Construction of Chlorobenzene-Utilizing Recombinants by Progenitive ...

APPLIED AND ENVIRONMENTAL. MICROBIOLOGY, OCt. 1987, p. 2470-2475 0099-2240/87/102470-06$02.00/0

Vol. 53, No. 10

Copyright © 1987, American Society for Microbiology

Construction of Chlorobenzene-Utilizing Recombinants by Progenitive Manifestation of a Rare Event L. KROCKEL AND D. D. FOCHT* Department of Soil and Environmental Sciences, University of California, Riverside, Califo)rnia 92521

Received 11 May 1987/Accepted 8 July 1987

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-0, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant-unlike its parent, R5-3-did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene. Benzene was metabolized through the MP pathway in CB1-9, while chlorobenzene was metabolized through the OP pathway.

Xenobiotics have become a major issue of environmental and public concern, since many of these widely used chemicals, particularly the chlorinated aromatic hydrocarbons, have been shown to be persistent in the environment. Biological cleanup of contaminated sites is thought to be the most convenient solution in terms of cost and management (16). The indigenous soil microflora, however, may be incapable of degrading these chemicals. Inoculation of soils with specific microorganisms which have been isolated from a different environment (e.g., sewage sludge) or which have been genetically altered to expand or modify substrate specificities could therefore be useful for biological decontamination. Microorganisms which utilize chlorinated aromatic hydrocarbons as sole carbon sources in the strictest sense (i.e., in the absence of supplemental 0.05% yeast extract) cannot normally be isolated by conventional enrichment culture techniques. Nevertheless, many aromatic-hydrocarbon-utilizing bacteria can cometabolize these compounds. The dead-end products formed from cometabolism in pure culture may be further metabolized to CO2, HCl, and H20 by other microbial species as noted for the mineralization of polychlorinated biphenyls in soil (3, 9). Thus the mobilization of the requisite gene pool into a single species would be a desirable goal for complete mineralization of a target compound. Recombinants are obtained by genetic engineering techniques or by natural genetic exchange between bacteria. The former method is very tedious and timeconsuming and is frequently limited by the lack of commercially available degradation products (e.g., 3-chlorocatechol and chloromuconic acid) for isolation of clones specific for given catabolic functions. Natural genetic exchange, on the other hand, involves very little labor, but requires consider*

able patience and luck, since the requisite organisms may either not exist or be present in such low numbers that the frequency of the desired genetic exchange event may be too low to be manifested over the observation period. Enrichment cultures of Alcaligenes sp. that grew on 1,4-dichlorobenzene (18) and 1,3-dichlorobenzene (5) required at least 10 and 6 months, respectively, for isolation. Nine months of continuous selection pressure in a chemostat was required before growth on chlorobenzene was achieved with strain WR1306, which was originally isolated from enrichment with benzene (17). Kellogg et al. (11) used the term molecular breeding to describe the natural genetic exchange and imposed selective pressure that could be combined to create a desired recombinant strain. They reasoned that such a methodology would result in more stable genotypes than those produced by genetic engineering, since the problems inherent in expressing genes under different structural and regulatory derivations would be obviated. Using this concept, Kilbane et al. (12) constructed a (2,4,5-trichlorophenoxy)acetic aciddegrading strain of Psetudomonas cepacia from one that was originally isolated by enrichment with (2,4-dichlorophenoxy)acetic acid; however, the other parent could not be identified, since it originated from the nonaxenic soil inoculum that was added to the chemostat. If two organisms together, but not separately, possess the catabolic enzymes for complete mineralization of a substrate, then it should theoretically be possible to construct a recombinant strain that will grow with that substrate as its sole carbon source. In practice, the frequency of genetic exchange between the two parents may be too low and the selection pressure inadequate to bring about the creation of the new progeny. Thus for any rare event of this type to be manifested, the progeny must increase in sufficient number to be detected. In contrast to all previous methods of

