Vol. 170, No. 4JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1495-15040021-9193/88/041495-10$02.00/0Copyright X 1988, American Society for Microbiology

Cloning and Expression of the Vibrio choleraeNeuraminidase Gene nanH in Escherichia coli

ERIC R. VIMR,l* LOIS LAWRISUK,1 JAMES GALEN,2 AND JAMES B. KAPER2Department of Veterinary Pathobiology, Microbiology Division, College of Veterinary Medicine, University of Illinois atUrbana-Champaign, Urbana, Illinois 61801,1 and Center for Vaccine Development, Division of Geographic Medicine,

Department of Medicine and Department of Microbiology and Immunology, School of Medicine, University of Maryland,Baltimore, Maryland 212012

Received 21 September 1987/Accepted 2 January 1988

A cosmid gene bank of Vibrio cholerae 395, classical Ogawa, was screened in Escherichia coli HB101 forexpression of the vibrio neuraminidase (NANase) gene ndnH (N-acylneuraminate glycohydrolase). Positiveclones were identified by their ability to cleave the fluorogenic NANase substrate 2'-(4-methylumbelliferyl)-cc-D-N-acetylneuraminic acid. Seven NANase-positive clones were detected after gcreening 683 cosmid isolateswith a rapid, qualitative plate assay method. The nanH gene was subcloned from one of the cosmids and waslocated within a 4.8-kilobase-pair BglII restriction endonuclease fragment. Evidence that nanH was theNANase structural gene was obtained by transposon mutagenesis and by purification and comnparison of thecloned gene product with the secreted NANase purified from the parent V. cholerae strain. The sequence of thefirst 20 amino-terminal amino acids of the secreted NANase purified from V. cholerae was determined byautomated Edman degradation and matched perfectly with the amino acid sequence predicted from nucleotidesequencing of nanH. The sequence data also revealed the existence of a potential signal peptide that wasapparently processed from NANase in both V. cholerae and E. coli. In contrast to V. chokrae, E. coli nanH+clones did not secrete NANase into the growth medium, retaining most of the enzyme in the periplasmiccompartment. Kinetic studies in V. cholerae showed that nanH expression and NANase secretion weretemporally correlated as cells in batch culture entered late-exponential-phase growth. Similar kinetics wereobserved in at least one of the E. coli nanH+ clones, suggesting that nanH expression in E. coli might becontrolled by some of the same signals as in the parent V. cholerae strain.

The sialic acids are a family of over 20 naturally occurringnine-carbon sugars that are differentiated by their degree andtype of N or 0 acylations of the parent compound, neura-minic acid (37). In addition to their unusual structuralfeatures, the sialic acids are exceptional in their nearlyunique phylogenetic distribution among species in the meta-zoan lineage that leads to humans (7). In this lineage thesialic acids are believed to play a pivotal role in a variety ofcomplex biological phenomena (7, 8, 37). Outside of thismetazoan lineage, sialic acid synthesis has been demon-strated conclusively in only a few bacterial genera. Theability of these bacteria to synthesize and express sialic acidsin cell surface polysaccharides and glycoconjugates is invari-ably correlated with pathogenicity for humans (7). In con-trast to the limited procaryotic distribution of sialic acidbiosynthetic capability, many bacterial species are able toenzymatically release sialic acids from glycoconjugates byproducing surface-associated or extracellular sialoglycohy-drolases. Since the sialic acids are usually found as terminalnonreducing sugar residues on host glycoconjugates, neur-aminidase (NANase; N-acylneuraminate glycohydrolase,EC 3.2.1.18) may function physiologically to supply bacteriawith free sialic acids that can be transported and degradedintracellularly for assmilation as carbon and energy sources.In mammalian hosts some procaryote NANases may play animportant role in pathogenesis as well (28, 36).As part of our investigation into the biological functions of

the sialic acids, we have been studying the physiology andregulation of a sialic acid catabolic system in Escherichiacoli (40, 41). These bacteria express at least two highly

* Corresponding author.

regulated activities: one is required for N-acylneuraminatetransport (nanl), and another is required for degradation ofsialic acids by an intracellular aldolase (nanA). Presumably,E. coli and other NANase-negative species are capable ofscavenging free sialic acids released from glycoconjugates inthe environment by NANase-positive bacteria. Thus, E. colido not themselves express an endogenous NANase (40). Toextend our analysis of nan catabolic gene regulation wesought to clone the NANase structural gene from Vibriocholerae, a species with many physiologic similarities to E.coli (3). Equally important to our decision to clone the V.cholerae nanH gene was the fact that the NANase secretedby this species has been studied extensively since 1947,when it was first discovered as an influenza virus receptor-destroying activity (5). Consequently, the biochemical prop-erties of vibrio NANase are well characterized (1, 11). Wealso became interested in the possible involvement ofNANase in the pathogenesis of disease due to V. choleraeand in the potential general role of microbial NANases in avariety of pathophysiological conditions. Therefore, cloningand characterization of a bacterial nanH gene is a first steptoward these anticipated studies.

In this communication we describe cloning of the NANasestructural gene from V. cholerae and demonstrate its expres-sion in E. coli. The fluorogenic assay we used to screen forNANase activity is sensitive and sufficiently general suchthat nanH genes from virtually any microbe may be isolatedby similar approaches. It may also be possible to adapt thisscreening protocol for identification of cloned eucaryoticnanH genes. We suggest that genes coding for NANasesin bacteria be termed N-acylneuraminate glycohydrolase

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(nanHf), to be consistent with our earlier designation for nansystem components (40).(A preliminary account of this work was presented previ-

ously [E. R. Vimr, L. Lawrisuk, J. Galen, and J. B. Kaper,Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, D90, p. 87]).

