Serratia marcescens and its extracellular nuclease

MiniReview

Serratia marcescens and its extracellular nuclease

Michael J. Benedik *, Ulrich StrychDepartment of Biochemical and Biophysical Sciences, University of Houston, Houston, TX 77204-5934, USA

Received 3 January 1998; revised 10 February 1998; accepted 30 March 1998

Abstract

Serratia marcescens produces an endonuclease with extraordinarily high specific activity that is released into the surroundingmedium. This enzyme has been the focus of studies on gene regulation, protein secretion, endonuclease action, and proteinstructure; it has also been found to have many applications in biotechnology. Here we briefly review these different facets ofresearch regarding the Serratia nuclease and summarize the current state of knowledge about this enzyme. z 1998 Federationof European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Serratia marcescens ; Nuclease; Extracellular secretion; DNA hydrolysis

1. Introduction

Students of microbiology often recall their intro-duction to Serratia marcescens in laboratory exer-cises where it was used because of its red pigment,prodigiosin. Variation in pigment formation is oftenused to demonstrate unstable phenotypes and thenatural variations found within a population [1].Surprisingly, the genetic and biochemical mecha-nisms leading to variegated production of prodigio-sin remain uncharacterized to date.

A second and often cited hallmark of Serratiaspp., which in fact characterizes the genus, is its ex-tracellular nuclease. This protein, of V26 kDa, is theproduct of the nucA gene. Although many bacteriasecrete extracellular proteins, and Serratia producesan abundance, extracellular nucleases are rare. Theyare limited to a small number of bacterial species

spread broadly throughout the eubacterial kingdom.Surprisingly, these extracellular nucleases are mostlyunrelated to one another by sequence or by mecha-nism and structure.

2. Why a nuclease?

What role might an extracellular nuclease play inthe biology of a microbe? In no case can we becertain of the answer, but reasonable speculationscertainly abound.

Scavenging is likely to be the predominant rolethese nucleases play. Unlike the luxurious life of Es-cherichia coli in a shaker £ask of LB medium, mostmicrobes are faced with a continual shortage of nu-trients and lead an ever-changing existence of fast orfamine. Extracellular degradatory enzymes are onemechanism microbes use to exploit their environ-ment and maximize availability of nutrients. Nucleicacid may not seem a particularly suitable carbon

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 3 4 - 1

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* Corresponding author. Tel. : +1 (713) 743-8377;Fax: +1 (713) 743-8351; E-mail: [email protected]

FEMS Microbiology Letters 165 (1998) 1^13

source, however under laboratory conditions it hasbeen demonstrated that Serratia is capable of slowgrowth on DNA as sole carbon source [2]. The ri-bose and deoxyribose are likely to be used directly toprovide energy. Equally important, scavenging nu-cleic acids for use as nucleotides in DNA andRNA synthesis may be an e¤cient way to conserveon the metabolically expensive synthesis of these pre-cursors.

The possible role of nuclease as a scavenging en-zyme becomes more apparent for Serratia when oneconsiders the other extracellular enzymes producedby this microbe. Among them exist multiple pro-teases [3,4], with at least one, the metalloprotease,being of great abundance and high speci¢c activity.Other proteases of varying speci¢city contribute tothe net proteolytic activity. Lipolysis is mediated bytwo separate enzymes, a general lipase or esterase[5,6] as well as a phospholipase [7]. Chitin degrada-tion is mediated by at least two chitinases as well aschitobiases [8,9]. Taken in combination, these en-zymes allow Serratia to fully degrade complete or-ganisms such as fungi and insects to simple metabo-lites. Other proteins are likely to be important formetabolite scavenging during an infection. TheHasA protein [10] works as an iron scavenging pro-tein, similarly the hemolysin [11] may allow releaseof sequestered iron from blood cells. Productionof bacteriocin [12,13] may improve the ability ofSerratia to compete in a mixed microbe environ-ment.

It has been suggested that nucleases may act asvirulence factors, for example the nuclease of Vibriocholerae has been postulated to play a role duringinvasion or establishment of an infection [14], how-ever no experiments have successfully demonstratedthis. In the lungs, exceptionally high concentrationsof DNA comprise the viscous component of bron-chial secretions. The ability to locally degrade thesesecretions, and allow bacteria to attach to cell surfa-ces hiding below, may assist in establishing a success-ful infection. The role of S. marcescens as an oppor-tunistic pathogen is being increasingly recognized[15], however it is not clear whether DNA poses aproblem in any of these infections nor whether thenuclease is important. One can imagine the nucleasewould contribute to infectivity under any conditionswhere copious cell lysis is occurring.

3. An overview of the enzyme

Nuclease was ¢rst puri¢ed from culture superna-tants of S. marcescens more than 30 years ago[16,17]. Since that time it has been the target of nu-merous biochemical studies. The enzyme is a sugar-nonspeci¢c hydrolase, i.e. capable of cleaving bothRNA and DNA in either double or single strandedform. It requires divalent cations, preferably Mg2�,displays a broad pH range from 6 to 10 (optimal at8^8.5) and has a wide temperature optimum between35³C and 44³C [16^19].

The nuclease enzyme has a high catalytic e¤ciencyrelative to other nucleases, about four times that ofstaphylococcal nuclease and approximately 34 timesgreater than DNase I. Kinetic studies have indicatedthat the rate limiting step for the reaction is an earlyone, association of the enzyme with its substrate and/or the cleavage of the resulting complex, not thedissociation of the product [20]. Recent studieshave demonstrated that nuclease activity on excesssubstrate is not limited by substrate inhibition [20];earlier reports to the contrary [21,22] failed to main-tain constant Mg2� to DNA ratios needed for max-imal activity [23]. The enzyme cleaves single or dou-ble stranded DNA and RNA with similar rates, solong as the substrate contains no fewer than ¢vephosphate residues [20]. Under in vitro conditions,however, nuclease has been shown to prefer GC-over AT-rich regions, as does DNase I. When pre-sented with a double stranded substrate, cleavage ofone strand is correlated with cleavage in the corre-sponding position of the complementary strandyielding a double strand break [24].

