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 10⁶ to 10⁸ 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 × 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.     

Transformation of mono- and dichlorinated phenoxybenzoates by phenoxybenzoate-dioxygenase in pseudomonas pseudoalcaligenes POB310 and a modified diarylether-metabolizing bacterium

Pseudomonas pseudoalcaligenes POB310 contains genes that encode phenoxybenzoate dioxygenase. The enzyme transforms mono- and dichlorinated phenoxybenzoates to yield protocatechuate that is used as a growth substrate and chlorophenols that are nonmetabolizable. Mass spectral analysis of (18)O metabolites obtained from the protocatechuate 3,4-dioxygenase-deficient mutant, POB310-B1, suggested that the reaction mechanism is a regioselective angular dioxygenation. A cloning vector containing reaction relevant genes (pD30.9) was transferred into Pseudomonas sp. strain B13 containing a modified ortho-cleavage pathway for aromatic compounds. The resultant Pseudomonas sp. strain B13-D5 (pD30.9) completely metabolized 3-(4-chlorophenoxy)benzoate. During growth on 3-phenoxybenzoate, strain B13-D5 (pD30.9) (K(s) = 0.70+/-0.04 mM, mu(max) = 0.45+/-0.03 h(-1), t(d) = 1.5 h, Y = 0.45+/-0.03 g bio- mass x g substrate(-1)) was better adapted to low substrate concentrations, had a faster rate of growth, and a greater yield than POB310 (K(s) = 1.13+/-0.06 mM, mu(max) = 0.31+/-0.02 h(-1), t(d) = 2.2 h, Y = 0.39+/-0.02 g biomass. g substrate(-1)).     

Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes.

A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation.     

Degradation of fluorobiphenyl by Pseudomonas pseudoalcaligenes KF707

The biphenyl-degrading bacterium Pseudomonas pseudoalcaligenes KF707 can use 2- and 4-fluorobiphenyl as sole carbon and energy sources. Accumulation of fluorinated catabolites was determined by fluorine-19 nuclear magnetic spectroscopy (¹⁹F NMR) and revealed that growth on 4-fluorobiphenyl yielded 4-fluorobenzoate and 4-fluoro-1,2-dihydro-1,2-dihydroxybenzoate as major fluorometabolites; 2-fluorobenzoate and 2-fluoromuconic acid were observed in 2-fluorobiphenyl-grown cultures. Pseudomonas pseudoalcaligenes KF707 was not able to use either 2- or 4-fluorobenzoate as a growth substrate. Thus, fluorobiphenyl is probably degraded via the classical Bph pathway to fluorobenzoate, which is partially transformed via the enzymes of benzoate catabolism. This is the first report of investigations on the growth of bacteria on fluorinated biphenyls and demonstrates that as with chlorobiphenyl degradation, mineralization of the compounds depends upon the bacterium's ability to effectively catabolize the halobenzoate intermediate.     

Cloning,nucleotide sequence,and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310

Pseudomonas pseudoalcaligenes strain POB310 degrades 3-and 4-carboxydiphenyl ether. The initial reaction involves an angular dioxygenation yielding an unstable hemiacetal that spontaneously decays to phenol and protocatechuate. We cloned a DNA fragment containing the gene encoding the initial, dioxygenase from an unstable, self-transmissible plasmid. Sequence analysis revealed two open reading frames encoding proteins with putative molecular masses of 46.3 and 33.6 kDa. The deduced amino acid sequences showed homologies to oxygenase and reductase subunits of aromatic ring-activating dioxygenases, and contained regions identical to consensus sequences that bind chloroplast-like and Rieske-type [2Fe2S] clusters suggesting that the initial dioxygenase is a class IA aromatic ring-activating dioxygenase system. Intitial dioxygenase activity was induced in bacteria grown in M9 minimal medium containing 3-or 40-carboxydiphenyl ether or phenol as carbon source, indicating that the regulation is dependent on the phenol pathway. The maximal specific activity was measured at the beginning of the exponential growth phase.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.     

两株真降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植权的降解率分别为７４．４％和９５．０％;在固太发酵中植酸的降解率为４０％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、ｐＨ和水分等影响因了也进行了初步的探讨。经初步鉴定,这两株菌中有一株为拟青霉（Ｐａｅｃｉｌｏｍｙｃｅｓ ｓｐ）,另一株为青霉（Ｐｅｎｉｃｉｌｌｉｕ     

Degradation of 3-phenoxybenzoic acid by a filamentous fungus Aspergillus oryzae M-4 strain with self-protection transformation

Applied Microbiology and Biotechnology - A novel filamentous fungus M-4 strain was isolated from soy sauce koji and identified as Aspergillus oryzae (Collection number: CGMCC 11645) on the basis of...     