MATERIALS AND METHODSBacterial strains, media, and plasmids. V. cholerae

CVD101 and JBK70 were derived from the 01 serotypeClassical Ogawa 395 and El Tor Inaba N16961 strains,respectively. Both strains are nontoxinogenic derivatives; V.cholerae CVD101 lacks the cholera toxin A subunit gene

(20), and strain JBK70 lacks both the A and B subunits (21).The E. coli host used in this study was strain HB101 [hsdS20(rB- mB-) recA13 ara-14 proA2 leuB6 lacYl galK2 xyl-5mtl-l thi-J supE44 rpsL20 (Smr)].

All bacterial strains were propagated at 37°C in Luriabroth (27) with vigorous aeration or on medium solidified byinclusion of agar to 1.5%. Media were supplemented withappropriate drugs at concentrations given previously (38).The plasmids constructed in this study are summarized inTable 1.Gene bank construction, mutagenesis, and sequencing.

Construction of the gene library of V. cholerae 395 chromo-somal DNA cloned into the cosmid vector pHC79 wasdescribed previously (34). In brief, chromosomal DNA par-tially digested with Sau3A was cloned into the BamHI site ofpHC79, and concatemers were packaged in vitro. Mini-kantransposon mutagenesis of cloned nanH was accomplishedby itfection of HB1O1(pCVD360) with X1105 by the proce-dure ofWay et al. (43). Kmr HB101 mutants were pooled forplasmid extraction by the method of Birnboim and Doly (4).Purified plasmid DNA was transformed back into HB101 asdescribed by Dagert and Ehrlich (10) by using a transforma-tion buffer containing 10 mM morpholinepropanesulfonicacid, 75 mM CaC12, and 0.5% glucose (pH 6.5). Kmrtransformants were selected on Luria agar medium contain-ing 50 jig of kanamycin per ml and screened for the NanH-phenotype by spotting the fluorogenic substrate 2'-(4-meth-ylumbelliferyl)-a-D-N-acetylneuraminic acid (MUNeuNAc)directly onto colonies, as described below.

Restriction endonucleases required for mapping and sub-cloning were purchased from New England Biolabs (Bev-erly, Mass.) or Boehringer-Mannheim Biochemicals (In-

dianapolis, Ind.) and used as recommended by these manu-facturers. Sequence analysis was carried out with [32P]dATP(specific activity, >800 Ci/mmol; 10 mCi/ml) from NewEngland Nuclear Corp. (Boston, Mass.) and the dideoxychain-termination procedure of Sanger et al. (35). Single-stranded M13mpi9 recombinant templates (44) were se-quenced with a DNA sequencing pack (no. 409) purchasedfrom New England Biolabs. Double-stranded sequencing ofpCVD363 by the supercoiled plasmid DNA sequencingtechnique of Chen and Seeburg (6) was accomplished withsynthetic oligonucleotide primers prepared on a Du Pontcoder 300 DNA synthesizer. Primers were synthesized byusing P-cyanoethyl phosphoramidite chemistry on a 1-,imolsynthesis scale. Uncoupled and deblocked primers were notpurified by high-pressure liquid chromatography or poly-acrylamide gel electrophoresis, and New England Biolabssequencing reagents were not modified for use in supercoiledsequencing reactions.

Plate assay for nanH expression. The 2'-a-glycoside of4-methylumbelliferone (MU) and N-acetylneuraminic acid,i.e., MUNeuNAc, is a fluorogenic substrate for many exo-neuraminidases (29). To screen the V. cholerae gene bankfor nanH expression, E. coli were transferred from microdi-lution plates by patching to Luria broth agar containingampicillin and grown overnight at 37°C. Petri plates werethen overlaid with 2 ml of molten 0.75% agar containingMUNeuNAc (1 mg/ml), 50 mM sodium acetate (pH 5.5), 150mM NaCl, and 4 mM CaCl2. NanH+ clones were detectedby their intense green-blue fluorescent halos when excitedwith 366-nm light from a hand-held source. After identifica-tion of fluorescent colonies, viable E. coli were recovereddirectly from the screening plates with a platinum needle andstreaked onto fresh medium.NANase assays. Unless otherwise indicated, NANase was

quantitated by measuring hydrolysis of MUNeuNAc in aTurner fluorometer as described by Myers et al. (29). Oneunit of enzyme released 1 nmol of MU (or sialic acidequivalent) in 1 min at 37°C. Where indicated, the competi-tive NANase inhibitor 2-deoxy-2,3-dehydro-N-acetylneur-aminic acid was used at 1 mM final concentration understandard assay conditions. In some experiments NANasewas measured by colorimetric determination of free sialicacid by the thiobarbituric acid method of Warren (42).Substrates used were sialyllactose from bovine colostrum or

TABLE 1. Plasmids used in this studya

Plasmiida Size (kb) Relevant markers Comments

pCVD315 3.9 Apr Derivative of pKO-l carrying the multiple cloning region fromM13mp19RF

pCVD360 -47 Apr NanH+ -41-kb Sau3A chromosomal fragment from V. cholerae Ogawa395 carrying the nanH gene inserted into the BamHI site of thecosmid pHC79

pCVD361 -49 Apr Kmr NanH- Mini-kan transpositional mutant of pCVD360 with a 1.9-kb DNAsegment carrying the kan gene inserted into the nanH gene

pSX46 7.5 Apr Tcr NanH 3.1-kb EcoRI fragment from pCVD360 inserted into the EcoRI siteof pBR322

pCVD363 8.9 Apr Kmr NanH- 5.0-kb EcoRI fragment from pCVD361 carrying the inactivatednanH gene inserted into the EcoRI site of pCVD315

pCVD364 8.7 Apr NanH+ 4.8-kb BgIII fragment from pCVD360 carrying the active nanHgene inserted into the BamHI site of pCVD315

pCVD365 10.6 Apr Kmr NanH- 6.7-kb BgIII fragment from pCVD361 carrying the inactivatednanH gene inserted into the BamHI site of pCVD315

a The construction of all plasmids is described in this study, with the exception of pCVD315. pCVD315 is a derivative of the promoter-screening vector pKO-l(24) in which bases 1 through 311 have been replaced with the multiple cloning region of M13mpl9RF (44) plus bases 29 through 185 of pBR322. Additionalmanipulations removed the PstI and AccI restriction sites located outside of the multiple cloning region. The construction of pCVD315 is described elsewhere(J. G. Galen, E. R. Vimr, L. Lawrisuk, and J. B. Kaper, manuscript in preparation).

