regulation of theserratia marcescensextracellular nuclease: positive control by a homolog of p2 ogr...

J. Mol. Biol. (1996) 256, 264–278

Regulation of the Serratia marcescens ExtracellularNuclease: Positive Control by a Homolog of P2 OgrEncoded by a Cryptic Prophage

Shida Jin 1, Yi-chi Chen 1, Gail E. Christie 2 and Michael J. Benedik 1*

The Serratia marcescens extracelluar nuclease is a secreted protein that is1Department of Biochemicaland Biophysical Sciences subject to growth phase and SOS control. Regulatory mutants affecting

nuclease expression have been isolated that define a new locus, nucC,University of Houstonessential for transcription of the nuclease gene nucA. The cloned nucC geneHouston, Texas 77204-5934

USA is able to activate efficient expression from the nucA promoter in Escherichiacoli, where it normally is poorly expressed. NucC is very similar to the2Department of Microbiology bacteriophage P2 Ogr protein, a transcriptional activator essential for P2

and Immunology, Medical late gene expression. NucC is able to replace P2 Ogr to support the growthCollege of Virginia, Virginia of P2 ogr− mutants in E. coli. Ogr is a poor activator of the nuclease promoterCommonwealth University in E. coli, but the related d gene product from satellite phage P4 is highlyRichmond, Virginia effective. The presence of genes encoding a lysozyme and a putative porin23298-0678, USA or holin in the nucC operon suggests that nucC may be part of a cryptic

prophage genome. The putative holin-like membrane protein is required inE. coli for extracellular secretion of the S. marcescens nuclease.

7 1996 Academic Press Limited

Keywords: gene regulation; bacteriophage P2; transcriptional activator;transposon mutagenesis; cryptic prophage*Corresponding author

Introduction

The transition of bacterial cells from exponentialgrowth to stationary phase is accompanied byglobal changes in gene expression that elicitadaptive responses affecting cellular physiology andmorphology. The synthesis of extracytoplasmicdegradatory proteins, toxins and bacteriocins isoften regulated by growth phase. Examples of theseinclude the Escherichia coli microcin B17 (Genilloudet al., 1989) and acid phosphatase (Atlung et al.,1989), the Serratia liquefaciens phospholipase(Givskov & Molin, 1992) and the Serratia marcescensextracellular nuclease (Chen et al., 1992). Thisnuclease is one of a number of extracellular proteinsexpressed by species of Serratia; others include thechitinases, lipases and marcescins. Many of thesesecreted proteins are regulated by substrateavailability as well as growth phase; the extracellu-lar nuclease, however, does not appear to be subjectto substrate or catabolite regulation.

The chitinases, lipases and marcescins sharesome common regulatory pathways with nuclease,and pleiotropic regulatory mutants have beenidentified that exert effects on all of these proteins(Winkler, 1968; Winkler & Timmis, 1973). One suchclass of mutants overexpresses these proteins(Winkler & Timmis, 1973; Chen et al., 1992) while

another class abolishes nuclease expression andreduces chitinase and lipase activity. The latter hasbeen shown to be in recA (Ball et al., 1990).Consistent with SOS control, a LexA binding site islocated in the nuclease gene nucA promoter region(Chen et al., 1992). SOS regulation has also beenshown for other extracellular proteins, such as thepectin lyase of Erwinia carotovora (McEvoy et al.,1992). SOS control of nuclease expression isindependent of the growth-phase regulation (Chenet al., 1992).

Secretion of nuclease is also growth-phaseregulated (Y. Suh, S. J., T. K. Ball & M. J. B.,unpublished results). Initially synthesized with atypical N-terminal signal sequence of 26 amino acidresidues (Ball et al., 1987), the mature extracellularnuclease (Biedermann et al., 1989) can be first foundas a periplasmic intermediate (Y. Suh, S. J., T. K. Ball& M. J. B., unpublished results). This initialtranslocation step across the inner membrane occursrapidly. Then it is secreted extracellularly in aslower step with a half-time of about one hour. Thissecond step occurs late in a culture. Pleiotropicmutations causing the overexpression of nucleasealso increase the efficiency of the extracellularsecretion step as if components of the secretorysystem were coordinately regulated (Y. Suh, S. J.,T. K. Ball & M. J. B., unpublished results).

0022–2836/96/070264–15 $12.00/0 7 1996 Academic Press Limited

Ogr Homolog Activates Serratia Nuclease Expression 265

In order to dissect the complex regulation ofS. marcescens extracellular nuclease, we have usedtransposon mutagenesis to generate mutants withaltered levels of nuclease expression. In this workwe describe the isolation of novel regulatorymutants that abolish expression of the nucA gene,and the subsequent cloning and characterization ofthe gene defined by these mutants. This regulatorygene, nucC, is closely related to the ogr gene oftemperate phage P2. The ogr gene encodes a basic72 amino acid residue zinc-binding protein (Lee &Christie, 1990) that is required for P2 late geneexpression (Sunshine & Sauer, 1975; Birkeland et al.,1988; Halling et al., 1990) and is thought to activateP2 late transcription through an interaction with thea subunit of host RNA polymerase (Sunshine &Sauer, 1975; Ayers et al., 1994).

Results

Isolation of mutants defective innuclease expression

The transposon Tn5 was used to create a pool ofinsertion mutants in S. marcescens strain SM6 andcolonies were screened for increased or decreasednuclease expression on DNase indicator plates. Fourmutants with increased nuclease expression havealready been described (Chen et al., 1992), whichappear to define two loci. These loci are most likelythe same as the two classes of nucSU mutantsdescribed earlier by Winkler & Timmis (1973). Fourmutants that abolish nuclease expression were alsoisolated (Sm6-Nuc17, SM6-Nuc50, SM6-Nuc57,SM6-Nuc168) and are the focus of this report.

Cultures of these four mutants have no detectablenuclease activity, either extracellularly or intracellu-larly, as analyzed by the microtiter dish assay(Table 1, column 1). These mutants also showedreduced nuclease expression when the nucleasegene is present on a multicopy plasmid (pNuc1R,Table 1, column 2), indicating that the mutationsaffect a trans-acting factor. Expression of nucleasewas also not induced by mitomycin C (data not

shown), which dramatically induces, expression ofnuclease in wild-type strains (Ball et al., 1990).Western blots of both the cellular and supernatantfractions from these mutants showed no nucleasesignal (data not shown), suggesting that themutations exert their effect at the level of nucleaseexpression and not activation or secretion.

A more accurate measure of transcription fromthe nuclease promoter in these mutants wasobtained using the transcriptional fusion plasmidpNuc2-LacZ (Ball et al., 1990; Chen et al., 1992) inwhich the lacZ gene is transcribed from the nucApromoter. The basal level of b-galactosidaseexpression from the plasmid does not differ in themutants as compared to wild-type, but there is noinduction by mitomycin C in the mutant strains(Table 1, columns 3 and 4). We presume that theconsistent basal expression is from a plasmidpromoter because these mutant strains produce nonuclease.

