Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100

A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. cepacia PT88. This mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments are identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T- phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.     

Purification and Properties of Component B of 2,4,5-Trichlorophenoxyacetate Oxygenase from Pseudomonas cepacia AC1100

Pseudomonas cepacia AC1100 degrades 2,4,5-trichlorophenoxyacetate (2,4,5-T), an herbicide and chlorinated aromatic compound. Although some progress has been made in understanding 2,4,5-T degradation by AC1100 by molecular analysis, little is known about the biochemistry involved. Enzymatic activity converting 2,4,5-T to 2,4,5-trichlorophenol in the presence of NADH and O(inf2) was detected in cell extracts of AC1100. Phenyl agarose chromatography of the ammonium sulfate-fractionated cell extracts yielded no active single fractions, but the mixing of two fractions, named component A and component B, resulted in the recovery of enzyme activity. Component B was further purified to homogeneity by hydroxyapatite and DEAE chromatographies. Component B had a native molecular weight of 140,000, and it was composed of two 49-kDa (alpha)-subunits and two 24-kDa (beta)-subunits. Component B was red, and its spectrum in the visible region had maxima at 430 and 560 nm (shoulder), whereas upon reduction it had maxima at 420 (shoulder) and 530 nm. Each mole of (alpha)(beta) heterodimer contained 2.9 mol of iron and 2.1 mol of labile sulfide. These properties suggest strong similarities between component B and the terminal oxygenase components of the aromatic ring-hydroxylating dioxygenases. Component A was highly purified but not to homogeneity. The reconstituted 2,4,5-T oxygenase, consisting of components A and B, converted 2,4,5-T quantitatively into 2,4,5-trichlorophenol and glyoxylate with the coconsumption of NADH and O(inf2).     

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.     

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia.

A pure culture of Pseudomonas cepacia, designated AC1100, that can utilize 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as its sole source of carbon and energy was isolated. An actively growing culture of AC1100 was able to degrade more than 97% of 2,4,5-T, present at 1 mg/ml, within 6 days as determined by chloride release, gas chromatographic, and spectrophotometric analyses. The ability of AC1100 to oxidize a variety of chlorophenols and related compounds is also reported.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia.

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.     

Effect of introducing genetically engineered microorganisms on soil microbial community diversity

Abstract Introducing the genetically engineered microorganism Pseudomonas cepacia AC1100 into soil microcosms resulted in elevated taxonomic diversity determined by phenotypic analyses of culturable isolates and genetic diversity determined by analysis of the heterogeneity of total microbial community DNA reannealing kinetics. The greatest impact occurred when P. cepacia AC1100 was introduced along with the herbicide 2,4,5-T, which P. cepacia AC1100 can degrade. The data suggests that both changes in the balance of populations and genetic recombination contributed to the increased diversity.     

Degradation of the chlorinated phenoxyacetate herbicides 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by pure and mixed bacterial cultures.

Combined cell suspensions of the 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)-metabolizing organism Pseudomonas cepacia AC1100, and the 2,4-dichlorophenoxyacetic acid (2,4-D)-metabolizing organism Alcaligenes eutrophus JMP134 were shown to effectively degrade either of these compounds provided as single substrates. These combined cell suspensions, however, poorly degraded mixtures of the two compounds provided at the same concentrations. Growth and viability studies revealed that such mixtures of 2,4-D and 2,4,5-T were toxic to AC1100 alone and to combinations of AC1100 and JMP134. High-pressure liquid chromatography analyses of culture supernatants of AC1100 incubated with 2,4-D and 2,4,5-T revealed the accumulation of chlorohydroquinone as an apparent dead-end catabolite of 2,4-D and the subsequent accumulation of both 2,4-dichlorophenol and 2,4,5-trichlorophenol. JMP134 cells incubated in the same medium did not catabolize 2,4,5-T and were also inhibited in initiating 2,4-D catabolism. A new derivative of strain AC1100 was constructed by the transfer into this organism of the 2,4-D-degradative plasmid pJP4 from strain JMP134. This new strain, designated RHJ1, was shown to efficiently degrade mixtures of 2,4-D and 2,4,5-T through the simultaneous metabolism of these compounds.     

Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites.

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.     

Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.     

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.     

Metabolism of Halophenols by 2,4,5-trichlorophenoxyacetic acid-degrading Pseudomonas cepacia.

Resting cells of 2,4,5-trichlorophenoxyacetic acid-grown Pseudomonas cepacia AC1100 were able to completely and rapidly dechlorinate several chlorine-substituted phenols, including 2,4,5-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol. Several other trichlorophenols were only partially dechlorinated. The evidence suggests that 2,4,5-trichlorophenol is an intermediate in the degradation of 2,4,5-trichlorophenoxyacetic acid by strain AC1100. Moreover, although strain AC1100 was isolated by selection for growth on a chlorinated aromatic compound, brominated and fluorinated analogs were efficiently dehalogenated by strain AC1100 resting cells, whereas an iodinated analog was poorly dehalogenated.     

Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.

Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself.     

