APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3439-3444 Vol. 56, No. 110099-2240/90/113439-06$02.00/0Copyright C 1990, American Society for Microbiology

Natural Plasmid Transformation in a High-Frequency-of-Transformation Marine Vibrio Strain

MARC E. FRISCHER, JENNIFER M. THURMOND, AND JOHN H. PAUL*

Department of Marine Science, University of South Florida, 140 7th Avenue South,St. Petersburg, Florida 33701-5016

Received 21 May 1990/Accepted 12 September 1990

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broadhost range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 x 10-9 and3.4 x 1O' transformants per recipient, respectively. Growth of plasmid transformants in nonselective mediumresulted in cured strains that transformed 6 to 42,857 times more frequently than the parental strain,depending on the type of transforming DNA. These high-frequency-of-transformation (Hff) strains weretransformed at frequencies ranging from 1.1 x 10-8 to 1.3 x 1O-4 transformants per recipient with plasmidDNA and at an average frequency of 8.3 x 10-5 transformants per recipient with homologous chromosomalDNA. The highest transformation frequencies were observed by using multimers of an R1162 derivativecarrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strainsdemonstrated that no plasmid DNA remained in the cured strains which may have provided homology to thetransforming DNA. Al transformants and cured strains could be differentiated from the parental strains bycolony morphology. DNA binding studies indicated that late-log-phase Hff strains bound [31H]bacteriophagelambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmidtransformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competentmutant (Hff strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could benaturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants maybe a means of plasmid acquisition by natural aquatic bacterial populations.

Until recently, gene exchange between bacteria wasthought to be largely a laboratory phenomenon. However, inthe past few years the potential for intra- and interspecificprocaryote gene exchange in natural environments has beendemonstrated (15, 18, 28, 33). Still, little is known of the fluxof genetic information through natural microbial communi-ties.Three mechanisms of gene exchange have been described

in bacteria as follows: (i) conjugation, which is the cell-contact-dependent plasmid exchange mediated by conjuga-tive plasmids (17); (ii) transduction, gene transfer mediatedthrough bacteriophages (35); and (iii) transformation, theprocess whereby a cell takes up and expresses genes en-coded by extracellular DNA (2). Natural transformation is anormal physiological process exhibited by a wide range ofbacteria (31, 32). Natural transformation is distinct fromartificial transformation, which is a widely used technique inmolecular biology for the induction of competence in cells bychemical, enzymatic, or physical means.There is considerable indirect evidence which suggests

that natural transformation may be a mechanism of genetransfer in aquatic environments (9). First, several marinebacterial isolates have been reported to be naturally trans-formable (16a, 33). Second, aquatic environments have beenshown to contain an abundance of dissolved DNA whichcould potentially act as transforming DNA (11).We have recently demonstrated natural plasmid transfor-

mation in a marine Vibrio sp. (16a). In this study, wedescribe a high-frequency-of-transformation (Hff) variant ofthis strain. Further, we report that a strain of Vibrio para-haemolyticus, a common marine pathogen responsible for

* Corresponding author.

shellfish poisoning, is also naturally transformable withplasmid DNA.

MATERIALS AND METHODSBacterial strains. The strains used in this study are listed in

Table 1. Strain DI-9 has been identified as a Vibrio sp. (16a).To verify the identity of transformants, HfT strains, and theparental Vibrio strain, biochemical taxonomic tests wereperformed (4) as well as phenotypic profiling using API 20Etest strips (Sherwood Medical, Plainview, N.Y.).

