Analysis of the SOS response in Salmonella enterica serovar typhimurium using RNA fingerprinting by arbitrarily primed PCR

Abstract

We report an analysis of a sample of the SOS response of Salmonella enterica serovar Typhimurium using the differential display of RNA fingerprinting gels of arbitrarily primed PCR products. The SOS response was induced by the addition of mitomycin C to an exponentially growing culture of serovar Typhimurium, and the RNA population was sampled during the following 2 h. These experiments revealed 21 differentially expressed PCR fragments representing mRNA transcripts. These 21 fragments correspond to 20 distinct genes. All of these transcripts were positively regulated, with the observed induction starting 10 to 120 min after addition of mitomycin C. Fifteen of the 21 transcripts have no homologue in the public sequence data banks and are therefore classified as novel. The remaining six transcripts corresponded to the recE, stpA, sulA, and umuC genes, and to a gene encoding a hypothetical protein in the Escherichia coli lysU-cadA intergenic region; the recE gene was represented twice by nonoverlapping fragments. In order to determine if the induction of these 20 transcripts constitutes part of a classical SOS regulon, we assessed the induction of these genes in a recA mutant. With one exception, the increased expression of these genes in response to mitomycin C was dependent on the presence of a functional recA allele. The exception was fivefold induced in the absence of a functional RecA protein, suggesting another layer of regulation in response to mitomycin C, in addition to the RecA-LexA pathway of SOS induction. Our data reveal several genes belonging to operons known to be directly involved in pathogenesis. In addition, we have found several phage-like sequences, some of which may be landmarks of pathogenicity determinants. On the basis of these observations, we propose that the general use of DNA-damaging agents coupled with differential gene expression analysis may be a useful and easy method for identifying pathogenicity determinants in diverse organisms.

FIG. 1

Standard RAP gel for a mitomycin C treatment of 2.0 μg/ml. In this experiment, the RT primer is EA10 (Table 1) and the cDNA amplification primer is OPN22. The time zero point is used as the untreated control. At each time point, two RNA samples were analyzed, using a total of 500 μg (Hi) or 250 μg (Lo) for each time point. In this particular experiment, bands A, B, and C were extracted and isolated as described in Materials and Methods.

FIG. 2

Confirmation of differential expression using a standard quantitative RAP-PCR gel. In this experiment, RNA was reverse transcribed by priming with random hexamers, and the subsequent PCR amplification was accomplished with a gene-specific primer pair. Molecular weight markers are on the extreme left and in the middle of the gel. The experiment for which results are shown on the left (with respect to the central molecular weight marker) was done on wild-type Salmonella serovar Typhimurium at 0, 0.5, and 2.0 μg of mitomycin C (MC)/ml. Samples were withdrawn and analyzed at the indicated time points. The right side of the figure shows the results of the same protocol carried out on a recA mutant strain. The data clearly indicate that the gene is not induced in the recA mutant, while other background bands remain unaffected.

FIG. 3

An experiment similar to that described for Fig. 2 was performed for the RW1 sequence. The transcript shows induction by mitomycin C (MC) (at 0.5 and 2.0 μg/ml) in the presence and absence of a functional RecA protein. The gene is clearly induced in the recA mutant strain and therefore is the first example of an SOS-induced gene with a regulatory structure different from that of the standard MC-induced regulon. Quantified induction levels are shown in Table 3.