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human al-acid glycoprotein. Activity was expressed ininternational units, where 1 U of NANase released 1 ,umol ofsialic acid in 1 min at 37°C.

,I-Lactamase assay. The chromogenic substrate PADAC{7 - (thienyl- 2 -acetamido) -3 -[2 -(4- N,N- dimethylaminophe-nylazo)-pyridinium methyl]-3-cephem-4-carboxylic acid} waspurchased from Calbiochem (La Jolla, Calif.) and used aspreviously described by Jones et al. (18) to measure ,B-lactamase activity. One unit of enzyme cleaved 0.1 ,mol ofsubstrate in 1 min at 37°C. Initial rates of PADAC hydrolysiswere measured with a Beckman DU-50 spectrophotometerequipped with a temperature-controlled cuvette changer.

Polyacrylamide gel electrophoresis. Native gels were pre-pared and run as described previously by Miller and Mac-Kinnon (26). Denaturing gel electrophoresis was carried outas described by Laemmli (22). The following protein stan-dards were purchased from Sigma Chemical Co. (St. Louis,Mo): P-galactosidase (116 kilodaltons [kDa]), phosphorylaseB (97.4 kDa), albumin (66 kDa), egg albumin (45 kDa), andcarbonic anhydrase (29 kDa).NANase purification and protein sequencing. In a typical

purification, 4 liters of V. cholerae CVD101 grown to theearly stationary phase was used as the source of enzyme.After cells were removed by low-speed centrifugation, theculture supernatant was brought to 40% saturation at 4°C bythe addition of solid ammonium sulfate. Precipitated proteinwas removed by centrifugation and discarded, and thesupernatant was next brought to 80% saturation with ammo-nium sulfate as above. After centrifugation, the pellet con-taining NANase was dissolved in 10 mM Tris buffer (pH7.2), which contained 100 mM NaCl and 4 mM CaC12, andthen dialyzed against multiple changes of the same buffer.Glycerol was added to the dialysate to give a 10% (vol/vol)solution, and bromphenol blue was added to 0.2% (finalconcentration). Up to 3 ml of crude enzyme was loaded intoa single trough of a 160- by 200- by 3-mm 7.5% polyacryl-amide native gel and electrophoresed at 4°C and 80 V untilthe tracking dye reached the bottom of the gel. The gel wasremoved and soaked in water for 15 min and then against twochanges of assay buffer (15 min each). To visualize NANase,a 1-cm-wide gel strip was overlaid with MUNeuNAc asdescribed above for the plate assay method. The fluorescentband was marked with razor blade cuts, and the regionacross the entire gel containing NANase was homogenizedin 10 ml of assay buffer with 25 strokes of a glass Douncehomogenizer (B pestle). Gel pieces were removed by cen-trifugation, and the supernatant was concentrated by vac-uum dialysis. Recoveries ranged from 50 to 80%, with 10- to20-fold purification. No loss of activity was observed for atleast 6 months when the enzyme was stored at 4°C.

Gel-purified NANase was prepared for automated Edmandegradation sequencing by dialysis against water, followedby lyophilization. Approximately 100 ,ug of the purifiedenzyme was sequenced by the University of Illinois Biotech-nology Center's Protein Sequencing Facility (Saw Kyin,Director; Noyes Laboratory, Urbana, Ill.) on an AppliedBiosystems model 470A Sequenator.Other procedures. Proteins were estimated with a modified

Lowry assay as described previously (23) by using a proteinstandard solution (Sigma). Periplasmic proteins were pre-pared by an osmotic shock procedure (30). Soluble proteinand membrane fractions were prepared by sonication asdescribed previously (41).

Materials. The ammonium salt of MUNeuNAc was thekind gift of Y. C. Lee (The Johns Hopkins University,Baltimore, Md.) and was made available to us by M. S.

Kuhlenschmidt (University of Illinois, Urbana). In someexperiments, MUNeuNAc was purchased from Sigma andwas found to be comparable to that supplied by Y. C. Lee(29). Bovine sialyllactose (sialosyl-a-2,3-lactose), human otl-acid glycoprotein, thiobarbituric acid, MU, and Clostridiumperfringens NANase (fraction VI) were purchased fromSigma. 2-Deoxy-2,3-anhydro-N-acetylneuraminic acid wasobtained from Boeringher-Mannheim. All other chemicalswere purchased from standard sources.

RESULTS

Isolation of the V. cholerae nanH gene. A gene bank ofchromosomal DNA derived from V. cholerae Ogawa 395was constructed using the cosmid cloning vector pHC79 andE. coli HB101, a strain with no endogenous NANase activ-ity. Seven of 683 E. coli Apr clones were positive forMUNeuNAc cleavage as determined by qualitative plateassays (Fig. 1). Positive clones were streaked from masterplates onto rich medium, and colonies were then tested forthe Leu- Pro- oxidase-negative phenotype of strain HB101.Clones were purified further by two single colony isolationsand retested for MUNeuNAc cleavage ability. Five of sevenof these clones were NanH-; the reason for loss of theNanH+ phenotype in these isolates is unknown. One stableNanH+ clone was used for further studies and was desig-nated HB101(pCVD360) (Table 1). The cosmid carried bythis stable NANase producer was approximately 47 kilo-bases (kb), as determined by measuring the sizes of theseven restriction fragments generated by digestion ofpCVD360 with EcoRI. This size was expected for DNAcloned into pHC79 and packaged into lambda phage headsfor transduction (17).To demonstrate that cleavage of MUNeuNAc, as mea-

sured by release of MU, was actually due to hydrolysis

FIG. 1. NANase plate assay. E. coli HB101 bearing cosmidswith inserts derived from V. cholerae was overlaid with 2 ml ofmolten agar containing MUNeuNAc and buffer. After incubation for1 h at room temperature, the plate was photographed with incident366-nm illumination. NanH+ clones are indicated by the lowercaseletters. One of these clones (b) contained cosmid pCVD360, whichwas used throughout this study, whereas a and c represent unstableclones (see the text).

