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

Abstract

A mutant of Salmonella enterica 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, Salmonella's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene pncA This insertion blocked most transcription of pncA, 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.IMPORTANCE 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.

Keywords: AspC; DNA replication; NAD metabolism; TyrB; integrated plasmid.

FIG 1

NAD in Salmonella is either synthesized de novo starting from aspartate and dihydroxyacetone phosphate (DHAP) or generated from Nm, NA, or quinolinic acid scavenged from outside the cell. Nicotinamide ribonucleoside (not shown) can also be taken up and converted into NMN (41). DNA ligase and other enzymes utilize NAD and break it down primarily into NMN. PRPP, phosphoribosyl pyrophosphate.

FIG 2

The aminotransferase reactions catalyzed by aspartate aminotransferase (AspC) and tyrosine aminotransferase (TyrB) have overlapping specificities at 37°C, but at 42°C only AspC is able to perform both enzymatic reactions.

FIG 3

Insertion of the virulence plasmid into the chromosome distorts read coverage from Illumina sequencing of genomic DNA. The coverage is scaled so that the average chromosomal coverage is the same between the strains. The dark top edge extends from the maximum coverage to the average coverage for the sequence represented by each pixel. Genomic DNA was harvested from stationary-phase cells after growth in LB at 37°C. The 11-kb MudJ transposons are inserted at two different positions within traH and delete 34 kb of pSLT plus 10 kb or 15 kb of adjacent chromosomal DNA.

FIG 4

Deletion of both pSLT replication origins is required for growth of the sppA::pSLT mutant on minimal glucose plates at 42°C. LB overnight cultures were diluted in buffered saline and spotted onto minimal glucose plates for 40 or 40,000 CFU per spot. The plates were incubated at 42°C for 24 h. The top line represents the wild-type Salmonella Typhimurium strain, and the second line represents the sppA::pSLT insertion mutant. The remaining strains include one or two deletions in the sppA::pSLT background. Each deletion inserted either a 1-kb Cm^r cassette (orange boxes) or a 1.5-kb Kan^r cassette (purple boxes) into the deleted region. The upper and lower images are from different parts of the same plate. Red arrows indicate replication genes. Dilutions were also spotted onto LB plates and grown at 30°C for colony counts (not shown).