APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 30-370099-2240/88/010030-08$02.00/0Copyright © 1988, American Society for Microbiology

Cloning and Gene Replacement Mutagenesis of aPseudomonas atlantica Agarase Gene

ROBERT BELAS,t* DOUGLAS BARTLETT, AND MICHAEL SILVERMAN

Agouron Institute, La Jolla, California 92037

Received 26 June 1987/Accepted 1 October 1987

An agarase gene (agrA) was isolated by cloning genomic DNA prepared from Pseudomonas atlantica. Theagarase activity in recombinant Escherichia coli was found in cell-free culture supernatants and could passthrough a 0.45-,um-pore-size membrane separating cells from agar, suggesting that the gene product wasexported in E. coli. The enzyme was specific for agar and agarose and did not digest alginate or carrageenan.Mutations generated by transposon mini-Mu dl(lacZ Kmr) were used to define the agrA coding region, as wellas the direction of transcription of the gene. A procedure was developed to produce a P. atlantica agrA mutant.This required construction of an agrA::kan insertion mutation in vitro and subsequent introduction of thedefect into the chromosome of P. atlantica by recombinational exchange. Transformation of P. atlantica withplasmids containing agrA::kan utilized a Tris-polyethylene glycol 6000-CaCl2 treatment for making competentcells. Replacement of wild-type agrA with agrA::kan resulted in loss of agarase activity. Uses of the agrA geneprobe and an Agr- mutant for environmental studies are discussed.

Bacteria able to digest agar have been assigned to thegenera Cytophaga (24), Streptomyces (11), and Pseudomo-nas (17, 18, 27). Phenotypically, the expression of theagarase activity is observed as a pitting and depression of theagar surrounding a colony of bacteria. In each of thesegenera, the breakdown of the agar is due to the activity of anextracellular enzyme, ,-agarase. Of the three principal gen-era characterized as agar-digesting bacteria, most researchhas centered on the agarolytic properties of the marinebacterium Pseudomonas atlantica.As diagrammed in Fig. 1, the pathway of agar breakdown

by P. atlantica involves the initial cleavage of agar at the(1--4)-1-D linkage by the extracellular enzyme P-agarase (I)(EC 3.2.1.81) resulting in neoagarotetraose [0-3,6-anhydro-ot-L-galactopyranosyl(1--3)- O- -D-galactopyranosyl(1--+4)-O-3,6-anhydro-ot-L-galactopyranosyl(1--*3)-D-galactose] (17, 18).This cleavage and subsequent reduction in size of theagarolytic products allows these tetrasaccharides and disac-charides to pass into the cell, while the much larger agaroligomer cannot cross the cell wall barrier. Neoagarote-traose is cleaved to produce the dimeric saccharide neoaga-robiose (0-3,6-anhydro-a-L-galactopyranosyl(1---3)-D-galac-tose). The enzyme responsible for this cleavage has beencalled neoagarotetraose hydrolase (9) or ,-agarase (II) byMorrice et al. (17) and is found in the culture supernatant.Additionally, 3-agarase (II) has endoenzymatic propertiesand can cleave the oligomeric agar to neoagaro-oligosaccha-rides of which neoagarobiose is the major product (17).Ultimately, the saccharide dimer is broken down by acell-bound enzyme, a-neoagarobiose hydrolase (6), to themetabolically useful carbon sources D-galactose and 3,6-anhydro-L-galactose.We are interested in exploring in molecular detail the

mechanisms which microorganisms employ to adapt tochanging and adverse conditions in the marine environment.Specifically, our research has focused on the strategies

* Corresponding author.t Present address: Department of Microbiology, Louisiana State

University, Baton Rouge, LA 70803-1715.

which bacteria have developed to acquire nutrients frominsoluble extracellular polymers such as agar. To this end,we cloned an agarase gene from P. atlantica, characterizedits expression in Escherichia coli, and constructed a mutantof P. atlantica defective in agarase production by utilizing agene replacement strategy. The adaptive advantage of aga-rase production and uses of the agarase gene probe arediscussed.