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catalyzed by a glycohydrolase, we carried out two sets ofexperiments. We first showed that MUNeuNAc cleavagewas inhibitable by 2-deoxy-2,3-anhydro-N-acetylneuraminicacid. This neuraminic acid derivative is a competitive inhib-itor of most neuraminidases (8). We observed >90% inhibi-tion of MUNeuNAc cleavage catalyzed by soluble extractsof HB101(pCVD360) and by extracts from all of the NanH+subclones (data not shown). The same level of NANaseinhibition was also observed with samples of culture super-natant from the parent strain Ogawa 395.We then tested for the ability of soluble extracts to release

free sialic acid from the oligosaccharide NANase substratesialyllactose, with free sialic acid detected colorimetricallyby the thiobarbituric acid assay method. Extracts from allNanH+ clones released sialic acid to the same extent as thatcatalyzed by samples of V. cholerae culture supernatant(data not shown). Further, sialic acid release was inhibitableby 2-deoxy-2,3-anhydro-N-acetylneuraminic acid. Extractsof HB101 were devoid of NANase activity when assayedwith MUNeuNAc or sialyllactose as the substrate. Weconclude that MUNeuNAc is a sensitive and specific indi-cator of NANase activity in our strains.

Subcloning and mapping of the vibrio nanH gene. Thelocation of the nanH gene within the approximately 40-kbSau3A insertion of pCVD360 was determined by insertionalinactivation. The nanH gene was interrupted with a 1.9-kbinsert encoding kanamycin resistance by using the X1105transducing phage which carries the mini-kan transposon. Ofthe 23 Apr Kmr pooled clones resulting from two X1105mutagenesis experiments, six proved to be NanH-. Plas-mids purified from these six NanH- clones were initiallyjudged to be identical based on restriction fragment profileanalysis with EcoRI. One such plasmid, pCVD361, was usedfor subcloning of the inactivated NANase gene.

Restriction fragment digestion profiles with EcoRI, BgllI,Sall, and PstI were used to compare pCVD360 withpCVD361 and determine the smallest restriction fragmentsthat might contain an intact nanH gene. Both Sall and PstIdigestions produced fragments greater than 12 kb frompCVD360, which increased by 1.9 kb after insertional inac-tivation in pCVD361 (data not shown). However, EcoRIproduced a 3.1-kb fragment from pCVD360 (5.0 kb inpCVD361), and BglII produced a 4.8-kb fragment frompCVD360 (6.7 kb in pCVD361). Therefore, EcoRI and BglIIdigestions of pCVD360 were subcloned into the pBR322derivative pCVD315 and transformed into HB101. Apr iso-lates were screened for NANase activity. No NanH+ cloneswere found in subclones containing the 3.1-kb EcoRI inser-tion. However, plasmids harboring the 4.8-kb Bgll fragment(designated pCVD364) expressed 858 U of NANase activityper mg of protein. Parallel constructions to subclone inacti-vated nanH EcoRI and BglII fragments from pCVD361 intoappropriately digested pCVD315 were also carried out toproduce pCVD363 and pCVD365, respectively. All plasmidconstructs are summarized in Table 1.

Restriction mapping of pCVD364 was facilitated by per-forming paired restriction analysis of both pCVD363 andpCVD365 versus pCVD364. Unique restriction sites presentwithin the inserted kanamycin resistance gene provided thereference sites against which V. cholerae restriction sitescould be easily mapped. A partial restriction map ofpCVD364 is shown in Fig. 2.

Expression of nanH cloned into E. coli. The production ofNANase from the various plasmid constructs described inTable 1 was investigated after transformation of individualconstructs into HB101. NANase activity from these sub-

BG A/H I

PV I

FIG. 2. Restriction map of selected sites in pCVD364. Thehatched triangle indicates the position of insertion of the kanamycinresistance gene from the mini-kan transposon into the nanH gene ofpCVD365. The initiation site and direction of transcription of nanH,as determined by partial sequence analysis of pCVD364, are repre-sented by a wavy line; for a detailed analysis of the neuraminidasesignal sequence, see Fig. 4. A complete description of the size ofnanH awaits completion of sequence analysis in progress. Ampicil-lin resistance, conferred by the bla gene, is shown by the stippledarrow. The hatched arrow represents the galK gene coding for thegalactokinase protein of E. coli and contains no promoter (galK isexpressed only from transcripts originating from upstream clonedDNA sequences). Abbreviations: BG II/H I, cloning junction be-tween the 4.8-kb BglII fragment from pCVD360 (bold line) clonedinto the unique BamHI site within the multiple cloning region ofpCVD315; H III, HindIII; PS I, PstI; PV I, PvuI; R I, EcoRl; SA I,Sall, and SS I, SstI (Sacl).

clones was qualitatively compared by visual inspection offluorescence with the plate assay procedure described inMaterials and Methods. Quantitative results were obtainedby measuring hydrolysis of MUNeuNAc with both wholecells and sonicated cell extracts.NANase activity was not observed in HB101 and

HB1O1(pCVD315), as expected for strains not carrying thenanH gene (Table 2). However, strains carrying intact nanHconstructs were observed to express 10- to 40-fold greaterNANase activity in cell extracts than when whole cells wereused. This would be expected if intracellular NANase weretranslocated into the periplasmic space of HB101 but notexported from the cell; the observed activity of whole cellsmay result from the poor diffusion of the MUNeuNAcsubstrate into the periplasmic space.When a 4.8-kb Bgll fragment carrying nanH from

pCVD360 was subcloned in pCVD315 to create pCVD364,an eightfold rise in the NANase activity of cellular extractswas observed relative to pCVD360 extracts. Since it hasbeen consistently observed that yields of purified plasmidsfrom pCVD315-derived constructs are 5 to 10 times greaterthan yields of pBR322-derived constructs, the eightfold risein NANase activity observed for pCVD364 is assumed toreflect similar increases in construct copy number.