Genetic characterization of mutants

Mutations in recA can abolish nuclease ex-pression, by virtue of increasing the LexAconcentration (Hines et al., 1988; Ball et al., 1990).Introduction of the E. coli recA gene on a multicopyplasmid into a recA::Tn5 mutant restores nucleaseexpression (Ball et al., 1990); no such complementa-tion was observed with these four mutants.Southern blotting (data not shown) confirmed thatthe Tn5 insertions were not within recA; nor werethey within the nuclease structural gene.

Two of the mutant alleles were cloned on the basisof the kanamycin resistance conferred by the Tn5.Genomic DNA of mutants Sm6-Nuc50 and SM6-Nuc168 was digested with EcoRI, which does notcut within Tn5, and ligated to pUC19 that had beencleaved by EcoRI and dephosphorylated.Kanamycin-resistant plasmids containing DNAfrom SM6-Nuc50 and SM6-Nuc168 were designatedp50E and p168E, respectively, and contained insertsof 18 to 20 kb.

Unique BamHI and SalI sites near the center ofTn5, just outside the kanamycin gene, were used togenerate subclones of each mutant carrying part ofthe transposon and flanking genomic DNA.Plasmid p50BK is a pACYC184 derivative carryingthe BamHI fragment from p50E that includes thegene for kanamycin resistance, while p50B carriesthe other part of Tn5. Similarly p168B and p168Scarry the non-resistant fragments generated bydigestion of p168E with BamHI or SalI. Using theseplasmids as template, a primer complementary tothe end of Tn5 was used to obtain the nucleotidesequence of the DNA adjacent to the Tn5 insertionsin the SM6-Nuc50 and SM6-Nuc168 mutants. Theinsertion site in SM6-Nuc50 is only 110 bp upstreamof that in SM6-Nuc168.

Because SM6-Nuc50 and SM6-Nuc168 have Tn5insertion sites very close to each other, it seemedlikely that the remaining Nuc− mutants hadinsertions in the same region. To test this

Table 1. Expression of the nuclease promoter in the Tn5insertion mutants

Nuclease b-Galactosidase

No NoStrain plasmid pNuc1R induction +Mitomycin C

SM6 240 2100 81 2300SM6-Nuc17 <3 240 63 100SM6-Nuc50 <3 30 81 160SM6-Nuc57 <3 10 100 130SM6-Nuc168 <3 30 67 130

Expression of nuclease and b-galactosidase from the nucleasepromoter, using pNuc1R or pNuc2-LacZ, respectively, is shownfor each mutant strain. To measure nuclease activity, freshovernight cultures were sonicated and 50 ml portions of eachsample were used in the microtiter dish nuclease assay (Ball et al.,1990). The values shown for nuclease correspond to the dilutionfactor required for equivalent activity. b-Galactosidase activity isshown in Miller units.

Ogr Homolog Activates Serratia Nuclease Expression266

Figure 1. Restriction map of the Bam3.3 fragment. The locations of the five open reading frames, as well as the overlapbetween NucE and NucD, are indicated above. Sites for restriction enzymes that cleave more than once are numberedfrom left to right. The bold line designates the region for which the new DNA sequence is presented in Figure 2. Sitesof Tn5 insertion are marked by ✽. The DNA fragments carried by the plasmids described in this work are shown below.The X indicates a frameshift mutation at that position.

hypothesis, genomic DNA from all four mutantstrains was digested with restriction enzymes andSouthern blots were prepared and probed using the1.5 kb SalI-BamHI fragment adjacent to the Tn5insertion in SM6-Nuc50. All mutants contained asingle Tn5 insertion in a 13 kb EcoRI fragment, a3.3 kb BamHI fragment, and a 1.2 kb PstI fragment(data not shown). Polymerase chain reaction (PCR)analysis using a primer within Tn5 and anotherfrom a region upstream of the SM6-Nuc50 insertionmapped the insertion site of Nuc 17 to about 20 bpupstream of the SM6-Nuc50 insertion site and theSM6-Nuc57 insertion site to within 100 to 110 bpupstream of the SM6-Nuc50 insertion site. Thus allfour transposons lie within a region of about 220nucleotides.

Isolation of a fragment encoding aregulatory locus

On the basis of the Southern blot analysis (seeMaterials and Methods), it seemed likely that the3.3 kb BamHI fragment in which all of the Tn5insertions were located was large enough to carrythe entire locus affected by the insertion of thetransposon. SM6 genomic DNA was digested withBamHI and fragments of size 2 to 5 kb were purifiedfrom LMP agarose and ligated with pUC18 DNAthat had also been cleaved with BamHI anddephosphorylated. Following transformation ofstrain MM294, colonies were probed with the 1.5 kbSalI-BamHI fragment of p50B. One positive clonewas recovered after screening 800 transformants.This strain grew on LB agar but had an unusualsmall, flat and very sticky colony morphology and

did not grow in broth. It did not express nuclease.Miniprep DNA was prepared from cells scraped offa plate and used to transform the SM6-Nuc50 andSM6-Nuc168 mutants. These transformants wererestored to a Nuc+ phenotype by the plasmid andwere able to grow in broth. Plasmid DNA isolatedfrom the S. marcescens transformants contained a3.3 kb BamHI insert, precisely the size of the bandseen in the wild-type SM6 Southern blot.

The plasmid-borne 3.3 kb BamHI fragment is ableto complement all four Nuc− Tn5 mutants, but doesnot confer a Nuc+ phenotype on E. coli. Thisindicates that the fragment does not contain anuclease structural gene but rather a regulatorylocus for nuclease expression. Because of theinhibitory effect that this cloned fragment has on thegrowth of E. coli, probably from a high level ofexpression, the 3.3 kb BamHI fragment was trans-ferred to the lower copy number plasmid pZ150 forfurther characterization.

DNA sequence analysis

The DNA sequence of a 2.95 kb region fromEcoRI to BamHI2 (Figure 1) was determined for bothstrands. The problem of compressed regions due tothe high G + C content of S. marcescens was solvedusing several strategies: inosine reactions were usedside by side with guanosine reactions, both strandswere carefully sequenced using multiple primersand subclones were generated so that reading couldstart close to the compressed regions. The sequenceof most compressed regions was resolved by thesemeans; however, several base-pairs outside thecoding regions remain uncertain.


Five open reading frames (ORFs) were found inthe sequenced region (Figure 1). Two of these lie onone strand and correspond to the S. marcescens rplSand trmD genes, encoding ribosomal protein L19and tRNA (m1G)methyltransferase, respectively.This sequence has been reported elsewhere (Jin &Benedik, 1994; Genbank no. L23334). The remainingthree ORFs were on the opposite strand; thenucleotide sequence from EcoRI to StuI2, containingthese three ORFs, is shown in Figure 2.