Corresponding author. 2470

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molecular breeding in a single chemostat, we present a new method that permits natural genetic exchange and subsequent selection of a novel genotype in a defined and reproducible manner, by the progenitive manifestation of a rare event. MATERIALS AND METHODS Bacterial strains. Isolation and identification of Pseludomonas alcaligenes C-0, which grows on 3-chlorobenzoate (3CB) as the sole carbon source, has been described previously (10). Pseudomonas putida R5-3 was isolated from activated sewage sludge by enrichment culture with toluene as the sole carbon source for growth. It was characterized by standard taxonomic characteristics (20). P. putida R5-3 is a gram-negative rod that is catalase positive, cytochrome oxidase positive, and motile by polar flagella, does not reduce nitrate, grows only aerobically, produces fluorescent pigment on King B but not King A agar, hydrolyzes arginine, does not hydrolyze gelatin, and produces neither acid nor gas from glucose. Liquid cultures were incubated at 27°C on a rotary shaker (120 rpm). Stock cultures were maintained on mineral medium with the selective carbon source, subcultured, and stored at 4°C. Chemicals. Toluene and benzene were obtained from Mallinckrodt, Inc., St. Louis, Mo. Chlorobenzene, 3chlorobenzoic acid, p- and m-chlorotoluene, p- and mxylene, and 1,4-dichlorobenzene (1,4-DCB) were purchased from Aldrich Chemical Co., Inc., Milwaukee, Wis. Catechol was supplied by Nutritional Biochemicals Corporation, Cleveland, Ohio. 4-Chlorocatechol was obtained from Chemicals Procurement Laboratories, Inc., College Park, N.Y. 3- and 4-Methylcatechol were purchased from Pfaltz and Bauer Inc., Waterbury, Conn. Catechols were purified by vacuum sublimation. All substituted catechols nevertheless contained catechol as an impurity as follows: 4chlorocatechol, 20%; 3-methylcatechol, 10%; 4 methylcatechol, M]) MP sp acta (K,,,

[>.M]) on:

Substrate

B

CB

on:

B

CB

15 (3.9) 74 6 (0.2) 10 32 (10.3) 44 3-Methylcatechol 59 31(8.5) 4-Methylcatechol a Expressed in nanomoles per minute per milligram of protein.

Catechol 4-Chlorocatechol

130 0 0 46

151 (9.3) 0 0 94

KROCKEL AND FOCHT

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APPL. ENVIRON. MICROBIOL.

TABLE 3. Specific activities of MP and OP in cell extracts of P. putida R5-3 Sp act (nmol/ml per mg of protein) of: Substrate

MP

Ba

Catechol

4-Chlorocatechol 3-Methylcatechol 4-Methylcatechol a

153 29 0 58

OP

4MBb 4,900 5,000 4,300 4,600

B

4MB

63 11 19 37

141 273 329

89

B, Benzene; 4MB, 4-methylbenzoate.

ized with pKFL2 and chromosomal DNA of P. alcaligenes C-0 but not with pKFL1 (not shown in Fig. 2). DISCUSSION P. putida CB1-9 is the first reported strain of this genus able to grow on 1,4-DCB. By comparison with Alcaligenes strain OBB65 (5), growth of CB1-9 was similar on 1,4-DCB but was faster on chlorobenzene, benzene, and toluene. Although strain WR1306 (17) grew twice as fast as CB1-9 on chlorobenzene, the former did not grow on 1,4-DCB, benzene, or toluene, and growth was suppressed in a chlorobenzene-saturated atmosphere. CB1-9 metabolizes chlorobenzene through the OP pathway in a manner similar to all other chlorobenzene utilizers (5, 17-19), but it is uniquely different from these strains in that it has both MP and OP activity. Benzene oxidation, in contrast to chlorobenzene metabolism, appears to be catalyzed by MP, because growth on either benzene or chlorobenzene induced greater activity on MP than OP with catechol as the substrate (Table 2). Moreover, growth on benzene has always resulted in the production of yellow MP-catalyzed products. MP activity in the parental strain, R5-3, was different from that found in CB1-9. Although both catechol dioxygenase activities were greater among 4MB-grown cells than benzene-grown cells of R5-3, the increase in MP activity was considerably greater when cells were grown on 4MB (Table 3). Moreover, the activity of MP on all four catechol substrates was similar with 4MB-grown cells, while it was vastly different with benzene-grown cells-particularly with respect to 3-methylcatechol and catechol. Others have found that MPs in P. putida grown on toluene (14) and methylbenzoates (4) are different. Chatfield and Williams (4) recently observed that the TOL plasmids in Pseudomonas spp. code for either two homologous or two nonhomologous MPs. Although plasmid pKFL3 of CB1-9 was 24 kb smaller than plasmid pKFL2 of R5-3, restriction digests of both TOL plasmids were very similar (Fig. 2). Consequently, the inability of CB1-9 to grow on methylbenzoates or xylenes may be related in part to the loss of the MP that has higher activity and lower substrate specificity and is coded for by a 24-kb portion of the pKFL3 plasmid. When the above observations were coupled with phenotypic observations, it is apparent that CB1-9 is more similar to R5-3 than to C-0. The genetic contribution of P. alcaligenes C-0 to the recombinant is not as clearly defined, although it obviously conferred the ability to grow on 3CB, which would be metabolized through 3-chlorocatechol by OP. Unfortunately, we could not compare the activity for this substrate by MP or OP among the three strains or with the other catechols, since it is not commercially available. According to recent concepts (1, 17), loss of MP activity is