MATERIALS AND METHODS

Bacteria, media, and culture conditions. E. coli strainswere maintained at 37°C in L broth (10 g of tryptone [DifcoLaboratories, Detroit, Mich.], 5 g of yeast extract [Difco], 10g of NaCl, 1,000 ml of H20) or on L agar (L broth solidifiedby the addition of 15 g of agar [Difco] per liter). Whenexpression of cloned agarase activity was measured, E. coliwas grown at room temperature or 30°C. Wild-type P.atlantica T6c (23) (a gift from D. White, University ofTennessee) and WY (from W. Yaphe, McGill University)were routinely grown at room temperature or 30°C in Difcomarine broth at 75% the recommended concentration (28g/1,000 ml of H20 and hereafter referred to as 2216 medium).2216 medium was solidified by the addition of 20 g of agar perliter. To test agarase breakdown of oligosaccharides otherthan agar, we amended 2216 broth with either alginate (30g/1,000 ml of 2216 broth) or carrageenan (15 g/1,000 ml of2216 broth). Alginate and carrageenan were obtained fromSigma Chemical Co., St. Louis, Mo. To assess the contri-bution of extracellular agarase production to the survival ofP. atlantica grown on agarose as a sole carbon source, weused 1.0% (wt/vol) agarose (Bethesda Research Laborato-ries, Inc., Gaithersburg, Md.) in seawater medium. Thecomposition of seawater medium was 12.1 g of Tris hydro-chloride, 2.78 g of Tris base, 1 g of NH4Cl, 0.2 g of MgCl2,0.1 g of K2P04 3H20, 1.1 mg of FeCI3 6H20, 500 ml ofseawater (La Jolla, Calif., coastal seawater), 500 ml of H20,pH 7.6. Antibiotic concentrations for selection of plasmids inE. coli were as follows (micrograms per milliliter): tetracy-cline, 10; kanamycin, 40; chloramphenicol, 50; and ampicil-

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CLONING OF AN AGARASE GENE FROM P. ATLANTICA

AGAR

NEOAGAROTETRAOSECH20H 0 CH20H 0

HO 00 H H h

HO~~~~~~~

\CH2 H (OH)2OH OH

,B-AGARASE (E)[NEOAGAROTETRAOSE HYDROLASE]

NEOAGAROBIOSE

CH20H 0

HO t

OHH \CH2 (OH)2

OH

OH

a -NEOAGAROBIOSE HYDROLASE

D-GALACTOSE 3,6 -ANHYDRO-L-GALACTOSE

CH20H O

HOt NH HOH

\C2CH (OH)2

OH

FIG. 1. Enzymology of agar digestion in P. atlantica. Oligomericagar is initially cleaved by the exported enzyme, 0-agarase (I). Thisenzyme cleaves agar at the P(1-*4) linkage, producing neoagarote-traose. This tetrameric saccharide is then broken down to neoaga-

robiose through the action of P-agarase (II), also referred to as

neoagarotetraose hydrolase.,-Agarase (II) is also capable of digest-ing oligomeric agar, cleaving it at the,B(1- 4) linkage and producingneoagarobiose. Neoagarobiose is cleaved at the 1--3 linkage bya-neoagarobiose hydrolase to produce D-galactose and 3,6-anhydro-L-galactose.

lin, 80. To maintain plasmids or drug resistance genes

inserted in genomic loci in P. atlantica, we used the follow-ing concentrations of antibiotics (micrograms per milliliter):kanamycin, 50; ampicillin, 100. Antibiotics were purchasedfrom Sigma or Calbiochem-Behring, La Jolla, Calif.

Cloning of P. atlantica agarase gene. Genomic DNA was

isolated from P. atlantica and partially digested with EcoRI.EcoRI fragments were ligated with T4 DNA ligase into theEcoRI site of plasmid pACYC184 (5) at 15°C for 3 to 4 h.Recombinant plasmids were recovered by transformationinto E. coli ED8654 (supE supF met hsdR hsdM+) madecompetent by the method of Mandel and Higa (14), andtransformants were selected on L agar containing tetracy-

cline. After incubation at 30°C for 48 h, transformants werescreened for agarolytic activity by visual inspection of thesurface of the agar plate. Colonies that pitted the agar orformed a shallow depression around the periphery of thecolony were confirmed to be agarase positive (Agr+) bystreaking on fresh media. Agar digestion also was verified byflooding the agar surface with Gran's iodine reagent (0.05 Miodine in 0.12 M KI) for 5 min and then examining thecolonies for zones of clearing (11). Gran's iodine reagentreacts with the intact agar oligosaccharide to form a reddishbrown color, while areas that have been enzymaticallydegraded by agarase are unstained and remain clear (see Fig.5 for an example of this reaction). From this screening,several agar-digesting E. coli clones were obtained, and theplasmid encoding the agarase activity designated as pBB917.Transposon mutagenesis. The agrA gene was first sub-

cloned. Plasmid pBB917 was digested with EcoRI plusHindlIl, and the resulting DNA fragments were ligated intoplasmid pKO1 (16) digested with the same restriction en-zymes. After ligation, transformation of ED8654, and selec-tion for plasmid-encoded ampicillin resistance, Agr+ colo-nies were identified by observing pitting and using a stainingprocedure as described above. Plasmids from these Agr+colonies harbored a single EcoRI-HindIII insert approxi-mately 4 kilobase pairs (kbp) in size. A single representativeharboring this plasmid, pBB918, was chosen for furtherstudy. Plasmid pBB918 was mutagenized with transposonmini-Mu dl by the muduction technique of Casadaban andco-workers (1, 3, 4, 7). Briefly, plasmid pBB918 DNA wasfirst transformed into E. coli P011681 [F- Mu dl 1681ara::(Mu cts)3 A(proAB argF lacIPOZYA)XIII strA]. Mini-Mu transposon Mu dl 1681(lacZ Kmi) (4) is a smaller version(15.8 kbp) of the Mu d transposon (3). A transformant(pBB918 in P011681) was heat induced, and the resultingbacteriophage lysate was used to infect Rec+ recipient strainMH3497 (lac gal rpsL Mu cts). The infected bacteria wereplated on L agar containing ampicillin (plasmid-encodedresistance) and kanamycin (transposon-encoded resistance).The resulting colonies were screened for loss of agaraseactivity. More than 150 independent Agr- mutants weredetected, and f-galactosidase activity was measured quali-tatively on MacConkey base agar (Difco) indicator platessupplemented with 1% (wt/vol) lactose. The location andorientation of the mini-Mu inserts in the mutated plasmidswas determined by restriction mapping utilizing the asym-metry of the EcoRI, HindIII, and PstI sites of the transposon(4, 7).Gene replacement mutagenesis. The 4-kbp EcoRI-HindIII