4. A family of nucleases

The Serratia enzyme belongs to a family of sugar-nonspeci¢c nucleases found in organisms rangingfrom bacteria to mammals. Other members of thisgroup of nucleases have been characterized fromBos taurus [25], Synecephalastrum racemosum [26],Saccharomyces cerevisiae [27], Anabaena sp.PCC7120 [28] and Streptococcus pneumoniae [29].They are generally homodimers with monomer mo-lecular masses ranging from 25 to 30 kDa. Sequenceanalysis revealed the presence of several conserved


M.J. Benedik, U. Strych / FEMS Microbiology Letters 165 (1998) 1^132

amino acid residues within the members of this fam-ily, suggesting a common reaction mechanism[28,30].

A comparison of Serratia nuclease to other endo-nucleases reveal di¡erences in their targets on poly-nucleotide substrates (Fig. 1). Serratia nucleaseand DNase I [31] both cleave to yield a 3P-OH and5P-phosphoryl whereas staphylococcal nucleasecleaves at the other side of the P bond [32]. Thechromophoric nuclease substrate deoxythymidine3P,5P-bis-(p-nitrophenyl-phosphate) however iscleaved di¡erently by DNase I and Serratia nuclease.DNase I cleaves the phosphodiester bond on the 3Pside [33], and Serratia nuclease attacks the 5P-side ofthe ribose sugar moiety of this particular substrateanalog. This may re£ect a functional di¡erence insubstrate recognition and cleavage by these enzymesor, more likely, may simply suggest some peculiar-ities of these enzymes' binding to this arti¢cial sub-strate.

5. Processing of the nuclease precursor

Nuclease is produced as a pre-protein of 266 ami-no acids with a signal peptide consisting of the ¢rst21 residues [18,34]. Two major isoforms are pro-duced by S. marcescens [35,36]. The ¢rst isoform,the 245 amino acid mature nuclease (Sm2) with amolecular mass of 26.7 kDa, is the result of typicalsignal sequence processing. The second isoform

(Sm1) is three amino acids shorter, lacking the ¢rstthree N-terminal amino acids Asp-Thr-Leu. Asmight be expected both isoforms share a high degreeof structural similarity and possess nearly identicalbiochemical properties, di¡ering only minutely intheir isoelectric points [35,37^40]. However, subtledi¡erences in their interaction with substrate, re-£ected in their base preferences, have recently beennoted [23,41].

In addition to these major isoforms, other iso-forms present only as minor species have beenidenti¢ed by capillary electrophoresis [42] and elec-trospray mass spectrometry [43^45]. These have fur-ther short deletions at the N-terminus. These iso-forms are not just products from S. marcescens ;the three amino acid deleted isoform Sm1 has alsobeen detected when nuclease is produced as a re-combinant protein in E. coli, albeit in smaller quan-tities [38^40].

Several mechanisms for the generation of theseisoforms have been proposed. The native moleculecould be processed by various membrane or periplas-mic proteases, present in both S. marcescens andE. coli, while passing through the cell envelope.Alternatively, the isoforms might be the result ofheterogeneous processing of the N-terminal signalpeptide by the leader peptidase. However Suh et al.[46] were unable to demonstrate that Sm2 couldbe processed or chased into the Sm1 form andconcluded that these isoforms likely represent alter-native signal peptide processing events. Their pro-duction is also temporally distinct, the Sm1 isoformis made later during the growth of a culture than isSm2.

The secretion of nuclease may also regulate itsactivity indirectly. It appears that disul¢de bridgeformation is a necessary requirement for enzyme ac-tivity. The pre-enzyme is probably inactive in thecytoplasm and only becomes active after secretionwhen the protein reaches the oxidizing environmentof the periplasm necessary to allow formation ofdisul¢des. Two disul¢de bridges between C9 andC13 in the N-terminal region, and C201 and C243near the C-terminus were ¢rst identi¢ed by Bieder-mann et al. [18] and were later con¢rmed throughhigh resolution peptide mapping [38^40]. Site-di-rected mutagenesis of the above residues revealedtheir importance for nuclease stability and activity.


Fig. 1. The cleavage sites for DNase I, staphylococcal nucleaseand Serratia nuclease on a polynucleotide substrate.

M.J. Benedik, U. Strych / FEMS Microbiology Letters 165 (1998) 1^13 3

A C9S mutant, for example, is ¢ve- to ten-fold lessstable than the wild-type enzyme and displays amore than 1000-fold reduction in nuclease activity[47]. Thus, disul¢de bridge formation can be consid-ered an essential step in regulating nuclease activity,and a crucial means of restricting its potentially det-rimental e¡ect to the extracellular milieu, in combi-nation with its removal from the cytoplasm by thesecretory machinery.

6. Structure of nuclease

The primary [34], secondary [30], tertiary [48] andquaternary structures [49] of nuclease are all wellcharacterized. The X-ray crystal structure of thefree monomer was ¢rst solved to 2.1 Aî resolution[48], a revised 1.7 Aî resolution structure was recentlypublished [50] and ¢nal re¢nement of the structure to0.92 Aî has just been completed (M. Miller and


Fig. 2. A ribbon diagram [70,71] depicting the nuclease dimer with N- and C-termini and the H89 residues in the active sites at the endof long K-helices.



Fig. 3. A nuclease monomer attacking B-DNA. The K-carbon tracing [48,72] shows that the enzymes' active site cleft and its substrate arecomplementary in shape and charge.