Constructions analogous to pCVD364 carrying insertion-ally inactivated nanH resulted in the loss of NANase activityof pCVD365, as expected. However, when the 3.1-kb EcoRIfragment from pCVD360, carrying the intact nanH gene, wassubcloned into pBR322 to create pSX46, no NANase activ-

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TABLE 2. NANase production in E. coli HB101

NANase sp actPlasmid NanH (U/mg of protein) in:phenotype'

Whole cells" Cell extractsc

None - <2 <2pCVD315 - <2 <2pCVD360 + 11 104pSX46 - <2 <2pCVD364 + + 21 858pCVD365 - <2 <2

a Results of plate assays where plus signs indicate degree of visualfluorescence relative to pCVD360. A minus sign indicates fluorescence wasindistinguishable from that of HB101 with no plasmids.

b Early-stationary-phase cells were collected by centrifugation, washed,and suspended in assay buffer at a fivefold concentration.

c Cells prepared as above were disrupted by sonication before the assay.

ity was observed. The simplest interpretation of this result isthat a nanH promoter is not located within 1 kb of the nanHtranslation start site (see below).NANase purification and identification of a potential signal

peptide-coding region in nanH. To prove that nanH was thestructural gene for NANase, we purified the exported formof this enzyme from V. cholerae CVD101 and determinedthe first 20 amino-terminal (N-terminal) amino acids forcomparison with DNA sequence data obtained frompCVD363. A relatively simple purification was accomplishedby fractionating concentrated extracellular proteins of V.cholerae on nondenaturing polyacrylamide gels. After elec-trophoretic separation, NANase was identified by its activityagainst MUNeuNAc included in a gel overlay. A singlefluorescent band was observed which had an Rf of 0.2 (Fig.3A, lane 1); a similar result was observed with NANase fromC. perfringens (lane 2). Elution of protein from the gel slicecontaining NANase activity resulted in recovery of a 90-kDapolypeptide (Fig. 3B, lane 3). This polypeptide representedone of the higher-molecular-weight polypeptides in concen-trated culture supernatant from strain CVD101 (Fig. 3B, lane4). The specific activity of the purified NANase was 9.2international units per mg of protein, a value comparable tothe specific activity of V. cholerae NANase sold by com-mercial suppliers, e. g., Calbiochem (La Jolla, Calif.). Thispreparation was not homogeneous by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (Fig. 3B, lane 3).However, more conservative excision of NANase fromsimilar gels resulted in electrophoretically pure enzyme (seeFig. 5, lane E, and Fig. 6, lane C). The data for the first 20amino acids obtained by automated Edman degradationmatched perfectly with the amino acid sequence predictedfrom nucleotide sequence information derived frompCVD363 (Fig. 4) and therefore allowed us to define thebeginning of the nanH structural gene (Fig. 2).Upstream from the first amino acid, alanine, in the ex-

ported form of the enzyme, we identified an in-phase openreading frame that could encode a 24-amino-acid signalpeptide if translated (Fig. 4). The predicted amino acidsequence of this putative signal contains all of the featuresexpected for a gram-negative bacterial signal peptide, asreviewed recently by Oliver (31). Five bases upstream fromthe N-terminal methionine codon in the potential signalpeptide-coding region is a potential ribosome-binding site(Fig. 4), suggesting that the signal peptide is probablytranslated in vivo. It is interesting to note that the bases inthis sequence are identical with those of the ribosome-binding site in the cholera toxin ctxAB operon (25). Prelim-

B.3 5 kDa

zws fM...

r: . ^ -116-974

-66

4-45

4. * -29

A.Rf 1 2

0.2-

FIG. 3. NANase purification. (A) Culture supernatant (20 RI)from V. cholerae CVD101 was electrophoresed in a 10% polyacryl-amide native gel and stained for activity as described in Materialsand Methods (lane 1). Migration of C. perfringens NANase (1 ,ug in20 ,ul of H20) is shown as a control (lane 2). Both enzymes migratedwith an Rf of 0.2 (see the text). (B) V. cholerae NANase was elutedfrom a native gel slice, and 6 jig of protein was fractionated in a 10%polyacrylamide denaturing gel, which was then stained with Coo-massie blue R-250 to visualize protein (lane 3). Concentrated culturesupernatant from the ammonium sulfate step was prepared asdescribed in Materials and Methods (lane 4). Lane 5 containsmolecular weight markers.

inary sequencing data of DNA upstream from this potentialribosome-binding site have not revealed promoterlike se-quences. We conclude from data in this and the previoussection that we have cloned the structural gene for NANasefrom V. cholerae. The data also strongly indicate the exist-ence of a signal peptide that may be necessary for transportof NANase into the periplasm of V. cholerae. This peptide is

*** *** *

AAT ATA AAG GGA GTA GAT ATG CGT TTC AAA AAC

Met Arg Phe Lys Asn

GTA AAG AAA ACC GCT TTA ATG CTT GCA ATG TTC

Val Lys Lys Thr Ala Leu Met Leu Ala Met Phe-------------------------------------------

GGT ATG GCG ACA AGC TTA AAC GCC GCA CTT TTT

Gly Met Ala Thr Ser Ser Asn Ala Ala Leu Phe

GAC TAT AAC GCA ACG GGT GAC ACT GAG TTT GAC

Asp Tyr Asn Ala Thr Gly Asp Thr Glu Phe Asp

AGT CCA GCC AAA CAG GGA

Ser Peo Ala Lys Gln Gly

FIG. 4. Nucleotide and amino acid sequence of the V. choleraeN-terminal and potential signal peptide-coding regions of nanH.Amino acids underlined by a solid line were determined by auto-mated Edman degradation and matched with the nucleotide se-quence of nanH. The DNA sequence for both strands was deter-mined through the mini-kan insertion in pCVD363. Data for thecoding strand are shown only to the point where amino acidsequence data were available. Amino acids underlined by a dashedline indicate the potential nanH signal peptide predicted from DNAsequencing. A potential ribosome-binding site is indicated by aster-isks.