fragment encoding the extracellular agarase activity frompBB918 was isolated by digesting pBB918 with EcoRI plusHindIII, electrophoretically separating the DNA fragmentson a 0.8% agarose gel, and electroeluting the 4-kbp fragmentfrom a gel slice in a Unidirectional Eluter (model UEA;International Biotechnologies, Inc., New Haven, Conn.). Toconstruct a recombinant with the agarase gene flanked byEcoRI sites, we required an EcoRI-HindIII linker. PlasmidpUC8 DNA (25) was digested with EcoRI plus HindIII andelectrophoretically separated on a 20% polyacrylamide gelby the procedure described by Maniatis et al. (15), and theresulting 36-base-pair (bp) EcoRI-HindIII fragment contain-ing a portion of the pUC8 polylinker was isolated by elec-troelution. Plasmid pBR322 was digested with EcoRI andthen held at 65°C for 15 min to inactivate the enzyme. TheEcoRI-HindIII linker, the EcoRI-HindIII agarase gene frag-ment, and the EcoRI-cut pBR322 were ligated together andtransformed into ED8654, selecting for ampicillin resistance

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and screening for agarase activity. This ligation yieldedpDB59 which contained agrA flanked by EcoRI sites. Theselectable marker to be introduced into agrA, the kanamycinresistance gene from pMB2190 (a gift from M. Berman), was

inserted into the Sall site in agrA. pMB2190 was completelydigested with Sall and pDB59 was partially digested (0.2units of Sall per p.g of DNA) with the same enzyme, theenzyme was heat inactivated, and the Sall-cut fragmentswere ligated. After transformation and selection for coloniesresistant to both ampicillin and kanamycin, agarase activitywas qualitatively measured to ensure that no agarolyticactivity was present. A single Agr- colony was selected, theplasmid (pDB60) was isolated and retransformed intoED8654, and the Agr- phenotype was verified. PlasmidpDB60 was confirmed to be the correct construction byrestriction enzyme analysis of fragments from known restric-tion endonuclease map sites.

Transfer of pDB60 into P. atlantica was accomplished bya modification of the transformation procedure described byFornari and Kaplan (8) originally used with Rhodopseudo-monas sphaeroides. A 20-ml culture of T6c was harvested atthe late log phase after 12 h of room temperature incubationwith vigorous shaking. The bacteria were pelleted by cen-

trifugation at 7,000 rpm in an SS34 rotor (Sorvall RC5B;Dupont Instruments, Newtown, Conn.) at 4°C and sus-

pended in 10 ml of ice-cold 500 mM Tris hydrochloride, pH7.2. The cells were centrifuged a second time, and the pelletwas suspended in 1.2 ml of ice-cold 100 mM Tris hydrochlo-ride containing 400 mM CaCl2. A 0.2-ml sample of compe-

tent cells was immediately mixed with 20 p.g of pDB60 DNAand an equal volume of 40% (wt/vol) polyethylene glycol6000 (Sigma). The solution was gently mixed to disperse thetwo phases, and the cell suspension was left on ice for 10min. The cells were heat shocked for 2 min at 30°C. 2216broth (1 ml) was added to the cell suspension, and themixture was incubated at 30°C for an additional 20 min, afterwhich another 3 ml of fresh 2216 broth was added. The cellswere shaken at 30°C for 9 h before a 1-ml sample was spreadon 2216 agar containing 50 p.g of kanamycin per ml. Afterincubation for 48 h at 30°C, one kanamycin-resistant, Agr+transformant was picked and inoculated into 5 ml of 2216broth without ampicillin selection (for the plasmid), but withkanamycin selection (for agrA: :kan). Because plasmidpDB60 segregated rapidly in the absence of selection, thisprocedure favored the loss of the plasmid and the retentionof the kan gene by homologous recombination to exchangethe chromosomal locus with agrA::kan. After two passages,each for 24 h at room temperature in medium with kanamy-cin selection alone, cells were diluted and spread on 2216agar. The resultant colonies were then screened for loss ofextracellular agarase activity, for kanamycin resistance, andfor ampicillin sensitivity. Approximately 0.1% of the colo-nies were Agr- Kanr Amps, and from these a single isolate,strain DB11, was saved for further analysis.DNA analytical methods. Small-scale isolation of plasmid