K. Krause, personal communication). This structuralresolution is among the highest reported to datefor proteins of this size. One characteristic of theenzyme is its unique endonuclease fold in the centerof which is a L-sheet made up of six antiparallelL-strands (Fig. 2). On one side, the fold is £ankedby a single dominant K-helix and a very long coiledloop, on the other side there is a helical domain

composed of three K-helices and two small antipar-allel L-strands. Electrostatic ¢eld calculations re-vealed a strongly polarized surface, which helps toattract and direct substrate to the binding pocket.Two ridges, containing positively charged aminoacids like R57, R87 and R131, £ank a deep cleftthat contains the catalytic center of the nuclease.Modeling predicts that these ridges could interact


Fig. 4. K-Carbon tracing [72] of the Serratia nuclease active center. A magnesium cation in the center (red) is bound to the amide oxygenof Asn119. Five water molecules (orange) are located in the sphere of the magnesium.


with about one full turn of B-DNA (Fig. 3). Prom-inent residues within this cleft of the enzyme areH89, N119 and E127 [48].

Early evidence for dimerization came from sedi-mentation velocity experiments [51,52] and crosslink-ing studies [53]. Subsequently dynamic light scatter-ing experiments and the analysis of X-raycrystallographic data con¢rmed nuclease is a homo-dimer that forms through complementary interac-tions between certain residues in the carboxy-termi-nal subdomain. The interface is stabilized by foursymmetric salt links and multiple hydrogen bondsbridging the monomers, dominated by interactionsbetween the two H184 residues from each monomer.In this model the active site of each of the monomersis located away from the interface, unobstructed inits ability to interact with the substrate. Computersimulations con¢rmed that dimerization does not af-fect those electrostatic properties of the surfacewhich contribute to the correct alignment of enzymeand substrate [54].

When encountering substrate molecules, the elec-trostatic ¢eld at the center surface of each monomerdirects the negatively charged polynucleotide to-wards a nucleotide binding site along two positivelycharged ridges that £ank the active site cleft. Theenzyme structure, in agreement with kinetic studies[20], suggests independent or non-cooperative actionof each of the dimers' active sites. The geometry ofthe structure predicts that the two active sites in thedimer do not act concurrently on a single substratemolecule, except perhaps in the case of a long sub-strate polymer that can wrap around to interact withboth catalytic sites. Despite the non-cooperativity ofthe two active sites, dimerization may have certainadvantages for nuclease; it increases the chance forthe enzyme to encounter substrate and it allows therequired enzyme rotation to occur in either directionwhen a substrate molecule is directed from the mid-section of the dipole towards the active center(s) atits ends [49].

7. Catalytic mechanism

Catalytically important amino acid residues of nu-clease have been identi¢ed by structural analysis, se-quence comparison, and by site-directed mutagene-

sis. Mutations of residues R57, R87, H89, N119 andE127 resulted in enzymes that were found to be cat-alytically inactive con¢rming that these residues con-stitute the active site of nuclease [30,48,55].

In view of their prominent location in the center ofthe active site of the nuclease model (Fig. 4), theamino acid residues H89 and E127 were primarytargets for mutational and biochemical investigations[48]. Mutant H89A is a¡ected exclusively in its kcat,but not in its Km, suggesting H89's immediate in-volvement in the actual hydrolysis of the phospho-diester bond [55]. One proposed reaction mechanism(Fig. 5A) predicts that H89 acts like a general base,which abstracts a proton from a water molecule,activating it for a nucleophilic attack on the phos-phorus atom adjacent to the scissile bond [33]. Inthis model N119, along with residue R57, stabilizesthe resulting phosphorane intermediate. Interestingly


Fig. 5. Two proposed models for the catalytic mechanism nucle-ase attack. H89 in the active center of the enzyme is perceived toact either as a general base (A) or a general acid (B) in a Mg2�-dependent hydrolysis reaction.


a recent structure determination of nuclease identi-¢ed N119 as being bound to Mg2� (Miller andKrause, personal communication). Therefore the sta-bilizing role of N119 is likely mediated by this Mg2�.This residue could have an additional role in posi-tioning the attacking water molecule relative to thephosphorus atom. A similar function has been dis-cussed for R57, acting through its guanidiniumgroup [33,55].

An alternative model (Fig. 5B) suggesting thatH89 might instead function as the general acid,protonating the leaving group, and E127 being thegeneral base [48,50,54] has also been proposed. How-ever the ability of H89A to cleave the arti¢cialchromophoric substrate deoxythymidine 3P,5P-bis-(p-nitrophenyl-phosphate), which does not requireprotonation of the leaving group, appears to contra-dict this model. Mutant H89A is inactive with thisnucleotide analogue, whereas an E127A mutant stillcleaves. This argues that E127 is dispensable for theinitial water activation [33], and therefore by defaultH89 is the opposite, at least for this arti¢cial sub-strate.

When the role of those amino acid residues thatare essential for nuclease activity, but are not directlyinvolved in the catalysis reaction, was studied bysite-directed mutagenesis, three additional residuesoutside of the active center were identi¢ed. R87,R131 and possibly also D86 mutants are mainly af-fected in their ability to bind nucleic acid substratebut not in their catalytic activity [55]. More speci¢-cally, R87 is thought to interact with the phosphategroup at the 3P-end of the ribose sugar [33], which isessential for cleavage near the 5P-end [20]. With re-gard to the nuclease model, all three residues arelocated in the putative substrate binding site of theenzyme, suitably positioned to assist in positioningdiverse nucleotide substrates into a conformationthat is accepted by the enzyme [48].

