Competition of plasmid-bearing Pseudomonas putida ... - Springer Link

3 downloads 0 Views 305KB Size Report
Abstract Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of ...
Appl Microbiol Biotechnol (1997) 48: 493±498

Ó Springer-Verlag 1997

ORIGINAL PAPER

A. E. Filonov á W. A. Duetz á A. V. Karpov R. R. Gaiazov á I. A. Kosheleva á A. M. Breure I. F. Filonova á J. G. van Andel á A. M. Boronin

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997

Abstract Plasmid-carrying Pseudomonas putida strains degrade naphthalene through di€erent biochemical pathways. The in¯uence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D ˆ 0:05 h)1) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive.

Introduction During the past decades, many bacterial strains capable of degrading polycyclic aromatic hydrocarbons (PAH) have been isolated and characterized. The biochemistry A. E. Filonov (&) á A. V. Karpov á R. Gaiazov I. A. Kosheleva á I. F. Filonova á A. M. Boronin Laboratory of Plasmid Biology, Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292, Russia Tel.: (095) 925 7448 Fax: (095) 923 3602 e-mail: [email protected] W. A. Duetz InstituÈt fuÈr Biotechnologie, ETH HoÈnggerberg, HTP, CH-8093, ZuÈrich, Switzerland A. M. Breure á J. G. van Andel Laboratory for Waste Materials and Emissions, National Institute of Public Health and Environmental Protection, Antonie van Leeuwenhoeklaan 9, P.O. Box 1,3720 BA Bilthoven, The Netherlands

of naphthalene catabolism in Pseudomonas strains has been well studied (Yen and Serdar 1988). In the various strains studied so far, the initial steps of naphthalene degradation (oxidation to salicylate) can be controlled by either plasmid or chromosomal genes. Salicylate can be oxidized further through di€erent metabolic pathways (ortho, meta or through gentisic acid) (Yen and Serdar 1988; Sayler et al. 1990; Dunn and Gunsalus 1973; Boronin et al. 1976, 1977a, b; Skryabin et al. 1980; Boronin 1992). It was shown earlier that, in batch culture with an excess of naphthalene in the medium, the bacteria catabolizing naphthalene via the meta pathway of catechol oxidation grow faster than those that catabolize it via the ortho pathway. The latter possess higher growth rates than the bacteria that utilize naphthalene via gentisic acid (Boronin et al. 1993). However, it is known that, in nature, because of adsorption to soil particles and low solubility, the aqueous concentration of hydrophobic substances like PAH may be very low, even in soil samples from areas that are heavily contaminated with PAH. Therefore, it could be expected that the af®nity for PAH may be a more important determinant for competitive behaviour in nature than the maximal obtainable growth rate. Under laboratory conditions, substrate-limited growth can be accomplished in chemostat culture. In this paper we compare the growth characteristics and the competition behaviour of naturally occurring and novel laboratory-constructed strains degrading naphthalene via di€erent biochemical pathways in chemostat culture under naphthalene limitation.

Materials and methods Bacterial strains and plasmids The bacterial strains used are listed in Table 1. The plasmids are described in Table 2. Strain Pseudomonas putida WD25 is a plasmid-free (mitomycin-C-cured) derivative of P. putida mt-2, reisolated from a chemostat culture after 2 months of cultivation under

494 Table 1 Bacterial strains. Nah growth on naphthalene (+ strain can grow on naphthalene; ) strain can not grow on naphthalene), Sal growth on salicylate (+ strain can grow on salicylate; ) strain can not grow on salicylate), Cys cysteine necessary (+ cysteine prototroph; ) cysteine auxotroph). Ortho catechol oxidation using catechol 1,2-dioxygenase, meta catechol oxidation using catechol 2,3-dioxygenase, Gentisic naphthalene catabolism via gentisic acid, Nah-sal naphthalene catabolism up to salicylate only; the pathways used by the strains were determined under batch cultivation.