essential to avoid routing of chlorocatechols through this pathway, since the ring fission products are not dehalogenated and are thus dead-end products. With respect to CB1-9, it is not necessary that MP activity be abolished in toto, but rather that the enzyme simply lack the broad substrate specificity (as induced in the methylbenzoate pathway) to oxidize chlorocatechols. The continuous amalgamated culture system presented herein offers a very quick, reliable, and convenient procedure for constructing desirable microorganisms from parental strains with different but complementary properties. This system differs from all others in that the two parental strains can be grown separately to maintain constant and equal (or unequal) cell densities of both. Moreover, separate selection pressures can be maintained in each continuous culture vessel by the use of different substrates. Also, the advantage of our method over the single-chemostat method is that it would be possible to maintain two organisms with different growth rates, so that washout of the slower-growing strain would not be a problem. Furthermore, competition for a common substrate utilized by both of them (benzoate in this case) is also eliminated. The principle of the CAC is based upon the assumption that a rare event becomes more probable as time accumulates. Concentrated mixed-cell suspensions (1010 cells per ml) of P. putida R5-3 and P. alcaligenes C-0 never resulted in recombinant clones that grew on chlorobenzene. Thus the frequency of genetic exchange between these species is a rare event, even rarer than the low frequency (10-6) reported between non-Hfr strains of E. coli (21). Moreover, only the tri-chemostat that was set at the lowest dilution rate failed to produce a recombinant which utilized chlorobenzene. Therefore the continued growth of the parental strains as they are amalgamated on the column increases the probability that each new progeny contributes to this rare event. Progenitive manifestation of the event, however, is necessary, and this is accomplished by unique selection pressure (e.g., utilization of chlorobenzene as a growth substrate) relative to both parental strains. The reaction bed column serves several purposes. First, the attachment of cells to the ceramic beads minimizes the probability of washout of the recombinant cells. Second, adsorption may enhance genetic exchange by immobilizing cells and concentrating them closer to one another. Third, a gradient of chlorobenzene concentrations is formed along the column in accordance with diffusion principles as it is cometabolized by P. putida R5-3. The establishment of the recombinant strain will occur when the concentration of chlorobenzene is optimal: hence potential problems relating to substrate toxicity are eliminated. Because of the novelty of this system, we cannot be certain of which of these factors is the most important in the progenitive manifestation of a new genotype. ACKNOWLEDGMENTS This work was supported in part by a grant from Occidental Chemical Corporation. We are grateful to H. Slater for his helpful commentary and discussion about chemostats and genetic exchange and to D. Shelton for the isolation and characterization of P. putida RS-3. LITERATURE CITED 1. Bartels, I., H.-J. Knackmuss, and W. Reineke. 1984. Suicide

inactivation of catechol-2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols. Appl. Environ. Microbiol. 47: 500-505.

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