8. Control of nuclease expression

Not surprisingly, nuclease production is regulated.However the parameters of its regulation are notinitially obvious. Nuclease expression is not substrateregulated, the addition of nucleic acid does not in-duce its expression nor does the addition of free

nucleotides repress it. It also is not catabolite regu-lated. Instead environmental signals control nucleaseexpression. First, transcription of nucA is growthphase regulated [56]. Transcription increases asgrowing cultures increase in density and approachsaturation. Preliminary results (Y. Suh and M. Be-nedik, unpublished) suggest this is modulated by afactor released by the bacteria into the growth me-dia, likely to be related to the HSL signaling mole-cules [57]. How this signal mediates its e¡ect on nucAtranscription has yet to be elucidated.

A transcriptional regulator which acts at the nucApromoter as an activator of transcription is theNucC protein. This protein is a member of the P2Ogr family of phage transcriptional activators [58]and likely interacts with the K subunit of RNA poly-merase. NucC binds to a region between 382 and351 upstream of the transcriptional start of nucA asdetermined by footprinting analysis and this regionincludes a copy of the TGT-N12-ACA activator rec-ognition motif. Deletion of the upstream TGT oblit-erates activation (G. Christie, personal communica-tion). Transcription of nucC appears also to begrowth phase regulated. The simplest model wouldhave extracellular density signals modulate produc-tion of NucC which in turn activates nucA transcrip-tion, however this suggestion awaits experimentaldemonstration.

Interestingly, nucC lies in an operon with two oth-er genes, nucD and nucE [58]. These bear a strongresemblance to phage proteins, speci¢cally NucE re-sembles holin proteins involved in releasing lysozymeto the peptidoglycan of Gram-negative bacteria andNucD is such a lysozyme. These proteins appear toplay no signi¢cant role in nuclease secretion; theirdeletion from the Serratia genome does not a¡ectnuclease secretion (U. Strych, W. Dai and M. Bene-dik, in preparation). The likely viral origin of thesegenes makes it easy to speculate that they originatedas part of a cryptic prophage.

A second environmental signal known to regulatenuclease expression is the bacterial SOS system [59].Nuclease production is increased strongly by agentswhich induce SOS controlled genes. A LexA bindingsite lies near the transcription start site of nucA [56],a second site lies upstream of the nucC operon [58]and controls its transcription. So nuclease appears tobe regulated dually by SOS through LexA repression



of transcription both at nucA itself as well as itsactivator gene nucC. Overexpression of LexA re-duces nuclease levels as do mutations in recA. Muta-tions in the LexA binding site of the nucA promoterincrease nuclease expression dramatically.

Pleiotropic regulatory mutants have also been de-scribed which increase nuclease expression [56,60].Recent work in our laboratory has shown thatmost of these act indirectly by partially inducingthe SOS system, therefore their role in regulatingnuclease expression is indirect (L. Guynn and M.Benedik, submitted).

There exist two potential start codons for the nu-clease open reading frame. Only the second is posi-tioned well with respect to a consensus ribosomebinding site. Site-directed mutations at these startcodons demonstrate that both can be used in vivo,however the ¢rst Met start is used with less than 10%the e¤ciency of the second. No translational regula-tion has been identi¢ed [56].

Sequence analysis of the nucA gene from ¢ve dif-ferent Serratia strains, not surprisingly, reveals ahigh degree of conservation (M. Benedik, unpub-lished). Although each strain has at least a singleamino acid change, no strain di¡ers by more thantwo amino acids from the consensus and all thesechanges are neutral. There is of course more varia-tion found at the DNA level, but the genes are stillabout 96% identical. None of the base substitutionswould be expected to alter gene expression or regu-lation.

9. Extracellular secretion of nuclease

Nuclease is found as an extracellular enzyme incultures of S. marcescens, as are many other pro-teins. Remarkably, Serratia uses a variety of secre-tory systems by which it exports proteins to thegrowth medium. The mechanism employed to exportnuclease remains obscure and has features suggestingit may be unique. The ability of Serratia to secretenuclease appears to be regulated, probably by hostcell physiology. Bacterial cultures at di¡ering celldensities display di¡erent kinetics and e¤ciencies ofnuclease secretion, these parameters can also bemodulated by growth medium, growth conditions,and host cell mutations [61].

Nuclease is initially synthesized as a precursor pro-tein carrying an N-terminal signal peptide [18]. Thissignal peptide mediates the export of nuclease to thebacterial periplasm using the standard general secre-tory system; inhibitors of E. coli envelope secretionalso block the export of nuclease to the periplasm[61].

Is nuclease intrinsically `greasy' leading to its ex-tracellular secretion? Early reports suggested thismay be the case. If nuclease is overexpressed in E.coli one is able to ¢nd signi¢cant enzymatic activityin the growth medium [34,62]. This appears to beespecially true when cultures are grown to high den-sities under conditions of very high expression suchas in a well aerated fermenter [62]. For productionpurposes this makes it possible to easily produce andpurify nuclease without lysing the bacterial cells. Infact, this property may be true for many typicallyperiplasmic enzymes and has been observed bothfor bacterial alkaline phosphatase and L-lactamase.It is likely that this is an artifact of overproductionand does not represent the normal `biological' situa-tion.

In a series of experiments performed at `normal'levels of nuclease expression, very di¡erent conclu-sions were formed. When nuclease is expressed in E.coli it is found predominantly in the bacterial peri-plasm whereas under similar conditions the enzymeis mostly extracellular when produced in S. marces-cens [61]. The periplasmic species is an intermediatein the secretion of nuclease from the cytoplasm tothe periplasm via the general secretory path, andthen extracellularly via an unknown mechanism.This second step is relatively slow and, dependingupon growth conditions, may take 5^60 min. Thisdi¡erence between E. coli and Serratia points to aspeci¢c mechanism existing for the extracellular stepof nuclease secretion by Serratia under these condi-tions.