Transforming DNA and gene probes. The plasmids used intransformation studies were the broad host range Inc P4plasmids pGQ3 and pQSR50. Both these plasmids encodefor resistance to the antibiotics kanamycin and streptomy-cin. pGQ3 is a derivative of pKT230 (3) that contains theEscherichia coli thymidine kinase gene (7, 16). pKT230 is aderivative of RSF1010 and pACYC177 (3). pQSR50 is aTnS-containing derivative of the plasmid R1162 (21). Por-tions of these plasmids were subcloned into the Riboprobevector pGEM3Z or pGEM4Z. [35S]RNA probes were pre-pared by transcription of the subcloned fragments with T7 orSP6 RNA polymerase, using [35S]UTP (1,320 uCi/mmol;NEN Research Products, Boston, Mass.) as described byPromega (Riboprobe system or Riboprobe Gemini system.Transcription of cloned DNA. Promega Technical Bulletin002. Promega Biotech, Madison, Wis. 1988). ChromosomalDNA from a spontaneous rifampin-resistant mutant of DI-9(RRVP3) was used as transforming DNA for the chromo-somal transformation assay.

Preparation of transforming DNA. Plasmids were amplifiedin E. coli cultures, using chloramphenicol and uridine asdescribed by Maniatis et al. (19). Large-scale plasmid DNApurification was performed by alkaline lysis as described byGriffith (14). To further separate plasmid from chromosomal

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TABLE 1. Naturally transforming bacterial strainsused in this study

Strain Source and characteristics

Vibrio sp. strain DI-9 ........ Isolate from Davis Island, Fla. (G.Stewart, University of South Flor-ida, Tampa, Fla.)

Vibrio sp. RRVP3 ............Spontaneous rifampin-resistant mutantof strain DI-9. Isolated onASWJP+PY + 500 jig of rifampinper ml (this study)

Vibrio sp. MF-1 .............. DI-9 naturally transformed with pGQ3Vibrio sp. MF-1C .............MF-1 cured of the plasmid pGQ3Vibrio sp. MF-3C .............MF-1 retransformed with pGQ3 and

curedVibrio sp. WJT-1 .............DI-9 transformed with pKT230 (16a)Vibrio sp. WJT-1C ...........WJT-1 cured of the plasmid pKT230V. parahaemolyticusUSFS 3420 .............. G. Stewart (University of South

Florida, Tampa, Fla.)

DNA, the plasmid extract was passed through a pZ523column (5 prime -- 3 prime Inc., West Chester, Pa.) (12, 34).

Plasmid multimers were prepared as described by Jeffrey etal. (16a). The degree of multimer formation was judged byvisualization on a 0.4% agarose gel with the fluorochromeHoechst 33258 (10). Chromosomal DNA was prepared bythe method of Marmur (20). All DNA concentrations weredetermined by the Hoechst 33258 method (25).

Culture conditions. Strain DI-9 and all the HIT strainswere grown in artificial seawater with 5 g of peptone per literand 1 g of yeast extract (ASWJP+PY[23]) per liter. Fortransformation assays, cells were grown to an optical densityof 0.8 at 600 nm at room temperature (26 + 2°C), whichcorresponds to approximately 2 x 109 cells per ml or late logphase (data not shown).

Filter transformation assay. In transformation assays, 1 mlof late-log-phase cell culture was immobilized onto a sterileNuclepore filter (47 mm; 0.2-[Lm pore size) (NucleporeCorp., Pleasanton, Calif.), keeping cells to a spot of no morethan 1.5-cm diameter. The filter was then transferred asep-tically cell-side-up onto an ASWJP+PY agar plate andoverlaid with 4 ,ug of pasteurized plasmid multimers sus-pended to a final volume of 100 ,ul in sterile deionized wateror 4.2 mM MgCl2. Pasteurization ofDNA was accomplishedby incubation at 75°C for 2 h. Cells were allowed to incubate16 h at room temperature. Following incubation, the filterwas transferred to 10 ml of ASWJP+PY and allowed toshake at approximately 200 rpm for 1 h. This was necessaryto resuspend the cell mat and to allow the cells to recoverbefore exposure to antibiotic selection. Cells were thenserially diluted and plated on selective (ASWJP+PY and 500pug of kanamycin per ml, 1,000 ,ug of streptomycin per ml)and nonselective media (ASWJP+PY) and enumerated after48 to 72 h of growth. Chromosomal filter transformationassays were done identically as plasmid filter transformationassays, except that 10 ,ug of homologous chromosomal DNAfrom RRVP3 was placed on the cell spot. Selection fortransformants was on ASWJP+PY containing 500 pug ofrifampin. All antibiotics used in these studies were pur-