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apparently removed before the release of NANase from V.cholerae into the extracellular medium, since it is missing inthe exported form of the enzyme.

Subcellular distribution of NANase in E. coli. It is fre-quently observed that genes coding for exported proteins,when expressed in E. coli, do not lead to release of productsinto the extracellular medium (31, 33). Experiments to detecthigh levels of extracellular NANase in E. coli nanH+ clonesby testing for MUNeuNAc cleavage were negative, indicat-ing that NANase was not being actively secreted by E. coli.Other control experiments with E. coli nanH+ soluble andmembrane subcellular fractions indicated that all of thecell-associated NANase was found in the soluble fraction.To determine whether NANase was expressed in the cyto-plasmic or periplasmic compartments, we prepared periplas-mic fractions from HB101(pCVD364) and HB101(pCVD365)by an osmotic shock procedure. HB101(pCVD364) is aNanH+ subclone and was seen to express a 90-kDa poly-peptide in the periplasmic fraction (Fig. 5, lane A). Thisstained polypeptide band was missing from the periplasmicfraction of HB101(pCVD365), a NanH- strain bearing amini-kan insertion into nanH (Fig. 5, lane B). The cytoplas-mic fraction prepared by sonication of osmotically shockedHB101(pCVD364) (Fig. 5, lane C) did not contain anyobvious increases in 90-kDa polypeptides, indicating thatmost of the nanH gene product in HB101(pCVD364) wastranslocated into the periplasmic compartment. The 29-kDapolypeptide in the cytoplasmic fraction of HB101(pCVD365)(Fig. 5, lane D) may represent a truncated form of NANase;alternatively, this polypeptide may be related to the expres-sion of the kanamycin resistance gene in pCVD365. Theresults in Fig. 5 suggest that, as with most other normallyexported gene products, expression of nanH in E. coli leadsto a product that is not efficently exported into the extracel-lular medium. Since most of the NANase expressed in E.coli was located in the periplasm, and the gene product wasthe same size as the enzyme exported by V. cholerae (Fig. 5,lane E), we conclude that E. coli may recognize and processthe potential signal peptide identified for nanH (Fig. 4).Thus, processing of this peptide would be necessary but notsufficient for NANase export into the medium by either E.coli or V. cholerae.

A B C D E F kDa

-116.97.4

-66

-45

.........,

FIG. 5. NANase localization in E. coli. All samples were frac-tionated by electrophoresis in a 10%o polyacrylamide denaturing geland visualized by protein staining. Lanes: periplasmic fraction fromosmotically shocked HB101(pCVD364) (lane A) and HB101(pCVD365) (lane B), soluble fraction from HB101(pCVD364) (laneC) and HB101(pCVD365) (lane D) after osmotic shock, purified V.cholerae NANase (lane E), molecular weight markers (lane F).

A B C D kDa

116

-9Z4-66

-45

-29

FIG. 6. NANase purified from E. coli. NANase from osmoticshock fluid of HB101(pCVD364) was gel purified as described inMaterials and Methods (lane B) and compared with NANase puri-fied from V. cholerae (lane C). Molecular weight markers are inlanes A and D. Electrophoresis conditions were as described in thelegend to Fig. 3.

To demonstrate that the 90-kDa polypeptide in HB101(pCVD364) was authentic NANase, we took advantage ofthe ability to detect activity in native gels. Periplasmic fluidfrom HB101(pCVD364) was fractionated on a 7.5% poly-acrylamide gel and stained for activity with MUNeuNAc. Asingle fluorescent band was observed at an Rf of 0.38, an Rfidentical to that observed with purified NANase from V.cholerae in this gel. The enzyme was eluted from a gel slicecontaining activity as before (Fig. 3) and fractionated on a10% polyacrylamide denaturing gel. A single 90-kDa bandwas observed (Fig. 6, lane B) which was identical in size tothe band obtained with purified V. cholerae NANase (Fig. 6,lane C). We conclude that the 90-kDa band in shock fluidfrom HB101(pCVD364) is NANase, and that the putativesignal peptide is removed in E. coli, since a molecular weightdifference of 2,600, the size predicted for the 24 amino acidsignal peptide, could have been detected relative to theprocessed vibrio enzyme.

Kinetics of nanH expression in V. cholerae and E. coli. Theproduction of extracellular NANase activity is increased atleast 10-fold in strains of V. cholerae growing in the station-ary phase relative to growth in the early exponential phase(Table 3). When sialic acid was added to cultures growing inearly exponential-phase, a five- to eightfold increase inNANase activity was observed after one cell doubling; latermeasurements of these cultures in the stationary phase againshowed elevated NANase activity relative to stationarycultures receiving no exogenous sialic acid, but activity wasonly increased twofold.The results are consistent with previous observations that

sialic acid can act as an inducer for V. cholerae NANase(13). However, these data further suggest that enzymeproduction may also correlate with the growth phase. Thus,in the absence of exogenous sialic acid, NANase productionin V. cholerae is stimulated when cells have entered thestationary phase. In contrast, when sialic acid is presentduring early log phase growth, immediate induction ofNANase activity occurs with elevated expression, maintain-ing itself into the stationary phase.To further investigate the growth phase dependence of

nanH expression in V. cholerae, exported NANase was

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TABLE 3. Induction of exported V. cholerae NANaseby sialic acid and growth phase