Two surprising observations have been made re-garding nuclease secretion. The ¢rst suggests a pro-moter speci¢city for extracellular secretion. Whennuclease is produced from a plasmid where transcrip-tion initiates from the lac promoter, the enzyme isfound predominantly in the periplasm, whereas ifexpressed from the native nucA promoter even onplasmid, the enzyme can be found extracellularly[63]. Careful monitoring of pulse-labeled protein



con¢rms this localization remains true for hoursafter labeling. The simplest explanation for thisdata suggests that precise temporal control of nucle-ase production may be important to allow e¤cientsecretion. Expression at the wrong phase of growthresults in protein trapped solely in the periplasm.The nucA promoter may be coordinately regulatedwith some facet of the secretion machinery and pro-tein production and secretion must be coupled forextracellular secretion to occur [61,63].

A second and perhaps more useful observation,nevertheless, yields a similar interpretation. Recallthat there are two predominant isoforms of the proc-essed mature nuclease. The Sm2 isoform is the nativeenzyme after removal of the signal peptide. Theslightly shorter Sm1 isoform lacks the ¢rst three N-terminal amino acids after processing of the signalpeptide. Suh et al. [61] were unable to demonstratethat Sm2 could be chased to the Sm1 form and sug-gest these species represent alternate signal peptideprocessing events. The appearance of these isoformsare not contemporaneous, rather the Sm2 form ap-pears predominantly during exponential growthwhereas a switch to the Sm1 isoform occurs as thecells enter stationary phase. In itself this would beunique for secreted proteins if it truly represents achange in signal peptide cleavage events. More strik-ing is the fate of these isoforms. The Sm2 isoform issecreted extracellularly whereas the Sm1 isoform re-mains trapped in the periplasm until much later,such as after overnight growth.

What does this all mean? The obvious conclusionis that for nuclease there is no simple signal presentin the protein which triggers its extracellular secre-tion. Expressing identical proteins from di¡erentpromoters, or virtually identical isoforms from thesame promoter yield species with very di¡ering fates,either trapped in the periplasm or secreted extracel-lularly. Rather some facet of spatial or temporal lo-calization may have an e¡ect on the ¢nal destinationof nuclease.

10. Nuclease and its many uses

With its high intrinsic activity and broad substratetolerance, nuclease has found many uses in a varietyof biotechnological applications. Puri¢ed nuclease

(under the tradename of Benzonase by A. BenzonPharma) is being sold commercially. Its primarymarket is for use in downstream processing. Typicaluses are to eliminate nucleic acid contaminationfrom puri¢ed proteins, commonly from recombinantDNA products, or to reduce viscosity for subsequentprocessing steps. The lysis of any signi¢cant quantityof bacteria leads to the release of nucleic acid con-taminants which greatly increases the viscosity of thesample and can lead to complications in subsequentpuri¢cation steps. The addition of nuclease is an in-expensive method to remove this contaminating nu-cleic acid. It has the added advantage of destroyingany recombinant DNA molecules that might other-wise contaminate the target protein of choice.

The ability to destroy DNA has led to use of theS. marcescens nuclease as a killer gene for the self-destruction of microorganisms released into the en-vironment. In general, nuclease requires secretionout of the cytoplasmic compartment to gain activity,however if expressed in the absence of a signal pep-tide, enough residual activity remains to kill cells byhydrolyzing their nucleic acids. Expressing the genefor mature nuclease, thereby preventing its export,results in a selective system for the destruction ofany bacterium carrying such a gene. Therefore therelease of genetically engineered organisms can becontrolled, environmental signals triggering the ex-pression of such a toxic cassette results in suicide.There are of course numerous such suicide systems,however the nuclease system has the added advant-age of destroying any recombinant DNA plasmids aswell, thereby alleviating fears concerning the releaseof such DNA [64].

There has been much interest in exploring noveluses for the Serratia nuclease in the former SovietUnion. Studies have shown nuclease can act as ane¡ective anti-viral agent and inhibit the replicationof both DNA and RNA viruses [65]. In fact it isextensively used to prevent viral paralysis in honey-bee hives. Along the same lines, Boeke and col-leagues [66] have developed an elegant antiretroviraltherapy based on delivery of nuclease by a modi¢edretroviral genome to block subsequent heterologousviral replication in the cell. Serratia nuclease wasfound to be too active and toxic to the host cell ;RNase H1 was found useful instead. However otherapplications for such a toxic agent could be envi-



sioned using a viral delivery system when the deathof a target cell is the desired outcome.

Nuclease has also been shown to have signi¢cant[67,68] anti-tumor properties, presumably by inter-fering with replication of dividing cells. Obviouslya suitable targeted delivery system is the limitingfactor for an e¡ective treatment, something alsolacking for many other molecules with similar anti-tumor properties. Of signi¢cant importance to theuse of nuclease in any therapeutic regime is its abilityto be tolerated by the mammalian immune system([69], M. Filimonova, personal communication).

11. Epilogue

Although under investigation for decades, interestin the Serratia nuclease has been resurgent for thepast 10 years. It remains an active subject of researchby many on secretion, mechanisms of nuclease actionand evolution, as well as for its unique propertiesuseful in many biotechnological applications withundoubtedly many more to come.

Acknowledgments

For the dozen years the Benedik lab has laboredon this topic many wonderful and stimulating stu-dents and post-doctoral fellows, past and presenthave made important contributions. To them wegive our sincere thanks. Drs. Mitch Miller andKurt Krause are gratefully acknowledged for theirassistance with ¢gures for this article. The Serratiacommunity at large, albeit small, has been helpful,encouraging, and always open with their ideas,which has made the time pass swiftly. Work on Ser-ratia has been supported by the NIH, NSF, TexasAdvanced Research Program and Welch Founda-tion, we thank them all.

References

[1] Grimont, P.A.D. and Grimont, F. (1978) The genus Serratia.Annu. Rev. Microbiol. 32, 221^248.

[2] Beliaeva, M.I., Kapranova, M.N., Vitol, M.Ia., Golubenko,I.A. and Leshchinskaia, I.B. (1976) Nucleic acids utilized as

the main source of bacterial nutrition. Mikrobiologiia 45,420^424.

