Molecular characterization of the alpha subunit of multicomponent phenol hydroxylase from 4-chlorophenol-degrading Pseudomonas sp. strain PT3

Multicomponent phenol hydroxylases (mPHs) are diiron enzymes that use molecular oxygen to hydroxylate a variety of phenolic compounds. The DNA sequence of the alpha subunit (large subunit) of mPH from 4-chlorophenol (4-CP)-degrading bacterial strain PT3 was determined. Strain PT3 was isolated from oil-contaminated soil samples adjacent to automobile workshops and oil stations after enrichment and establishment of a chlorophenol-degrading consortium. Strain PT3 was identified as a member of Pseudomonas sp. based on sequence analysis of the 16S rRNA gene fragment. The 4-CP catabolic pathway by strain PT3 was tentatively proposed to proceed via a meta-cleavage pathway after hydroxylation to the corresponding chlorocatechol. This hypothesis was supported by polymerase chain reaction (PCR) detection of the LmPH encoding sequence and UV/VIS spectrophotometric analysis of the culture filtrate showing accumulation of 5-chloro-2-hydroxymuconic semialdehyde (5-CHMS) with λ_max 380. The detection of catabolic genes involved in 4-CP degradation by PCR showed the presence of both mPH and catechol 2,3-dioxygenase (C23DO). Nucleotide sequence analysis of the alpha subunit of mPH from strain PT3 revealed specific phylogenetic grouping to known mPH. The metal coordination encoding regions from strain PT3 were found to be conserved with those from the homologous dinuclear oxo-iron bacterial monooxygenases. Two DE(D)XRH motifs was detected in LmPH of strain PT3 within an approximate 100 amino acid interval, a typical arrangement characteristic of most known PHs.     

Effect of gacS and gacA mutations on colony architecture, surface motility, biofilm formation and chemical toxicity in Pseudomonas sp. KL28

GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to H2O2, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and H2O2 resistance, which are important traits for its capacity to survive in particular niches.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA(ind)) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpA(ind) contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the K(d) value for indole decreased from 1.5 mm to 78 microm. Investigation of the major reaction products of HbpA(ind) with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpA(ind) mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpA(ind), there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.     

Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600.

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression of subfragments in tac expression vectors, and identification of polypeptide products in maxicells was used to demonstrate that the polypeptides observed are produced from the six open reading frames identified in the sequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth on phenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. This system, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used to demonstrate that all six polypeptides are required for growth on phenol.     

Ssg, a putative glycosyltransferase, functions in lipo- and exopolysaccharide biosynthesis and cell surface-related properties in Pseudomonas alkylphenolia

In the presence of vaporized p-cresol, Pseudomonas alkylphenolia KL28 forms specialized aerial structures (SAS). A transposon mutant of strain KL28 (C23) incapable of forming mature SAS was isolated. Genetic analysis of the C23 mutant revealed the transposon insertion in a gene (ssg) encoding a putative glycosyltransferase, which is homologous to the Pseudomonas aeruginosa PAO1 PA5001 gene. Deletion of ssg in KL28 caused the loss of lipopolysaccharide O antigen and altered the composition of the exopolysaccharide. Wild-type KL28 produced a fucose-, glucose- and mannose-rich exopolysaccharide, while the mutant exopolysaccharide completely lacked fucose and mannose, resulting in an exopolysaccharide with glucose as the major component. The mutant strain showed reduced surface spreading, pellicle and biofilm formation, probably due to the cumulative effect of lipopolysaccharide truncation and altered exopolysaccharide composition. Our results show that the ssg gene of KL28 is involved in both lipopolysaccharide and exopolysaccharide biosynthesis and thus plays an important role in cell surface properties and cell-cell interactions of P. alkylphenolia.     

Simultaneous degradation of atrazine and phenol by Pseudomonas sp. strain ADP: effects of toxicity and adaptation

The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na(2)-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to approximately 35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.     

Cloning and expression of naphthalene dioxygenase genes from Comamonas sp. MQ for indigoids production

The naphthalene dioxygenase (NDO) genes were cloned from Comamonas sp. MQ and successfully expressed in Escherichia coli BL21 (DE3) (designated as ND_IND). The whole cells of recombinant strain ND_IND possessed relatively high transformation efficiency towards indole and most indole derivatives. According to the UV–vis and HPLC–MS analyses, the major products derived from the indoles could be indigo with different substituent groups. Furthermore, strain ND_IND was able to produce 205 mg/l indigo from 300 mg/l indole with a specific production rate of 8.4 mg/(g dry cell weight h). The effects of phenol, pyridine and quinoline on indigo production were determined, which indicated that phenol and pyridine had little inhibition on indigo production while quinoline would result in a 32% decrease in indigo yield. The present study proposed the potential application of recombinant strain ND_IND in indigoid pigments production, and offered the promise of applying strain ND_IND for the production of indigo using indole-containing wastewater as the raw materials.     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonads by kinetic parameters