NANase sp act (U/ml perunit of A6W)b Induction

Strain' Growth phase rto(lUninduced Induced ratio (I/U)(U) (I

CVD101 Exponentialc 23 167 8Stationaryd 239 376 2

JBK70 Exponentialc 7 32 5Stationaryd 254 495 2

a Cells from overnight cultures of CVD101 and JBK70 were collected bycentrifugation, washed, and suspended to an A6. of 0.1 in 5 ml of freshmedium.

b Average of duplicate assays from a single experiment.c At an A6w of 0.3, sialic acid (1 mg/ml, final concentration) (induced) or

water equal to the volume of inducer solution (uninduced) was added to theexponentially growing cultures. After one cell doubling, cells were removedby microcentrifugation of 1-ml samples, and NANase was measured in theresulting culture supernatants.d Two hours after the exponential-phase measurement, culture supernatant

NANase was measured to give the stationary-phase values.

measured in cultures of CVD101 growing in the absence ofsialic acid (Fig. 7). Increased NANase secretion was de-tected in the late exponential phase and remained relativelyconstant for at least several hours in the stationary phase(Fig. 7). At later times, e.g., after 62 h of incubation (Fig. 7),NANase activity could no longer be detected in the culturemedium of strain CVD101, suggesting that enzyme activitywas lost as a consequence of physical or proteolytic inacti-vation. Both NANase expression and secretion were tem-porally correlated; thus, NANase did not exist as an intra-cellular pool before its secretion into the medium. This wasshown by measuring NANase in extracts of CVD101 fromsamples taken before the apparent growth phase induc-tion (Fig. 7). This result extends previous experiments by

6.04.0

2.0

o 1.0CD

5 0.6r 0.4

2 0.2

a 0.10 0.06

0.04

0.020.01

180

1600

140 (D

120 ED

100 >

80 ,

60 a)CZ

40 z

z20

1 3 5 7 9 11 62FIG. 7. Kinetics of nanH expression in V. cholerae. Cells from

overnight cultures of V. cholerae CVD101 were collected by cen-

trifugation, and a portion was inoculated into fresh medium to givea starting A6. of 0.04. After a lag of 2 h, cell growth was monitoredby following the increase in A6. of appropriately diluted culturesamples (0). NANase was measured in culture supernatants afterremoving cells by centrifugation for 5 min in a microfuge (0).

Kabir et al. (19), who showed that low levels of cell-boundNANase existed in classical vibrio strains at all stages of cellgrowth. We conclude that NANase secretion by V. choleraeis an efficient event, and that in addition to induction bysialic acid, enzyme expression is modulated by a signalactive in the late exponential phase.

E. coli strain HB1O1(pCVD360) did not export NANaseinto the culture medium (see above) (Fig. 5). Therefore, todetermine whether NANase production in E. coli was alsoregulated by the growth phase, we measured enzyme activ-ity in cell extracts. In contrast to V. cholerae strains, E. coliHB1O1(pCVD360) contained significant cell-bound NANaseat early time points during exponential-phase growth, de-clining rapidly thereafter (Fig. 8). We interpret this result toreflect the phenotypic lag of the stationary-phase cells usedfor initial inoculations, which contained high levels ofNANase. Enzyme induction does not seem to occur in theearly exponential phase, a situation similar to that observedwith V. cholerae stains. The precipitous decline in cell-bound activity during early-exponential-phase growth maytherefore represent a dilution of endogenous enzyme duringcontinued cell growth with subsequent induction of NANaseproduction as cultures approach the stationary phase. Tocontrol for the possibility that the apparent induction ofNANase in HB1O1(pCVD360) was due to a plasmid effect,such as changes in copy number, we determined the specificactivities of P-lactamase and NANase in cell extracts fromcultures at different stages of the growth cycle. As expectedfor an enzyme whose synthesis is not regulated duringgrowth, P-lactamase specific activity in HB1O1(pCVD360)was relatively constant throughout the course of the exper-iment (Fig. 8). This result suggests that any major differencesin NANase specific activity are not due to an increase inplasmid copy number. In contrast to ,-lactamase, NANasespecific activity increased rapidly as cells passed from thelate exponential phase into the early stationary phase. Weconclude that nanIH expression in E. coli is regulated by amechanism that is correlated with growth phase, and that

00CD

0

C1)

a-a)

E

a)CD

zz

Incubation Time (Hours)

FIG. 8. Kinetics of nanH and bla expression in E. coli. Cellsfrom an overnight culture of HB101(pCVD360) were collected bycentrifugation, washed, and suspended to a starting A6. as shown(0). At indicated times during growth, chloramphenicol and sodiumazide were added (final concentrations of 100 ,ug/ml and 0.2%,respectively) to parallel 75-ml cultures. Cells were collected bycentrifugation and washed, and extracts were prepared by sonica-tion. Appropriate dilutions were assayed in duplicate for intracellu-lar NANase (0) and P-lactamase (A).

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this phenomenon is analogous to that observed for V.cholerae. A decision on whether the phenomena in bothspecies involve identical molecular regulatory componentswill require further experimentation.

DISCUSSION

NANases are widely distributed in nature; they havebeen found in viruses, bacteria, protozoa, fungi, and eucary-otic metazoan cells (7); however, the physiological functionsof these enzymes have not been fully elucidated and maybe quite different in the various species. Influenza virusNANases are strongly implicated in the dissemination ofthese viruses in susceptible hosts (2). NANases of bacterialand protozoal origin, in addition to their probable role in cellmetabolism, have been implicated in pathogenesis. NANaseexpressed by Trypanosoma cruzi, the etiologic agent ofChagas' disease, may be causally related to the virulence ofthis protozoan (9), and in infections caused by the gram-positive bacillus Erysipelothrix rhusiopathiae much of thehost cell and tissue destruction may be attributable toNANase expression (15).

In V. cholerae, it has been suggested that NANase mayfacilitate cholera toxin binding to host intestinal epithelialcells by converting cell surface polysialogangliosides to GM1monosialoganglioside toxin receptors (19). Kabir et al. (19)and Staerk et al. (39) have presented epidemiological andexperimental evidence that NANase in V. cholerae is asso-ciated with increased virulence of this organism. CloningnanH from the V. cholerae chromosome and analyzing thekinetics and steps involved in NANase production providesinitial information essential to resolving the role of NANasein V. cholerae pathogenesis.