The non-redundant protein database wassearched for proteins similar to ORF1 (nucE), ORF2(nucD) and ORF3 (nucC) with the program BLASTP(Altschul et al., 1990). nucE, which encodes aputative membrane protein with two transmem-brane domains and a positively charged Cterminus, has weak sequence similarity to the holingene S of phage f21 (not shown). Like the f21 holin,NucE exhibits structural but not sequence similarityto the other members of the phage holin family.Holins, exemplified by the S protein of phage l,are small phage-encoded proteins essential forendolysin or lysozyme to gain access to thepeptidoglycan layer (Young, 1993). A structuralalignment of NucE and l S protein is presented inFigure 3A. NucE does not have the dual start motiftypical of many of the phage holins and is notespecially lethal on multicopy plasmids. WhetherNucE is in fact a member of the holin family or isanother type of presumed membrane proteinremains to be determined.

The second ORF (NucD) overlaps the upstreamnucE gene by 16 nt. The NucD protein sequenceshowed significant similarity to several knownphage lysis proteins, with strongest homologies tothe P22 protein 19 and to the f21 lysozymes. Thefirst of these is shown in Figure 3B. There are twopotential AUG start codons for NucD; both arepreceded by reasonable Shine-Dalgarno sequences.Translation from these two possible sites wouldlead to peptides that differ by nine amino acidresidues in length. Strikingly, there is a leader regionfound in NucD that resembles a putative signalpeptide according to von Heijne’s (1986) rule, whichwould be of normal length using the second startbut would be somewhat long using the first startcodon. This was surprising in that no lysozyme todate has been shown to have a signal peptide. AphoA translational fusion to the NucD aminoterminus suggests that it has the ability to localizePhoA such that it is active, presumably by acting asa signal sequence (data not shown).

The third ORF begins after a 72 nt gap. This ORF,designated nucC, showed striking similarity to afamily of transcriptional activators encoded by theP2-related temperate bacteriophages. Members ofthis family include the Ogr protein of phage P2(Sunshine & Sauer, 1975; Birkeland & Lindqvist,1986; Christie et al., 1986), the B protein of phage 186(Kalionis et al., 1986; Dibbens & Egan, 1992), and thed proteins of satellite phage P4 (Halling et al., 1990)and retronphage fR73 (Sun et al., 1991). Theseproteins are functionally as well as structurally

related; they are required for phage late genetranscription and all have been shown to supportgrowth of P2 ogr− mutants and to activate a clonedP2 late promoter in trans (see Christie & Calendar,1990). A comparison of these proteins with NucC isshown in Figure 3C. NucC has 66% amino acididentity to P2 Ogr. All seven residues that areinvariant among members of this family are sharedby NucC; this includes the four conserved cysteineresidues that are thought to comprise a Cys4-coordinated zinc-binding domain, Cys-X2-Cys-X22-Cys-X4-Cys (Lee & Christie, 1990).

The nucC gene encodes an activator ofnuclease expression

In order to localize the nuclease activator functionon the 3.3 kb BamHI fragment, we examined theability of various subclones of this region totrans-activate expression of the nuclease gene. Aspreviously reported (Ball et al., 1987), the nucleasepromoter does not normally function in most strainsof E. coli. However, low level expression of thenuclease promoter can be detected in strainMC1000.1, where it is both growth phase regulatedand SOS inducible (Chen et al., 1992). WhenMC1000.1 was cotransformed with the nucleaseplasmid pAC9Nuc1 and a compatible plasmidcarrying the 3.3 kb BamHI fragment, nucleaseexpression increased dramatically (Table 2). De-letion of sequences upstream of ClaI2 and down-stream of XbaI (Figure 1) did not eliminatetransactivation of nuclease expression (although theupstream deletion reduced it), indicating that thenuclease activator gene mapped to the regioncontaining the three open reading frames identifiedby sequence analysis. On the basis of its homologyto a known transcriptional activator, the nucC geneproduct was the logical candidate for an activator ofthe nucA nuclease gene promoter. nucD and nucEwere deleted from pZ150Bam3.3 by removal of thefragment between the second and third internalNcoI sites to yield pZ150Bam3.3DNco2-3. Thisplasmid still retained the ability to activate nucleaseexpression from MC1000.1 (pAC9Nuc1) with 80units of nuclease activity found in the cellsupernatant compared to less than three units fromthe control. The plasmid pZ150Bam3.3DNco2-3was also capable of restoring the defect in theS. marcescens SM6Nuc-Tn5 mutants, as shown inTable 3. SM6 transformed with this plasmid had athreefold higher nuclease expression than SM6alone, and all four mutants had nuclease expressionrestored to the same level as the SM6 transformant.This confirms that NucC is responsible for thenuclease activator function.

NucC also activated the cloned nuclease pro-moter in E. coli. When plasmids carrying nucCexpressed from a heterologous promoter (lac or tac)were introduced into E. coli MC1000.1 carryingeither the nuclease plasmid pAC9Nuc1 or thenucP-lacZ transcriptional fusion pNuc2-LacZ, trans-activation of the nuclease promoter was observed

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Figure 3. A, Structural similarity of NucE (ORF1) to phage l holin S. + , − , charged residues, = = = , potentialtransmembrane domains; tttt, potential b-turns. B, Comparison of the amino acid sequences of NucD (ORF2) and P22gene 19 protein. =, identical residues; :, similar residues; . . . . , gaps; and **, stop codons. C, Alignment of NucC (ORF3)to the P2 Ogr family of transcriptional activators. Amino acid sequences of NucC, P2 Ogr, 186 B, fR73d, and each halfof P4 d are aligned; only amino acid differences are shown. —, identical residues, . . . , gaps.

(Table 4). This activation required expression ofNucC; when nucC was cloned in antisenseorientation to the promoter no activation of nucleaseexpression was observed either from SM6-Nuc50 orfrom pAC9Nuc1 in MC1000.1 (data not shown).NucC activation of nuclease expression is notspecific to E. coli strain MC1000.1 but is alsoobserved in other E. coli genetic backgrounds (datanot shown).

To confirm the size of NucC, pulse labeling exper-iments were performed using pSE380TacNucC. The

results, shown in Figure 4, correlate well with thepredicted molecular weight of 8.3 kDa.

NucC is functionally homologous to the P2Org and P4 d proteins

The extensive similarity between nucC and theogr gene strongly supports the conclusion thatNucC is a transcriptional activator. Since othermembers of this family of activators are to someextent functionally interchangeable, we testedwhether this was also the case for NucC. E. coli

Table 2. Effect of the 3.3 kb BamHI fragment on nucleaseexpression and secretion in E. coli

Nuclease b-Lactamase

Plasmids Sup Cell Sup Cell

pAC9 + pZ150Bam3.3 <1 <1 N.D. N.D.pAC9Nuc1 + pZ150 <1 3 <0.1 12pAC9Nuc1 + pZ150Bam3.3 140 30 2.4 16

Overnight LB cultures of MC1000.1 carrying the indicatedplasmids were separated into supernatant (Sup) and whole cellfractions (Cell) by centrifugation. The cell pellet was resus-pended in an equal volume and, lysed by sonication. Nucleasewas assayed as in Table 1 and b-lactamase was assayed asdescribed in Materials and Methods, and is expressed asDA240 min−1 × 100. N.D., not done.