chased from Sigma Chemical Co. (St. Louis, Mo.).Verification of plasmid transformation. Presumptive plas-

mid transformants, identified as antibiotic-resistant colonies,were transferred to charged nylon circles and grown on thefilter until colonies were visible (24 to 48 h). Colonies were

lysed on the filters by the method of Buluwela et al. (6), andthe DNA was denatured and fixed by the method described

by Maniatis et al. (19). We found that the combination ofthese two procedures yielded a more distinct hybridizationsignal than either method alone (data not shown). Filterswere hybridized overnight with [35S]RNA probes at 42°Cessentially by the method of Church and Gilbert (8) andmodified as described by Promega Technical Bulletin 002.Filter washing consisted of one wash in 2x SSC (0.3 MNaCl, 0.03 M sodium citrate [pH 7.0] containing 1 mMdithiothreitol) for 5 min at room temperature followed bythree 60-min washes at 65°C in PSE (0.25 M sodium phos-phate, 2% sodium dodecyl sulfate, 1 mM EDTA, pH 7.4) andthree 30-min washes in PES (40 mM sodium phosphate, 1%SDS, 1 mM EDTA, pH 7.4) at 65°C. Filters were dried, andhybridization was detected by autoradiography. Colonieswere also subcultured, and the plasmid DNA was extractedby the miniprep method of Maniatis et al. (19). Transformingplasmid DNA could be identified in the transformants bySouthern blotting and probing with [35S]RNA gene probes asdescribed above (Fig. 2).

Curing of plasmids from natural plasmid transformants.DI-9 plasmid transformants were cured of their plasmids bygrowth in ASWJP+PY without antibiotics. After five suc-cessive 24-h transfers to fresh medium, the culture wasserially diluted and plated onto nonselective medium. Aftergrowth, the colonies were replica plated to selective me-dium. Colonies which failed to grow in the presence ofantibiotics were selected.DNA binding studies. [3H]DNA was prepared by end

labeling HindIlI-digested lambda DNA with all four [3H]deoxynucleoside triphosphates as previously described (24).To measure DNA uptake rates of DI-9 and WJT-1C, cultureswere diluted to approximately 2 x 108 cells per ml in freshlyautoclaved, sterile filtered ASWJP+PY in an acid-washedpolymethylpentene flask. End-labeled [3H]DNA was addedto the diluted culture (0.25 ,uCi/ml, 3 ng/ml). Triplicate 2-mIsamples were filtered onto Nuclepore filters (pore size, 0.2,um) at time intervals ranging from 0 min to 4 h. After thesample had passed through the filter with mild vacuum (10mm Hg), the filters were immediately washed with 3 ml ofsterile filtered ASWJP containing 10 ,ug of calf thymus DNAper ml to prevent further DNA binding of labeled [3H]DNA.Filters were then placed in scintillation vials containing 0.5ml of 0.5 M Protosol (NEN Research Products, Boston,Mass.) to solubilize the filter. After the filters were com-pletely dissolved, 25 ,lI of glacial acidic acid and 10 ml ofEcoscint 0 scintillation counting fluid (National Diagnostics,Manville, N.J.) was added to the vial, and the radioactivityassociated with the filter was determined by liquid scintilla-tion counting. Nonspecific binding of DNA by mediumparticulates was assessed in cell-free controls. Nonspecificbinding generally contributed less than 10% of overall bind-ing compared with the 30-min time points with cells present.Background counts were routinely subtracted from all ex-periments.