In this study, we have isolated a cosmid clone from achromosomal library of V. cholerae which expressedNANase activity in E. coli. Transposon mutagenesis local-ized this activity to a 4.8-kb BglII fragment, and the preciselocation of the start of the nanH structural gene was deter-mined by comparison of the N-terminal amino acid sequencewith the predicted sequence derived from DNA sequencedata. Interestingly, a 3.1-kb EcoRI fragment which shouldcontain the entire structural gene (as shown by preliminaryDNA sequence data) did not express NANase activity whencloned separately. Since the LiNA sequence data of the 5'end revealed a ribosomal binding site but no promoterlikesequences, it is possi'ble that the nanH gene is part of anoperon and is transcribed from a promoter further upstream.Ongoing DNA sequence studies and RNA transcriptionalanalyses should elucidate the transcriptional control of thenanH gene.

Nucleotide sequencing of the nanH N-terminal protein-coding region identified a potential signal peptide (Fig. 4).The predicted amino acid sequence for this peptide is similarto that of other gram-negative signal peptides (31), with theexception of two unusual features. One is the presence offour methionine residues; most signal peptides that havebeen sequenced contain no more than three (31). Anotherunusual feature is the potential for four positively chargedamino acid residues in the N-terminal portion of the peptide.We are unaware of any gram-negative signal peptide thatcontains four charged residues, although this number isfound in gram-positive signal peptides (31). Whether theseunusual features of the nanHl signal peptide have any func-tional significance is not known. However, if the leadersequence in nanH is translated in vivo, as seems likely given

the spacing of a ribosome-binding site (Fig. 4), then ourresults indicate efficient processing of NANase in both E.coli and V. cholerae. This interpretation suggests that theNANase secretion pathway may normally include the peri-plasmic compartment. Since NANase export into the extra-cellular medium by V. cholerae was efficient (Fig. 7), andbecause NANase export by E. coli did not proceed past theperiplasmic compartment, we conclude that E. coli may lackfunctions necessary for transport across the outer membranewhich are normally present in V. cholerae. This situationwould be analogous to a recent study demonstrating exportof E. coli heat-labile toxin in V. cholerae (16). By comparingtoxin secretion in this system to toxin gene expression in E.coli, Hirst and Holmgren (16) concluded that the toxinsecretion pathway included a transient stage in the periplasmin which toxin subunits A and B presumably assembledbefore their export across the outer membrane as holotoxin.The intriguing possibility of an outer membrane secretionmachinery in V. cholerae was left as an open question. Theease and sensitivity of the NANase fluorometric assayshould facilitate experiments designed to gain evidence foror against an outer membrane translocation machinery. Thefact that V. cholerae exports a number of toxins and hydro-lytic enzymes suggests that this species may be generallyuseful for investigating outer membrane translocation mech-anisms (14).The observations in this communication (Table 3, Fig. 7

and 8) and those made by others (13), suggest that nanHexpression is controlled by sialic acid availability andgrowth-phase-dependent mechanisms. The former controlmechanism is similar to other catabolic genes that areinduced by their substrates or by substrate-related mole-cules. Control of nani expression by sialic acid availabilityis therefore analogous to sialic acid induction of nanA andnanT in E. coli (40, 41). Recently, we reported the discoveryof nanH in the salmonellae (Vimr et al., Abstr. Annu. Meet.Am. Soc. Microbiol. 1987), including Salmonella typhimu-rium LT-2. Previous results demonstrated that this specieswas able to utilize sialic acid as a carbon and energy source(40), indicating the presence of a nan system. These obser-vations may be pertinent to the recent discovery of cholera-like toxin in the salmonellae (12, 32), as they further suggestthat NANase may function to increase toxin receptor den-sity in susceptible hosts. We have since shown that NANaseproduction in LT-2 is also modulated by sialic acid, suggest-ing that nanH expression may be regulated similarly to nanAand nanT in this species. A functionally related nan systemalso appears to exist in V. cholerae (13). It is relevant to notethat, similar to our results in Fig. 7 and 8, NANase export inthe salmonellae was also maximal as cells entered thestationary phase (E. R. Vimr and L. Lawrisuk, manuscriptin preparation). Thus, a variety of microorganisms respondto sialic acid availability by inducing multiple components ofa nan catabolic system, and it will be interesting to define thegenetic controls operating in these systems, since one of thenan system components is an extracellular enzyme that mayhave a significant involvement in pathogenesis.To our knowledge, the results presented in this communi-

cation represent the first successful cloning of a nanH genefrom any bacterial species. Evidence that we have clonedthe NANase structural gene is based on enzyme expressionin E. coli, purification and comparison of the V. cholerae-derived NANase with enzyme from E. coli, insertion muta-genesis, and congruence of the NANase N-terminal aminoacid sequence with the sequence predicted from nucleotidesequence analysis. Taken together, the results of these

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NEURAMINIDASE EXPRESSION IN E. COLI 1503

complementary approaches offer compelling evidence thatwe have identified the nanH structural gene from V. chol-erae and characterized its expression in an E. coli host.Using the cloned V. cholerae nanH gene, we are now usinga variety of animal models to establish the significance ofNANase in disease due to V. cholerae.

ACKNOWLEDGMENTS

We are grateful to Y. C. Lee and M. S. Kuhlenschmidt forsupplying much of the MUNeuNAc used in this research. We thankS. Kyin for expertly sequencing NANase.

This work was supported by Public Health Service grant 1 RO1A123039 and Biomedical Research Support grant RR05460 (E.R.V.)and grant A119716 (J.B.K.) from the National Institutes of Health.L.L. was supported by a U.S. Department of Agriculture predoc-toral training grant in the molecular basis of infectious diseases(84-GRAD-9-0062).

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