JOURNAL OF BACTERIOLOGY, Nov. 1987, p. 4878-4883 Vol. 169, No. 110021-9193/87/114878-06$02.00/0Copyright © 1987, American Society for Microbiology

Plasmid pCBI Carries Genes for Anaerobic Benzoate Catabolism inAlcaligenes xylosoxidans subsp. denitrificans PN-1

CHERYL K. BLAKEt AND GEORGE D. HEGEMAN*Microbiology Group, Department of Biology, Indiana University, Bloomington, Indiana 47405

Received 27 April 1987/Accepted 17 July 1987

Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does notferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa andPseudomonas stutzeri strains. A restriction endonuclease map was constructed.

The fate of aromatic compounds in aerobic and anaerobicenvironments is of considerable interest because enormousquantities of natural and synthetic aromatic compounds arereleased into the environment annually. Although microor-ganisms are capable of degrading an impressive variety oforganic compounds, some aromatic compounds are de-graded slowly or not at all (17). These compounds, whichmay not be toxic in the usual concentrations found in soil orwater, may be converted to more toxic forms or may beconcentrated through the food chain (7).

It is important to understand the biochemical and ecolog-ical basis of molecular recalcitrance to biodegradation. Thepresence or absence of molecular oxygen is an importantfactor affecting the degradation of aromatic compounds. Themechanisms by which the benzene nucleus is degraded in thepresence of molecular oxygen are understood well (8, 9, 16).During aerobic degradation, oxygen is used both in respira-tion as the ultimate electron acceptor and as a reactant in thehydroxylation and cleavage of the aromatic ring (10, 37). Incontrast to the large amount of information available on theaerobic processes of aromatic catabolism, relatively little isknown about the anaerobic processes (5, 35, 49). One classof bacteria capable of anaerobic degradation of aromaticcompounds is the denitrifying bacteria. Pseudomonas sp.strain PN-1 was described by Taylor et al. (41, 43) as the firstpure culture capable of anaerobic catabolism of aromaticcompounds under denitrifying conditions. The organism wasdesignated Pseudomonas sp. strain PN-1 on the basis of itsmorphology, Gram stain reaction, oxidase reaction, motilitycharacteristic of polar flagellation, and a G+C content of67.3 mol% (41). Interestingly, this organism has the ability todehalogenate and mineralize anaerobically several halo-aromatic compounds (42) and to mineralize anaerobicallyseveral methoxy-aromatic compounds, e.g., vanillic acid(40). This mineralization of methoxy-aromatic compoundscontrasts with other described anaerobic bacteria whichutilize only the methyl group of a methoxylated aromaticcompound, presumably in a transmethylation reaction (1, 13,25, 44). The mechanism used by strain PN-1 to cleave thephenylmethylether bond is not known, but methanol is notdetected in cultures grown on vanillic acid (40), as might beexpected if cleavage were analogous to the aerobic process.

* Corresponding author.t Present address: Center for Applied Microbiology, University

of Texas, Austin, TX 78712-1095.

Two alternatives have been proposed for the mechanismof ring cleavage by the denitrifying bacteria: hydration orreduction of the ring before cleavage. Reduction of thearomatic ring under anaerobic conditions had been shown inthe photosynthetic organism Rhodopseudomonas palustris(12, 19; P. L. Dutton and W. C. Evans, Biochem. J. 109:5-6,1968). Williams and Evans (47, 48) provided support for areductive pathway in a denitrifying bacterium, a Paracoccussp. (incorrectly identified as a Moraxella sp.; W. C. Evans,personal communication). Cyclohexane carboxylate, 2-hy-droxycyclohexane carboxylate, cyclohexanone, and adipateisolated from extracts of anaerobically grown benzoatecultures were identified by gas-liquid and thin-layer chroma-tography. These compounds (except adipate), which werecapable of supporting growth or were utilized by theParacoccus sp., were the same as those found by Dutton andEvans (12) for R. palustris growing photosynthetically onbenzoate under anaerobic conditions. Thus, Evans proposeda reductive mechanism for ring cleavage in the denitrifyingbacteria which is similar to that shown for photosyntheticbacteria (14). The difference between the two pathways is inthe decarboxylation step in the pathway suggested for thedenitrifying bacteria. The ring cleavage products for thedenitrifying bacteria and the photosynthetic bacteria areadipate and pimelate, respectively.