Table 3. Plasmid-encoded NucC restores nucleaseexpression in the Tn5 insertion mutants

Nuclease expression

Strain No plasmid pZ1540Bam3.3DNco2-3

SM6 10 30SM6-Nuc17 <3 100SM6-Nuc50 <3 30SM6-Nuc57 <3 30SM6-Nuc168 <3 30

Late stationary phase cultures were sonicated to releasenuclease, which was measured by the microtiter dish assay asdescribed for Table 1.


Table 4. Activation of the nucA promoter in E. coli byNucCPlasmids Nuclease b-Galactosidase

pAC9Nuc1 + pZ150 3pAC9Nuc1 + pTA108lacNucC 160pNuc2-LacZ + pACYC184 32pNuc2-LacZ + pACYC184TacNucC 980

Cultures of E. coli MC1000.1 carrying the indicated plasmidswere grown up overnight in LB, without IPTG induction.Nuclease activity was measured as described for Table 1, andb-Galactosidase activity was measured as described by Miller(1972).

Figure 4. Identification of the nucC gene product.Cultures of JM101 bearing pSE380 or pSE380TacNucCwere pulse-labeled with [35S]methionine, and proteinswere separated by SDS-PAGE as described in Materialsand Methods.

strains were transformed with pAC9Nuc1, whichcarries the nuclease (nucA) gene under control of itsown promoter, and a compatible plasmid express-ing NucC, P2 Ogr or P4 d. The resultingtransformants were tested for nuclease activity andfor the ability to support growth of a P2 phagedeleted for ogr. Plasmid pRF5 (Christie et al., 1986)expresses high levels of Ogr under control of the lpL promoter, which can be regulated by the tsrepressor expressed from pRK248cIts (Bernard &Helinski, 1979). Plasmid pBJ14 expresses P4 d froma modified T7A1 promoter that contains two copiesof the lac operator (Julien & Calendar, personalcommunication). As can be seen in Table 5, theS. marcescens NucC protein can replace P2 Ogr intrans to support growth of P2 D15. Reciprocalactivation of the cloned nuclease promoter by Ogrwas not observed. There were negligible levels ofnuclease following thermal induction of Ogr instrain MC1000.1 (Table 5). Expression of Ogr wasconfirmed by complementation of P2 D15 and bydirect observation of a protein band the approxi-mate size of Ogr on a Coomassie-blue-stainedprotein gel after thermal induction of MC1000.1(pAC9Nuc1 + pRF5 + pRK248cIts). In contrast, P4 dprotein expressed from pBJ14 induced high levels ofnuclease expression (Table 5). The lacIq allele ofE. coli strain JM101 was used to allow regulatedexpression of d from pBJ14. The functionalsubstitution of these activators clearly demonstratesthat NucC is a member of the Ogr family.

The nucE , nucD and nucC genesform an operon

Three lines of evidence suggest that nucE, nucDand nucC form an operon. First, the Tn5 insertionsin the nuclease-deficient mutants are all upstreamof nucC, in nucD, yet abolish nucC expression.Secondly, expression of NucC from plasmidscarrying various upstream regions indicated thatthe nucC promoter lies between BamHI1 and ClaI2

(Figure 2). Deletion of upstream sequence fromBamHI1 to ClaI2 (pZ150Cla2Xba or pZ150Cla2Bam2)reduced, but did not eliminate, NucC activitycompared to pZ150Bam3.3, as assayed by trans-acti-vation of nuclease activity from pAC9Nuc1 inMC1000.1. In addition, plasmid pACYC184NucC,which carries an NcoI-RsaI fragment that includesthe entire nucC gene and 100 nucleotides upstream,

shows no NucC activity. This is also consistent withthe location of the promoter further upstream.Thirdly, transcriptional fusions to lacZ were used tomap the promoter upstream of the ClaI2 site.Plasmid pCla2Sal1-LacZ carries the region fromClaI2 to SalI1 fused to lacZ while pRlSal1-LacZcarries a fragment that extends 480 nt upstream tothe EcoRI site. These constructs were tested forb-galactosidase activity in wild-type S. marcescensSM6, the nuclease deficient mutants Nuc17 andNuc50, and the nuclease overexpression mutantsSU93 and SU132 (Chen et al., 1992). As shown inTable 6, pCla2Sal1-LacZ has only backgroundb-galactosidase activity whereas in pR1Sal1-LacZwas about 16-fold above background. Thus thepromoter lies between the EcoRI and the ClaI2 sites.A construct that contains the entire upstreamsequence back to the BamHI site does not exhibitany further increase in NucC activation ability (datanot shown). Together these data support theconclusion that nucE, nucD and nucC form anoperon (the nucC operon) that is transcribed from apromoter between nt 1 and 480 of the sequenceshown in Figure 2. Normal expression from thispromoter was also seen in the nuclease overexpress-ing mutant SU132, but a sixfold increase in activitywas observed in the overexpressing mutant SU93(Table 6). This suggests that the nuclease over-expression phenotype of SU93 is probably anindirect consequence of NucC overexpression dueto the mutation in this strain.


Table 5. Functional complementation between NucC, Ogr and dStrain Plasmids Nuclease Growth of P2D15

MC1000.1 <1 −MC1000.1 pAC9Nuc1 6 −MC1000.1 pAC9Nuc1 + pRF5 + pRK248cItsa 10 +MC1000.1 pAC9Nuc1 + pTA108lacNucC 160 +JM101 <1 −JM101 pAC9Nuc1 3 −JM101 pAC9Nuc1 + pBJ14 70 +JM101 pAC9Nuc1 + pBJ14( + IPTG)b 200 +

Co-transformants of the nuclease gene and nucC, ogr, or d were measured for nuclease activityfrom stationary phase cultures as described for Table 1 and scored for the ability to plate P2ogr− under inducing conditions. pRF5 has P2 ogr under lpL control. pBJ14 expresses P4 d froma T7AI promoter carrying two copies of the lac operator.

a Plate was incubated at 40°C to allow thermal induction.b Induction with 0.1 mM IPTG; cells were sick and the cell density was much lower than

without induction.