DNA was done as described by Silhavy et al. (21). Forlarge-scale plasmid DNA preparations and CsCl purification,the alkaline methods described by Maniatis et al. (15) andBirnboim and Doly (2) were used. All restriction enzymes

were obtained from Boehringer Mannheim Biochemicals,Indianapolis, Ind. T4 DNA ligase was purchased from NewEngland BioLabs, Inc., Beverly, Mass. All enzymes were

used according to the directions provided by the supplier.Transfer of DNA fragments to nitrocellulose (Schleicher &Schuell, Inc., Keene, N.H.) was performed by the method ofSouthern (22), and plasmid DNA was radioactively labeled

with [cx-32P]dCTP (New England Nuclear Corp., Boston,Mass.) by the recommended procedure of the supplier.

RESULTS

Cloning of an agarase gene. P. atlantica T6c was selectedfor cloning the agarase gene because agarolytic activity waseasily observed when colonies were incubated on 2216 agarplates for 24 to 48 h at room temperature or 30°C. Theagarase activity was present in cell-free culture supernatantsand when cells were grown on 0.45-p.m-pore-size nitrocellu-lose filters placed over 2216 agar, indicating that this enzymewas extracellular and not associated with the outer mem-brane, in agreement with previously reported observations(17, 18, 28). The Agr+ phenotype was seen as a pitting ordepression in the agar surrounding the colony. The agaraseactivity could also be observed after flooding the agar plateswith Gran's iodine reagent (see Materials and Methods),which stained the uncleaved agar oligosaccharide a darkreddish brown while a zone of clearing was seen around theperiphery of agarase-producing (Agr+) colonies. The clearzone was due to cleavage of the oligosaccharide into smallermolecules which did not stain by this procedure (11).To clone the gene encoding agarase activity, P. atlantica

genomic DNA was isolated, CsCl purified, and partiallycleaved with EcoRI. The EcoRI fragments were ligated toEcoRI-digested plasmid pACYC184. The ligation mixturewas transformed into E. coli ED8654, and the cells werespread on L agar plates with tetracycline for selection of theplasmid-borne drug resistance. After 24 h of incubation at370C, approximately 3,500 transformants were examined byeye for indication of pitting (agarase activity) around colo-nies. No Agr+ colonies were identified, but after furtherincubation at 300C for an additional 18 h, eight Agr+ colonieswere located. The Agr+ colonies were streaked on fresh Lagar containing tetracycline, and overnight incubation at300C confirmed that each pitted the surrounding agar andgave a clear zone with iodine staining. When incubated attemperatures higher than 35°C, none of the Agr+ clonesvisually pitted the agar nor could any clearing be seen afterflooding the plates with Gran's iodine reagent. These datasuggest that the production, export, or activity of the extra-cellular agarase is affected at tempertures above 300C. Plas-mid DNA from each of the Agr+ clones was prepared, and arestriction endonuclease map for each of the eight cloneswas determined. All eight Agr+ clones were identical basedon restriction site maps, and therefore one, pBB917, wasselected for further analysis. The map of pBB917 is shown inFig. 2.Export of agarase by recombinant E. coli was then tested.

ED8654 harboring pBB917 was grown on top of a nitrocel-lulose membrane (0.45-p.m-pore diameter, BA85; Schleicher& Schuell) laid on an L agar surface. After several genera-tions, the membrane with the bacteria was removed from theagar, and the surface was flooded with iodine reagent. Aprominent zone of clearing indicating that enzyme activityhad diffused from the cells was observed in the area beneaththe membrane. Further, culture supernatants devoid of cellshad an agarase activity that was demonstrable when asample was placed on an agar surface and that surfaceflooded with iodine reagent after a few hours of incubation toallow for the enzyme to degrade the agar. These data suggestthat the cloned agarase activity was extracellular and wasexported past the outer membrane of E. coli. The specificityof the cloned agarase was also determined by growing therecombinant and P. atlantica on medium containing algi-

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CLONING OF AN AGARASE GENE FROM P. ATLANTICA

A N A

C~

p pBBB |7N 11.2 kbp

B

Tet r

FIG. 2. Plasmid constructions. The 7.2-kbp EcoRl bacterialDNA insert from P. atlantica was inserted into pACYC184 to makepBB917 (agrA). The solid black area indicates the segment of DNAencoding agarase activity, while the hatched area represents addi-tional P. atlantica DNA not associated with the agrA locus. A4.0-kbp fragment containing agrA was subcloned by restrictingpKO1 and pBB917 with EcoRI plus HindlIl to generate pBB918. Toconstruct pDB59, pBB918 and pUC8 were digested with EcoRI plusHindllI, pBR322 was restricted with EcoRI, and the EcoRI-HindIlIfragments plus the EcoRI-cut plasmid were ligated with T4 DNAligase. Partial Sall digestion of pDB59 and pMB2190 followed byligation generated the agrA::kan gene replacement, pDB60, whichwas ultimately used to produce a null mutation in the agrA locus inthe P. atlantica genome (strain DB11). Restriction sites are as

follows: A, Accl; B, BamHl; C, ClaI; H, Hindlll; N, Nrul; P, PstI;R, EcoRI; and S, Sall. Antibiotic resistances are designated as Bla

(P-lactamase) for ampicillin, Kanr for kanamycin, and Tetr fortetracycline. Plasmids are not drawn to scale, and some restrictionsites in later constructions are not shown for reasons of clarity.

nate, carrageenan, and agarose. Both E. coli with the clonedagarase and P. atlantica digested agarose but failed todegrade either alginate or carrageenan.