In contrast to the Paracoccus sp., strain PN-1 is unable toutilize any of the compounds identified in the study ofWilliams and Evans as intermediates (47). Consequently,Taylor et al. (41) suggested an alternative to the reductivepathway for strain PN-1 in which the aromaticity of the ringis lost by the addition of three molecules of water to the ring.Predictions based on chemical reactivity suggest that thisreaction is unlikely under anaerobic conditions (2; G. Bak-ker, Ph.D. dissertation, Delft University of Technology,Delft, The Netherlands, 1977). However, by using 180labeled water, Vogel and Grbit-Galit (45) have obtainedevidence that in a methanogenic culture, an early step in theanaerobic degradation of benzene and toluene involves theincorporation of oxygen from water.The finding of the reduced organic compounds identified

by Williams and Evans (47) suggests that a reductive mech-anism is operational in the Paracoccus sp., but these com-pounds have not been shown to be directly involved in thepathway used by that Paracoccus sp. or by strain PN-1.Enzymes catalyzing reactions of the anaerobic pathwayhave not been identified in the denitrifying bacteria, and it isnot known whether intermediates are activated as coenzyme

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TABLE 1. Description of strains used in this study

Strain Descriptiona Source (reference)

PN-1 Wild type, pCBI, A. xylosoxidans subsp.Nals denitrificans PN-1 ATCC

43374 (41; this work)PAO11 Trp-, Nalr, pMO821 B. W. HollowayPA280 Nalr Derivative of P. aeruginosa

PRS279, ATCC 17645 (38)PS222 Nalr Derivative of P. stutzeri

PRS221, ATCC 17588 (38)PS317 Nalr Derivative of P. stutzeri

PRS316, ATCC 17682 (38)PS319 Nalr Derivative of P. stutzeri

PRS318, ATCC 17684 (38)PS322 Nalr Derivative of P. stutzeri

PRS321, ATCC 17687 (38)PA281 Nalr, pCBI Derivative of PA280PS223 Nalr, pCBI Derivative of PS222PS318 Nalr, pCBI Derivative of PS317PS323 Nalr, pCBI Derivative of PS322

a Nals or Nalr, Sensitivity or resistance, respectively, to 20 .ig of nalidixicacid per ml; Trp-, tryptophan requiring.

A thioesters, as appears to be the case in the photosyntheticbacterium R. palustris (31) and in three denitrifying strains ofPseudomonas sp. (32).

In this paper, we report the results of morphological,biochemical, and nutritional testing of strain PN-1 whichhave led us to suggest that this strain be reclassified asAlcaligenes xylosoxidans subsp. denitrificans PN-1 (22, 23).In addition, we report the finding of a plasmid in strain PN-1which is functional in the degradation of aromatic com-pounds under denitrifying conditions. This plasmid, desig-nated pCBI, can be transferred by conjugation to otherdenitrifying species, thus conferring upon them the novelability to degrade benzoate anaerobically.(A report of this work was presented by C. K. Blake at the

Microbial Metabolism Symposium, University of Minne-sota, St. Paul, Minn., 13 to 16 July 1986. This report is basedon a portion of a thesis to be submitted by C. K. Blake toIndiana University.)

MATERIALS AND METHODS

Biological materials and conditions. The strains used in thisstudy are listed in Table 1. A. xylosoxidans subsp.denitrificans PN-1 (ATCC 43374) (formerly Pseudomonassp. strain PN-1) was the gift of B. F. Taylor (RosenstielSchool of Marine and Atmospheric Science, University ofMiami, Miami, Fla.). Unless otherwise indicated, cells werecultured in standard mineral medium (SMM), which wasessentially the mineral medium of Doudoroff (11) with thefollowing changes. The phosphate buffer was prepared as a5x stock solution with 25.6 g of K2HPO4 per liter and 7.5 gof KH2PO4 per liter. The pH of the stock solution wasadjusted to 9.0 with 10 N KOH, sodium benzoate (6.94 mM)was added as a growth substrate, and KNO3 (20 mM) wasadded as an electron acceptor for anaerobic cultures. For asolid medium, 15 g of agar was added. The final pH of themedium was 8.2 to 8.5. Cultures were incubated at 30°C.Screw-cap test tubes were filled entirely with the liquidmedium to allow anaerobic growth. GasPak jars (BBLMicrobiology Systems, Cockeysville, Md.) were used toattain anaerobiosis for cultures on plates.