High level expression of the nucC operonis lethal

We have shown that deleting the region betweenthe second and the third NcoI sites from the clonedBam3.3 fragment yields a plasmid that expressesnucC from its own promoter and complements thedefect in the Tn5 nuclease expression mutants.However, we were unable to transform the Tn5mutant strains with any of the plasmids expressingnucC from the lac or tac promoter. This suggestedthat unregulated expression of nucC might be lethalin these strains. We therefore attempted toco-transform SM6-Nuc50 and SM6-Nuc168 withpACYC184TacNucC and a plasmid encoding lacIq,pSE380BspMI. Transformants were obtained; thesecolonies were quite sick but had a Nuc+ phenotypeon DNA Test Agar plates. This was reminiscent ofthe high expression lethality exhibited by theoriginal Bam3.3 fragment when cloned in E. coli. Totest whether the combination of NucC and the3.3 kb BamHI fragment was the cause of thissynthetic lethality, we attempted to transformMC1000.1 with compatible plasmids carrying theentire 3.3 kb BamHI fragment and nucC under Ptaccontrol. While transformants were viable withpACYC184 and the pZ150Bam3.3 plasmid or withpACYC184TacNucC and pZ150, no transformantswere obtained with pZ150Bam3.3 and pA-

CYC184TacNucC. This suggests that NucC activatesexpression of a function in the Bam3.3 clone that isalso lethal to E. coli, while overexpression of NucCalone is not lethal. Since overexpression of NucCwas also lethal in the SM6 mutants, which carrypolar Tn5 insertions in nucD, we presume that it ishigh level expression of the upstream nucE genethat is responsible for this lethality.

Similarities in regulation of the nucC operonand the nuclease gene

The lethality of high level expression of nucC inS. marcescens or in E. coli bearing the Bam3.3fragment suggested autogenous regulation of thenucC operon by NucC. The cloned nucC promoterwas therefore assayed directly for activation byNucC in E. coli. MC1000.1 was cotransformed withthe NucC plasmid pACYC184TacNucC and thePnucC-lacZ fusion plasmid pR1Sal1-LacZ. As can beseen in Table 6, lacZ expression from PnucC was12-fold higher in the presence of NucC. Thus NucCactivates expression not only of the nucleasepromoter but of its own promoter as well.

The nuclease gene is also under SOS control. Wetherefore investigated whether the nucC operonmight be similarly regulated. SM6 bearing thePnucC-lacZ transcriptional fusion plasmid pR1Sal1-LacZ was grown in LB medium at 30°C with or

Table 6. Expression of PnucC-lacZ transcriptional fusionsStrains Plasmids b-Galactosidase

SM6 <5SM6-Nuc50 <5SM6 pCla2Sal1-LacZ <5SM6 pR1Sal1-LacZ 85SM6-Nuc50 pCla2Sal1-LacZ <5SM6-Nuc50 pR1Sal1-LacZ 90SM6-Nuc17 pR1Sal1-LacZ 78SM6-SU93 pR1Sal1-LacZ 540SM6-SU132 pR1Sal1-LacZ 82MC1000.1 pR1Sal1-LacZ + pACYC184 70MC1000.1 pR1Sal1-LacZ + pACYC184TacNucC 940

Cultures were grown to stationary phase in LB medium and b-galactosidaseactivity was measured as described by Miller (1972).


Figure 5. SOS induction of PnucC-lacZ transcriptionalfusion in wild-type S. marcescens SM6. SM6 (pR1Sal1-LacZ) was grown in LB after a 1:400 dilution of anovernight culture at 30°C. For SOS induction mitomycinC was added at A600 = 0.4 to a final concentration of0.1 mg/ml. b-galactosidase activity is expressed in Millerunits.

could be a binding site for another protein thatregulates both promoters, such as the putativerepressor identified in the SU132 mutant (Chenet al., 1992).

Requirement for NucE but not NucD inextracellular secretion of the nucleasefrom E. coli

When we were studying activation of nucleaseexpression in E. coli, we made the strikingobservation (Table 2) that the Bam3.3 fragment notonly activates nuclease expression in E. coli but thatnuclease activity is found extracellularly. This is notdue to general cell lysis; the majority of b-lactamaseactivity in the same cultures was found in thecellular fraction and not in the supernatant. Deletionand frameshift mutations of the nucC operon wereused to map the function responsible for thisobserved nuclease secretion.

The SM6-Nuc50 and SM6-Nuc168 mutantshave Tn5 insertions in the nucD gene. Whentransformed with the NucC-expressing plasmidpZ150Bam3.3Nco2-3, these strains expressed andsecreted nuclease extracellularly. Since both thechromosome and plasmid lack an intact nucD gene,this suggested that activation of the chromosomalnucE gene by the plasmid-encoded NucC might beresponsible for secretion.

E. coli MC1000.1 carrying the nuclease plasmidpAC9Nuc1 was transformed with several nucoperon plasmids and assayed for nuclease activity.pZ150Bam3.3DR1 deletes the region upstream ofEcoRI, but still contains the entire nucC operon.pZ150Bam3.3Nco2-3 carries a deletion of nucD andnucE. pZ150Bam3.3 (BglII) carries a frameshiftmutation introduced into nucE at the BglII siteby filling in the sticky ends and ligating.Nuclease secretion was assayed by comparing thenuclease activity in the supernatant and whole cellfractions of stationary phase cultures. Theframeshift mutation at the BglII site or the deletionof nucE and nucD both abolish nuclease secretion(Table 7) but retain NucC activation ability andthus are not just polar on a downstream func-tion. Thus, nucE appears to be required forsecretion of nuclease. A more complete analysis ofthe role of this holin-like protein in nucleasesecretion, both in E. coli and S. marcescens, will bepresented elsewhere (Jin & Benedik, unpublishedresults).

without 0.1 mg/ml mitomycin C added at a celldensity of A600 = 0.4. Cells were monitored forgrowth and aliquots were assayed at various timesfor b-galactosidase activity. The results, plotted inFigure 5, clearly demonstrate that the PnucC-lacZtranscriptional fusion is inducible by mitomycin C.Like the nuclease gene itself, therefore, the nucCoperon is under SOS control.

The similar regulation of nucA and the nucCoperon suggested that there might exist commonmotifs in the DNA sequence around the promoterregion. The alignment in Figure 6 shows that bothsequences have LexA binding sites and also share aconserved sequence just upstream of the nucleasepromoter −35 region. The distances between theSOS boxes and upstream conserved sequences aresimilar, as are the distances from the start of thegene. The LexA binding sites and +1 transcriptionalstart in nucA were experimentally confirmed inprevious studies (Chen et al., 1992). The upstreamconserved sequence could formally be a NucCrecognition or binding site, although it does notresemble the sequences in P2 and P4 late promotersto which other members of this family of activatorsbind (Julien and Calendar, 1995). Alternatively, it

Figure 6. Comparison of the upstream regulatory regions of the nucC operon and nuclease (nucA) gene. The LexAbinding site is underlined, as is a conserved upstream sequence. The putative −10 and −35 regions are marked for thenucA promoter as is the start site (+1 is at the A residue) of nucA transcription.


Table 7. Extracellular secretion of S. marcescens nucleasefrom E. coli MC1000.1

Nuclease

Plasmids Sup Cell

pAC9Nuc1 + pZ150Bam3.3 130 30pAC9Nuc1 + pZ150Bam3.3DRl 120 30pAC9Nuc1 + pZ150Bam3.3(BglII) 80 240pAC9Nuc1 + pZ150Bam3.3DNco2-3 80 490

The co-transformants were grown in LB broth to stationaryphase and the cultures fractionated into supernatant (Sup) andwhole cells (Cell). The cell pellet was resuspended in an equalvolume of sonication buffer prior to sonication. Nuclease activitywas measured by the microtiter dish assay.