Physiological and cellular responses of the 2,4-D degrading bacterium,Burkholderia cepacia YK-2, to the phenoxyherbicides 2,4-D and 2,4,5-T

Our previous research has demonstrated that novel 43-kDa DnaK and 41-kDa GroEL proteins are synthesized in Burkholderia sp. YK-2 in response to sublethal concentrations of 2,4-D stress [Cho et al. (2000) Curr Microbiol 41:33-38]. In this study, we have extended this work to examine the cellular responses of strain YK-2 to stresses induced in response to the phenoxyherbicides 2,4-D or 2,4,5-T. Strain YK-2 exhibited a more sensitive response to 2,4,5-T stress than to 2,4-D stress, as shown in physiological and morphological changes, suggesting a greater cytotoxic effect of 2,4,5-T. SEM analyses revealed the presence of perforations and irregular rod forms with wrinkled surfaces for cells treated with either herbicide. These irregularities were found more frequently for 2,4,5-T-treated cells than for 2,4-D-treated cells. Analysis of cellular fatty acids showed similar effects in the shifts of total cellular fatty acid composition in response to 2,4-D and 2,4,5-T. Strain YK-2 could degrade 2.25 m M 2,4-D completely during 28 h of incubation with transient production of 2,4-dichlorophenol as a metabolite; however, 2,4,5-T was not catabolized at any of the concentrations tested. BIOLOG and 16S rDNA analyses revealed that strain YK-2 was 98% similar to the Burkholderia cepacia species cluster; therefore, we have designated this strain as B. cepacia YK-2.     

Cloning, characterization, and sequence analysis of the clcE gene encoding the maleylacetate reductase of Pseudomonas sp. strain B13.

A 3,167-bp PstI fragment of genomic DNA from Pseudomonas sp. strain B13 was cloned and sequenced. The gene clcE consists of 1,059 nucleotides encoding a protein of 352 amino acids with a calculated mass of 37,769 Da which showed maleylacetate reductase activity. The protein had significant sequence similarities with the polypeptides encoded by tcbF of pP51 (59.4% identical positions), tfdF of pJP4 (55.1%), and tftE of Burkholderia cepacia AC1100 (53.1%). The function of TcbF as maleylacetate reductase was established by an enzyme assay.     

Cleavage of Treponema denticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent on PrtP

Analysis of potential virulence factors of oral spirochetes focuses on surface and secreted proteins. The Treponema denticola chymotrypsin-like protease (CTLP) is implicated in degradation of host cell molecules and contributes to tissue invasion. The CTLP complex, composed of the 72-kDa PrtP protein and two auxiliary proteins with molecular masses of approximately 40 and 30 kDa, is also involved in localization and oligomerization of the T. denticola major surface protein (Msp). The larger auxiliary protein was reported to be encoded by an open reading frame (ORF2) directly upstream of prtP. The deduced 39-kDa translation product of ORF2 contains a sequence matching the N-terminal sequence determined from one of the CTLP complex proteins. No proteins with significant homology are known, nor was information available on the third protein of the complex. DNA sequence analysis showed that ORF2 extended an additional 852 bp upstream of the reported sequence. The complete gene, designated prcA, encodes a predicted N-terminally-acylated polypeptide of approximately 70 kDa. Isogenic mutants with mutations in prtP, prcA, and prcA-prtP all lacked CTLP protease activity. The prcA mutant lacked all three CTLP proteins. The prcA-prtP mutant produced only a C-terminally-truncated 62-kDa PrcA protein. The prtP mutant produced a full-length 70-kDa PrcA. Immunoblot analysis of recombinant PrcA constructs confirmed that PrcA is cleaved to yield the two smaller proteins of the CTLP complex, designated PrcA1 and PrcA2. These data indicate that PrtP is required for cleavage of PrcA and suggest that this cleavage may be required for formation or stability of outer membrane complexes.     

Identification of an operon involved in sulfolipid biosynthesis in Rhodobacter sphaeroides.

Two new mutants of Rhodobacter sphaeroides deficient in sulfolipid accumulation were isolated by directly screening mutagenized cell lines for polar lipid composition by thin-layer chromatography of lipid extracts. A genomic clone which complemented the mutations in these two lines, but not the previously described sulfolipid-deficient sqdA mutant, was identified. Sequence analysis of the relevant region of the clone revealed three, in tandem open reading frames, designated sqdB, ORF2, and sqdC. One of the mutants was complemented by the sqdB gene, and the other was complemented by the sqdC gene. Insertional inactivation of sqdB also inactivated sqdC, indicating that sqdB and sqdC are cotranscribed. The N-terminal region of the 46-kDa putative protein encoded by the sqdB gene showed slight homology to UDP-glucose epimerase from various organisms. The 30-kDa putative protein encoded by ORF2 showed very striking homology to rabbit muscle glycogenin, a UDP-glucose utilizing, autoglycosylating glycosyltransferase. The 26-kDa putative protein encoded by the sqdC gene was not homologous to any protein of known function.     

Molecular analysis of an essential gene upstream of rpoN in Rhizobium NGR234

Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.