Identification methods. Cell form, Gram stain reaction,motility, and flagellar morphology were determined by usingcells grown anaerobically on SMM with benzoate. Motilityand cell form were determined with wet-mount techniques,and flagellar morphology was determined by the stainingmethod of Mayfield and Inniss (28) and by electron micros-copy. The accumulation of poly-p-hydroxybutyrate wasdetermined by growing the cells in a nitrogen-limited SMM[0.02% (NH4)2SO4] (38) with 0.2% acetate as a carbonsource. Poly-,-hydroxybutyrate granules were detected bythe staining method of Hugh and Gilardi (20) and by chem-ical analysis (26). Catalase activity was detected by theproduction of bubbles upon addition of 0.5 ml of aerobicallygrown cultures to 0.5 ml of 3% hydrogen peroxide. Theoxidase test was performed by removing growth from thesurface of an agar plate and smearing it onto a piece of filterpaper moistened with a freshly prepared solution oftetramethyl-p-phenylenediamine hydrochloride (24). To testfor starch hydrolysis, a plate containing SMM, benzoate,and 0.2% soluble starch was inoculated with strain PN-1.After incubation, the plate was flooded with an iodinesolution (38). To test for gelatin hydrolysis, a plate contain-ing SMM, benzoate, and 0.4% gelatin was inoculated withstrain PN-1. After incubation, the plate was flooded with asolution of 15% HgCl2 in 20% concentrated HCl (34). Tween80 (polyoxyethylene sorbitan monooleate) esterase activitywas tested by inoculating strain PN-1 on a plate containingSMM, benzoate, 1% Tween 80, 0.5% NaCl, and 0.01%CaCl2 (33).The conversion of nitrate to nitrite was determined by the

Trommsdorff test (15). Denitrification was determined bychecking the medium for residual nitrate and nitrite and byobserving gas production.The ability of strain PN-1 to grow on 139 carbon sources

was tested by auxanography (29) or by growth in liquidculture or both. The auxanographic method involved theinoculation of SMM plates with washed cells of strain PN-1.Approximately 1 to 4 mg of the test compounds was added tothe surface of the agar plate. The plates were incubatedaerobically or anaerobically in GasPak jars. All carbonsources which gave ambiguous results by the auxanographicmethod and all aromatic substrates were checked for theirability to support growth by the inoculation of PN-1 intoliquid cultures containing SMM and a carbon source at aconcentration of 0.025, 0.05, or 0.1%. The nonaromaticsubstrates were tested aerobically; the aromatic substrateswere tested anaerobically as well.Pseudomonas aeruginosa PAO11 was the gift of B. W.

Holloway (Department of Genetics, Monash University,Clayton, Victoria, Australia). Strain PAO11 was maintainedon nutrient agar (Difco Laboratories, Detroit, Mich.) con-taining 50 pug of kanamycin (Sigma Chemical Co., St. Louis,Mo.) per ml. Strain PAO11 carries the plasmid pMO821, aderivative of R91-5 which has lost carbenicillin resistanceand carries the transposon TnS, specifying kanamycin resis-tance. This IncPlO plasmid can be transferred from P.aeruginosa to other gram-negative bacteria at a high fre-quency but replicates poorly or not at all in those organisms.The P. aeruginosa and Pseudomonas stutzeri strains were

maintained aerobically in nutrient broth (Difco) or on nutri-ent agar plates. Anaerobic cultures containing nonaromaticsubstrates were grown on standard mineral base plus KNO3(20 mM) at a pH of 6.8 (11). Anaerobic cultures containingaromatic substrates were grown on SMM. All Pseudomonasspp. cultures were incubated at 370C.

Nalidixic acid-resistant mutants of each of the five Pseu-

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domonas strains were obtained by selecting resistant colo-nies on heavily inoculated nutrient agar plates containing 20,ug of nalidixic acid (Sigma) per ml.Mating. For transfer of the plasmid pMO821 into strain

PN-1, the two strains were mixed on an SMM plate warmedto 30°C containing 10 mM sodium succinate and 0.01%(wt/vol) yeast extract (Difco). The culture was incubatedaerobically at 30°C. After 72 h, the cells were suspended inphosphate-buffered saline warmed to 30°C (36). Mutants ofstrain PN-1 carrying TnS were selected on SMM platescontaining 10 mM sodium succinate and 50 ,g of kanamycinper ml. These cultures were incubated anaerobically at 30°C.

Matings of strain PN-1 and the Pseudomonas spp. weredone as described above, except that the selection forexconjugants was performed on SMM plates containing 6.94mM sodium benzoate and 20 jig of nalidixic acid per ml.Cultures were incubated anaerobically at 37°C.