Ebright, 1994), which also makes specific contacts(albeit at different residues) with the C terminus ofa (Zou et al., 1992; Tang et al., 1994).

It is unlikely that NucC is the only regulatoryfactor, besides LexA, that controls nuclease ex-pression. Two classes of S. marcescens mutants havebeen identified that overexpress nuclease (Winkler,1968; Chen et al., 1992). One of these, SM6-SU93,also increases expression from the nucC promoter(Table 6). The SU93 gene product is thus a candidatefor a putative repressor of the nucC operon.Mutations in this gene are likely to cause nucleaseoverexpression as a consequence of NucC over-expression, although the SM6-SU93 product couldalso act directly as a repressor of nucA expression.Since the nucC operon is autogenously regulated ina positive fashion by NucC itself and high levelexpression of this operon is toxic, the existence of arepressor for the nucC operon would not besurprising. The SM6-SU132 mutant, on the otherhand, may define a negative regulator that actsdirectly on the nucA gene. It does not appear to acton the nucC operon. Alternatively, the SM6-SU132product may act on some other regulator of nucleaseexpression. Surprisingly, SOS induction acts on bothnucC and nucA (Ball et al., 1990). This may reflect anincreased requirement for NucC to allow maximalexpression of nuclease under SOS-induced con-ditions, or genes of the nucC operon may play otheras yet unidentified roles in S. marcescens.

The nucC gene lies distal to two open readingframes that resemble phage lysis genes. The nucEgene encodes a putative holin or transmembraneprotein, while the nucD gene has sequencesimilarity to phage endolysin or lysozyme proteins.There are three lines of evidence to suggest thatnucE, nucD, and nucC form an operon. First, the Tn5insertion sites of the mutants are located in nucD;SM6-Nuc50 has its Tn5 insertion site 435 bpupstream and SM6-Nuc168 325 bp upstream of thenucC start. The insertion sites of SM6-Nuc17 andSM6-Nuc57 are even further upstream. However,nuclease expression is restored solely by theintroduction of nucC in trans, suggesting that theTn5 insertions are polar on nucC expression.Second, deletion of the BamHI1 to ClaI2 region fromthe Bam3.3 fragment significantly reduced ex-pression of the nuclease activation function,indicating there is a promoter for nucC in theBamHI1-ClaI2 fragment. Thirdly, transcriptionalfusions of lacZ to the ClaI2-SalI1 and EcoRI-SalI1

fragments demonstrate that there is no promoterbetween ClaI2 and SalI1, while expression wasobtained if the additional 400 bp back to the EcoRIsite was added. Taken together, these data supportthe conclusion that nucE, nucD, and nucC lie in anoperon that is transcribed from a promoter betweenEcoRI and ClaI2.

Several lines of evidence suggest that unregulatedexpression of the nucC operon is lethal. The nucCgene cannot be transformed into S. marcescensstrains when expressed from the lac or tacpromoters, unless in the presence of lacIq at high

Discussion

This study has identified a positive regulator ofthe S. marcescens extracellular nuclease gene nucAthrough characterization of a group of mutantsdefective in nuclease expression. The four mutants,SM6-Nuc17, SM6-Nuc50, SM6-Nuc57 and SM6-Nuc168, all have a Tn5 insertion in a common 3.3 kbBamHI fragment. This BamHI fragment, whencloned, can activate in trans the nucA nuclease genepromoter in E. coli. Deletion analysis localized theactivator function, NucC, to an open reading frameof 225 bp. Pulse-labeling experiments confirmed thesynthesis of a protein of approximate Mr 9 kDa,which is consistent with the predicted size of8.3 kDa for the product of the gene.

NucC shares 66% identity with the phage P2 lategene control protein, Ogr. It is functionallyinterchangeable with members of the Ogr family ofproteins, which also includes the P4 and fR73 dgenes and the phage 186 B gene (Bertani & Six, 1988;Christie & Calendar, 1990). The nucC gene was ableto complement a P2 ogr− phage and allow it to formplaques. The d gene of P4 was able to transactivatenuclease gene expression in E. coli, although Ogrshowed little or no activation of the cloned nucleasegene.

How does NucC activate transcription of thenucA and nucC promoters? Other members of theOgr family bind to specific promoter sequencesupstream of the −35 region of phage late promoters(Julien & Calendar, 1995) and are thought to interactdirectly with the a subunit(s) of host RNApolymerase. Mutations affecting two adjacentresidues in the C-terminal domain of a specificallyblock P2 late transcription, and this block can besuppressed by mutations in P2 ogr or P4 d (Sunshine& Sauer, 1975; Halling et al., 1990; Ayers et al., 1994).NucC probably functions in a similar manner,although specific residues in a affecting the actionof NucC have not yet been identified. It is likely thatinteractions between the a subunit and theseactivators enhance the affinity of RNA polymerasefor the promoter, and may also promote confor-mational changes in RNA polymerase that stimulateinitiation. Such a mechanism has been proposed foractivation of the lac P1 promoter by the E. colicatabolite repression protein, CRP (Busby &


copy. Even these transformants formed sicklyflattened colonies, which nevertheless were Nuc+ onnuclease indicator plates. Healthy Nuc+ transfor-mants of S. marcescens were only obtained whennucC was expressed from its own promoter. Thisreflects the strict regulation of nucC that must existin the normal physiology of S. marcescens growth. InE. coli, NucC demonstrated synthetic lethality onlyin the context of the entire Bam3.3 fragment, whichalso contains the two cryptic phage lysis genes,nucD and nucE. This suggests that it is activation ofnucEDC expression by NucC rather than over-expression of NucC itself that leads to the observedlethality. Overexpression of NucC is also lethal inthe Tn5 mutants whose insertion sites are withinnucD, suggesting that it is the nucE gene thataccounts for this synthetic lethal phenotype.Overexpression of this holin-like protein may leadto increased cell permeability and cell death. On ahigh copy number plasmid like pUC18 the BamHI3.3 kb fragment alone is lethal in E. coli whereas itis not lethal in S. marcescens. This difference couldbe explained by the existence of a repressor for thenucEDC operon in S. marcescens but not in E. coli.The mutant gene in the SU93 overexpressing strainmight be a candidate for such a factor. It is alsounlikely that the synthetic lethality is merely due tooverproduction of nuclease, since the Bam3.3fragment is lethal in E. coli in the absence of thenucA gene.

