Integration of the pSLT Plasmid into the Chromosome Results in a Temperature-Sensitive Growth Defect Due to Aberrant DNA Replication.

A mutant of serovar Typhimurium was isolated that simultaneously affected two metabolic pathways as follows: NAD metabolism and DNA repair. The mutant was isolated as resistant to a nicotinamide analog and as temperature-sensitive for growth on minimal glucose medium. In this mutant, 's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene This insertion blocked most transcription of , which reduced uptake of the nicotinamide analog. The pSLT insertion mutant also exhibited phenotypes associated with induction of the SOS DNA repair system, including an increase in filamentous cells, higher exonuclease III and catalase activities, and derepression of SOS gene expression. Genome sequencing revealed increased read coverage extending out from the site of pSLT insertion. The two pSLT replication origins are likely initiating replication of the chromosome near the normal replication terminus. Too much replication initiation at the wrong site is probably causing the observed growth defects. Accordingly, deletion of both pSLT replication origins restored growth at higher temperatures. In studies that insert a second replication origin into the chromosome, both origins are typically active at the same time. In contrast, the integrated pSLT plasmid initiated replication in stationary phase after normal chromosomal replication had finished. The gradient in read coverage extending out from a single site could be a simple but powerful tool for studying replication and detecting chromosomal rearrangements. This technique may be of particular value when a genome has been sequenced for the first time to verify correct assembly.