Plasmid isolation and gel conditions. Plasmids were iso-lated by the alkaline lysis method (27). Separations weredone by electrophoresis in 0.7% agarose at 50 V with Trisborate buffer (27). Lambda bacteriophage DNA cut withHindIlI (Bethesda Research Laboratories, Inc., Gaithers-burg, Md.) served as the size marker. DNA bands werevisualized by soaking gels in 0.5 jig of ethidium bromide perml.

Restriction endonuclease analyses. Plasmids (0.2 to 1.0 pugof DNA) were restricted in a 20-,ul digestion mixture con-taining 50 mM Tris hydrochloride (pH 7.5), 10 mM MgCl2, 1mM dithiothreitol, 50 mM NaCl, 10 ,ug of RNase A (Sigma),and 4 U of restriction enzyme HindIlI or PstI. The digestionmixtures with the restriction enzyme XhoI, BamHI, SphI, orNcoI were mnixed as described above but contained 100 mMNaCl. Mixtures were incubated at 37°C for 1 h. All restric-tion enzymes were from New England BioLabs, Inc., Bev-erly, Mass.

Determination of copy number. The minimum number ofcopies of plasmid pCBI per cell was estimated as follows.Plasmid DNA from 1012 cells of strain PN-1 was purified byequilibration in a cesium chloride-ethidium bromide gradi-ent. The ratio of the amount of plasmid DNA per cell to themolecular weight of the plasmid was used to determine thenumber of plasmid molecules per cell.

RESULTS

Strain description. Strain PN-1 is a gram-negative rodapproximately 1.4 by 0.6 ,m in size. The cells are motilewith one to four lateral flagella. Strain PN-1 is oxidase andcatalase positive. It does not hydrolyze starch, gelatin, orTween 80. Poly-i-hydroxybutyrate is present as shown bystaining and chemical analysis.None of the following sugars which were tested as carbon

and energy sources were utilized by strain PN-1: D-glucose,L-arabinose, D-xylose, D-fructose, D-mannitol, D-mnannose,D-ribose, D-galactose, D-lyxose, D-fuCose, L-fuCose, L-sorbose, D-raffinose, L-rhamnose, D-melibiose, D-trehalose,cellobiose, lactose, glycogen, dulcitol, salicin, maltose, su-crose, melezitose, inulin, adonitol, sorbitol, meso-erythritol,meso-inositol, N-acetyl-D-glucosamine, D-glucosamine, D-gluconate, or 2-ketogluconate.Of the organic acids and alcohols, only acetate and ethanol

were utilized. The following organic acids and alcohols werenot utilized: glycolate, propionate, DL-glycerate, DL-lactate,n-butyrate, isobutyrate, n-valerate, caproate, heptanoate,caprylate, pelargonate, caprate, n-propanol, isopropanol,ethylene glycol, propylene glycol, and glycerol.

Succinate, fumarate, and citrate were utilized. No growthoccurred on the other tested di- and tricarboxylic acids,including oxalate, malonate, DL-malate, citraconate, glutar-ate, adipate, pimelate, suberate, azelate, sebacate, aconit-ate, and cis,cis-muconate. The two tested keto acids, pyruv-ate and 2-ketoglutarate, supported the growth of strain PN-1.

Benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, hydro-cinnamate, and benzoylformate were utilized as growthsubstrates by strain PN-1 under aerobic and anaerobicconditions. Vanillate, vanillin, and gentisate were utilizedonly under anaerobic conditions. The following aromaticcompounds were not utilized by strain PN-1: salicylate,homovanillate, acetovanillone, homovanillyl alcohol,protocatechuate, 4-anisate, phenol, catechol, 3-toluate,anthranilate, 4-aminobenzoate, hippurate, 2,4-dihydroxyben-zoate, phenylacetate, homoprotocatechuate, veratrate, DL-mandelate, 4-hydroxy-3-methoxycinnamate, and phthalate.Some differences in carbon source utilization exist be-

tween strain PN-1 and Alcaligenes strains (22, 23). Theability of A. xylosoxidans subsp. denitrificans to utilize thetested organic acids, dicarboxylic acids, and amino acids isvariable within the species. Although strain PN-1 is mnorelimited nutritionally than Alcaligenes strains, it otherwisefits the species description (22, 23).Mating of strains PAOll and PN-1. Strain PN-1 was mated

with strain PAO11 carrying the suicide vector pMO821.After conjugation, kanamycin resistance in strain PN-1 wasselected for by plating on minimal medium (SMM) contain-ing succinate and kanamycin. All of the resulting kanamycin-resistant clones of strain PN-1 had lost the ability to grow onbenzoate under denitrifying conditions.The result that no kanamycin-resistant clones of strain