Strain (plasmid)

Phenotype

Donors BS394(pBS2) BS394(pBS3) BS394(NAH7)

Pathway

References

Nah

Sal

Cys

+ + +

+ + +

) ) )

Ortho Meta Meta

Boronin et al. 1977b Boronin et al. 1977b Collection of Laboratory of Plasmid Biology

+ ) )

+ + +

Ortho ± ±

Boronin et al. 1976 Boronin et al. 1976 This study

+ + +

+ + +

Ortho Gentisic Meta

Boronin et al. 1976 Skryabin et al. 1980 Dunn and Gunsalus 1973

+ + + ) +

+ + + + +

Ortho Meta Meta Nah-sal Meta

This This This This This

Recipients BS203 ) BS206 ) WD25 ) Wild-type isolates BS203(NPL-1) + BS291(pBS4) + PpG7(NAH7) + Laboratory-constructed strains BS203(pBS2) + BS206(pBS3) + WD25(NAH7) + WD25(pBS2*)a + WD25(pBS3) +

study study study study study

a Plasmid pBS2* in the strain WD25 is the mutant of original plasmid pBS2 without ability to encode the Sal+ phenotype

Table 2 Naphthalene-catabolic plasmids. C23O catechol; 2,3-dioxygenase; NC non-conjugative plasmid Designation

Size (kb)

Incompatibility group

Conjugal transfer frequency

C23Oa activity

Host

References

NAH7 NPL-1 PBS2 PBS3 PBS4

83 100 130 180 173

P9 P9 P7 P7 P7

10)4±10)3 10)3 a 10)5±10)3 10)5±10)4 NC

+ ) ) + )

P. P. P. P. P.

Dunn and Gunsalus 1973 Boronin et al. 1976 Boronin et al. 1977b Boronin et al. 1977b Skryabin et al. 1980

a

putida PpG7 putida BS203 putida BS238 ¯uorescens BS243 ¯uorescens BS291

Can be expressed only in Sal+ strains

succinate limitation. Strains WD25(NAH7), WD25(pBS3), WD25(pBS2*), BS203(pBS2) and BS206(pBS3) were constructed by conjugal transfer of respective plasmids into recipients WD25, BS203 and BS206 (see Table 1). It should be noted that strain WD25, harbouring plasmid pBS2*, does not exhibit salicylate hydroxylase activity. Salicylate therefore accumulates in the culture liquid during growth of strain WD25(pBS2) on naphthalene. Media The growth medium was a mineral salt solution according to Evans et al. (1970), bu€ered (50 mM phosphate bu€er) at pH 7.0. Concentrations of all nutrients in this medium were decreased by a factor of 4. Continuous cultivation All experiments were done at dilution rates of 0.05 h)1 in 100-ml glass fermenters shaped like concical ¯asks, with a small head-space volume and stirred magnetically (160 rpm). Medium was fed in at the bottom of the vessel through a needle. Naphthalene was fed to the fermenter in the gas phase (2.0 mg h)1) by blowing air (6.0 l h)1) through a sealed glass vial with naphthalene crystals. This air supply also satis®ed the oxygen requirements of the microorganisms. The fermenter was equipped with two stainless-steel ba‚es and was kept at 28 °C. The vial was put into a water bath at 20 °C to prevent naphthalene crystallization in the exhaust pipe. Once a day the ¯ask with the crystals was weighed to an accuracy of 0.1 mg.

Assays The number of viable cells colony-forming units were determined by plating culture dilutions on peptone/agar plates. Concentrations of naphthalene and salicylate in the medium were determined spectrophotometrically (k ˆ275 nm and 295 nm respectively) and by HPLC (Volkering et al. 1992). Dry weight was determined using absorbance values as described previously (Duetz et al. 1991). Activities of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase were determined by the methods described (Shamsuzzaman and Barnsley 1974; Nakazawa and Yokota 1973). Conjugal transfer Conjugal transfer of plasmids with naphthalene selection was carried out on agar plates. Auxotrophic P. putida mutants were used as donor strains according to Cane and Williams (1982). Plasmid DNA isolation Plasmid DNA isolation was described previously (Boronin et al. 1993; Duetz et al. 1991). Competition experiments The experimental protocol was described earlier (Duetz and van Andel 1991). The experiments were started at ratios 1:1 and 1:3 of the competing strains (as estimated by the concentration of living

495 Table 3 The discriminating features of the strains used to distinguish them in competition experiments during mixed cultivation Strains

Tests NA 30 °C

WD25(NAH7) WD25(pBS3) BS203(NPL-1) BS203(pBS2) BS206(pBS3) PpG7(NAH7) BS291(pBS4)

PA

2-MN

mTE

KB Nal Neo 50 50

Sp 50

Km 50

Sm 50

Tc 50

Cb 500

+ SM,Y + SM, Y + SM, Y + SM, Y + RS, G + SM, Y + SM, W

+

) B ) B ) B ) B ) B +

+ WY + WY )

