A novel pathway for the biodegradation of 3-nitrotoluene in Pseudomonas putida

Abstract: 3-Nitrotoluene was degraded when incubated with the resting cells of Pseudomonas putida OU83. Most of the 3-nitrotoluene (70%) was metabolized via reduction of the nitro group to form 3-aminotoluene (3-AT). A minor portion (30%) was degraded through a novel pathway involving oxidation of 3-NT to form 3-nitrophenol through a series of intermediary metabolites: 3-nitrobenzyl alcohol, 3-nitrobenzaldehyde and 3-nitrobenzoic acid. Degradation of 3-nitrophenol occurred with the formation of a transient intermediary metabolite, hydroxynitroquinone, which was further degraded with the near stoichiometric release of nitrite into the medium. 3-Nitrotoluene-induced cells showed increased oxygen consumption with 3-nitrotoluene, 3-nitrobenzaldehyde, 3-nitrobenzoate, and 3-nitrophenol as substrates in comparison to uninduced cells. Cell extracts prepared from strain OU83 contained benzylalcohol dehydrogenase and benzaldehyde dehydrogenase activities. The experimental evidence suggests a novel pathway for the degradation of 3-NT in which C-1 elimination is catalyzed by a cofactor-independent deformylase, rather than a decarboxylase or dioxygenase.     

Characterization of temperature-sensitive and lipopolysaccharide overproducing transposon mutants of Pseudomonas putida CA-3 affected in PHA accumulation

A library of 20 000 transposon (Tn5) mutants of the gram-negative bacterium Pseudomonas putida CA-3 was generated and screened for adverse affects in polyhydroxyalkanoates (PHA) accumulation. Two mutants of interest were characterized phenotypically. CA-3-126, a mutant disrupted in a stress-related protein Clp protease subunit ClpA, demonstrated greater decreases in PHA accumulation compared with the wild type at reduced and elevated temperatures under PHA-accumulating growth conditions. CA-3-M, which is affected in the aminotransferase class I enzyme, accumulated reduced levels of PHA relative to the wild type and had lower growth yields on all carbon sources tested. Mutant CA-3-M produced up to 10-fold higher levels of lipopolysaccharide relative to the wild type and exhibited 1.2-fold lower aminotransferase activity with phenylalanine as a substrate compared with the wild-type strain. The composition of the lipopolysaccharide produced by the mutant differed from that produced by the wild-type strain. Growth and PHA accumulation by CA-3-M was the same as the wild type when the nitrogen concentration in the medium was increased to 265 mg N L⁻¹.     

Mechanism of cyanide and thiocyanate decomposition by an association of Pseudomonas putida and Pseudomonas stutzeri strains

The intermediate and terminal products of cyanide and thiocyanate decomposition by individual strains of the genus Pseudomonas, P. putida strain 21 and P. stutzeri strain 18, and by their association were analyzed. The activity of the enzymes of nitrogen and sulfur metabolism in these strains was compared with that of the collection strains P. putida VKM B-2187^T and P. stutzeri VKM B-975^T. Upon the introduction of CN⁻ and SCN⁻ into cell suspensions of strains 18 and 21 in phosphate buffer (pH 8.8), the production of NH ₄ ⁺ was observed. Due to the high rate of their utilization, NH₃, NH ₄ ⁺ , and CNO⁻ were absent from the culture liquids of P. putida strain 21 and P. stutzeri strain 18 grown with CN⁻ or SCN⁻. Both Pseudomonas strains decomposed SCN⁻ via cyanate production. The cyanase activity was 0.75 μmol/(min mg protein) for P. putida strain 21 and 1.26 μmol/(min mg protein) for P. stutzeri strain 18. The cyanase activity was present in the cells grown with SCN⁻ but absent in cells grown with NH ₄ ⁺ . Strain 21 of P. putida was a more active CN⁻ decomposer than strain 18 of P. stutzeri. Ammonium and CO₂ were the terminal nitrogen and carbon products of CN⁻ and SCN⁻ decomposition. The terminal sulfur products of SCN⁻ decomposition by P. stutzeri strain 18 and P. putida strain 21 were thiosulfate and tetrathionate, respectively. The strains utilized the toxic compounds in the anabolism only, as sources of nitrogen (CN⁻ and SCN⁻) and sulfur (SCN⁻). The pathway of thiocyanate decomposition by the association of bacteria of the genus Pseudomonas is proposed based on the results obtained. Original Russian Text ? N.V. Grigor’eva, T.F. Kondrat’eva, E.N. Krasil’nikova, G.I. Karavaiko, 2006, published in Mikrobiologiya, 2006, Vol. 75, No. 3, pp. 320–328.     