RESULTS

Table 2 shows the results of plasmid multimer transforma-tion of Vibrio strain DI-9 and V. parahaemolyticus. Trans-formation of Vibrio strain DI-9 occurred with plasmid mul-timers ofpQSR50 and pGQ3 at frequencies of 3.5 x 10-9 ands1.44 x 10-9 transformants per recipient, respectively(Table 2). A strain of V. parahaemolyticus (USFS 3420) wasalso naturally transformed with plasmid DNA at a frequencyof 9.7 x 10`0 transformants per recipient with pQSR50plasmid multimers.

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NATURAL PLASMID TRANSFORMATION IN A VIBRIO SP. 3441

TABLE 2. Natural transformation filter assay of wild-type and HfT marine strains with plasmid multimer DNA

Strain DNA Transformation' Transformation frequency HfT/DI-9 ratio

Wild-type strainVibrio strain DI-9 pGQ3 + <1.4 x 10-9b

pQSR50 + 3.5 x 10-9

V. parahaemolyticusUSFS 3420 pGQ3 <1.0 x 10o

pQSR50 + 1.9 x io-9

HfT Vibrio strainsMF-lC pGQ3 + 0.9 X 10-8 to 6.6 x 10-8 6-47WJT-1C pGQ3 + 2.3 x 10-8 to 6.7 x 10-8 16-48MF-3C pGQ3 + 1.3 x 10-8 9MF-1C pQSR50 + 0.2 x 10-5 to 1.1 x 10-5 571-3,143WJT-1C pQSR50 + 0.01 x 10-4 to 1.5 x 1O-4 286-42,857MF-3C pQSR50 + 2.5 x 10-6 714a +, Transformation was detected; -, transformation could not be detected.b Transformation could be detected only after liquid enrichment (16a).

Isolation ofHfT strains. Several DI-9(pGQ3) transformantswere cured of their plasmids by successive growth in non-selective medium. The transformants and cured strainspossessed a colony morphology distinct from that of theoriginal (parental) strain DI-9 (Fig. 1). The biochemicalphenotype profile of all the cured strains was identical to thatof DI-9 as determined by the API 20E test strip (SherwoodMedical). DI-9 and all the cured strains are rod shaped,motile (a single polar flagellum), oxidase positive, and able toferment glucose.

FIG. 1. Colony morphology of Vibrio strain DI-9 (A), transform-ant strain WJT-1 (B), and cured strain WJT-1C (C). Colonies were

grown 7 days on artificial seawater agar supplemented with 5 g ofpeptone per liter and 1 g of yeast extract per liter.

The results of natural plasmid transformation assays of theplasmid-cured strains appear in Table 2. When reexposed toplasmid multimers, these cured strains transformed at sig-nificantly higher frequencies than the parental strain DI-9.Transformation frequencies of the cured strains MF-1C,WJT-1C, and MF-3C ranged from 9 x 10-9 to 6.7 x 10-8transformants per recipient with pGQ3 multimers or were 6-to 48-fold greater than the transformation rate of the parentalstrain DI-9. The cured strains transformed at frequenciesranging from 1 x 10-6 to 1.5 x 1O-4 transformants perrecipient with pQSR50 plasmid multimers or 286 to 42,857more efficiently than the parental strain DI-9. These strainsare referred to as HfT strains. Plasmid transformants couldbe identified by Southern hybridization (Fig. 2) or directly bycolony hybridization with the appropriate labeled gene probe(data not shown).To rule out the possibility that these high transformation

frequencies were caused by homology resulting from trans-forming plasmid DNA remaining in the HfT strains, a dotblot of a total DNA preparation from the HfT strain MF-1Cwas hybridized with a probe (pJHP II) made to the originaltransforming DNA (Fig. 3). Concentrations of DNA dottedon the filter were sufficient to detect a single copy of theplasmid inserted into the chromosome. No hybridizationwas found with the parental strain DI-9 or the cured strainMF-1C, whereas strong hybridization occurred with theDI-9(pGQ3) transformant MF-1 (Fig. 3). These results indi-cate that no sequences from the transforming DNA remainedin the cured strains.