Many extracellular proteins from heterologousbacteria fail to be secreted into the growth mediumwhen their structural genes are expressed in E. coli(Ball et al., 1987; d’Enfert et al., 1987; Lindeberg &Collmer, 1992). By co-transforming E. coli carryingthese genes with a library of chromosomal DNAfragments from the original bacteria, accessorygenes essential for secretion have been identified.These include the pullulanase secretory genes fromKlebsiella oxytoca and the out genes for pectinase ofErwinia chrysanthemi (d’Enfert et al., 1987; Lindeberg& Collmer, 1992). When a low copy numberplasmid carrying the nucEDC operon was trans-formed into MC1000.1 (pAC9Nuc1), it not onlyincreased nuclease expression but also allowedsecretion of nuclease into the growth medium,which does not normally occur in E. coli. Deletionand frameshift analysis showed that NucE isinvolved in this process. The lysozyme-like NucD isapparently not required for nuclease secretion inS. marcescens because the Tn5 insertions in nucDgene had no effect on nuclease secretion whenNucC was provided in trans. Final confirmation ofthis conclusion awaits generation of a non-polarnucE knockout in S. marcescens.

The operon organization of nucC, nucD and nucEsuggests the coordinated expression of the nucleaseand its secretory accessory gene. This coordinationis not uncommon for extracellular proteins; it is alsopresent in expression and secretion of the K. oxytocapullulanase and E. chrysanthemi pectate lyases(Vidal-Ingigliardi et al., 1991; Nasser et al., 1992) andtheir secretory genes.

NucE has two putative alpha helix transmem-brane domains and a positively charged Cterminus. The latter seems important for nucleasesecretion in E. coli, because the BglII frameshift is atthe C terminus. Although there is no sequencesimilarity, NucE shows structural similarity tophage holins, which are used to release lysozymeduring phage lysis (Young, 1993). Unlike lS,however, NucE expression is not lethal at low tomoderate levels. NucE also differs in that it carriesa highly basic N terminus. The cellular location ofNucE has not yet been determined but, like mostholins, it seems not to have a signal peptide. NucEdoes not appear to have the dual start motif foundin many phage holins (Young, 1993) that results inone peptide acting to inhibit the holin function ofthe slightly longer holin peptide. Sequence gazingdoes not yield any single nucleotide change thatwould reconstruct this motif. In addition to phageholins, NucE also shows structural similarity toother membrane channels such as the mechanosen-sitive channel of E. coli, MscL (Sukharev et al., 1994).MscL is only 136 amino acid residues in length andcontains a highly hydrophobic core of two domainsand a hydrophilic carboxyl terminus with netpositive charge of 3. MscL is thought to allow smallsolutes to pass rapidly in order to maintain osmoticregulation. Whether NucE functions as a holin or achannel is unclear at this point. Further experimentsto elucidate the mode of action of NucE are inprogress.

It is known that certain E. coli B and K-12 strainshave functional P2 ogr genes present on a cryptic P2prophage in the host chromosome (Slettan et al.,1992; Barreiro & Ljungquist, 1992). However, thenucE and -D genes from the putative S. marcescenscryptic prophage are clearly not derived from P2.While NucC resembles Ogr, NucE and NucD showno similarity to the P2 lysis functions Y and K(Ziermann et al., 1994). The fact that the nucleasetranscriptional activator and putative secretoryprotein belong to a family of phage proteins raisesthe question of whether the nuclease gene is alsoderived from a phage. This seems unlikely, sincenucleases similar to the S. marcescens extracellularnuclease can be found in other evolutionarily verydistant organisms. Muro-Pastor et al. (1992) re-ported an extracellular non-specific nuclease ofsimilar size from the Gram-negative cyanobac-terium Anabaena sp. PCC7120, which shows 70%similarity and 24% identity to the Serratia nuclease.A more distantly related nuclease was also found inthe mitochondrial inner membrane of Saccharomycescerevisiae (Vincent et al., 1988). The presence of arelated nuclease in such diverse organisms asenteric bacteria, photosynthetic cyanobacteria and aeukaryotic yeast suggests that the nuclease gene isan ancient one and is unlikely to have been spreadby a phage after the separation of enteric bacteria,cyanobacteria and yeast. However, the Serratianuclease may have co-opted cryptic prophage genesfor its regulation and secretion. This may representthe first example of cryptic prophage genes used for


a normal cellular function, that of nucleaseexpression and secretion, which is a hallmark ofSerratia species.

Materials and Methods

Bacterial strains, plasmids, medium andgrowth conditions

The E. coli and S. marcescens strains and plasmids usedin these studies are described in Table 8. Both E. coli andS. marcescens were routinely grown in LB medium, at37°C and 30°C, respectively. Antibiotics were used ata concentration of 100 mg/ml ampicillin, 25 mg/mlkanamycin, 10 mg/ml tetracycline and 30 mg/ml chloram-phenicol for E. coli, and 500 mg/ml ampicillin, 100 mg/mlkanamycin, 200 mg/ml tetracycline and 200 mg/ml chlo-ramphenicol for S. marcescens. DNase Test Agar (Gibco orDifco) was supplemented with 80 mg/ml methyl green foruse as nuclease indicator medium. Mitomycin C wasadded to a final concentration of 0.1 mg/ml to cultures inmid log phase and growth was allowed to continue asdesignated or left overnight. For pulse labeling, cells weregrown in Mops (3'-[N-morpholino]propane-sulfonicacid), pH 8.0, minimal medium (Neidhardt et al., 1974),

supplemented with 0.7% glycerol and 20 mg/ml of allamino acids except L-methionine.

Mutagenesis

Transposon mutagenesis of strain SM6 using l as adelivery vehicle for Tn5 has been described previously(Hines et al., 1988) and mutants with altered nucleaseexpression were identified by Chen et al. (1992).

DNA techniques

Restriction endonucleases and other DNA modificationenzymes were used as recommended by the suppliers.Plasmid DNA for sequence analysis was prepared usingPromega Magic Mini Prep columns and denatured withalkali before sequencing. M13 single-stranded DNA wasprepared as described by Sambrook et al. (1989). DNAfragments used as probes for colony hybridization andSouthern blotting were excised from LMP agarose, heatdenatured, and labeled by priming with randomhexanucleotides using the Promega Prime-a-Gene label-ing system. Southern hybridization was carried out asdescribed by Sambrook et al. (1989).

