Comparative Analysis of the Genetic Basis of Branched Nonylphenol Degradation by Sphingobium amiense DSM 16289T and Sphingobium cloacae JCM 10874T.
ABSTRACT: Branched nonylphenol (BNP), a degradation product of nonylphenol polyethoxylates, exerts estrogenic effects on various organisms. The genes underlying BNP degradation by Sphingobium amiense DSM 16289T were analyzed by complete genome sequencing and compared with those of the versatile BNP-degrading Sphingobium cloacae JCM 10874T. An opdA homolog (opdADSM16289) encoding BNP degradation activity was identified in DSM 16289T, in contrast with JCM 10874T, possessing both the opdA homolog and nmoA. The degradation profile of different BNP isomers was examined by Escherichia coli transformants harboring opdADSM16289, opdAJCM10874, and nmoAJCM10874 to characterize and compare the expression activities of these genes.
Project description:Sphingobium cloacae JCM 10874T can degrade phenolic endocrine-disrupting chemicals, nonylphenol, and octylphenol. Here, we report the complete genome sequence of the JCM 10874T strain.
Project description:The nonylphenol-degrading bacterium Sphingomonas sp. strain NP5 has a very unique monooxygenase that can attack a wide range of 4-alkylphenols with a branched side chain. Due to the structural similarity, it can also attack bisphenolic compounds, which are very important materials for the synthesis of plastics and resins, but many of them are known to or suspected to have endocrine disrupting effects to fish and animals. In this study, to clarify the substrate specificity of the enzyme (NmoA) for bisphenolic compounds, degradation tests using the cell suspension of Pseudomonas putida harboring the nonylphenol monooxygenase gene (nmoA) were conducted. The cell suspension degraded several bisphenols including bisphenol F, bisphenol S, 4,4'-dihydroxybenzophenone, 4,4'-dihydroxydiphenylether, and 4,4'-thiodiphenol, indicating that this monooxygenase has a broad substrate specificity for compounds with a bisphenolic structure.
Project description:In contrast to many steroid hormones and cholesterol, mammalian bile salts are 5β-steroids, which leads to a bent structure of the steroid core. Bile salts are surface-active steroids excreted into the environment in large amounts, where they are subject to bacterial degradation. Bacterial steroid degradation is initiated by the oxidation of the A-ring leading to canonical Δ<sup>4</sup>-3-keto steroids with a double bond in the A-ring. For 5β-bile salts, this Δ<sup>4</sup>-double bond is introduced into 3-keto-bile salts by a 5β-Δ<sup>4</sup>-ketosteroid dehydrogenase (5β-Δ<sup>4</sup>-KSTD). With the Nov2c019 protein from bile-salt degrading <i>Sphingobium</i> sp. strain Chol11, a novel 5β-Δ<sup>4</sup>-KSTD for bile-salt degradation belonging to the Old Yellow Enzyme family was identified and named 5β-Δ<sup>4</sup>-KSTD1. By heterologous production in <i>Escherichia coli</i>, 5β-Δ<sup>4</sup>-KSTD function could be shown for 5β-Δ<sup>4</sup>-KSTD1 as well as the homolog CasH from bile-salt degrading <i>Rhodococcus jostii</i> RHA1. The deletion mutant of <i>5β-Δ<sup>4</sup>-kstd1</i> had a prolonged lag-phase with cholate as sole carbon source and, in accordance with the function of 5β-Δ<sup>4</sup>-KSTD1, showed delayed 3-ketocholate transformation. Purified 5β-Δ<sup>4</sup>-KSTD1 was specific for 5β-steroids in contrast to 5α-steroids and converted steroids with a variety of hydroxy groups regardless of the presence of a side chain. 5β-Δ<sup>4</sup>-KSTD1 showed a relatively low <i>K</i> <sub>m</sub> for 3-ketocholate, a very high specific activity and pronounced substrate inhibition. With respect to the toxicity of bile salts, these kinetic properties indicate that 5β-Δ<sup>4</sup>-KSTD1 can achieve fast detoxification of the detergent character as well as prevention of an overflow of the catabolic pathway in presence of increased bile-salt concentrations.
Project description:The phosphotriesterase from <i>Sphingobium</i> sp. TCM1 (<i>Sb</i>-PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1'-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. <i>Sb</i>-PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>, of <i>Sb</i>-PTE toward the test phosphate triesters ranged from ∼10 to 10<sup>5</sup> M<sup>-1</sup> s<sup>-1</sup>. The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.
