Use of an s-triazine nitrogen source to select for and isolate a recombinant chlorobenzoate-degrading Pseudomonas

Abstract A novel mating system, which uses the s -triazine nitrogen source (cyanuric acid, CA) as a selective nutritional marker, was developed to evaluate mobility and facilitate identification of chlorobenzoate (CBA) degradation genes in Pseudomonas aeruginosa strain JB2. Matings were done between strain JB2, which is unable to grow with CA, and a CA-utilizer Pseudomonas sp. strain D on 2-CBA-CA and 3-CBA-CA media. Isolates were recovered only from the 2-CBA-CA and were all identified as strain D derivatives. Subtractive hybridization analysis of genomic DNAs from the parental strains and a selected isolate, Pseudomonas sp. strain JPL, was used to determine that the latter organism had acquired approx. 16.1 kb of DNA from strain JB2.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2.

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.     

Integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular cloning for the identification and functional characterization of mobile ortho-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2.

Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the alpha and beta subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151-2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa approximately 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB(JB2) appeared to differ from that reported for OhbAB(142) primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa(-) mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Integration of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and Molecular Cloning for the Identification and Functional Characterization of Mobile ortho-Halobenzoate Oxygenase Genes in Pseudomonas aeruginosa Strain JB2

Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the α and β subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151–2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa ≈ 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB_JB2 appeared to differ from that reported for OhbAB₁₄₂ primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa⁻ mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution.     

Metabolic engineering of bacteria for environmental applications: construction of Pseudomonas strains for biodegradation of 2-chlorotoluene

In this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct Pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. The assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the TOD system of Pseudomonas putida F1 (todC1C2BA), which affords the bioconversion of 2-chlorotoluene into 2-chlorobenzaldehyde by virtue of its residual methyl-monooxygenase activity on o-substituted substrates. A second catabolic segment encoded the entire upper TOL pathway from pWW0 plasmid of P. putida mt-2. The enzymes, benzyl alcohol dehydrogenase (encoded by xylB) and benzaldehyde dehydrogenase (xylC) of this segment accept o-chloro-substituted substrates all the way down to 2-chlorobenzoate. These TOL and TOD segments were assembled in separate mini-Tn5 transposon vectors, such that expression of the encoded genes was dependent on the toluene-responsive Pu promoter of the TOL plasmid and the cognate XylR regulator. Such gene cassettes (mini-Tn5 [UPP2] and mini-Tn5 [TOD2]) were inserted in the chromosome of the 2-chlorobenzoate degraders Pseudomonas aeruginosa PA142 and P. aeruginosa JB2. GC-MS analysis of the metabolic intermediates present in the culture media of the resulting strains verified that these possessed, not only the genetic information, but also the functional ability to mineralise 2-chlorotoluene. However, although these strains did convert the substrate into 2-chlorobenzoate, they failed to grow on 2-chlorotoluene as the only carbon source. These results pinpoint the rate of the metabolic fluxes, the non-productive spill of side-metabolites and the physiological control of degradative pathways as the real bottlenecks for degradation of certain pollutants, rather than the theoretical enzymatic and genetic fitness of the recombinant bacteria to the process. Choices to address this general problem are discussed.     

Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a sphingomonas strain that degrades various polycyclic aromatic hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Bacteria--degraders of polycyclic aromatic hydrocarbons, isolated from soil and bottom sediments in salt-mining areas

Fifteen bacterial strains capable of utilizing naphthalene, phenanthrene, and biphenyl as the sole sources of carbon and energy were isolated from soils and bottom sediments contaminated with waste products generated by chemical and salt producing plants. Based on cultural, morphological, and chemotaxonomic characteristics, ten of these strains were identified as belonging to the genera Rhodococcus, Arthrobacter, Bacillus, and Pseudomonas. All ten strains were found to be halotolerant bacteria capable of growing in nutrient-rich media at NaCl concentrations of 1-1.5 M. With naphthalene as the sole source of carbon and energy, the strains could grow in a mineral medium with 1 M NaCl. Apart from being able to grow on naphthalene, six of the ten strains were able to grow on phenanthrene; three strains, on biphenyl; three strains, on octane; and one strain, on phenol. All of the strains were plasmid-bearing. The plasmids of the Pseudomonas sp. strains SN11, SN101, and G51 are conjugative, contain genes responsible for the degradation of naphthalene and salicylate, and are characterized by the same restriction fragment maps. The transconjugants that gained the plasmid from strain SN11 acquired the ability to grow at elevated NaCl concentrations. Microbial associations isolated from the same samples were able to grow at a NaCl concentration of 2.5 M.     

