Strain AC1100 Research Articles

Burkholderia phenoliruptrix sp. nov., to Accommodate the 2,4,5-Trichlorophenoxyacetic Acid and Halophenol-Degrading Strain AC1100

Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid. We performed a polyphasic-taxonomic study to determine its taxonomic position. The G+C content of strain AC1100 was 62.6 mol%. On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the beta-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity). DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species. Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp. nov. is proposed. The type strain is AC1100T (= LMG 22037T = CCUG 48558T).

Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS–PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K m values of 1±0.1 μM, 32±5 μM and 4±2 μM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K i=27 μM. When other polychlorinated substrates were studied, IC 50 values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O 2 consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.

A fusion promoter created by a new insertion sequence, IS1490, activates transcription of 2,4,5-trichlorophenoxyacetic acid catabolic genes in Burkholderia cepacia AC1100.

Transposition and transcriptional activation by insertion sequences in Burkholderia cepacia AC1100 were investigated. Two closely related new elements, IS1413 and IS1490, were identified and characterized. These elements are not highly related to other insertion sequences identified in AC1100 or other B. cepacia isolates. Based on their structures and the sequences of the inverted terminal repeats and the putative transposase protein, the insertion elements (IS elements) are similar to IST2 of Thiobacillus ferrooxidans and several related elements. All the IS elements that have been identified in this strain are found in multiple copies (10 to 40), and they have high-level promoter activity capable of stimulating transcription from a distance up to 500 bp from a target gene. Strain AC1100 was originally isolated after prolonged selection for the ability to utilize the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole carbon source. Three IS elements are located near the first gene of the 2,4,5-T catabolic pathway, tftA. IS1490 inserted 110 bp upstream of tftA and created a fusion promoter responsible for constitutive transcription of the gene. Our results confirm the hypothesis that IS elements play a central role in transcription of 2,4,5-T genes and likely have stimulated rapid evolution of the metabolic pathway.

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight

Many microorganisms in nature have evolved new genes which encode catabolic enzymes specific for chlorinated aromatic substrates, allowing them to utilize these compounds as sole sources of carbon and energy. An understanding of the evolutionary mechanisms involved in the acquisition of such genes may facilitate the development of microorganisms with enhanced capabilities of degrading highly chlorinated recalcitrant compounds. A number of studies have been based on microorganisms isolated from the environment which utilize simple chlorinated substrates. In our laboratory, a selective technique was used to isolate microorganisms capable of degrading highly chlorinated compounds, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as sole sources of carbon and energy. This article summarizes the genetic and biochemical information obtained regarding the pathway of degradation, the mechanism of recruitment of new genes, and the organization of the degradative genes. In addition, we discuss the potential practical application of such microorganisms in the environment.

Identification of a Meta-Cleavage Pathway for Metabolism of Phenoxyacetic Acid and Phenol in Pseudomonas cepacia AC1100

Pseudomonas cepacia strain AC1100 grows luxuriantly on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) but does not utilize phenoxyacetic acid. After long-term selective pressure on phenoxyacetic acid, mutants designated as strain PAA, capable of utilizing phenoxyacetic acid as well as phenol, emerged spontaneously at a frequency of 1×10−8. A deletion mutant strain PT88, which is devoid of a part of 2,4,5-T metabolic pathway, generated neither phenoxyacetic acid utilizing nor phenol-utilizing mutants. The wild type (Wt) strain AC1100 and all its mutants utilized benzoate viaortho-cleavage pathway. However, only mutant strain PAA harbored the whole set of enzymes required for utilization of phenol viameta-cleavage pathway. The results suggest that Wt strain AC1100 carries silent genes for meta-cleavage pathway which are expressed in strain PAA enabling it to utilize phenoxyacetic acid and phenol. Gene activation is presumed to be due to the translocation of insertion elements.

Pulmonary exposure of mice to “engineered” pseudomonads influences intestinal microbiota populations

Microbial biotechnology applications have prompted research into their potential impacts on human health and the environment. In this study, a mouse model was used to evaluate indirect effects (e.g., alteration of the intestinal microbiota) of pulmonary exposure to representative biotechnology agents (Pseudomonas aeruginosa strain AC869 and Pseudomonas cepacia strain AC 1100) selected for their ability to degrade hazardous chemicals. CD-I® mice were challenged intranasally with approximately 103 or 107 colony-forming units (cfu) of strain AC869 or 108 cfu of strain AC1100. At time intervals, clearance of the microorganisms and effects on resident microbiota were determined. When the low (103 cfu) dose was administered, strain AC869 was not recovered from the small intestine but was detectable in the cecum and lungs 3 h after treatment and persisted in the nasal cavity intermittently for 14 d. Treatment of animals with 107 cfu of strain AC869 resulted in detection 14 d following treatment. Strain AC869 challenge modified the small intestinal anaerobe count and cecal obligately anaerobic gram-negative rods (OAGNR) and lactobacilli. Following exposure, Pseudomonas cepacia strain AC1100 persisted in the lungs for 7 d and was recovered from the small intestine, cecum, and nasal cavity 2 d following treatment. Strain AC1100 treatment impacted the small intestinal anaerobe count, OAGNR counts, and reduced lactobacilli numbers. Strain AC1100 also altered the cecal OAGNR and lactobacilli. Therefore, pulmonary treatment of mice with Pseudomonas aeruginosa or cepacia affects the balance of the protective intestinal microbiota, which may cause further negative health effects (e.g., harbored pathogen multiplication, opportunistic pathogen invasion, bacterial translocation).

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.

Repeated sequences including RS 1100 from Pseudomonas cepacia AC1100 function as IS elements

Several lines of evidence were obtained that the previously identified, repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences for a newly identified 3400 bp repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T − strain PT88 by a ColE1 :: Tn 5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38–39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5- t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5- t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5- t genes in AC1100 are presently under investigation.

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.

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

A pure culture of Pseudomonas cepacia AC1100 was able to degrade and grow in presence of 2,4,5-trichlorophenoxyacetic acid in soil. At optimum temperature (30 degrees C) and moisture content (15 to 50% [wt/vol]) strain AC1100 could degrade as much as 95% of 2,4,5-trichlorophenoxyacetic acid at high concentration (1 mg/g of soil) within 1 week.