Table 8. Strains, phage and plasmidsStrains/phage/plasmids Descriptions Source

JM101 supE thi D(lac proAB)F'[traD36 proAB+ lacIq lacZDM15] Vieira & Messing (1982)MC1000.1 DNase Test Agar-insensitive derivative of MC1000 Chen et al. (1992)MM294 supE44 hsdR endA1 pro thi Meselson & Yuan (1968)SM6Nuc17, 50, 57, 168 Nuclease defective Tn5 mutants of SM6 This workSM6 Serratia marcescens 1ab wild-type Ball et al. (1987)SU93, SU132 Nuclease overexpressing Tn5 mutants of SM6 Chen et al. (1992)P2 D15 P2 ogr-att-int deletion 15 Birkeland et al. (1988)p168B BamHI fragment of p168E w/o Km gene in pACYC184 This workp168E Kmr EcoRI fragment of SM6-Nuc168 with Tn5 in pUC19 This workp168S SalI fragment of p168E w/o Km gene in pACYC184 This workp50B BamHI fragment of p50E w/o Km gene in pACYC184 This workp50BK Kmr BamHI fragment of p50E in pACYC184 This workp50E Kmr EcoRI fragment of SM6-Nuc50 with Tn5 in pUC19 This workpAC9 Kmr derivative of pACYC177 with pUC9 polylinker Devine et al. (1989)pAC9Nuc1 Kmr Nuc+ 3.0 kb EcoRI fragment in pAC9 at EcoRI site This workpACYC184 Tcr Camr p15 replicon Chang & Cohen (1978)pACYC184NucC 0.5 kb NcoI-EcoRI of pSE380TacNucC This workpACYC184TacNucC 2.3 kb SphI-HindIII of pSE380TacNucC This workpBJ14 Apr P4 d under T7AI promoter B. Julien & R. CalendarpBluescriptKSII(+) Apr ColE1 high copy replicon StratagenepCla2Sal1-LacZ ClaI2-SalI1 into pNuc2-LacZ This workpNuc1R 3.0 kb EcoRI fragment carrying nucA in pUC18 Ball et al. (1987)pNuc2-LacZ LacZ+ Pnuc-lacZ transcriptional fusion Ball et al. (1990)pR1Sal1-LacZ EcoRI-SalI1 into pNuc2-LacZ transcriptional fusion vector This workpRF5 Apr P2 Ogr+ (under lPL ) Christie et al. (1986)pRK248cIts Tcr cIts, source of cI Bernard & Helinski (1979)pSE380 Apr Ptac lacIq overexpression vector Brosius (1992)pSE380DBspMI BspMI deletion of tac promoter, source of LacI This workpSE380TacNucC NucC under tac promoter This workpTA108 Apr pSC101 replicon with pUC8 polylinker T. SilhavypTA108PlacNucC 0.5 kb ClaI-HindIII of pSE380TacNucC into pTA108 This workpUC18/pUC19 Apr high copy number ColE1 replicon Yanisch-Perron et al. (1985)pUC18Bam3.3 3.3 kb BamHI fragment in pUC18 This workpZ150 pBR322 derivative with f1 origin Zagursky & Berman (1984)pZ150Bam3.3 3.3 kb BamHI fragment in pZ150 This workpZ150Bam3.3(BglII) Frameshift at BglII site of C terminus of NucE This workpZ150Bam3.3DNco2-3 Deletion of NcoI2-NcoI3 in Bam3.3 This workpZ150Cla2Bam2 2.5 kb ClaI2-BamHI2 fragment of Bam3.3 This workpZ150Cla2Xba 1.7 kb ClaI2-XbaI fragment of Bam3.3 This workpZ150Nuc4 1.4 kb RsaI fragment in pZ150 Lab collectionpZ150TacNucC 2.3 kb HindIII-SphI of pSE380TacNucC This work


DNA sequencing

To determine the DNA sequence of this region, severalrestriction fragments from the 3.3 kb BamHI fragmentwere subcloned into M13mp18 and mp19 using compat-ible sites within the fragment and the polylinker. The M13universal primer as well as additional synthetic oligonu-cleotide primers were used to determine the completesequence on both strands between the EcoRI and BamHI2

sites on the 3.3 kb BamHI fragment (Figure 1). DNAsequencing was performed using the USB Sequenase 2.0kit, and inosine reactions were used to reduce com-pression. The sites of the Tn5 insertions in the SM6-Nuc50and SM6-Nuc168 mutants (Chen et al., 1992) weredetermined from the plasmids p50BK, p50B, p168B, andp168S, using a synthetic oligonucleotide complementaryto the ends of Tn5.

Pulse labeling

JM101 bearing pSE380 or pSE380TacNucC were grownin MOPS minimal medium to an A600 of 0.4 to 0.6.Cultures were induced with 0.4 mM IPTG (isopropl-b-D-thiogalactopyranoside) for 35 minutes prior to theaddition of 100 mCi of [35S]methionine. After threeminutes of labeling, cold methionine (1 mg/ml) wasadded and incubation was continued for ten minutes.Cells were killed by adding KCN to 0.1 M. A 0.2 mlsample was sonicated to release the cellular contents andmixed with 0.4 ml acetone to precipitate proteins. Thepellet was resuspended in SDS-PAGE loading buffer andfractionated by electrophoresis on a 15% polyacrylamidegel containing 0.1% SDS as described by Dreyfusset al. (1984). After drying, the gel was subjected toautoradiography.

Enzyme assays

Nuclease activity was determined from halo size onDNase Test Agar medium. This indicator medium isespecially sensitive and can detect nuclease levels lessthan 10−3 of that of uninduced SM6. A microtiter dishassay, which has been described (Ball et al., 1990), wasused for more quantitative analysis. Nuclease activitiesare presented as the dilution factor required to show noloss of fluorescence during a standard incubation period.All samples for any single experiment were measuredtogether in a single dish. b-Galactosidase activity wasassayed by the method of Miller (1972). b-Lactamaseactivity was measured by adding a 10 ml sample (culturesupernatant or sonic extract) to 90 ml 50 mM potassiumphosphate (pH 6.6) with 0.75 mg/ml ampicillin. Thedecrease of absorbance at 240 nm was monitored usingthe kinetics program on a Milton Roy Spectronic 3000spectrophotometer.

Transformation and electroporation

E. coli was transformed as described previously (Chunget al., 1989). S. marcescens was transformed by electropo-ration. A 10 ml sample of cells (A600 0.8) was pelleted at7700 g for 15 minutes at 4°C and washed three times incold sterile double-distilled water. After the addition ofDNA the cells were pulsed at 2.5 kV for 5 ms. A 1 mlportion of LB was added and the cells were incubated at30°C for one hour to allow time for the expression ofantibiotic resistance prior to plating on selective media.

Analysis of DNA and protein sequences

The GCG software (Devereux et al., 1984) was used foranalysis of DNA and protein sequences. The completenucleotide sequences of the S. marcescens nucC, nucD, andnucE genes have been deposited in GenBank underaccession number U11698. The accession number of theS. marcescens trmD and rplS genes is L23334.

AcknowledgementsWe thank Dr Ry Young at Texas A & M for critical

comments and suggestions, and Drs R. Calendar andB. Julien for providing plasmid pBJ14. This work wassupported by grant GM36891 from the National Institutesof Health and a Texas Advanced Research Program award(no. 36521178) to M.J.B. and American Cancer Societygrant no. NP-869A to G.E.C. Certain equipment used inthis work was purchased through an NSF equipmentgrant (no. DIR9109294).

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Edited by M. Gottesman

(Received 11 September 1995; accepted 13 November 1995)

Note added in proof: The nucC gene described in this work has also been found to regulate bacteriocin28b expression in S. marcescens. The conclusions of this publication are similar to ours (Ferrer, S., Viejo,M. B., Guasch, J. F., Enfedaque, J. & Regue, M. (1996). J. Bacteriol. In the press).

regulation of theserratia marcescensextracellular nuclease: positive control by a homolog of p2 ogr...

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