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.     

Cloning of Pseudomonas sp. strain CBS3 genes specifying dehalogenation of 4-chlorobenzoate.

The degradation of 4-chlorobenzoate (4-CBA) by Pseudomonas sp. strain CBS3 is thought to proceed first by the dehalogenation of 4-CBA to 4-hydroxybenzoate (4-HBA), which is then metabolized following the protocatechuate branch of the beta-ketoadipate pathway. The cloning of the 4-CBA dehalogenation system was carried out by constructing a gene bank of Pseudomonas sp. strain CBS3 in Pseudomonas putida. Hybrid plasmid pPSA843 contains a 9.5-kilobase-pair fragment derived from the chromosome of Pseudomonas sp. strain CBS3. This plasmid confers on P. putida the ability to dehalogenate 4-CBA and grow on 4-CBA as the only source of carbon. However, pPSA843 did not complement mutants of P. putida unable to grow on 4-HBA (POB-), showing that the genes involved in the metabolism of 4-HBA were not cloned. Subcloning of Pseudomonas sp. strain CBS3 genes revealed that most of the insert is required for the dehalogenation of 4-CBA, suggesting that more than one gene product is involved in this dehalogenation.     

Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [¹⁴C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1_a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4.

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.     

Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.     

Molecular detection and diversity of polycyclic aromatic hydrocarbon-degrading bacteria isolated from geographically diverse sites

Nineteen polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from environmental samples in Kuwait, Indonesia, Thailand, and Japan by enrichment with either naphthalene or phenanthrene as a sole carbon source. Sequence analyses of the 16-S rRNA gene indicated that at least seven genera (Ralstonia, Sphingomonas, Burkholderia, Pseudomonas, Comamonas, Flavobacterium, and Bacillus) were present in this collection. Determination of the ability of the isolates to use PAH and its presumed catabolic intermediates suggests that the isolates showed multiple phenotypes in terms of utilization and degradation pathways. The large subunit of the terminal oxygenase gene (phnAc) from Burkholderia sp. strain RP007 hybridized to 32% (6/19) of the isolates, whilst gene probing using the large subunit of terminal oxygenase gene (pahAc) from Pseudomonas putida strain OUS82 revealed no pahAc-like genes amongst the isolates. Using three degenerated primer sets (pPAH-F/NR700, AJ025/26, and RieskeF/R), targeting a conserved region with the genes encoding the large subunit of terminal oxygenase successfully amplified material from 6 additional PAH-degrading isolates. Sequence analyses showed that the large subunit of terminal oxygenase in 4 isolates was highly homologous to the large subunit of naphthalene dioxygenase gene from Ralstonia sp. strain U2. However, we could not obtain any information on the oxygenase system involved in the naphthalene and/or phenathrene degradation by 7 other strains. These results suggest that PAH-degrading bacteria are diverse, and that there are still many unidentified PAH-degrading bacteria.     

Community shifts in a seeded 3-chlorobenzoate degrading membrane biofilm reactor: indications for involvement of in situ horizontal transfer of the clc-element from inoculum to contaminant bacteria

Pseudomonas putida BN210, carrying the self- transferable clc-element encoding degradation of 3-chlorobenzoate on the chromosome, was used as inoculum in different membrane biofilm reactors treating 3-chlorobenzoate-contaminated model wastewater. Analysis of the bacterial population in the effluent and in the biofilm showed the loss of BN210 beyond detection from the reactors and the appearance of several novel 3-chlorobenzoate mineralizing bacteria mainly belonging to the beta-proteobacteria. In contrast, in non-inoculated reactors, no 3-chlorobenzoate degradation was observed and no 3-chlorobenzoate degraders could be recovered. Southern blots hybridization of genomic DNA using clc-element-specific probes and FIGE analysis indicated the presence of the complete clc-element in one or more copies in the isolates. Moreover, the isolates could transfer the clc genes to Ralstonia metallidurans recipients. Two representative reactor isolates, Ralstonia sp. strains KP3 and KP9 demonstrated a higher growth rate on 3-chlorobenzoate than strain BN210 in batch cultures. When BN210, KP3 and KP9 were simultaneously inoculated in a membrane reactor supplied with 3-chlorobenzoate, strain KP3 outcompeted the two other strains and remained the major 3-chlorobenzoate degrading population in the reactor. Our data suggest that in situ horizontal transfer of the clc-element from the inoculum to contaminant bacteria in the reactors was involved in the establishment of novel 3-chlorobenzoate degrading populations that were more competitive under the defined reactor conditions than the inoculum strain.     

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .