Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms.

Two genetically engineered microorganisms (GEMs), Pseudomonas sp. strain B13 FR1(pFRC20P) (FR120) and Pseudomonas putida KT2440(pWWO-EB62) (EB62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. FR120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3CB) and 4-methyl benzoate (4MB); EB62 contains a derivative TOL plasmid-encoded degradative pathway for toluene experimentally evolved so that it additionally processes 4-ethyl benzoate (4EB). Experiments assessed survival of the GEMs, their ability to degrade target substrates, and lateral transfer of plasmid-encoded recombinant DNA. GEMs added at initial densities of 10(6) to 10(7) bacteria per ml of activated sludge declined to stable population densities of 10(4) to 10(5) bacteria per ml. FR120 degraded combinations of 3CB and 4MB (1 mM each) following 3 days of adaptation in the microcosms. Indigenous microorganisms required an 8-day adaptation period before degradation of 4MB was observed; 3CB was degraded only after the concentration of 4MB was much reduced. The indigenous microbial community was killed when both compounds were present at concentrations of 4.0 mM. However, in parallel microcosms containing FR120, the microbial community maintained a normal density of viable cells. Indigenous microbes readily degraded 4EB (2 mM), and EB62 did not significantly increase the observed rate of degradation. In filter matings, transfer of pFRC20P, which specifies mobilization but not transfer functions, from FR120 to P. putida UWC1 was not detectable (< 10(-7) transconjugants per donor cell).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of commensal relationships on the spatial structure of a surface-attached microbial consortium

A flow cell-grown model consortium consisting of two organisms, Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1), was studied. These bacteria have the potential to interact metabolically because Pseudomonas sp. B13(FR1) can metabolize chlorobenzoate produced by Burkholderia sp. LB400 when grown on chlorobiphenyl. The expected metabolic interactions in the consortium were demonstrated by high performance liquid chromatography (HPLC) analysis. The spatial structure of the consortium was studied by fluorescent in situ rRNA hybridization and scanning confocal laser microscopy. When the consortium was fed with medium containing a low concentration of chlorobiphenyl, microcolonies consisting of associated Burkholderia sp. LB400 and Pseudomonas sp. B13(FR1) bacteria were formed, and separate Pseudomonas sp. B13(FR1) microcolonies were evidently not formed. When the consortium was fed citrate, which can be metabolized by both species, the two species formed separate microcolonies. The structure development in the consortium was studied online using a gfp -tagged Pseudomonas sp. B13(FR1) derivative. After a shift in carbon source from citrate to a low concentration of chlorobiphenyl, movement of the Pseudomonas sp. B13(FR1) bacteria led to a change in the spatial structure of the consortium from the unassociated form towards the associated form within a few days. Experiments involving a gfp -based Pseudomonas sp. B13(FR1) growth activity reporter strain indicated that chlorobenzoate supporting growth of Pseudomonas sp. B13(FR1) is located close to the Burkholderia sp. LB400 microcolonies in chlorobiphenyl-grown consortia.     

Degradation of 2-chlorobenzoate by in vivo constructed hybrid pseudomonads

Abstract 5-Chlorosalicylate degrading bacteria were obtained from the mating between Pseudomonas sp. strain WR401 and Pseudomonas sp. strain B13. Further selection of the hybrid organisms for growth on 2-chlorobenzoate allowed the isolation of strains such as JH230. During growth on 2-chlorobenzoate stoichiometric amounts of chloride were released. Steps in the pathway for 2-chlorobenzoate degradation were determined by simultaneous adaptation studies, assays of enzymes in cell extracts and cooxidation of the analogous substrate 2-methylbenzoate. Results indicate that 2-chlorobenzoate was degraded to 3-chlorocatechol. Ring cleavage of 3-chlorocatechol was by a catechol 1,2-dioxygenase to from 2-chloro- cis, cis - muconate. Further degradation runs via 4-carboxymethylenebut-2-en-4-olide.     

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P in continuous culture

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells.     

Low-Frequency Horizontal Transfer of an Element Containing the Chlorocatechol Degradation Genes from Pseudomonas sp. Strain B13 to Pseudomonas putida F1 and to Indigenous Bacteria in Laboratory-Scale Activated-Sludge Microcosms

The possibilities for low-frequency horizontal transfer of the self-transmissible chlorocatechol degradative genes (clc) from Pseudomonas sp. strain B13 were investigated in activated-sludge microcosms. When the clc genes were transferred into an appropriate recipient bacterium such as Pseudomonas putida F1, a new metabolic pathway for chlorobenzene degradation was formed by complementation which could be selected for by the addition of mono- or 1,4-dichlorobenzene (CB). Under optimized conditions with direct donor-recipient filter matings, very low transfer frequencies were observed (approximately 3.5 × 10⁻⁸ per donor per 24 h). In contrast, in matings on agar plate surfaces, transconjugants started to appear after 8 to 10 days, and their numbers then increased during prolonged continuous incubation with CB. In activated-sludge microcosms, CB-degrading (CB⁺) transconjugants of strain F1 which had acquired the clc genes were detected but only when strain B13 cell densities of more than 10⁵ CFU/ml could be maintained by the addition of its specific growth substrate, 3-chlorobenzoate (3CBA). The CB⁺ transconjugants reached final cell densities of between 10² and 10³ CFU/ml. When strain B13 was inoculated separately (without the designated recipient strain F1) into an activated-sludge microcosm, CB⁺ transconjugants could not be detected. However, in this case a new 3CBA-degrading strain appeared which had acquired the clc genes from strain B13. The effects of selective substrates on the survival and growth of and gene transfer between bacteria degrading aromatic pollutants in a wastewater ecosystem are discussed.     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs)

Abstract In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph -module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.     