Table 3 shows the result of chromosomal transformationstudies with the wild-type and HfT strains. WJT-1C wastransformed at a frequency of 8.3 x 1i-0 transformants perrecipient (Table 3). This represents a 244-fold increase inchromosomal transformation frequency compared with thatof the parent DI-9. This increase in transformation efficiencycannot be explained by the presence of plasmid DNAresiding in the HfI strains, since Vibrio strain DI-9 chromo-somal DNA has no homology to these plasmids (Fig. 3).[3H]DNA binding studies. Figure 4 shows the results of a

typical [3H]DNA uptake experiment with parent strain DI-9and HfT strain WJT-1C. Binding rate comparisons withineach experiment revealed that mid-log-phase cultures ofboth strains bound heterologous DNA similarly (Fig. 4A),while late-log-phase cultures of WJT-1C bound heterologousDNA approximately twice as fast as DI-9 during the first 30

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FIG. 2. Autoradiogram of Southern transfer of strain DI-9, trans-formant strain WJT-1, and Hft strain WJT-1C probed with [35S]labeled Riboprobe RNA probe pJHP1. Lanes: 1, undigestedpKT230; 2, XhoI-digested pKT230; 3, undigested DI-9 plasmidDNA preparation; 4, XhoI-digested DI-9 plasmid DNA preparation;5, undigested WJT-1 plasmid preparation; 6, XhoI-digested WJT-1plasmid preparation; 7, undigested WJT-1C plasmid preparation; 8,XhoI-digested WJT-1C plasmid preparation. The faint signals inlanes 3 and 7 are due to contamination from lanes 2 and 6.

min of exposure to labeled DNA (Fig. 4B). Uptake ratesvaried dramatically between experiments, but the ratio ofthe short-term (30-min) uptake rates of DI-9 and WJT-1Cremained fairly constant. Incubation temperature before andduring exposure to DNA may greatly affect the DNA uptakerate. DI-9 and the HfT strains will grow at temperaturesranging from 15 to 39°C, with optimum growth rates at 37°C.Uptake studies performed at 28°C yielded 6 to 2,383 timesthe uptake rate of those performed at room temperature (20to 23°C). Mid-log-phase cultures of both strains possessedsimilar DNA uptake rates. The ratio of WJT-1C to DI-9uptake rate for mid-log-phase cells was 0.918 ± 0.219 (0.1 <P < 0.025, n = 5). In contrast, the initial uptake rates oflate-log-phase cultures were approximately twofold greater(2.1 + 0.59) for the HfT strain than for the parental DI-9(0.02 < P < 0.05, n = 4 [Table 4]).

DISCUSSIONIn this study, we have isolated a HIT variant of the marine

Vibrio strain DI-9 which can be naturally transformed withplasmid and homologous chromosomal DNA. We have alsodemonstrated natural plasmid transformation in a V. para-

haemolyticus strain. This suggests that natural plasmidtransformation may be a genus-wide phenotype of Vibrio.The parental Vibrio strain DI-9 was previously shown to

transform with the broad host range plasmid pKT230 andpGQ3 at a frequency of 0.3 x 10-8 to 3.1 x 10-8 transfor-

1 2 3 4FIG. 3. Dot blot of total DNA from parental, transformant, and

HfT Vibrio strains. Row A, DI-9 (2,000 [lane 1], 4,000 [lane 2], and6,000 [lane 3] ng); row B, pKT230 plasmid (5 [lane 1], 10 [lane 2], 15[lane 3], and 50 [lane 4] ng); row C, MF-1 (2,000 [lane 1], 4,000 [lane2], and 6,000 [lane 3] ng); row D, MF-1C (2,000 [lane 1], 4,000 [lane2], and 6,000 [lane 3] ng). Probed with 35S-labeled Riboprobe RNAprobe pJHPII.