)

+

)

+

)

)

)

+

)

+

)

)

+

)

+

)

+

+

)

)

)

)

)

)

+

+ FL, G +

+

+

+

+

)

)

)

+

)

)

)

+

) B

) YG

)

+

)

)

)

+

4 °C

+ ) YG + ) YG + + YG + + YG + ) FL, BG + + YG + + YG

+ + ) B + + +

+ Bacterial growth, ) the absence of bacterials growth, YG yellow-green colonies, BG, blue-green colonies, FL ¯uorescing colonies, SM smooth colonies, RS rough-surface colonies, W white colonies, Y yellow colonies, B brown pigmentation in the absence of bacterial growth G green colonies, WY white-yellow colonies, NA nutrient agar; PA peptone agar, 2-MN, Evans agar with 2-methylnaphthalene; mTE, m-toluate-containing Evans agar, KB Nal King's B agar with nalidixic acid, Neo neomycin, Sp spectinomycin Km kanamycin Sm streptomycin, Tc tetracyclin, Cb carbenicillin. The ®nal concentrations of antibacterial agents in the nutrient agar are shown in lg/ml. cells in 1 ml chemostat culture). Diluted samples were plated on peptone/agar. The ratio of competing strains was determined by testing no fewer than 100 isolated colonies on selective media by the replica plating method. Competing strains were discriminated using the distinctive features given in Table 3. In the case of identity of phenotypic characteristics, the competing strains were discriminated by the size of isolated plasmid DNA. Isolation of plasmid DNA was carried out as described previously (Duetz et al. 1991). Quanti®cation of the growth-rate advantage of competing strains from population dynamics Mathematical analysis of the population dynamics was carried out as described previously (Duetz and van Andel 1991; Duetz et al. 1991). Brie¯y, competition in a chemostat at a dilution rate D between two strains present at population fraction pi with variable growth rate li (i ˆ 1; 2) is described by equations: dpi =dt ˆ …li ÿ D†pi ; p1 ‡ p2 ˆ 1;

i ˆ 1; 2;

p1 l1 ‡ p2 l2 ˆ D:

…1† …2†

The assumption that l1 =l2 ˆ constant allows to an analytical solution to be found and the constant-growth-rate ratio C ˆ l1 =l2 to be estimated by a non-linear least-squares ®tting procedure (Duetz et al. 1991). Let us suppose that the growth rate l can be described by the Monod equation S l ˆ lmax ; …3† S ‡ KS where lmax and KS are considered to be constant. At low substrate concentration Eq. 3 can be simpli®ed: l  Slmax =KS ˆ aS

for S  KS ;

…4†

where a ˆ lmax =KS

…5†

In this case the growth-rate ratio C ˆ l1 =l2 has the following form: C ˆ l1 =l2 ˆ a1 =a2 ˆ constant because factors a1 and a2 are constant according to Eq. 5.

…6†

Now one can use the ratio C for indirect comparison of the competitive abilities of strains. Let us consider the example of three strains, 1, 2 and 3, with estimated growth ratios C1;2 ˆ a1 =a2 and C2;3 ˆ a2 =a3 for pairs of strains, (1, 2) and (2, 3). Then the constant growth ratio C1,3 for pair (1, 3) can be calculated as C1;3 ˆ a1 =a3 ˆ …a1 =a2 †…a2 =a3 † ˆ C1;2 C2;3 :

…7†

This relationship can also be used in a more general case when l ˆ aF …S†, where factor a is speci®c for a given strain and some function F(S) is the same for all strains.

Results The absorbance of individual strains at the same dilution rate in steady-state chemostat culture was proportional to the amount of naphthalene fed (the rate of ¯ushing naphthalene-saturated air through the culture). The residual concentration of naphthalene in the culture liquid in all experiments was less than 0.5 lg ml)1, suggesting that bacterial growth was limited by naphthalene. The growth parameters of the strains investigated are given in Table 4. After a steady state had been reached in the chemostat culture of the individual strains, the cultures were mixed in a third chemostat (see Materials and methods) and the dynamics of their ratio was observed. Mathematical analysis of the experimental data for the population change over time yielded values for the ratio (C) of the growth rates of competing strains (Table 5). C values of zero were obtained in competition experiments with strain BS291(pBS4), catabolizing naphthalene via gentisic acid, indicating that growth of this strain stopped completely because all the substrate supplied was utilized by another competing strain.