PN-1 could degrade benzoate anaerobically suggested thepresence of a catabolic plasmid in strain PN-1 that wasexcluded by the incoming vector plasmid, pMO821. Agarosegel electrophoresis showed the presence of a plasmid in thewild-type strain PN-1, pMO821 in PAGli, and a DNA bandcorresponding in electrophoretic mobility to pMO821 whichwas obtained from the kanamycin-resistant exconjugants ofstrain PN-1 (data not shown). A band corresponding to theplasmid found in the wild-type strain PN-1 was not found inthe exconjugants. The plasmid in strain PN-1 was designatedpCBI.

Transfer of plasmid pCBI. To determine whether all of thegenes necessary for the anaerobic catabolism of benzoatewere carried on plasmid pCBI, strain PN-1 was mated with20 nalidixic acid-resistant denitrifying strains of bacteria,including four P. aeruginosa strains and 16 P. stutzeristrains. Exconjugants were selected by incubating culturesanaerobically on SMM plates containing benzoate, nitrate,and nalidixic acid. The identity of all resulting clones wasconfirmed by biochemical and nutritional testing, sincestrain PN-1 typically produces nalidixic acid-resistant mu-tants at a frequency of 10-8.Four P. stutzeri strains and one P. aeruginosa strain

acquired the ability to grow under denitrifying conditionswith benzoate as a carbon and energy source. Agarose gelelectrophoresis of the plasmid DNA from the parental andexconjugant strains showed the acquisition of a plasmid bythe exconjugants. The size and restriction patterns of theplasmids found in the exconjugants were identical to those ofplasmid pCBI from strain PN-1 (data not shown).

Acquired substrate utilization. The aerobic and anaerobicutilization of benzoate, vanillate, pimelate, and adipate bystrains PN-1, PA280, PA281, PS222, and PS223 was tested.Strain PN-1 utilized benzoate aerobically and anaerobically

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ViindUl\ V A Kbp 0X\C12/ °

FIG. 1. Restriction endonuclease map of pCBI.

and utilized vanillate anaerobically. Parental P. aeruginosaPA280 utilized benzoate and vanillate aerobically and uti-lized adipate aerobically and anaerobically. The exconjugantPA281 gained the ability to utilize benzoate and vanillateanaerobically. Parental P. stutzeri PS222 utilized none of thesubstrates; the exconjugant PS223 gained the ability toutilize benzoate anaerobically.

Restriction endonuclease map of pCBI. The size of plasmidpCBI was determined by digestion of the plasmid with sixdifferent enzymes. The sizes of the resulting fragments were

compared to molecular weight standard markers andsummed for each digest. Plasmid pCBI was determined to beapproximately 17.4 kilobase pairs (kbp) in length. A mini-mum of 26 copies of pCBI per cell was estimated. Arestriction map of the plasmid showing the restriction sites ofthe enzymes NcoI, XhoI, and HindIII has been constructed(Fig. 1).

DISCUSSION

Our interest in this organism arises from its ability tocatabolize aromatic compounds under denitrifying condi-tions. The biochemistry, enzymology, and genetics of thepathway of anaerobic catabolism of aromatic compounds bydenitrifying bacteria are largely unknown. We have renamedstrain PN-1 as A. xylosoxidans subsp. denitrificans PN-1 andhave shown that the ability of strain PN-1 to catabolizebenzoate anaerobically is conferred by a resident catabolicplasmid which can be transferred to other denitrifying or-

ganisms.Presence of a catabolic plasmid. Transposon mutagenesis

was undertaken to obtain stable mutants blocked in theanaerobic benzoate pathway. Since all of the kanamycin-resistant mutants had lost the ability to grow anaerobicallyon benzoate and had also lost a plasmid which was present inthe wild-type strain PN-1, the presence of a plasmid in themutants corresponding to pMO821 suggests that pMO821and pCBI are in the same incompatibility group (IncP10)and, unexpectedly, that pMO821 can be maintained in strainPN-1.