Cross-regulation of biphenyl- and salicylate-catabolic genes by two regulatory systems in Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 grows on biphenyl and salicylate as sole sources of carbon. The biphenyl-catabolic (bph) genes are organized as bphR1A1A2(orf3)A3A4BCX0X1X2X3D, encoding the enzymes for conversion of biphenyl to acetyl coenzyme A. In this study, the salicylate-catabolic (sal) gene cluster encoding the enzymes for conversion of salicylate to acetyl coenzyme A were identified 6.6-kb downstream of the bph gene cluster along with a second regulatory gene, bphR2. Both the bph and sal genes were cross-regulated positively and/or negatively by the two regulatory proteins, BphR1 and BphR2, in the presence or absence of the effectors. The BphR2 binding sequence exhibits homology with the NahR binding sequences in various naphthalene-degrading bacteria. Based on previous studies and the present study we propose a new regulatory model for biphenyl and salicylate catabolism in strain KF707.     

Synthesis of poly(3-hydroxyalkanoates) by mutant and recombinant Pseudomonas strains

We have studied the accumulation kinetics and physical characteristics of the poly(3-hydroxyalkanoates) (PHAs) formed by several Pseudomonas strains, mutants and recombinants. Although PHA synthesis generally begins only after an essential nutrient such as N, P, S or Mg becomes limiting, we have identified at least one strain (P. putida KT2442) that begins producing PHA during the exponential growth phase. This PHA is chemically and physically identical to that produced by P. oleovorans GPol, the strain in which we first identified PHA. Analysis of the PHA formed by a mutant strain defective in PHA degradation (P. oleovorans GPo500) revealed that the molecular mass (M_w), the monomer composition and thermal characteristics were similar to that of the PHA of the wild-type parent strain P. oleovorans GPo1. The pha locus of P. oleovorans encodes enzymes that are involved in PHA biosynthesis and degradation. It has been subcloned to study the two PHA polymerases separately in a PHA^– mutant (GPp104) derived from P. putida KT2442. The recombinant strains accumulated lower PHA levels than the wild-type strains, and the M_w of these polymers were lower than those produced by the wild-type P. oleovorans and parent strain. The monomer composition of the two PHAs formed by the two PHA polymerases differed, indicating that the PHA polymerases have different substrate specificities for the incorporation of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers into PHA. Despite these differences, the PHAs formed were essentially indistinguishable from wild-type PHAs with respect to their thermal characteristics.Correspondence to: B. Witholt     

Oxidation of polychlorinated biphenyls by Pseudomonas sp. strain LB400 and Pseudomonas pseudoalcaligenes KF707.

Biphenyl-grown cells and cell extracts prepared from biphenyl-grown cells of Pseudomonas sp. strain LB400 oxidize a much wider range of chlorinated biphenyls than do analogous preparations from Pseudomonas pseudoalcaligenes KF707. These results are attributed to differences in the substrate specificity of the biphenyl 2,3-dioxygenases from both organisms.     

Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains

Four bacterial strains (CA26, CA28, CA37, and CA45), which all were able to use aniline, 3-chloroaniline (3-CA), and 4-chloroaniline (4-CA) as sole sources of carbon, nitrogen and energy, were isolated after enrichment in aerated soil columns and identified as Pseudomonas acidovorans strains. In addition strains CA26 and CA45 were able to degrade 2-chloroaniline (2-CA) at very low rates. At 25°C strain CA28 was grown on aniline and 3-CA with generation times of 3.0 and 7.7 h, respectively, and exhibited complete mineralization of these substrates in degradation rates of 2.25 mmol aniline and 1.63 mmol 3-CA g^-1 of biomass per hour, respectively. Degradation of 4-CA occurred at 1.54 mmol 4-CA g^-1 of biomass per hour and a generation time of 18.7 h but, in contrast, was not complete due to formation of minor amounts of chlorohydroxymuconic semialdehyde, a meta-cleavage product of 4-chlorocatechol. The initial attack on the substrate, the formation of corresponding chlorocatechols from 3-CA and 4-CA, was found to be the rate-limiting degradation step. Evidence for two different aniline-oxygenase systems in strain CA28 with distinct activity pattern on chlorinated and nonsubstituted anilines was demonstrated by oxygen uptake rate experiments with aniline and chloroaniline pregrown cells. Further degradation was shown to be initialized by catechol dioxygenases.Non-standard abbreviations CA chloroaniline - DCA dichloroaniline - ECM enrichment and cultivation medium - CFU colony forming unit     

Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Degradation of 2-methylbenzoic acid by Pseudomonas cepacia MB2.