496 Table 4 Growth characteristics of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways under chemostat cultivation. A540 absorbance at 540 nm, path length 1 cm; cfu number of colony-forming units; Yield the eciency of carbon assimilation, (g dry weight/g naphthalene)

Table 5 Results of competition experiments. C the ratio of the growth rate of the losing strain to that of the winning strain (in continuous culture at a limiting concentration of naphthalene)

Strains

A540

Meta pathway (catechol 2,3-dioxygenase) WD25(pBS3) 0.36 WD25(NAH7) 0.36 BS206(pBS3) 0.33 PpG7(NAH7) 0.36 Ortho pathway (catechol 1,2-dioxygenase) BS203(NPL-1) 0.29 BS203(pBS2) 0.50 Gentisic pathway BS291(pBS4) 0.27 Oxidation of naphthalene to salicylic acid WD25(pBS2) 0.08

No. 1 2 3 4 5 6 7 8 9 10

10)8 viability cfu

Yield (g/g)

Plasmid/chromosomal control

1.8 4.3 4.7 6.8

0.60 0.60 0.56 0.60

Plasmid Plasmid Plasmid Plasmid

4.8 9.1

0.48 0.84

Mixed Mixed

3.1

0.44

Plasmid

0.8

0.14

Plasmid

Competing strains WD25(NAH7) meta WD25(pBS3) meta WD25(NAH7) meta BS206(pBS3) meta BS206(pBS3) meta BS203(NPL-1) ortho BS203(pBS2) ortho BS203(pBS2) ortho BS206(pBS3) meta PpG7(NAH7) meta

In most cases, after 3±5 days one strain outgrew the other. A typical competition pattern is shown in Fig. 1. In one experiment the competition pattern of strains PpG7(NAH7) and WD25 (NAH7) was not straightforward (Fig. 2). The two strains harbour the same plasmid encoding naphthalene degradation but have a di€erent chromosomal background. During the ®rst 7 days, strain PpG7(NAH7) outgrew strain WD25(NAH7) (C ˆ 0:51). Subsequently, the pattern reversed and, by day, 10 strain WD25(NAH7) had almost totally outcompeted strain PpG7(NAH7). Notably, the strain thus selected di€ered from the original one by the absorbance values under the same steady-state chemostat conditions (0.36 for the original and 0.46 for the selected strain). This di€erence was retained after prolonged conservation (12 months) in non-selective conditions. The selected strain was designated WD25(NAH7)win. The data from all competition experiments listed in Table 5 were used to determine the ability to compete relative to the most competitive strain BS203(pBS2) (Table 6). When no data for direct competition between the strains investigated were available, the ratio of the growth rates was calculated from other experiments

PpG7(NAH7) meta PpG7(NAH7) meta WD25(pBS3) meta BS203(NPL-1) ortho BS203(pBS2) ortho BS203(pBS2) ortho BS291(pBS4) gentisic BS206(pBS3) meta BS291(pBS4) gentisic BS203(pBS2) ortho

Winning strain

C

1 part: PpG7(NAH7) 2 parts: WD25(NAH7)win WD25(pBS3) = PpG7(NAH7)

0.51 0.00 1.00

WD25(pBS3)

0.65

BS203(NPL-1)

0.18

BS203(pBS2)

0.30

BS203(pBS2)

0.80

BS203(pBS2)

0.00

BS203(pBS2)

0.10

BS206(pBS3)

0.00

BS203(pBS2)

0.58

Fig. 1 Competition between Pseudomonas putida WD25(NAH7) and P. putida WD25(pBS3) in chemostat culture under naphthalene limitation at a dilution rate of 0.05 h)1

497

Fig. 2 Competition between P. putida WD25(NAH7) and P. putida PpG7(NAH7) in chemostat culture under naphthalene limitation at a dilution rate of 0.05 h)1 Table 6 Ratios of growth rates of competing strains and the most competitive strain BS203(pBS2). The pathways are de®ned as in Table 1 Strain (plasmid)

Pathway

C

Way data obtained

BS203(pBS2) BS203(NPL-1) WD25(pBS3) PpG7(NAH7) WD25(NAH7) BS206(pBS3) BS291(pBS4)

Ortho Ortho Meta Meta Meta Meta Gentisic

1.00 0.80 0.58 0.58 0.38 0.20 0.00

By de®nition C6 C2C10 C10 C3C2C10 (C5+C8)/2 C7

C the ratio of the growth rate of the particular strain to that of the most competitive strain BS203(pBS2). Ci the ratio of growth rates from the ith experiment in Table 5

using Eq. (7). For example, the relative growth rate (0.58) for strain WD25(pBS3) was calculated as a product of the constant growth rate ratios from experiments 2 and 10 from Table 5 (1.00 and 0.58 respectively). Results of experiments 5 and 8 (Table 5), with the same pair of strains, were averaged.