Transfer of pCBI. Strain PN-1 was mated individually with20 other denitrifying strains of bacteria to determine whether

all the genes necessary for anaerobic ring cleavage werecarried on the plasmid. P. aeruginosa strains carry genes foraerobic catabolism of aromatic compounds, including benzo-ate, but the P. stutzeri strains do not. Exconjugant strains ofboth species were able to catabolize benzoate anaerobically,suggesting that the genes carried on the plasmid are sufficientto convert benzoate to intermediates of central metabolismand that the genes of the aerobic aromatic pathway areapparently not necessary. The results of agarose gel electro-phoresis suggest that the presence of plasmid pCBI isassociated with the phenotype of anaerobic benzoate catab-olism and that no rearrangements of this plasmid occurred inthe exconjugants.

Additional substrates. Having the plasmid pCBI present intwo additional species of bacteria provided the opportunityto learn more about what genes are carried on the plasmid.Adipate is the putative ring cleavage product of the reductivebenzoate pathway proposed by Evans (14). Pimelate is thering cleavage product of anaerobic benzoate catabolism inthe photosynthetic organism R. palustris (12, 19). The re-sults show that the exconjugants did not gain the ability togrow on adipate or pimelate, suggesting that they are notintermediates of the pathway. Strain PN-1 grows anaer-obically but not aerobically on vanillate, a methoxylatedaromatic compound. Vanillate was selected as a growthsubstrate to determine whether genes for anaerobic demeth-ylation in strain PN-1 were carried on pCBI.

Interestingly, P. aeruginosa PA281 acquired the ability todegrade vanillate anaerobically, but P. stutzeri PS223 didnot. This finding suggests that plasmid pCBI does not carrythe genes necessary for handling of the methoxyl group.Aerobically, oxygenases are utilized to demethoxylate aro-matic compounds (4). However, P. aeruginosa appears tohave not only a mechanism for aerobic demethoxylation, butalso an alternate mechanism for handling the methoxyl groupwhich does not require molecular oxygen or oxygenases.This finding is surprising because the wild-type organismuses aromatic compounds only under aerobic conditions.Anaerobic cleavage of phenylmethylethers has been

shown to occur in several species of bacteria (1, 13, 25, 44),but the evidence favors a transmethylation reaction ratherthan a demethoxylation involving methanol formation. Itwould be interesting to know whether this type of activitywith methoxylated aromatic compounds occurs in strainsPN-1 and PA280.

Plasmid pCBI. Catabolic plasmids carrying the pathwaysfor the degradation of aliphatic and aromatic hydrocarbons,aromatic compounds, and simple organic compounds havebeen shown in Pseudomonas spp. as well as in other generaof bacteria. These plasmids provide a means of developingmuch greater metabolic diversity for a population of bacteriawithout the need to maintain chromosomal genes for thesepathways in each bacterium. Not surprisingly then, most ofthese catabolic plasmids, which vary in size from 15 to over500 kbp, are self-transmissible.

Plasmid pCBI is a small plasmid (17.4 kbp) carrying thegenes necessary for the degradation of benzoate underanaerobic conditions and for transfer of the plasmid to otherdenitrifying bacterial strains. This conjugative ability and thesmall size of pCBI bring into question the size of the codingregion available for the transfer functions, as well as thepathway. The number of genes necessary for transfer func-tions in plasmids of gram-negative bacteria is not wellestablished. In the case of the F plasmid in Escherichia coli,21 genes of known function plus 7 genes of unknownfunction have been shown to be involved in transmissibility

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(21). In Pseudomonas plasmids RP4 and R18, five and eightcistrons, respectively, have been identified (3, 18, 39). Mostof the known catabolic self-transmissible plasmids are con-siderably larger than pCBI, e.g., TOL and CAM at 117 and-500 kbp, respectively (30, 46). The smallest of the reportedcatabolic self-transmissible plasmids is pEG (37 kbp), whichis involved in styrene degradation (6). It is likely that few, ifany, genes other than those needed for replication, transfer,and the catabolic pathway are present on the plasmid. Thisfinding, plus the relatively high minimum copy number of theplasmid (26 copies per cell), allows the use of genetictechniques to elucidate the mechanism of ring cleavage instrain PN-1.

ACKNOWLEDGMENTS

We thank Barrie F. Taylor for providing strain PN-1, Bruce W.Holloway for the gift of P. aeruginosa PAO11, and Barry Poliskyand Gary Janssen for valuable discussion and advice.

This work was supported by research grant PCM-8314087 fromthe National Science Foundation.

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