Agarase::mini-Mu dl(lacZ Kmr) fusions. The initial EcoRIclone, pBB917, contained a 7.2-kbp insertion. Because thisinsert was sufficiently large to encode several proteins, aportion of DNA from the original insert was subcloned intoanother vector, thereby further localizing the agrA gene aswell as facilitating additional molecular investigations. Plas-mid pBB917 was digested with EcoRI plus HindIII, and theEcoRI-HindIII fragments were ligated to pK01 (16) digestedwith the same enzymes. Plasmid pK01 was chosen becauseit does not contain a promoter which directs transcriptionacross the EcoRI or HindlIl sites (16). Thus, agarase activityencoded by this plasmid would most likely be the result oftranscription from the P. atlantica promoter located up-stream from the gene encoding agarase activity and not dueto readthrough into the agarase gene from promoters locatedin the plasmid. The mixture was transformed into competentED8654, and the cells were spread on L agar containingampicillin. Screening for Agr+ colonies was conducted asdescribed above. Several Agr+ colonies were found, andrestriction analysis revealed that each carried the 4.0-kbpEcoRI-HindIII insert from pBB917. One representative wasselected, and the plasmid was designated pBB918.To define the agarase-coding region and to identify the

direction of transcription of the agarase gene, we usedtransposon mutagenesis with mini-Mu dl(lacZ Kmr). Muta-genesis with mini-Mu dl results in transcriptional fusion ofthe lacZ (encoding ,B-galactosidase) gene of the transposonto the target gene. If the insertion of the transposon is in thecorrect orientation with respect to transcription from thepromoter of the target gene, the lacZ gene will be transcribedand ,B-galactosidase will be produced. If the insertion is inthe opposite orientation, no 3-galactosidase will be pro-duced.A total of 150 Agr- transposon-generated mutants were

isolated and analyzed for the approximate site of transposoninsertion as determined by restriction site mapping. Of theoriginal 150, 35 agrA::mini-Mu insertions were chosen fordetailed mapping to ascertain the site of insertion of thetransposon as well as the orientation of the mini-Mu. 1-Galactosidase activity was detected by streaking mutants onMacConkey agar supplemented with lactose. Mini-Mu inser-tions which gave the Agr- phenotype mapped within a1.5-kbp region of pBB918 (Fig. 3). Mini-Mu transposoninsertions 1, 5, 31, 40, 16, 28, 62, 67, and 37 were Agr-LacZ+ and oriented to position the lacZ of the transposon incorrect alignment with an upstream (left on Fig. 3) promoterassociated with the cloned agrA locus. Other transposoninsertions giving the Agr- LacZ- phenotype were mapped.These were located in the 1.5-kbp region and had theopposite orientation of insertion (data not shown). Insertions6 and 72, and 63 and 26 were Agr+, and the latter pair werealso aligned to allow transcription of lacZ from the agrApromoter. However, little or no 1-galactosidase activity wasmeasured in mutants 63 and 26, indicating that transcriptionfrom the upstream promoter terminates somewhere betweenmini-Mu insertions 37 and 63. Transposon insertions 77 and35 were both Agr- LacZ-. Restriction analysis revealed thatthe mini-Mu element was inserted in the opposite orientationand so was not aligned with an agrA-associated promoter.The result of this analysis indicated that the beginning of theagarase-coding region was ca. 150 bp upstream of theleftward NruI site and was defined by transposon insertions72 and 77. The 3' terminus of the gene was defined by

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Agr+ I AgrI-~~ ~ ~ ~ ~~~~-_~~~~~~~~~I

I~r IDB-8 in aMu*uZD jD )flwD sI

pBB918:: iniMumiini)(-M(i

I I I

R A N A N A H

TRANSCRIPTION

0 2 3 4

kbp

FIG. 3. Restriction map, locations of mini-Mu dl(lacZ Kmr) transposon insertions, and direction of transcription of agrA. Plasmid pBB918contains an agarase-expressing (solid black area) EcoRI-HindIII fragment from P. atlantica cloned into the EcoRI-HindIII site of plasmidvector pKO0. Symbols above the map represent location of mini-Mu transposon insertions positioned by analyzing Hindlll and EcoRI plusPstI digests of corresponding plasmids. The position of transposon insertions was accurate to approximately ±50 bp. Mutants 6 and 72, and63 and 26 produced agarase activity; all other transposon insertions destroyed agarase activity. Direction of transcription was analyzed byascertaining orientation of mini-Mu insertion and 3-galactosidase activity (see Materials and Methods). Restriction sites are as follows: A,AccI; C, ClaI; H, HindIJI; N, NruI; R, EcoRI; and S, SalIl.

transposons 35 and 63. From these results and additionalmapping data, we conclude that a region of 1.5 kbp isnecessary for expression of agarase activity and that thecoding region is transcribed in the direction shown in Fig. 3.