苯系物降解菌Pseudomonas putida SW-3的筛选及其降解苯的研究

【目的】以苯、甲苯和苯乙烯为唯一碳源,从工业石油废水中筛选苯系物降解菌,分析其降解特性,探讨底物间相互作用对降解情况的影响。【方法】经生理生化和16S r RNA基因分析进行菌种鉴定,采用顶空气相色谱法测定苯系物含量,通过细胞的疏水性、乳化能力、排油圈及透射电镜观察分析菌株降解特性。【结果】经鉴定该菌为Pseudomonas putida,命名为SW-3菌株。最适降解条件下,单位菌体对苯、甲苯和苯乙烯的最大降解速率分别为0.072、0.035和0.019 g/(L·h),苯系混合物的总降解率达79.99%。底物降解实验表明,苯可促进甲苯和苯乙烯的降解,而苯乙烯则能抑制甲苯的降解。菌株的吸附、摄取和降解特性的研究发现,菌株SW-3在自身分泌的表面活性剂的协助下以耗能的方式运输苯。【结论】菌株SW-3具有产生表面活性剂和降解苯系物的能力,且底物间的相互作用能够显著影响菌株对不同底物的降解。     

蚯蚓对细菌降解土壤中菲的作用

通过30d室内培养试验,分别研究了接种蚯蚓(E)、细菌(B)以及同时接种细菌和蚯蚓(BE)对土壤中菲降解的影响.结果表明: 在土壤中菲的初始污染浓度为50 mg*kg-1的条件下,各处理间菲的降解率差异显著,其降解率的大小顺序依次为:BE》B》E》CK(对照); 在150 mg*kg-1菲的初始污染浓度下,BE处理中菲的降解率高达98.86%,显著高于CK和E处理.B处理中细菌的双加氧酶活性在3种菲初始污染浓度下没有显著差异,而BE处理中双加氧酶的活性随着土壤中菲的初始污染浓度的升高而增加.在相同菲污染浓度下BE处理中蚯蚓体内的菲含量明显高于E处理.表明蚯蚓能够通过生物富集作用降低土壤中菲的浓度,而蚯蚓与细菌的相互作用能够进一步促进土壤中菲的降解.     

Broad spectrum and narrow spectrum mercury resistance encoded by different plasmids of a Pseudomonas putida strain

Abstract Pseudomonas putida strain H harbours two plasmids of different sizes. It was demonstrated that the large plasmid pPGH1 confers a broad spectrum resistance to mercurials, whereas the small plasmid pPGH2 confers a narrow spectrum one. Under the influence of the small plasmid the resistance of cells against poisoning with 2,4-di-chlorophenol or o -cresol increases in comparison to cells without this plasmid. Both plasmids proved to be not self-transmissible, but pPGH1 is transferable by mobilisation by means of the IncP-1 vectors R68.45 or RP1.     

Modification of phospholipid composition in Pseudomonas putida A ATCC 12633 induced by contact with tetradecyltrimethylammonium

AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.     

Identification and properties of an inducible phenylacyl-CoA dehydrogenase in Pseudomonas putida KT2440