Discussion The diversity of degradative plasmids (Boronin 1992; Sayler et al. 1990) controlling the catabolism of naphthalene via various pathways (Boronin et al. 1993; Yen and Serdar 1988) probably, results from the fact that di€erent plasmids provide selective advantages to their bacterial hosts under di€erent environmental conditions. One of the factors that may play an important role in this respect is the anity for the limiting substrate. In this study, we compared the growth and competition behaviour of a number of wild-type and recombinant strains under naphthalene limitation in chemostat culture.

The maximal yield coecient was obtained for strain BS203(pBS2) harbouring the ortho pathway of catechol cleavage (Table 4). Strain BS203(NPL-1), also utilizing the ortho pathway, reveals lower absorbance and cell viability values in comparison not only with BS203(pBS2), but also with PpG7(NAH7) harbouring the meta pathway of catechol cleavage. These ®ndings suggest that growth characteristics in chemostat culture under naphthalene limitation are determined not only by the pathway of xenobiotic oxidation used but probably by other factors also, such as variation between di€erent systems of substrate transport, di€erences in regulation of catabolic gene expression and so on. It seems quite important to know the relative significance of host bacteria and plasmids in xenobiotic degradation to construct new e€ective degrader strains. Introduction of the same plasmid into di€erent hosts, as in the case of NAH7 plasmid in WD25 and PpG7 strains, and pBS3 plasmid in WD25 and BS206 strains, resulted in approximately equal absorbance and yield values (Table 4). In the case of plasmid pBS2 in strain WD25, the lower eciency of carbon assimilation can be explained by the above-mentioned loss of ability of the plasmid to carry out salicylate oxidation after conjugal transfer into strain WD25. On the other hand, the same host, BS203, carrying di€erent plasmids NPL±1 and pBS2, reveals an approximately twofold di€erence in growth characteristics. Comparison of all competition experiments (Table 6) suggests that the plasmid-bearing strains in which naphthalene is degraded by catechol 1,2-dioxygenase for catechol oxidation (ortho pathway) were the most selectively advantageous under naphthalene limitation. Less competitive were the strains with plasmids in which naphthalene catabolism is controlled by catechol 2,3dioxygenase (meta pathway). The least competitive under these conditions was the strain BS291 carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisate. The good competition behaviour of the strains degrading naphthalene through the ortho pathway is surprising in the light of results of earlier batch experiments using strains bearing the same plasmids (Boronin et al. 1993). The highest speci®c growth rate was found for strains harbouring plasmids that control naphthalene catabolism through the meta-pathway of catechol oxiortho dation (lmeta max > lmax ). These results are in accordance with the maximum growth depending on the phenolassimilation pathways obtained for Alcaligenes eutrophus JMP 134 growing on phenol in chemostat culture (MuÈller and Babel 1996). According to Eq. 5, the selective advantage of the strains degrading naphthalene via the ortho pathway of catechol cleavage over those that catabolize naphthalene via the meta pathway can be meta ortho expressed as lmeta < lortho . Taking into max =KS max =KS meta ortho account lmax > lmax , one can conclude that KSmeta > KSortho . The ®nding suggests that strains degrading naphthalene by the ortho pathway of catechol

498

cleavage have lower saturation constants KS than those using the meta pathway. The results of competition experiments (Table 5) show that particular strains constructed by creating new plasmid-bacterium combinations can successfully compete with wild-type strains under conditions of naphthalene limitation and overgrow them in mixed chemostat culture. For instance, the most competitive strain BS203(pBS2) out-competed the naturally occurring strain BS203(NPL-1). Also, strain WD25(NAH7)win has a selective advantage over the wild-type strain PpG7(NAH7). This may be due to adaptation of wildtype microorganisms to particular natural conditions di€ering from chemostat culture. The competition experiment shown in Fig. 2 allows one to suggest the occurrence of spontaneous mutants capable of more ecient assimilation of naphthalene in chemostat culture. Such adaptive mutant derivatives of their respective strains in prolonged chemostat cultivation were described earlier as ®tness mutants (Cox and Gibson 1974; Dykhuizen and Hartl 1983; Helling et al. 1981). The possibility that such adaptive changes occur should be taken into account when results of long-term competition experiments in a chemostat are interpreted. Acknowledgements We are grateful to V. D. Selivanov for technical assistance. This study was supported by the National Institute of Public Health and Environmental Protection (The Netherlands), Federation of European Microbiological Societies, by grants from the Russian State Research and Technical Programme ``Novel Methods in Bioengineering'' (the main trend ``Environmental Biotechnology'') and from the Russian Fundamental Research Foundation.