We report the isolation of Pseudomonas cepacia MB2, believed to be the first microorganism to utilize 2-methylbenzoic acid as the sole carbon source. Its growth range included all mono- and dimethylbenzoates (with the exception of 2,5- and 2,6-dimethylbenzoates) and 3-chloro-2-methylbenzoate (but not 4- or 5-chloro-2-methylbenzoate) but not chlorobenzoates lacking a methyl group. 2-Chlorobenzoate, 3-chlorobenzoate, and 2,3-, 2,4-, and 3,4-dichlorobenzoates inhibited growth of MB2 on 2-methylbenzoate as a result of cometabolism to the corresponding chlorinated catechols which blocked the key enzyme catechol 2,3-dioxygenase. A metapyrocatechase-negative mutant, MB2-G5, showed accumulation of dimethylcatechols from 2,3- and 3,4-dimethylbenzoates, and phenols were detected in resting-cell transformation extracts bearing the same substitution pattern as the original substrate, presumably following thermal degradation of the intermediate dihydrodiol. 2-Methylphenol was also found in extracts of the mutant cells with 2-methylbenzoate. These observations suggested a major route of methylbenzoate metabolism to be dioxygenation to a carboxy-hydrodiol which then forms a catechol derivative. In addition, the methyl group of 2-methylbenzoate was oxidized to isobenzofuranone (by cells of MB2-G5) and to phthalate (by cells of a separate mutant that could not utilize phthalate, MB2-D2). This pathway also generated a chlorinated isobenzofuranone from 3-chloro-2-methylbenzoate.     

Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13.

Twenty-five gram-negative bacteria and one gram-positive bacterium capable of growing on poly(3-hydroxyoctanoic acid) [P(3HO)] as the sole source of carbon and energy were isolated from various soils, lake water, and activated sludge. Most of the isolates degraded only P(3HO) and copolymers of medium-chain-length (MCL) hydroxyalkanoic acids (HA). Except for the gram-positive strain, which was able to hydrolyze P(3HO) and poly(3-hydroxybutyric acid) [P(3HB)], no isolate was able to degrade polymers of short-chain-length HA, such as P(3HB) or poly(3-hydroxyvalerate) [P(3HV)]. All strains utilized a large variety of monomeric substrates for growth. All gram-negative strains, but not the gram-positive strain, accumulated poly(hydroxyalkanoic acids) (PHA), consisting of MCL HA, if they were cultivated under accumulation conditions. One strain, which was identified as Pseudomonas fluorescens GK13 (biovar V), was selected and the extracellular P(3HO) depolymerase of this strain was purified from the culture medium of P(3HO)-grown cells by chromatography with Octyl-Sepharose CL4B and by gel filtration with Superose 12. The relative molecular weights of the native and sodium dodecyl sulfate-treated enzymes were 48,000 and 25,000, respectively. The purified enzyme hydrolyzed P(3HO), copolymers of MCL HA, and para-nitrophenyl esters of fatty acids. P(3HB), P(3HV), and characteristic substrates for lipases, such as Tween 80 or triolein, were not hydrolyzed. The P(3HO) depolymerase of P. fluorescens GK13 was insensitive to phenylmethylsulfonyl fluoride and dithioerythritol, unlike other PHA depolymerases. The dimeric ester of 3-hydroxyoctanoic acid was identified as the main product of enzymatic hydrolysis of P(3HO).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of 2-bromobenzoic acid by a strain of Pseudomonas aeruginosa.

A strain of Pseudomonas aeruginosa producing 2-bromobenzoic acid, designated 2-BBZA, was isolated by enrichment culture from municipal sewage. It degraded all four 2-halobenzoates as well as certain 3-halo- and dihalobenzoates, though none of the 4-halobenzoates supported growth of this organism. 3-Hydroxybenzoate and 3-chlorocatechol were respective inhibitors of salicylate and catechol oxidation: when each was added separately to resting cells incubated with 2-bromobenzoate, salicylate and catechol were found. Oxygen uptake data suggest that the same dehalogenase may be involved in the oxidation of 2-bromo-, 2-chloro-, and 2-iodobenzoates.