Agarase gene probe. DNA probes have been used toidentify genes in pathogenic bacteria (13, 26), and the clonedagrA gene could be used in the identification and enumera-tion of agarase-producing marine pseudomonads. To evalu-ate the usefulness of agrA as a hybridization probe, genomicDNA from agarase-producing P. atlantica ATCC 19262,WY, and T6c was cleaved with various restriction endonu-cleases and transferred to nitrocellulose as described above.The DNA was then hybridized to radioactively labeledplasmid pDB59 (see Fig. 1 and below) containing agrA, andthe unhybridized probe was removed by washing the nitro-cellulose at conditions of both high (>85% homology) andlow (>20% homology) stringency. The resulting autoradio-grams showed that the cloned agrA hybridized to the paren-tal strain (T6c) and also to P. atiantica WY. The hybridizedDNA fragments were identical for each strain when cut withthe same enzyme, indicating that both T6c and WY possessidentical or very similar agarase genes (data not shown).Hybridization to DNA from strain ATCC 19262 was not seeneven though low-stringency conditions and prolonged auto-radiogram exposures (7 days at -80°C) were used. Weinterpret this to indicate that among the agarase-producingbacteria taxonomically identified as P. atlantica, there existssubstantial divergence of the agarase genes at the DNAsequence level. Such evolutionary divergence, as shownwith the cloned agrA probe, could prove to be very valuablein the taxonomic characterization of subgroups within thosebacteria currently identified as P. atlantica.Gene replacement mutagenesis. It has been reported that

strains of P. atlantica contain two or more agarase activities(17, 18): one considered to be the principal extracellularendoenzyme responsible for the 1(1->4) cleavage of theoligosaccharide, termed 3-agarase (I), and the other possess-ing minor endo-,(1--->4) activity, as well as an exoenzymaticactivity, called P-agarase (II) (17, 18). To ascertain that thecloned gene encoded the major extracellular agarase activity

of P. atlantica, we replaced the chromosomal agrA locuswith a mutant agrA gene containing a selectable drug resis-tance gene insertion. This involved inactivation of agrA byinserting a kan gene in the coding sequence followed byintroduction of the agrA::kan plasmid into P. atlantica inwhich the wild-type locus was displaced by homologousrecombination (10, 12, 20).To facilitate construction of the replacement, we isolated

the 4-kbp EcoRI-HindIII agrA fragment from pBB918 byelectroelution from an EcoRI-HindIII digest of the plasmidelectrophoretically separated on an agarose gel. A 36-bpEcoRI-HindIII portion of the multiple cloning site frompUC8 (25) was also isolated from a polyacrylamide gel (seeMaterials and Methods). The linker was needed so that the4-kbp EcoRI-HindIll fragment could be ligated into theEcoRI site on pBR322. These two fragments were mixedtogether with pBR322 cut with EcoRI and ligated, and themixture was transformed into ED8654. The structure of theresulting Agr+ plasmid, pDB59, was confirmed by restric-tion enzyme digestion of plasmid DNA and analysis ofDNAfragments after electrophoretic separation on agarose gels.Restriction fragment mapping and analysis of transposoninsertions indicated that a convenient Sall restriction siteexisted approximately at the midpoint of the agrA codingregion. To inactivate agrA in pDB59, we chose to introducethe kanamycin resistance gene from pMB2190. ThepMB2190 kanamycin resistance gene (kan) is flanked bymultiple cloning sites, including Sall sites (Fig. 2). Bothplasmids were digested with Sall, the enzyme was heatinactivated, and the DNA mixture was ligated and trans-formed into ED8654, selecting for kanamycin and ampicillinresistances. Loss of agarase activity was screened by visualinspection of nonpitting colonies and by lack of clearingaround colonies after flooding plates with Gran's iodinereagent. The efficacy of the construction was confirmed byisolation of plasmid DNA, subsequent restriction enzymedigestion, and DNA fragment analysis on agarose gels. Thisplasmid containing the kanamycin resistance gene insertedin agrA (agrA::kan) was designated pDB60.pDB60 was introduced into P. atlantica by a transforma-