[3] Braun, V. and Schmitz, G. (1980) Excretion of a protease bySerratia marcescens. Arch. Microbiol. 124, 55^61.

[4] Bromke, B.J. and Hammel, J.M. (1979) Regulation of extra-cellular protease formation by Serratia marcescens. Can.J. Microbiol. 25, 47^52.

[5] Heller, K. (1979) Lipolytic activity copuri¢ed with the outermembrane of Serratia marcescens. J. Bacteriol. 140, 1120^1122.

[6] Li, X.Y., Tetling, S., Winkler, U.K., Jaeger, K.E. and Bene-dik, M.J. (1995) Gene cloning, sequence analysis, puri¢cationand secretion by Escherichia coli of an extracellular lipasefrom Serratia marcescens. Appl. Environ. Microbiol. 61,2674^2680.

[7] Givskov, M., Olsen, L. and Molin, S. (1988) Cloning andexpression in Escherichia coli of the gene for extracellularphospholipase A1 form Serratia liquefaciens. J. Bacteriol.170, 5855^5862.

[8] Jones, J.D., Grady, K.L., Suslow, T.V. and Bedbrook, J.R.(1986) Isolation and characterization of genes encoding twochitinase enzymes from Serratia marcescens. EMBO J. 5, 467^473.

[9] Monreal, J. and Reese, E.T. (1969) The chitinase of Serratiamarcescens. Can. J. Microbiol. 15, 689^696.

[10] Leto¡e, S., Ghigo, J.M. and Wandersman, C. (1994) Secretionof the Serratia marcescens HasA protein by an ABC trans-porter. J. Bacteriol. 176, 5372^5377.

[11] Schiebel, E., Schwarz, H. and Braun, V. (1989) Subcellularlocation and unique secretion of the hemolysin of Serratiamarcescens. J. Biol. Chem. 264, 16311^16320.

[12] Foulds, J.D. and Shemin, D. (1969) Properties and character-istics of a bacteriocin from Serratia marcescens. J. Bacteriol.99, 655^660.

[13] Guasch, J.F., Enfedaque, J., Ferrer, S., Gargallo, D. and Reg-ue, M. (1995) Bacteriocin 28b, a chromosomally encoded bac-teriocin produced by most Serratia marcescens biotypes. Res.Microbiol. 146, 477^483.

[14] Focareta, T. and Manning, P.A. (1991) Distinguishing be-tween the extracellular DNases of Vibrio cholerae and devel-opment of a transformation system. Mol. Microbiol. 5, 2547^2555.

[15] Hejazi, A. and Falkiner, F.R. (1997) Serratia marcescens.J. Med. Microbiol., 46, 903^912.

[16] Eaves, G.N. and Je¡ries, C.D. (1963) Isolation and propertiesof an exocellular nuclease of Serratia marcescens. J. Bacteriol.85, 273^278.

[17] Nestle, M. and Roberts, W.K. (1969) An extracellular nucle-ase from Serratia marcescens. I. Puri¢cation and some proper-ties of the enzyme. J. Biol. Chem. 244, 5213^5218.

[18] Biedermann, K., Jepsen, P.K., Riise, E. and Svendsen, I.(1989) Puri¢cation and characterization of a Serratia marces-cens nuclease produced by Escherichia coli. Carlsberg Res.Commun. 54, 17^27.

[19] Nestle, M. and Roberts, W.K. (1969) An extracellular nucle-ase from Serratia marcescens. II. Speci¢city of the enzyme.J. Biol. Chem. 244, 5219^5225.



[20] Friedho¡, P., Meiss, G., Kolmes, B., Pieper, U., Gimadutdi-now, O., Urbanke, C. and Pingoud, A. (1996) Kinetic analysisof the cleavage of natural and synthetic substrates by theSerratia nuclease. Eur. J. Biochem. 241, 572^580.

[21] Filimonova, M.N., Krause, K.L. and Benedik, M.J. (1994)Kinetic studies of the Serratia marcescens extracellular nucle-ase isoforms. Biochem. Mol. Biol. Int. 33, 1229^1236.

[22] Nyeste, L., Sevella, B., Hollo, J. and Bekes, F. (1976) Kineticstudy of extracellular nuclease of Serratia marcescens. Eur.J. Appl. Microbiol. 2, 161^168.

[23] Filimonova, M.N., Gubskaya, V., Nuretdinov, I., Benedik,M.J., Bogomolnaya, L., Andreeva, M. and Leshchinskaya,I.B. (1997) Isoforms of Serratia marcescens nuclease. Therole of Mg�� ions in mechanism of hydrolysis. Biokhimiia62, 1148^1154.

[24] Meiss, G., Friedho¡, P., Hahn, M., Gimadutdinow, O. andPingoud, A. (1995) Sequence preferences in cleavage ofdsDNA and ssDNA by the extracellular Serratia marcescensendonuclease. Biochemistry 34, 11979^11988.

[25] Cote, J. and Ruiz-Carrilo, A. (1993) Primers for mitochon-drial DNA replication generated by endonuclease G. Science261, 765^769.

[26] Chen, L.Y., Ho, H.C., Tsai, Y.C. and Liao, T.H. (1993) De-oxyribonuclease of Synecephalastrum racemosum ^ Enzymaticproperties and molecular structure. Arch. Biochem. Biophys.303, 51^56.

[27] Vincent, R.D., Hofmann, T.T. and Zassenhaus, H.P. (1988)Sequence and expression of NUC1, the gene encoding themitochondrial nuclease in Saccharomyces cerevisiae. NucleicAcids Res. 16, 3297^3312.