Project description:13-lipoxygenases (13-LOX) catalyze the dioxygenation of various polyunsaturated fatty acids (PUFAs), of which α-linolenic acid (LeA) is converted to 13-S-hydroperoxyoctadeca-9, 11, 15-trienoic acid (13-HPOT), the precursor for the prostaglandin-like plant hormones cis-(+)-12-oxophytodienoic acid (12-OPDA) and methyl jasmonate (MJ). This study aimed for characterizing the four annotated <i>A. thaliana</i> 13-LOX enzymes (LOX2, LOX3, LOX4, and LOX6) focusing on synthesis of 12-OPDA and 4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl] cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid (OCPD). In addition, we performed interaction studies of 13-LOXs with ions and molecules to advance our understanding of 13-LOX. Cell imaging indicated plastid targeting of fluorescent proteins fused to 13-LOXs-N-terminal extensions, supporting the prediction of 13-LOX localization to plastids. The apparent maximal velocity (V<i><sub>max</sub></i> <sub><i>app</i></sub>) values for LOX-catalyzed LeA oxidation were highest for LOX4 (128 nmol·s<sup>-1</sup>·mg protein<sup>-1</sup>), with a K<sub>m</sub> value of 5.8 µM. <i>A. thaliana</i> 13-LOXs, in cascade with 12-OPDA pathway enzymes, synthesized 12-OPDA and OCPD from LeA and docosahexaenoic acid, previously shown only for LOX6. The activities of the four isoforms were differently affected by physiologically relevant chemicals, such as Mg<sup>2+</sup>, Ca<sup>2+</sup>, Cu<sup>2+</sup> and Cd<sup>2+</sup>, and by 12-OPDA and MJ. As demonstrated for LOX4, 12-OPDA inhibited enzymatic LeA hydroperoxidation, with half-maximal enzyme inhibition at 48 µM. Biochemical interactions, such as the sensitivity of LOX toward thiol-reactive agents belonging to cyclopentenone prostaglandins, are suggested to occur in human LOX homologs. Furthermore, we conclude that 13-LOXs are isoforms with rather specific functional and regulatory enzymatic features.
Project description:We report the complete genome sequence of <i>Veillonella nakazawae</i> JCM 33966<sup>T</sup> (=CCUG 74597<sup>T</sup>). This bacterium is a member of the oral <i>Veillonella</i> and has the potential to be anticariogenic as an oral probiotic seed.
Project description:<i>Sphingobium barthaii</i> KK22<sup>T</sup> is a high-molecular-weight polycyclic aromatic hydrocarbon-degrading soil bacterium that has been investigated in biotransformation, microbial ecology, and DNA damage studies. The complete genome sequence of <i>S. barthaii</i> revealed four closed circular sequences, including two chromosomes, a megaplasmid, and a smaller plasmid, by hybrid assembly using short- and long-read sequencing technologies.
Project description:In plants, cis-jasmone (CJ) is synthesized from ?-linolenic acid (LA) via two biosynthetic pathways using jasmonic acid (JA) and iso-12-oxo-phytodienoic acid (iso-OPDA) as key intermediates. However, there have been no reports documenting CJ production by microorganisms. In the present study, the production of fungal-derived CJ by Lasiodiplodia theobromae was observed for the first time, although this production was not observed for Botrytis cinerea, Verticillium longisporum, Fusarium oxysporum, Gibberella fujikuroi, and Cochliobolus heterostrophus. To investigate the biosynthetic pathway of CJ in L. theobromae, administration experiments using [18,18,18-<sup>2</sup>H<sub>3</sub>, 17,17-<sup>2</sup>H<sub>2</sub>]LA (LA-d5), [18,18,18-<sup>2</sup>H<sub>3</sub>, 17,17-<sup>2</sup>H<sub>2</sub>]12-oxo-phytodienoic acid (cis-OPDA-d5), [5',5',5'-<sup>2</sup>H<sub>3</sub>, 4',4'-<sup>2</sup>H<sub>2</sub>, 3'-<sup>2</sup>H<sub>1</sub>]OPC 8:0 (OPC8-d6), [5',5',5'-<sup>2</sup>H<sub>3</sub>, 4',4'-<sup>2</sup>H<sub>2</sub>, 3'-<sup>2</sup>H<sub>1</sub>]OPC 6:0 (OPC6-d6), [5',5',5'-<sup>2</sup>H<sub>3</sub>, 4',4'-<sup>2</sup>H<sub>2</sub>, 3'-<sup>2</sup>H<sub>1</sub>]OPC 4:0 (OPC4-d6), and [11,11-<sup>2</sup>H<sub>2</sub>, 10,10-<sup>2</sup>H<sub>2</sub>, 8,8-<sup>2</sup>H<sub>2</sub>, 2,2-<sup>2</sup>H<sub>2</sub>]methyl iso-12-oxo-phytodienoate (iso-MeOPDA-d8) were carried out, revealing that the fungus produced CJ through a single biosynthetic pathway via iso-OPDA. Interestingly, it was suggested that the previously predicted decarboxylation step of 3,7-didehydroJA to afford CJ might not be involved in CJ biosynthesis in L. theobromae.
Project description:Nickel is an essential component of many eukaryotic and prokaryotic metallo-enzymes. Due to its employment in many industrial applications, wastewaters from industrial plants often contain millimolar concentrations of Ni2+ that are toxic and life-threatening for many organism. Several lines of preliminary evidence suggest that members of the genus Sphingobium are able to grow in the presence of high concentrations of metal ions. We have isolated a novel Sphingobium strain (sp. ba1) able to grow in the presence of high concentrations (up to 20 mM) of NiCl2. Sequencing of its genome allowed the identification of several genes coding for proteins potentially involved in efflux-mediated resistance mechanisms. Here we use the RNA-seq approach to analyze the response of the Sphingobium sp. ba1 strain to high concentrations (10 mM) of Ni ions. Transcriptomic data show the differential expression of about one-hundred and twenty genes, most of which are up-regulated and encode proteins such as membrane proteins and components of metal efflux systems, enzymes involved in oxidative stress responses (catalases, peroxidases) and signal transduction systems.