mants per recipient (16a). In the present study, the broadhost range plasmids pGQ3 and pQSR50 were used as trans-forming DNA. The HfT strains transformed at significantlygreater frequencies than the wild type with both plasmidsand homologous chromosomal DNA. Transformation fre-quencies of the HtT strains varied by 4 orders of magnitude,depending upon the transforming DNA employed. Use ofpQSR50 plasmid multimers as transforming DNA consis-tently resulted in the highest transformation frequencies bynearly 2 orders of magnitude. This was surprising sincepGQ3 and pQSR50 are derived from plasmids which arethought to be identical (RSF1010 and R1162, respectively)(13). Because pQSR50 carries the transposon TnS, it wasconceivable that the transposon played a role in the in-creased transformation efficiency observed for this plasmid.It was speculated that illegitimate recombination eventsencoded for by transposition genes could have accounted forthe higher frequency of transformation with pQSR50. Trans-posons which mediate their own conjugal transfer have beenreported previously (5), although this phenomenon has notbeen reported for transformation. Thus, illegitimate recom-bination events may have circumvented the need for homol-ogy required with normal recA type-mediated recombina-tion, which is believed to occur in natural transformation(32). However, transformation of WJT-1C occurred with

TABLE 3. Natural filter transformation of wild-type andHfT marine strains with Rifr chromosomal DNA

from RRVP3

TreatmentaStrain Rifr DNA + Frequency

CT DNA Rifr DNA DNase I

DI-9 2.7 x 10-7 6.1 x 10-7 2.2 x 10-7 3.4 x 10-7WJT-1C 3.3 x 10-7 8.3 x 10-5 1.1 x 10-7 8.3 x 10-5

" CT DNA, Calf thymus chromosomal DNA; Rif" DNA, chromosomalDNA from RRVP3; Rif' DNA + DNase I, chromosomal DNA from RRVP3and DNase I (100 Kunitz).

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NATURAL PLASMID TRANSFORMATION IN A VIBRIO SP. 3443

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strains were not caused by plasmid DNA either remaining.8 A. free in the cell or incorporated in the chromosome of the HfT1.6- T (cured strains) which could have provided DNA homology1.4- _ _ -I for plasmid transformation. There was no homology be-

1.2- r _ _ tween the transforming chromosomal DNA and any of the1.0- ..- ~plasmids employed.0.8-- The nature of the physiological difference between the0.6; |HfT strain and the parental strain DI-9 that allows efficient0.4t -. transformation is not yet known. This variant may possess a0.4-- mutation which affects the DNA binding or uptake mecha-0.2 nism(s), because the HfT strains bound [3H]DNA at about0.0

3 s twice the rate of the parental strain DI-9. However, a0 30 60 90 1202.0 twofold increase in binding rate might not account for the

1 B. increase by several orders of magnitude observed in trans-formation frequency for the HfT strains. Further DNA1.6-, binding studies with labeled biologically active chromosomal.4- , and plasmid DNA may reveal larger differences in binding1.2- , rates between these strains.

1.0- , Although we have not identified the genetic basis of the0.8- , difference between these strains, it seems reasonable that0.6-, the HfIT phenotype is the result of a spontaneous mutation to0.4-/ a competent phenotype. The above observations are not0.2 -, consistent with a physiological condition, since the high

o______o_______________________________ frequency of transformation and morphological phenotype0.0

3 s s are heritable and stable (no reversal of a physiological0 30 60 90 120condition to the DI-9 phenotype has been observed). Fur-

Time (min) ther, the frequency of initial transformation of DI-9 (approx-imately 10-9) is consistent with the frequency of occurence

i. 4. Typical binding curves of [3H]lambda DNA by parental of spontaneous mutations. Although a back mutation to theDI-9 ( ) and HfI strain WJT-1Cs-- - -). (A) Mid-log- DI-9 phenotype may occur, there is no way to select for suchcells; (B) late-log-phase cells.

a mutant. If one assumes a back mutation rate similar to theinitial mutation rate (10-9), 109 colonies would need to be

