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The production of extracellular nuclease is one of the defining attributes of Serratia marcescens and other Serratia species. However, nuclease is only one of a number of extracellular proteins expressed by species of Serratia; others include proteases, chitinases, lipases, and a bacteriocin called marcescin. Unlike these other secreted proteins, the extracellular nuclease does not appear to be subject to substrate or catabolite regulation. Nuclease is instead regulated by growth phase (4, 16), as is the synthesis of many other extracytoplasmic degradatory proteins, toxins, and bacteriocins (1, 8, 10, 11).

Many of the extracellular proteins of Serratia nevertheless share some common regulatory pathways with nuclease, and pleiotropic regulatory mutants that exert effects on nuclease, lipase, chitinase, and marcescin have been identified (4, 12, 17, 18). One such mutant, which abolishes nuclease production and reduces chitinase and lipase activity, has been shown to be in the Serratia recA gene (2). Consistent with SOS control, a LexA binding site is located in the nuclease gene nucA promoter region (4). SOS regulation has also been shown for other extracellular proteins, such as the pectin lyase of Erwinia carotovora (14). Other alleles abolishing nuclease as well as bacteriocin production define a novel regulatory locus, nucC, which is closely related to the ogr gene of temperate phage P2 (7, 13). The nucC operon is also growth phase and SOS regulated independently of nucA.

Yet another class of pleiotropic mutants overexpresses these proteins (4, 17, 18). These overexpression mutants (called nuc^su [17]) display no noticeable growth defect. They remain SOS inducible and are growth phase regulated. These alleles appear to fall into at least two categories based on their relative levels of nuclease overexpression. However, the nature of these mutations and the genes they represent have previously not been determined. The molecular characterization of these mutants is the focus of this work.

Mutant description. We previously described (4, 12) the isolation of transposon Tn5 insertion mutations made in the wild-type S. marcescens strain SM6 that displayed altered nuclease halo sizes on DNase indicator plates. Among these were mutations that abolish expression of the nuclease (nucA) gene; these mutations map to the transcriptional activator gene nucC (13) as well as to recA. Other mutations displaying increased nuclease halo sizes were also described previously (4); these include mutants nuc^su93 and nuc^su95, which express nearly 50-fold more nuclease than SM6, and nuc^su161, which expresses about 10-fold as much nuclease as SM6. Similar mutants had previously been described, and one of these, W1050 (U. Winkler), which we use routinely, is derived from SM6(F' prolac) and carries a chemically induced pleiotropic mutation causing the overexpression of nuclease as well as other extracellular enzymes (17, 18).

DNA sequence analysis. Sequence data obtained with a primer to the end of Tn5 revealed that two of the mutants, nuc^su93 and nuc^su95, indeed have Tn5 insertions in the same gene and at essentially the same site. This gene has significant sequence identity to Escherichia coli xerC (5). XerC is a subunit of the XerCD site-specific recombinase that serves to monomerize chromosomes and plasmids prior to cell division (3). The DNA flanking the insertion in nuc^su161 shows significant sequence identity to the E. coli uvrD gene. UvrD is DNA helicase II, which unwinds double-stranded DNA and is important in DNA replication, recombination, and repair (9). In E. coli, the xerC and uvrD genes are closely linked.

View larger version (8K): [in this window] [in a new window]   FIG. 1.   Map of S. marcescens xerC-uvrD region. The gene order is identical to that of E. coli, and reading frames are oriented as indicated by the gray arrowheads. Sites of Tn5 insertions are marked by inverted triangles; restriction sites are as shown. The region which has been fully sequenced is indicated by the dashed arrow with kilobase markers shown below the line.

Measuring SOS induction. Why do Tn5 insertions in xerC and uvrD lead to nuclease overexpression? The simplest explanation is that these mutants are partially induced for their SOS response, resulting in induction of nucA transcription, since the nucA promoter is SOS inducible (4). E. coli strains with mutations in either xerC or uvrD show partial SOS induction (15a).

Suppression of the nuclease overexpression phenotype. Since S. marcescens xerC and uvrD have a high degree of similarity to their E. coli homologs, we tested whether the E. coli genes carried on multicopy plasmids could suppress the phenotype. Plasmid pSD105 carrying E. coli xerC and pET11d-H2wt carrying E. coli uvrD (9) were transformed into the different mutant and control strains. In Table 1, nuclease activity produced by these transformants is shown. SM6 containing these plasmids had no change in nuclease activity, whereas nuc^su93, nuc^su95, and W1050 containing pSD105 now produced wild-type levels of nuclease. Plasmid pET11d-H2wt had no effect on nuclease activity of strain W1050. From this, we conclude that the W1050 mutation is also in xerC. We were unable to obtain stable transformants of nuc^su93 or nuc^su95 with this plasmid. The nuc^su161 mutant containing pET11d-H2wt also produced wild-type nuclease levels, but pSD105 had no effect in this strain. These data show that the E. coli homologs specifically suppress the nuclease overexpression phenotype; therefore, the nuclease overexpression phenotype is in fact due to the Tn5 insertion mutations and not to a secondary mutation elsewhere.

Conclusions. In this work, we demonstrate that most nuclease overexpression mutants (nuc^su) are likely due to an indirect effect on transcription of nucA by the SOS system. The mutant phenotype is a result of partial induction of the SOS system caused by the mutation. Here we have sequenced two genes in which such mutations have been identified, xerC and uvrD. These genes are very similar to their E. coli homologs (5), and complementation data show that the E. coli genes are able to functionally replace the defective S. marcescens genes. The ability of E. coli XerC to repress the nuclease overexpression in W1050 suggests that it is also a xerC mutant.

Nucleotide sequence accession number. The DNA sequence has been deposited in GenBank (accession no. AF028736).