[28] Muro-Pastor, A.M., Flores, E., Herrero, A. and Wolk, C.P.(1992) Identi¢cation, genetic analysis and characterization of asugar-non-speci¢c nuclease from the cyanobacterium Anabae-na sp. PCC 7120. Mol. Microbiol. 6, 3021^3030.

[29] Puyet, A., Greenberg, B. and Lacks, S.A. (1990) Genetic andstructural characterization of endA. A membrane-bound nu-clease required for transformation of Streptococcus pneumo-niae. J. Mol. Biol. 213, 727^738.

[30] Friedho¡, P., Gimadutdinow, O. and Pingoud, A. (1994)Identi¢cation of catalytically relevant amino acids of the ex-tracellular Serratia marcescens endonuclease by alignment-guided mutagenesis. Nucleic Acids Res. 22, 3280^3287.

[31] Liao, T. (1975) Deoxythymidine 3P,5P-di-p-nitrophenyl phos-phate as a synthetic substrate for bovine pancreatic deoxyri-bonuclease. J. Biol. Chem. 250, 3721^3724.

[32] Cuatrecasas, P., Wilchek, M. and An¢nsen, C.B. (1969) Theaction of staphylococcal nuclease on synthetic substrates. Bio-chemistry 8, 2277^2284.

[33] Kolmes, B., Franke, I., Friedho¡, P. and Pingoud, A. (1996)Analysis of the reaction mechanism of the non speci¢c endo-nuclease of Serratia marcescens using an arti¢cial minimalsubstrate. FEBS Lett. 397, 343^346.

[34] Ball, T.K., Saurugger, P.N. and Benedik, M.J. (1987) Theextracellular nuclease gene of Serratia marcescens and its se-cretion from Escherichia coli. Gene 57, 183^192.

[35] Filimonova, M.N., Dementiev, A.A., Leshchinskaya, I.B., Ba-kulina, G.Y. and Shliapnikov, S.V. (1991) Isolation and char-

acterization of extracellular nuclease of Serratia marcescens.Biokhimiia 56, 508^520.

[36] Yonemura, K., Matsumoto, K. and Maeda, H. (1983) Isola-tion and characterization of nucleases from a clinical isolate ofSerratia marcescens kums 3958. J. Biochem. (Tokyo) 93,1287^1295.

[37] Bannikova, G.E., Blagova, E.V., Dementiev, A.A., Morguno-va, E.Y., Mikchailov, A.M., Shlyapnikov, S.V., Varlamov,V.P. and Vainshtein, B.K. (1991) Two isoforms of Serratiamarcescens nuclease. Crystallization and preliminary X-rayinvestigation of the enzyme. Biochem. Mol. Biol. Int. 24,813^822.

[38] Pedersen, J., Filimonova, M.N., Roepstor¡, P. and Bieder-mann, K. (1993) Characterization of Serratia marcescens nu-clease isoforms by plasma desorption mass spectrometry. Bio-chim. Biophys. Acta 1202, 13^21.

[39] Pedersen, J., Filimonova, M.N., Roepstor¡, P. and Bieder-mann, K. (1995) Nuclease isoforms of natural and recombi-nant strains of Serratia marcescens. Comparative character-istics of plasma desorption mass spectrometry. Biokhimiia60, 450^461.

[40] Pedersen, J., Filimonova, M.N., Roepstor¡, P. and Bieder-mann, K. (1995) Serratia marcescens nucleases. II. Primarystructure analysis by peptide mapping combined with plas-ma-desorption mass spectrometry. Bioorg. Khim. 21, 336^344.

[41] Filimonova, M.N., Garusov, A., Smetanina, T., Andreeva,M., Bogomolnaya, L. and Leshchinskaya, I.B. (1996) Iso-forms of Serratia marcescens nuclease. Comparative analysisof substrate speci¢city. Biokhimiia 61, 1800^1806.

[42] Pedersen, J., Pedersen, M., Soeberg, H. and Biedermann, K.(1993) Separation of isoforms of Serratia marcescens nucleaseby capillary electrophoresis. J. Chromatogr. 645, 353^361.

[43] Pedersen, J., Andersen, G., Roepstor¡, P., Filimonova, M.N.and Biedermann, K. (1995) Serratia marcescens nucleases.I. Comparison of native and recombinant nucleases using elec-trospray mass spectrometry. Bioorg. Khim. 21, 330^335.

[44] Pedersen, J., Andersen, J., Roepstor¡, P., Filimonova, M.N.and Biedermann, K. (1993) Characterization of natural andrecombinant nuclease isoforms by electrospray mass spec-trometry. Biotechnol. Appl. Biochem. 18, 389^399.

[45] Pedersen, J., Andersen, J., Roepstor¡, P., Filimonova, M.N.and Biedermann, K. (1995) Characteristics of isoforms of Ser-ratia marcescens from electrospray mass spectrometry. Biokhi-miia 60, 462^469.

[46] Suh, Y., Alpaugh, M., Krause, K.L. and Benedik, M.J. (1995)Di¡erential secretion of isoforms of Serratia marcescens ex-tracellular nuclease. Appl. Environ. Microbiol. 61, 4083^4088.

[47] Ball, T.K., Suh, Y. and Benedik, M.J. (1992) Disul¢de bondsare required for Serratia marcescens nuclease activity. NucleicAcids Res. 20, 4971^4974.

[48] Miller, M.D., Tanner, J., Alpaugh, M., Benedik, M.J. andKrause, K.L. (1994) 2.1 Aî structure of Serratia endonucleasesuggests a mechanism for binding to double-stranded DNA.Nature Struct. Biol. 1, 461^468.

[49] Miller, M.D. and Krause, K.L. (1996) Identi¢cation of theSerratia endonuclease dimer: Structural basis and implica-tions for catalysis. Protein Sci. 5, 24^33.



