Identification and characterization of SnrA, an inducible oxygen-insensitive nitroreductase in Salmonella enterica serovar Typhimurium TA1535.

The biological activity of many nitrosubstituted compounds, many of which are produced commercially or have been identified as environmental contaminants, is dependent on metabolic activation catalyzed by nitroreductases. In the current study, we have cloned a nitroreductase gene, Salmonella typhimurium nitroreductase A (snrA), from S. enterica serovar Typhimurium strain TA1535, and characterized the purified gene product. SnrA is 240 amino acids in length and shares 87% sequence identity to the Escherichia coli homolog, E. coli nitroreductase A (NfsA). SnrA is the major nitroreductase in S. enterica serovar Typhimurium strain TA1535 and catalyzes nitroreduction through a ping-pong bi-bi mechanism in a NADPH and flavine mononucleotide (FMN) dependent manner. SnrA exhibits extremely low levels of FMN reductase activity but the nitroreductase activity of SnrA is competitively inhibited by exogenously added FMN. Treatment of TA1535 with paraquat resulted in induction of nitroreductase activity, suggesting that SnrA is a member of the S. enterica serovar Typhimurium SoxRS regulon associated with cellular defense against oxidative damage. Examination of the microbial genomes databases shows that SnrA homologs are widely distributed in the microbial world, being present in isolates of both Archea and Eubacteria. Southern hybridization and PCR failed to detect the snrA gene in the closely related S. enterica serovar Typhimurium strain TA1538. S. enterica serovar Typhimurium strains TA1535 and TA1538 and their derivatives are commonly used in mutagenicity testing. Differences in metabolic capacity between these two strains may have implications for the interpretation of mutagenicity data.

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1.

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT.

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.

The oriT region of the conjugative transfer system of plasmid pCU1 and specificity between it and the mob region of other N tra plasmids.

The oriT region of the conjugative IncN plasmid pCU1 has been localized to a 669-bp sequence extending from pCU1 coordinates 8.48 to 9.15 kb. The nucleotide sequence of this region was determined. The region is AT-rich (69% AT residues), with one 19-bp and one 81-bp sequence containing 79% or more AT residues. Prominent sequence features include one set of thirteen 11-bp direct repeats, a second set of two 14-bp direct repeats, six different inverted repeat sequences ranging from 6 to 10 bp in size, and two sequences showing 12 of 13 nucleotides identical to the consensus integration host factor binding sequence. Specificity between this oriT and mobilization (mob) functions encoded by the N tra system was demonstrated. This specificity is encoded by the region lying clockwise of the BglII site at coordinate 3.3 on the pCU1 map. Two N tra plasmids isolated in the preantibiotic era were unable to mobilize recombinant plasmids carrying the oriT region of pCU1 or to complement transposon Tn5 mutations in the mob region of the closely related plasmid pKM101.