Agr+

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CLONING OF AN AGARASE GENE FROM P. ATLANTICA

So/I So/I+EcoRI &coRI+Hind[o o 0

CL F _D QF-D X a

21.3-

5.154.28-3.53-

2.03-1.90-

1.58- -

1.33 -

1 2 3 4 5 6 7 8 9FIG. 4. Hybridization of radioactively labeled probe pDB60 to

genomic DNA from wild-type P. atlantica T6c, agrA::kan genereplacement strain DB11, and DNA from agrA::kan plasmid pDB60.The kanamycin resistance gene is carried on a ca. 1.5-kbp Sallfragment as seen in lanes 1 and 3. Two bands appear in lane 9 as aresult of a HindIll site situated within the kan gene. Molecularweight markers (phage lambda DNA restricted with EcoRI plusHindIlI) used to determine size of hybridized fragments are indi-cated on left of autoradiogram and are expressed in kilobase pairs.

tion procedure. Two separate developments allowed us touse such an approach. First, while most CaCl2-based trans-formation procedures do not produce competent P. atlan-tica, a modification of a transformation procedure originallyused for R. sphaeroides (8) was successful. Second, anonmucoid variant cell type (crenated colony morphology)was found to be 100 times more competent than the originalparental strain by the above procedure. P. atlantica wastransformed as detailed in Materials and Methods. Efficiencyof transformation was low, ca. 1.5 x 10-7 transformants perrecipient cell; however, the efficiency could be increased100-fold if plasmid DNA was isolated from P. atlantica andthe cells were transformed with this DNA. This resultsuggests that a host restriction-modification system exists inP. atlantica which lowers the transformation frequency ofplasmid DNA from E. coli.

Cells transformed with pDB60 were both kanamycin andampicillin resistant, indicating that the ColEl-based plasmidcould be maintained in P. atlantica. However, in the ab-sence of selection the plasmid was rapidly lost. To isolate theagrA::kan recombinant, a single transformant colony wasinoculated in 5 ml of 2216 broth containing kanamycin butwithout selection for the plasmid-borne ampicillin resis-tance, and the bacteria were then passed to fresh 2216 brothtwice in 48 h at room temperature. A sample was spread on2216 agar and incubated for 72 h at 30°C, and the plate wasexamined for nonpitting colonies. Approximately 0.1% ofthe colonies were Agr-, and all those were kanamycinresistant and ampicillin sensitive, indicating that pDB60 hadbeen lost and that the genomic copy of agrA had beenreplaced by the plasmid copy harboring the kanamycinresistance gene. A representative Amps Kanr Agr- P. atlan-tica, designated DB11, was incubated overnight in 2216broth at 30°C, genomic DNA was isolated and digested withseveral restriction endonucleases, and the fragments wereelectrophoretically separated on an agarose gel and then

transferred to nitrocellulose by the method of Southern (22).Figure 4 shows an autoradiogram of wild-type P. atlanticagenomic DNA, along with DB11 genomic DNA and pDB60DNA, digested with Sall, Sall plus EcoRI, and EcoRI plusHindIII. Hybridization was to radioactively labeled pDB60.Comparison of lanes 2 and 3, 5 and 6, and 8 and 9 indicatedthat the genomic copy of agrA had been replaced by agrA::kan. As a result of replacement of agrA with agrA::kan,most of the exported agarase activity was lost in DB11 ascompared with that in wild-type P. atlantica (Fig. 5). How-ever, it was observed that a minor agarase activity waspresent in DB11. This minor agarolytic activity may be theresult of the second agarase, ,B-agarase (II), as reported byMorrice et al. (17, 18).

Construction of a gene replacement in the wild-type agrAenabled us to test the possibility that the extracellularagarase conferred a survival advantage to P. atlantica byproviding the bacteria with the ability to break down andutilize agar as a sole carbon source. Wild-type strain T6c andthe Agr- gene replacement strain DB11 were inoculatedonto seawater medium solidified with 1.0% agarose. After 7days of incubation at room temperature, the plates wereexamined and colony size and morphology were compared.The wild-type bacteria produced colonies which were largerand more mucoid than those of the Agr- strain. Although thewild-type strain grew better and produced more exopolysac-charide, the Agr- strain could give rise to colonies on amedium with agar as the sole carbon source. Apparently, theminor agarase activity produced by the Agr- mutant wassufficient to sustain growth on agar. Further long-termmicrocosm expenrments are needed to assess the relativecontribution of the agarases of P. atlantica, but our resultssuggest that growth on agar is impaired in a mutant defectivein agrA.

DISCUSSION

The primary agarolytic activity of P. atlantica is due to theenzyme ,-agarase (I) (EC 3.2.1.81). This protein is anextracellular enzyme capable of being secreted through thebacterial cell wall and into the surrounding environment,where it acts to break down agar oligosaccharides found invarious red algae, thus providing a carbon source for thebacteria. ,B-Agarase cleaves agar at the ,(1--4) linkage,producing tetrameric saccharide molecules which are poten-

FIG. 5. Agarase activity in wild-type P. atlantica T6c (top)and agrA::kan gene replacement strain DB11 (bottom). Bacteriawere incubated overnight at 30°C. Agar was flooded with Gran'siodine (Materials and Methods), and the zone of clearing resultingfrom ,B-agarase (I) activity was examined. Minor clearing surround-ing DB11 may be due to 0-agarase (II) activity.