[50] Lunin, V.Y., Levdikov, V.M., Shlyapnikov, S.V., Blabova,E.V., Lunin, V.V., Wilson, K.S. and Mikhailov, A.M.(1997) Three-dimensional structure of Serratia marcescens nu-clease at 1.7 Aî resolution and mechanism of its action. FEBSLett. 412, 217^222.

[51] Filimonova, M.N., Baratova, L.A., Vospel'nikova, N.D.,Zheltova, A.O. and Leshchinskaia, I.B. (1981) Serratia mar-cescens endonuclease. Properties of the enzyme. Biokhimiia46, 1660^1666.

[52] Friedho¡, P., Gimadutdinow, O., Ruter, T., Wende, W., Ur-banke, C., Thole, H. and Pingoud, A. (1994) A procedure forrenaturation and puri¢cation of the extracellular Serratia mar-cescens nuclease from genetically engineered Escherichia coli.Protein Express. Purif. 5, 37^43.

[53] Filimonova, M.N., Benedik, M.J., Urazov, N., and Leshchin-skaya, I.B. (1998) Serratia marcescens nucleases are polydis-persive at pH optimum. Appl. Biochem. Microbiol. (in press).

[54] Antosiewicz, J., Miller, M.D., Krause, K.L. and McCammon,J.A. (1997) Simulation of electrostatic and hydrodynamicproperties of Serratia endonuclease. Biopolymers 41, 443^450.

[55] Friedho¡, P., Kolmes, B., Gimadutdinow, O., Wende, W.,Krause, K.L. and Pingoud, A. (1996) Analysis of the mecha-nism of the Serratia nuclease using site-directed mutagenesis.Nucleic Acids Res. 24, 2632^2639.

[56] Chen, Y.-C., Shipley, G.L., Ball, T.K. and Benedik, M.J.(1992) Regulatory mutants and transcriptional control of theSerratia marcescens extracellular nuclease gene. Mol. Micro-biol. 6, 643^651.

[57] Eberl, L., Winson, M.K., Sternberg, C., Stewart, G.S., Chris-tiansen, G., Chhabra, S.R., Bycroft, B., Williams. P., Molin,S. and Givskov, M. (1996) Involvement of N-acyl-L-hormo-serine lactone autoinducers in controlling the multicellular be-haviour of Serratia liquefaciens. Mol. Microbiol. 20, 27^136.

[58] Jin, S., Chen, Y.C., Chrisitie, G.E. and Benedik, M.J. (1996)Regulation of the Serratia marcescens extracellular nuclease:Positive control by a homologue of P2 Ogr encoded by acryptic prophage. J. Mol. Biol. 256, 264^278.

[59] Ball, T.K., Wasmuth, C.R., Braunagel, S.C. and Benedik,M.J. (1990) Expression of Serratia marcescens extracellularproteins requires RecA. J. Bacteriol. 172, 342^349.

[60] Winkler, U. and Timmis, K. (1973) Pleiotropic mutations inSerratia marcescens which increase the synthesis of certainexocellular proteins and the rate of spontaneous prophageexcision. Mol. Gen. Genet. 124, 197^206.

[61] Suh, Y., Jin, S., Ball, T. K. and Benedik, M. J. (1996) Two-step secretion of extracellular nuclease in Serratia marcescens.J. Bacteriol. 178, 3771^3778.

[62] Biedermann, K., Fiedler, H., Larsen, B., Riise, E., Emborg, C.and Jepsen, P. (1990) Fermentation studies of the secretion ofSerratia marcescens nuclease by Escherichia coli. Appl. Envi-ron. Microbiol. 56, 1833^1838.

[63] Chen, Y.-C., Suh, Y., Riise, E., Kartman, B., Jin, S. andBenedik, M.J. (1996) Inhibition of Serratia marcescens nucle-ase secretion by a truncated nuclease peptide. Gene 72, 9^16.

[64] Ahrenholtz, I., Lorenz, M.G. and Wackernagel, W. (1994) Aconditional suicide system in Escherichia coli based on theintracellular degradation of DNA. Appl. Environ. Microbiol.60, 3736^3751.

[65] Pan¢lova, Z.I. and Salganik, R.I. (1983) Isolation of Serratiamarcescens mutants superproducers of endonuclease by expo-sure to nitrosomethylurea in a synchronized culture. Mikro-biologiia 52, 974^978.

[66] Schumann, G., Cannon, K., Ma, W.P., Crouch, R.J. andBoeke, J.D. (1997) Antiretroviral e¡ect of a gag-RNase HIfusion gene. Gene Ther. 4, 593^599.

[67] Kurinenko, B.M., Beliaeva, M.I., Cherepneva, I.E. and Ku-prianova-Ashina, F.G. (1977) Permeability of dextran-boundnuclease through the vascular barrier and tumor cell mem-brane. Vopr. Onkol. 23, 86^90.

[68] Kurinenko, B.M., Beliaeva, M.I., Cherepneva, I.E. and Vies-ture, Z.A. (1977) Antitumor action of Serratia marcescensnuclease covalently bound to soluble dextrans. Vopr. Onkol.23, 94^98.

[69] Black, K.L., Ciacci, J.D., Ammirati, M., Selch, M.T. andBecker, D.P. (1993) Clinical trial of Serratia marcescens ex-tract and radiation therapy in patients with malignant astro-cytoma. J. Clin. Oncol. 11, 1746^1750.

[70] Merritt, E.A. and Bacon, D.J. (1997) Raster3D: PhotorealisticMolecular Graphics. Methods Enzymol 277, 505^524.

[71] Kraulis, P.J. (1991) MOLSCRIPT: A program to produceboth detailed and schematic plots of protein structures.J. Appl. Crystallogr. 24, 946^950.

[72] Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M.(1991) Improved methods for building protein models in elec-tron density maps and the location of errors in these models.Acta Crystallogr. A47, 110^119.



Serratia marcescens and its extracellular nuclease

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