Phenol-degrading pseudomonads possessing different phenol hydroxylases (PH) were evaluated by the values of apparent half-saturation constant for phenol-oxygenating activity (K ( S )), maximum specific growth rate (mu (max)), lag-time length (lambda), inhibition constant (K ( I )) and growth yield factor (Y ( X/S )). Strains of the same PH type showed similar kinetic parameters: single-component PH (sPH) harbouring strains had higher values of K ( S ) and lower values of mu (max) than the strains having multicomponent PH (mPH). However, the values of K ( I ) and the dependencies of the lag-time length on initial phenol concentration were strain-specific. The elevated ratio between specific activities of catechol 1,2-dioxygenase (C12O) and muconate cycloisomerase in sPH-strains caused irreversible accumulation of a high amount of exogenous cis,cis-muconate (CCM) which resulted in decreased Y ( X/S ) values. Co-presence of sPH and mPH genes did not give the strains PC16 and P69 any extra advantage and according to determined kinetic parameters only one PH was active during phenol degradation. At the same time simultaneous functioning of catechol ortho and meta cleavage pathways (strain PC20) resulted in higher mu (max) and Y ( X/S ) values. Evaluation of strains showed that the type of PH determined the efficiency of phenol degradation, whereas the tolerance to elevated phenol concentrations was strain-specific.     

Enzymatic synthesis oF L-tryptophan from D,L-2-amino-delta2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, S-carbamyl-L-cysteine amidohydrolase, and Escherichia coli L-tryptophanase

L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-delta2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5'-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from DL-ATC and indole.     

Purification and Characterization of a Novel Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038

We report here the characterization of the catalytic component (ISP(NAR)) of a new naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The genes encoding the two subunits of ISP(NAR) are not homologous to their previously characterized counterparts in Pseudomonas. The deduced amino acid sequences have only 33 and 29% identity with the corresponding subunits in Pseudomonas putida NCIB 9816-4, for which the tertiary structure has been reported.     

Degradation of chlorophenols by a defined mixed microbial community

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

Degradation of chlorophenols by a defined mixed microbial community.

Synthetic sewage containing phenol, acetone, and alkanols plus 4-chlorophenol or a mixture of isomeric chlorophenols is completely degraded by a defined mixed culture with Pseudomonas sp. strain B13 as a chlorocatechol-dissimilating member of the community. Total degradation of the organic carbon was indicated by release of stoichiometric amounts of chloride and low content of dissolved organic carbon in the cell-free effluents. During adaptation to high loads of chlorophenols the initial meta-cleavage activity was completely replaced by ortho-cleavage activity of type I and II. In the fully acclimated culture, hybrid strains such as Alcaligenes sp. strain A7-2 were detected, which are more competitive than Pseudomonas sp. strain B13 with respect to chlorophenol degradation.     

产脂肪酶细菌RYXP的诱变选育及突变株产酶条件优化

以"凤丹"牡丹根际土壤中分离筛选到的产脂肪酶菌株Pseudomonas sp. RYXP作为出发菌株,对其进行了紫外线诱变选育,并采用单因素试验和正交试验方法对活性最强正突变株的产脂肪酶基本特性进行了测定。结果表明,出发菌株Pseudomonas sp. RYXP的紫外线诱变最佳条件为:15 W紫外灯30 cm距离照射1 min;将产脂肪酶活性最强的正突变菌株编号为RYXP-3,单因素试验表明RYXP-3产脂肪酶适宜的碳源为玉米淀粉,适宜氮源为豆饼粉,适宜的磷酸二氢钾含量是0.3%,适宜的初始pH值为7;正交试验表明RYXP-3的最佳的产酶培养基成分组成是:玉米淀粉7%,豆饼粉3%,磷酸二氢钾0.3%,初始pH值为8。在优化方案A1B2C2D3产酶条件下,突变株RYXP-3最高的产酶活性达到56.1 U/mL。突变菌株RYXP-3可作为产油牡丹"凤丹"专用促生菌肥开发的备选资源菌株。     

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.     

Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp

An internal loop airlift reactor (ILALR) is developed and studied for biodegradation of phenol/m-cresol as single and dual substrate systems under batch and fed batch operation using an indigenous mixed microbial strain, predominantly Pseudomonas sp. The results showed that the culture could degrade phenol/m-cresol completely at a maximum concentration of 600mgl(-1) and 400mgl(-1), respectively. Batch ILALR study has revealed that phenol has been preferentially degraded by the microbial culture rather than m-cresol probably owing to the toxic effect of the later. Sum kinetic model evaluated the interaction between the phenol/m-cresol in dual substrate system, which resulted in a high coefficient of determination (R(2)) value >0.98). The fed batch results showed that the strain was able to degrade phenol/m-cresol with maximum individual concentrations 600mgl(-1) each in 26h and 37h, respectively. Moreover for fed batch operation, degradation rates increased with increase in feed concentration without any lag in the degradation profile.