmotic context. Theseexperiments suggest that RP location besides optimazing protein biosynthesis, providesthe ideal macromolecular crowding conditions.