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tially capable of passing into the bacterial cell, where addi-tional enzymes in the agar digestion pathway degrade thepolysaccharides into metabolically usable D-galactose and3,6-anhydro-L-galactose. We showed here that the primarysecreted agarase of P. atlantica is encoded by a genedesignated agrA, which is approximately 1.5 kbp in size asdetermined by transposon mutagenesis and restriction map-ping of the cloned gene. Further, it was demonstrated thatreplacement of the wild-type agrA with an in vitro-modifiedlocus containing a kanamycin resistance gene results in lossof the primary exported agarase activity.The size of the coding region and direction of agrA

transcription were assessed in a 4.0-kbp subclone on thepromoterless vector pK01 (16). It was necessary to use thisplasmid to prevent readthrough from plasmid promoters thatcould interfere with transcription directed by the agrA-associated promoter. Transposon mini-Mu dl(lacZ Kmi)was used to mutagenize the agrA in E. coli and to producetranscriptional fusions between the target gene (agrA) andthe lacZ gene of the transposon. Since the lacZ in thistransposon does not contain a promoter, any transcriptionthrough this gene and resulting ,-galactosidase activity isdue to a promoter located upstream from the transposoninsertion. Thus, the P-galactosidase activity of mini-Mudl(lacZ Kmr) fusions to agrA on pBB918 was due totranscription from a promoter associated with agrA. Thirty-five different transposon insertions were used to define thesize of the coding region and the direction of transcription ofagrA. This analysis defined a maximum coding region ofapproximately 1,500 bp and predicted, based on an averagesize of an amino acid as 110 molecular weight, that the agrAgene could encode for a protein of 55,000 molecular weight.Morrice et al. (18) have reported that the molecular weight of,B-agarase (I) from strain ATCC 19292 (NCMB 301) is 32,000.However, probing the genome of strain ATCC 19262 withthe agrA gene cloned from strain T6c indicated that thehomolog of agrA is not present in strain ATCC 19262.Therefore, comparison of the sizes of agarase is not justifiedbecause the gene encoding agarase in these two strains maybe different.To verify that the cloned gene did in fact encode the

primary extracellular agarase activity in P. atlantica, wereplaced wild-type agrA with an in vitro construction thatplaced a kanamycin resistance gene in the middle of the agrAcoding region. This approach has been successfully used toconstruct mutants in E. coli (12), Rhizobium species (20),and Pseudomonas syringae (19). Agr- P. atlantica did notpossess the exported agarase activity, did not pit the agar,and did not show the large zone of clearing around Agr+colonies after the agar was stained with Gran's iodinereagent. There was evidence, however, of a low-level aga-rase activity indicated by a narrow zone of clearing imme-diately adjacent to the Agr- colonies. This agarolytic activ-ity was much weaker than that of the primary exportedagarase in that the colonies were not observed to pit the agareven after prolonged incubation, and it seemed to be moreclosely associated with the cells (Fig. 5). This secondary,cell-associated agarase activity may be due to the enzymedescribed by Morrice et al. (17, 18) as P-agarase (II) and byGroleau and Yaphe (9) as neoagarotetraose hydrolase. Thedata presented support this conclusion since the only agardigestion that was observed was immediately adjacent to thebacterial colony, while Agr+ colonies produce a wide zoneof agar digestion. The minor agarase(s) observed in the Agr-mutant may be important in the utilization of agar becausegrowth and production of exopolysaccharide, although im-

paired in the mutant, did occur on seawater medium withagarose.We applied recombinant DNA technology and other ge-

netic methods such as gene replacement mutagenesis to themarine bacterium P. atlantica. Although our goal was toexamine the role that extracellular enzymes play in thesurvival of bacteria in the ocean, these methods could alsobe useful for analyzing the functions of other gene systems inP. atlantica and related bacteria. The cloned agarase genewas used to construct a strain with a precisely defineddefect, but this gene was useful for other aspects of ecolog-ical research. The agarase gene was used to probe thegenomes of several strains of P. atlantica. It was apparentthat all strains classified as P. atlantica do not contain thehomolog of the agrA gene and that such probes could beused to study genetic drift between isolates of this species orthe genetic diversity of agar-digesting bacteria in general.Furthermore, species-specific DNA probes might be used toanalyze the presence and topological distribution of partic-ular organisms attached to animate and inanimate surfaces inthe marine environment. This would be particularly advan-tageous if the objects of investigation were difficult tocultivate in the laboratory or if disturbance of the communitystructure must be avoided. Finally, the cloned gene could beexploited to overproduce the agarase enzyme which couldbe used to dissect the chemical structure of complex poly-saccharides such as agar or possibly to produce commer-cially useful by-products.

ACKNOWLEDGMENTS

This research was supported by a contract from the Office ofNaval Research (ONR N00014-83-K-0079).We thank Miriam Wright and Marcia Hilmen for excellent tech-

nical assistance.

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