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.     

Genome Sequence of the Polychlorinated-Biphenyl Degrader Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.     

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.     

Epoxide formation on the aromatic B ring of flavanone by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes KF707

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2' and C3' on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2',3'-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.     

Epoxide Formation on the Aromatic B Ring of Flavanone by Biphenyl Dioxygenase of Pseudomonas pseudoalcaligenes KF707

Prokaryotic dioxygenase is known to catalyze aromatic compounds into their corresponding cis-dihydrodiols without the formation of an epoxide intermediate. Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 showed novel monooxygenase activity by converting 2(R)- and 2(S)-flavanone to their corresponding epoxides (2-(7-oxabicyclo[4.1.0]hepta-2,4-dien-2-yl)-2, 3-dihydro-4H-chromen-4-one), whereby the epoxide bond was formed between C2′ and C3′ on the B ring of the flavanone. The enzyme also converted 6-hydroxyflavanone and 7-hydroxyflavanone, which do not contain a hydroxyl group on the B-ring, to their corresponding epoxides. In a previous report (S.-Y. Kim, J. Jung, Y. Lim, J.-H. Ahn, S.-I. Kim, and H.-G. Hur, Antonie Leeuwenhoek 84:261-268, 2003), however, we found that the same enzyme showed dioxygenase activity toward flavone, resulting in the production of flavone cis-2′,3′-dihydrodiol. Extensive structural identification of the metabolites of flavanone by using high-pressure liquid chromatography, liquid chromatography/mass spectrometry, and nuclear magnetic resonance confirmed the presence of an epoxide functional group on the metabolites. Epoxide formation as the initial activation step of aromatic compounds by oxygenases has been reported to occur only by eukaryotic monooxygenases. To the best of our knowledge, biphenyl dioxygenase from P. pseudoalcaligenes KF707 is the first prokaryotic enzyme detected that can produce an epoxide derivative on the aromatic ring structure of flavanone.     

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.     

Pseudomonas pseudoalcaligenes KF707 upon biofilm formation on a polystyrene surface acquire a strong antibiotic resistance with minor changes in their tolerance to metal cations and metalloid oxyanions

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO(2) (-), SeO(3) (2-), TeO(3) (2-)) and three metal cations (Cd(2+), Ni(2+), Al(3+)) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria-Bertani) medium were thicker (23 mum) than biofilms grown in minimal (SA saccarose-arginine) medium (13 mum). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50-100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO(3) (2-) to elemental Te(0) or SeO(3) (2-) to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO(3) (2-) and SeO(3) (2-) is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.     

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 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.     

Induction of maleate hydratase in Pseudomonas pseudoalcaligenes

Maleate hydratase (malease, EC 4.2.1.31) activity in P. pseudoalcaligenes is induced when grown on3-hydroxybenzoate. The specific malease activity was constant during the logarithmic phase in a batch culture containing 3-hydroxybenzoate as the carbon source, when 3-hydroxybenzoate-grown cells were used as inoculum. When yeast extract-grown cells were used as inoculum, the specific malease activity was correlated with growth. In both instances the specific malease activity dropped rapidly as soon as growth ceased. Maleate did not serve as a growth substrate for this microorganism, but a mutant able to grow on maleate was selected. The specific malease activity of maleate-grown cells of this mutant was not higher than the basal level of induction of malease activity.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

Control of the pyrimidine biosynthetic pathway in Pseudomonas pseudoalcaligenes

The five de novo enzyme activities unique to the pyrimidine biosynthetic pathway were found to be present in Pseudomonas pseudoalcaligenes ATCC 17440. A mutant strain with 31-fold reduced orotate phosphoribosyltransferase (encoded by pyrE) activity was isolated that exhibited a pyrimidine requirement for uracil or cytosine. Uptake of the nucleosides uridine or cytidine by wild-type or mutant cells was not detectable; explaining the inability of the mutant strain to utilize either nucleoside to satisfy its pyrimidine requirement. When the wildtype strain was grown in the presence of uracil, the activities of the five de novo enzymes were depressed. Pyrimidine limitation of the mutant strain led to the increase in aspartate transcarbamoylase and dihydroorotate dehydrogenase activities by more than 3-fold, and dihydroorotase and orotidine 5-monophosphate decarboxylase activities about 1.5-fold, as compared to growth with excess uracil. It appeared that the syntheses of the de novo enzymes were regulated by pyrimidines. In vitro regulation of aspartate transcarbamoylase activity in P. pseudoalcaligenes ATCC 17440 was investigated using saturating substrate concentrations; transcarbamoylase activity was inhibited by P_i, PP_i, uridine ribonucleotides, ADP, ATP, GDP, GTP, CDP, and CTP.     

Cross-regulation in development of neuroendocrine and immune systems

Cross-regulatory effects of immune and neuroendocrine systems on their appearance and functioning occur during a whole life period. At different stages of ontogenesis, the functions of these systems are diverse. In perinatal ontogenesis hormones, neuropeptides and neurotransmitters control the processes of growth and differentiation of various embryo tissues, particularly lymphoid. In the postnatal period, their functions are mostly in homeostasis maintaining of the immune system in response to changes of the environment. Conversely, transmitters of the immune system, such as cytokines, whose synthesis is increased in inflammation, and thymic peptides, program the development of the neuroendocrine system of the embryo. The perinatal period is crucial for final appearance of these systems. Changes in one of the interacting systems, caused by negative environmental factors at this stage, usually provoke changes in other developing systems for a long period. Plasticity of physiological systems in perinatal development allows the organism to adapt to changed conditions. However, these changes can limit physiological functions in interacting systems and induce the appearance of various pathologies in postnatal life.     

Purification and Characterization of Maleate Hydratase from Pseudomonas pseudoalcaligenes

Maleate hydratase (malease) from Pseudomonas pseudoalcaligenes has been purified. The purified enzyme (98% pure) catalyzes the stereospecific addition of water to maleate and citraconate (2-methylmaleate), forming d-(+)-malate and d-(+)-citramalate, respectively. 2,3-Dimethylmaleate was also a substrate for malease. The stability of the enzyme was dependent on the protein concentration and the addition of dicarboxylic acids. The purified enzyme (89 kDa) consisted of two subunits (57 and 24 kDa). No cofactor was required for full activity of this colorless enzyme. Maximum enzyme activity was measured at pH 8 and 45 degrees C. The K(m) for maleate was 0.35 mM, and that for citraconate was 0.20 mM. Thiol reagents, such as p-chloromercuribenzoate and iodoacetamide, and sodium dodecyl sulfate completely inhibited malease activity. Malease activity was competitively inhibited by d-malate (K(i) = 0.63 mM) and d-citramalate (K(i) = 0.083 mM) and by the substrate analog 2,2-dimethylsuccinate (K(i) = 0.025 mM). The apparent equilibrium constants for the maleate, citraconate, and 2,3-dimethylmaleate hydration reactions were 2,050, 104, and 11.2, respectively.