A novel acyl-CoA dehydrogenase that initiates beta-oxidation of the side chains of phenylacyl-CoA compounds by Pseudomonas putida was induced by growth with phenylhexanoate as carbon source. It was identified as the product of gene PP_0368, which was cloned and overexpressed in Escherichia coli. This phenylacyl-CoA dehydrogenase was found to be dimeric with a subunit molecular mass of 66 kDa, to contain FAD and to be active with phenylacyl-CoA substrates having side chains from four to at least 11 carbon atoms. The same enzyme was induced by the aliphatic alkanoate octanoate. The optimal aliphatic substrates for the enzyme were palmitoyl-CoA and stearoyl-CoA, a property shared with mammalian very-long-chain acyl-CoA dehydrogenases. The FAD in the enzyme was reduced by aromatic and aliphatic substrates, with changes to the oxidation-reduction potential. Chemical reduction by dithionite ion and oxidation by ferricyanide ion showed that the enzyme can accept four electrons: two to reduce the flavin and two to slowly reduce an unknown acceptor, which in its reduced form interacts with the oxidized flavin in a charge-transfer complex. The experiments identify for the first time an acyl-CoA dehydrogenase that oxidizes the activated forms of aromatic acids similar to those used to first demonstrate the biological beta-oxidation of fatty acids.     

Involvement of the plasmid pPGH1 in the phenol degradation of Pseudomonas putida strain H

Pseudomonas putida strain H (wildtype) was shown to harbour two plasmids with a molecular mass of about 50 kb and 200–220 kb, respectively. Evidence is presented that the larger one, pPGH1, is involved in the phenol degradation via the meta-cleavage pathway.     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Cloning and sequencing of rpoH and identification of ftsE-ftsX in Pseudomonas putida PpG1.

The rpoH gene encoding the heat-shock sigma factor of Pseudomonas putida was cloned by using its ability to complement the temperature-sensitive growth of the Escherichia coli rpoH mutant. The cloned DNA contained an open reading frame for a 284 amino acid sequence exhibiting high homology to the sigmaH proteins of P. aeruginosa and E. coli. Moreover, homologs to the cell division genes ftsX and ftsE were found immediately upstream of the rpoH gene.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

The role of fatty acid biosynthesis and degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in Pseudomonas putida

Abstract The relationship between fatty acid metabolism and PHA biosynthesis in P. putida is described. Detailed ¹H and ¹³C NMR studies were performed to investigate the structures of poly(3-hydroxyalkanoates) (PHAs) formed from carbohydrates and fatty acids. On the basis of these results, it is proposed that during growth on glucose the 3-hydroxyacyl-acyl carrier protein intermediates of the de novo fatty acid biosynthetic pathway are diverted to PHA biosynthesis. Similarly, further evidence is presented that during cultivation on fatty acids, intermediates of the β-oxidation cycle serve as precursors of PHA biosynthesis.     

Degradation of tetradecyltrimethylammonium by Pseudomonas putida A ATCC 12633 restricted by accumulation of trimethylamine is alleviated by addition of Al 3+ ions

Aims: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis’ acid in the medium influences its biodegradability. Methods and Results: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l^?1 of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0·1 mmol l^?1 AlCl₃ added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al³⁺ : TMA complex. Conclusions: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl₃. Significance and Impact of the Study: The use of Lewis’ acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.     

Molecular characterization of the Pseudomonas putida 2,3-butanediol catabolic pathway

Abstract The 2,3-butanediol dehydrogenase and the acetoin-cleaving system were simultaneously induced in Pseudomonas putida PpG2 during growth on 2,3-butanediol and on acetoin. Hybridization with a DNA probe covering the genes for the E1 subunits of the Alcaligenes eutrophus acetoin cleaving system and nucleotide sequence analysis identified acoA (975 bp), acoB (1020 bp), acoC (1110 bp), acoX (1053 bp) and adh (1086 bp) in a 6.3-kb genomic region. The amino acid sequences deduced from acoA , acoB , and acoC for E1α ( M _r 34639), E1β ( M _r 37268), and E2 ( M _r 39613) of the P. putida acetoin cleaving system exhibited striking similarities to those of the corresponding components of the A. eutrophus acetoin cleaving system and of the acetoin dehydrogenase enzyme system of Pelobacter carbinolicus and other bacteria. Strong sequence similarities of the adh translational product (2,3-butanediol dehydrogenase, M _r 38361) were obtained to various alcohol dehydrogenases belonging to the zinc- and NAD(P)-dependent long-chain (group I) alcohol dehydrogenases. Expression of the P. putida ADH in Escherichia coli was demonstrated. The aco genes and adh constitute presumably one single operon which encodes all enzymes required for the conversion of 2,3-butanediol to central metabolites.