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-D?bler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.     

Involvement of a chlorobenzoate-catabolic transposon, Tn5271, in community adaptation to chlorobiphenyl, chloroaniline, and 2,4-dichlorophenoxyacetic acid in a freshwater ecosystem.

A chlorobenzoate-catabolic transposon (Tn5271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying Tn5271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of Tn5271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate- and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets Tn5271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.     

Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on dehydrogenation of 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid

The dehydrogenation of substituted 3,5-cyclohexadiene-1,2-diol-1-carboxylic acids by dihydrodihydroxybenzoic acid dehydrogenases from benzoate grown cells of Alcaligenes eutrophus and Pseudomonas sp. B 13 and 3-chlorobenzoate grown cells of the latter organism was examined. No significant differences (Km and Vrel values) were detected for the enzymes from both organisms. The same dihydrodihydroxybenzoic acid dehydrogenase is formed in Pseudomonas sp. B13 during growth on benzoate as well as on 3-chlorobenzoate. The lower turnover rates of 3- and 5-chlorodrodihydroxybenzoic acid compared to dihydrodihydroxybenzoic acid are counterbalanced by an increase in specific activity. With the exception of 4-substituted dihydrodihydroxybenzoic acids exhibiting relative high Km values, only slight sterical and electronic substituent effects are evident. Reaction rates were never reduced to a critical level.     

Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms.

Pseudomonas sp. strain B13 FR1(pFRC20P) is a genetically engineered microorganism (GEM) which is able to degrade chloro- and methylaromatics through a constructed ortho cleavage pathway. The fate of the GEM and its ability to degrade substituted aromatic compounds in two different aquatic sediments was investigated by using a microcosm system which consisted of intact layered sediment cores with an overlying water column. The GEM survived in Lake Plussee and in Rhine river sediments at densities of approximately 10(5) bacteria per g (dry weight) (1 to 5% of the total CFU) throughout a 4-week period of investigation. According to several criteria, the microcosm system was stable and healthy throughout the experiment and the addition of the GEM did not affect the total number of extractable CFU (I. Wagner-Döbler, R. Pipke, K. N. Timmis, and D. F. Dwyer, Appl. Environ. Microbiol. 58:1249-1258, 1992). When compared with uninoculated controls, the presence of the GEM enhanced the rate of degradation of a mixture of 3-chlorobenzoate and 4-methylbenzoate (25 microns each) which had been added to the water column of the sediment cores.     

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.     

Nitrogen control of atrazine utilization in Pseudomonas sp. strain ADP

Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas(-) mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas(-) mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.     

Degradation of chloro- and methyl-substituted benzoic acids by a genetically modified microorganism

Degradation of 3-chlorobenzoic acid (3CB), 4-chlorobenzoic acid (4CB), and 4-methylbenzoic acid (4MB) as single substrates (carbon sources) and as a substrate mixture were studied in batch and continuous culture using the genetically modified microorganism Pseudomonas sp. B13 FR1 SN45P. The strain was able to mineralize the single compounds as well as the substrate mixture completely. Conversion of the three compounds in the substrate mixture proceeded simultaneously. Maximum specific substrate conversion rates were calculated to be 0.9 g g(-1) h(-1) for 3 CB and 4CB and 1.1 g g(-1) h(-1) for 4MB. Mass balances indicated the transient accumulation of pathway intermediates during batch cultivations. Hence, the rate limiting step in the degradative pathway is not the initial microbial attack of the original substrate or its transport through the cell membrane. Degradation rates on 3CB were comparable to those of the parent strain Pseudomonas sp. B13. The stability of the degradation pathways of strain Pseudomonas sp. B13 FR1 SN45P could be demonstrated in a continuous cultivation over 3.5 months (734 generation times) on 3CB, 4MB, and 4CB, which were used as single carbon sources one after the other.     

Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(pPOB) and two modified Pseudomonas strains.