References Boronin AM (1992) Diversity of Pseudomonas plasmids: To what extent? FEMS Microbiol Lett 100: 461±468 Boronin AM, Starovoitov II, Borisoglebskaya AN (1976) Plasmid of Pseudomonas putida which controls initial stages of naphthalene oxidation. Dokl Akad Nauk SSSR 228: 962±965 Boronin AM, Borisoglebskaya AN, Starovoytov II (1977a) Mutants of the plasmid NPL-1, controlling the oxidation of naphthalene. Dokl Akad Nauk SSSR 235: 494±496 Boronin AM, Kochetkov VV, Starovoitov II, Skryabin GK (1977b) Plasmid pBS2 and pBS3, controlling the oxidation of

naphthalene in bacteria of the genus Pseudomonas. Dokl Akad Nauk SSSR 237: 1205±1208 Boronin AM, Filonov AE, Gayazov RR, Kulakova AN, MshenÂsky YN (1993) Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture. FEMS Microbiol Lett 113: 303±308 Cane PA, Williams PA (1982) The plasmid-coded metabolism of naphthalene and 2-methylnaphthalene in Pseudomonas strains: phenotypic changes correlated with structural modi®cation of the plasmid pWW60-1. J Gen Microbiol 128: 2281±2290 Cox EC, Gibson TC (1974) Selection for high mutation rates in chemostats. Genetics 77: 169±184 Duetz WA, Andel JG van (1991) Stability of TOL plasmid pWWO in Pseudomonas putida mt-2 under non-selective conditions in continuous culture. J Gen Microbiol 137: 1369±1374 Duetz WA, Winston M, Andel JG van, Williams PA (1991) Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate. J Gen Microbiol 137: 1363±1368 Dunn NW, Gunsalus J (1973) Transmissible plasmid coding early enzymes of naphthalene oxidation in Pseudomonas putida. J Bacteriol 114: 974±979 Dykhuizen D, Hartl M (1983) Selection in chemostats. Microbiol Rev 47: 150±168 Evans CGT, Herbert D, Tempest DW (1970) The continuous cultivation of microorganisms. 2. Construction of a chemostat. Methods Microbiol 2: 277±327 Helling RB, Kinney T, Adams J (1981) The maintenance of plasmid-containing organisms in populations of Escherichia coli. J Gen Microbiol 123: 129±141 MuÈller RH, Babel W (1996) Growth rate-dependent expression of phenol-assimilation pathways in Alcaligenes eutrophus JMP 134 ± the in¯uence of formate as an auxilliary energy source on phenol conversion characteristics. Appl Microbiol Biotechnol 46: 156±162 Nakazawa T, Yokota T (1973) Benzoate metabolism in Pseudomonas putida (arvilla) mt-2: demonstration of two benzoate pathways. J Bacteriol 115: 262±267 Sayler GS, Hooper SW, Layton AC, King JMH (1990) Catabolic plasmids of environmental and ecological signi®cance. Microb Ecol 20: 1±20 Shamsuzzaman KM, Barnsley EA (1974) The regulation of naphthalene oxygenase in Pseudomonads. J Gen Microbiol 83: 165± 170 Skryabin GK, Kochetkov VV, Eremin AA, Perebityuk AN, Starovoitov II, Boronin AM (1980) New naphthalene-biodegrading plasmid pBS4. Dokl Acad Nauk SSSR 250: 212± 215 Volkering F, Breure AM, Sterkenburg A, Andel JG van (1992) Microbial degradation of polycyclic aromatic hydrocarbons: e€ect of substrate availability on bacterial growth kinetics. Appl Microbiol Biotechnol 36: 548±552 Yen K-M, Serdar CM (1988) Genetics of naphthalene catabolism in Pseudomonads. Crit Rev Microbiol 15: 247±268