2, the Tn5-free precursor of pQSR50, at an even greater examined for detection of a revertant by colony morphology.ency (data not shown). Transformation studies with Although naturally competent strains of other genera haveL010, the precursor of pGQ3, are presently in progress. been reported (1, 26), this is the first report of a mutation toe efficiency of transformation was over 2 orders of a competent phenotype. Possession of the parental VibrioLitude greater with chromosomal DNA for the HfT strain DI-9 and the competent mutant provides us with ants than the parental DI-9 strain. This indicates that the model system to explore the differences between competentphenotype was not associated with a condition that and noncompetent bacterial strains.ved efficient plasmid transformation (i.e., plasmid up- Despite the fact that studies of the mechanisms of geneticor recircularization). These results also demonstrate exchange are among the earliest reports in the field ofhe high transformation frequencies observed in the HfIT microbial genetics, little is known of the flux of genetic

material through microbial communities or the importance ofBL4Sor-trmbidigatsf3HDNAbgenetic exchange in the environment. Recent interest in geneBLE 4. Short-term binding rates of [3H]DNA by wild-type trnfrpoesswhinaulmcoblcmuiishs(DI-9) and Hfr (WJT-1C) marine Vibrio strains transfer processes within natural microbial communities has

been stimulated largely by the concern over the use of)eriment DNA binding rate (ng of DNA/109 cells/min) genetically engineered microorganisms in the environment.no.' DI-9 WJT-1C WJT-lC/DI-9 In response to this concern, there has been a renewed

interest in investigating the potential for natural transforma-)g phase" tion to occur in the environment. Continued investigations of

0.233 0.166 0.800 gene transfer in the environment as well as efforts to study

0.007 0.008 1.130 the genetics of bacteria other than E. coli has resulted in a1.350 1.040 0.770 growing list of bacteria which have been found to be natu-1.410 1.670 1.180 rally transformable (27, 31). The majority of competent

organisms which have been described are capable of beingog phasec transformed by homologous chromosomal DNA from the

0.0006 0.0016 2.67 same or closely related species (31). Homology of transform-0.0200 0.0430 2.15 ing DNA to recipient DNA is considered necessary for0.0040 0.0092 2.29 stable chromosomal integration by a homologous recombi-1.1280 1.4300 1.28 nation event (32). In addition, for some organisms, notably

.periments 1 to 3 and 6 to 8 were performed at room temperature, and Haemophilus influenza, there is a requirement for sequencements 4, 5, and 9 were performed at 28'C. homology in the initial binding of transforming DNA (29, 30).erage ratio of WJT-lC/DI-9 = 0.918 0.219, t= 0.837 (0.1 < P" < In general, there seems to have been evolutionary pressureerage ratio of WJT-lC/DI-9 = 2.10 + 0.588, t x 3.735 (0.02 <P < to accept only homologous DNA as transforming material.

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3444 FRISCHER ET AL.

This is consistent with the idea that natural transformationmechanisms arose as a mechanism for DNA repair (22).Although the majority of competent bacteria are only

transformable with homologous DNA, many microorgan-isms are transformable with heterologous plasmid DNA as

well (27). Less-specific natural transformation systems may

allow for the spread of plasmids within natural microbialcommunities. Thus, the finding that a high frequency oftransformation mutation can occur in a marine bacteriumsuggests that natural transformation by these mutants couldbe a means of plasmid dissemination in marine and aquaticenvironments. Potentially, plasmids could be transferredboth intra- and interspecifically via an extracellular route.Secondly, HfT strains may have wide application in theassessment of factors important in transformation in theenvironment. These strains can be used as recipients inmicrocosm studies for the determination of environmentalfactors important for gene transfer via natural transformation.

ACKNOWLEDGMENTS

This work was supported in part by the National Science Foun-dation grant OCE 8817172 to J.H.P. and by a Clearwater PowerSquadron research grant to M.E.F.We thank Richard Meyers of University of Texas in Austin for

providing us with several of the plasmids used in this study. Also,we thank Wade Jeffrey for the strain WJT-1 and his critical reviewof an early version of the manuscript.

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