Pseudomonas pseudoalcaligenes POB310(pPOB) and Pseudomonas sp. strains B13-D5(pD30.9) and B13-ST1(pPOB) were introduced into soil microcosms containing 3-phenoxybenzoic acid (3-POB) in order to evaluate and compare bacterial survival, degradation of 3-POB, and transfer of plasmids to a recipient bacterium. Strain POB310 was isolated for its ability to use 3-POB as a growth substrate; degradation is initiated by POB-dioxygenase, an enzyme encoded on pPOB. Strain B13-D5 contains pD30.9, a cloning vector harboring the genes encoding POB-dioxygenase; strain B13-ST1 contains pPOB. Degradation of 3-POB in soil by strain POB310 was incomplete, and bacterial densities decreased even under the most favorable conditions (100 ppm of 3-POB, supplementation with P and N, and soil water-holding capacity of 90%). Strains B13-D5 and B13-ST1 degraded 3-POB (10 to 100 ppm) to concentrations of <50 ppb with concomitant increases in density from 10(6) to 10(8) CFU/g (dry weight) of soil. Thus, in contrast to strain POB310, the modified strains had the following two features that are important for in situ bioremediation: survival in soil and growth concurrent with removal of an environmental contaminant. Strains B13-D5 and B13-ST1 also completely degraded 3-POB when the inoculum was only 30 CFU/g (dry weight) of soil. This suggests that in situ bioremediation may be effected, in some cases, with low densities of introduced bacteria. In pure culture, transfer of pPOB from strains POB310 and B13-ST1 to Pseudomonas sp. strain B13 occurred at frequencies of 5 x 10(-7) and 10(-1) transconjugant per donor, respectively. Transfer of pPOB from strain B13-ST1 to strain B13 was observed in autoclaved soil but not in nonautoclaved soil; formation of transconjugant bacteria was more rapid in soil containing clay and organic matter than in sandy soil. Transfer of pPOB from strain POB310 to strain B13 in soil was never observed.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl.

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Simultaneous degradation of chloro- and methyl-substituted aromatic compounds: competition between Pseudomonas strains using the ortho and meta pathway or the ortho pathway exclusively

Pseudomonas sp. D7-4 and Pseudomonas sp. B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chlorobenzoate alone. The competition behaviour of these strains was compared in batch and in chemostat cultures. Despite misrouting of metabolites, strain WR201 was competitive, in a lot of the competition experiments, with mixtures of these substrates. Only in a narrow range of the mixing ratio of chloro- and methylbenzoate was the presence of both the meta and ortho pathways a disadvantage for competitiveness. Outside these ranges other attributes, such as high growth rates or short lag periods, of a respective strain were even more essential for one strain to outcompete another. Received: 13 February 1998 / Received revision: 28 April 1998 / Accepted: 30 April 1998     

Metabolism of both 4-chlorobenzoate and toluene under denitrifying conditions by a constructed bacterial strain.

T1, a dentrifying bacterium originally isolated for its ability to grow on toluene, can also metabolize 4-hydroxybenzoate and other aromatic compounds under denitrifying conditions. A cosmid clone carrying the three genes that code for the 4-chlorobenzoate dehalogenase enzyme complex isolated from the aerobic bacterium Pseudomonas sp. strain CBS3 was successfully conjugated into strain T1. The cloned enzyme complex catalyzes the hydrolytic dechlorination of 4-chlorobenzoate to 4-hydroxybenzoate. Since molecular oxygen is not required for the dehalogenation reaction, the transconjugate strain of T1 (T1-pUK45-10C) was able to grow on 4-chlorobenzoate in the absence of O2 under denitrifying conditions. 4-Chlorobenzoate was dehalogenated to 4-hydroxybenzoate, which was then further metabolized by strain T1. The dehalogenation and metabolism of 4-chlorobenzoate were nitrate dependent and were coupled to the production of nitrite and nitrogen gas. 4-Bromobenzoate was also degraded by this strain, while 4-iodobenzoate was not. Additionally, when T1-pUK45-10C was presented with a mixture of 4-chlorobenzoate and toluene, simultaneous degradation of the compounds was observed. These results illustrate that dechlorination and degradation of aromatic xenobiotics can be mediated by a pure culture in the absence of oxygen. Furthermore, it is possible to expand the range of xenobiotic substrates degradable by an organism, and it is possible that concurrent metabolism of these substrates can occur.     

Motility and chemotaxis of Pseudomonas sp. B4 towards polychlorobiphenyls and chlorobenzoates

The polychlorinated biphenyl (PCB)-degrading Pseudomonas sp. B4 was tested for its motility and ability to sense and respond to biphenyl, its chloroderivatives and chlorobenzoates in chemotaxis assays. Pseudomonas sp. B4 was attracted to biphenyl, PCBs and benzoate in swarm plate and capillary assays. Chemotaxis towards these compounds correlated with their use as carbon and energy sources. No chemotactic effect was observed in the presence of 2- and 3-chlorobenzoates. Furthermore, a toxic effect was observed when the microorganism was exposed to 3-chlorobenzoate. A nonmotile Pseudomonas sp. B4 transformant and Burkholderia xenovorans LB400, the laboratory model strain for PCB degradation, were both capable of growing in biphenyl as the sole carbon source, but showed a clear disadvantage to access the pollutants to be degraded, compared with the highly motile Pseudomonas sp. B4, stressing the importance of motility and chemotaxis in this environmental biodegradation.