Complete genome sequence of Paraburkholderia sp. strain 22B1P capable of utilizing 3-chlorobenzoate as a carbon source.

Paraburkholderia sp. strain 22B1P utilizes 3-chlorobenzoate as a carbon source. Complete genome sequencing of strain 22B1P revealed two chromosomes and two plasmids. The genes involved in the conversion of 3-chlorobenzoate to 3-chlorocatechol and those involved in the conversion of 3-chlorocatechol to 3-oxoadipate were located on chromosomes 2 and 1, respectively.

Isolation and Genomic Analysis of 3-Chlorobenzoate-Degrading Bacteria from Soil.

The compound 3-chlorobenzoate (3-CBA) is a hazardous industrial waste product that can harm human health and the environment. This study investigates the physiological and genetic potential for 3-chlorobenzoate (3-CBA) degradation. Six 3-CBA Gram-negative degraders with different degradation properties belonging to the genera Caballeronia, Paraburkholderia and Cupriavidus were isolated from the soil. The representative strains Caballeronia 19CS4-2 and Paraburkholderia 19CS9-1 showed higher maximum specific growth rates (µmax, h-1) than Cupriavidus 19C6 and degraded 5 mM 3-CBA within 20-28 h. Two degradation products, chloro-cis,cis-muconate and maleylacetate, were detected in all isolates using high-performance liquid chromatography and mass spectrometry. Genomic analyses revealed the presence of cbe and tfd gene clusters in strains 19CS4-2 and 19CS9-1, indicating that they probably metabolized the 3-CBA via the chlorocatechol ortho-cleavage pathway. Strain 19C6 possessed cbe genes, but not tfd genes, suggesting it might have a different chlorocatechol degradation pathway. Putative genes for the metabolism of 3-hydroxybenzoate via gentisate were found only in 19C6, which utilized the compound as a sole carbon source. 19C6 exhibited distinct characteristics from strains 19CS4-2 and 19CS9-1. The results confirm that bacteria can degrade 3-CBA and improve our understanding of how they contribute to environmental 3-CBA biodegradation.

The structure-function relationship of bacterial transcriptional regulators as a target for enhanced biodegradation of aromatic hydrocarbons.

The sheer persistence and dissemination of xenobiotic aromatic hydrocarbons contaminants demand sustainable solutions for degradation. Therefore, major pathways of microbial catabolism of aromatic hydrocarbons under aerobic conditions are reviewed and analysed to elicit enhanced biodegradation of aromatic hydrocarbons, via the structure-function relationship of bacterial transcriptional regulators. The initial step of the catabolism occurs via the incorporation of molecular oxygen into the aromatic ring by a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD) enzyme system or monooxygenase system forming different central intermediates such as catechols, protocatechuates, gentisates, and (hydroxy)benzoquinols. The central or lower pathways involve the ring cleavage of central intermediates to tricarboxylic acids. These metabolic pathways are tightly regulated, where the inducer or substrate-specific transcriptional regulation of aromatic catabolic pathways depend on the specific regulatory proteins that acts on a specific promoter in response to a respective inducer signal. These regulatory systems have been grouped according to the regulatory proteins and their families, and identified based on their conserved motifs and their modes of DNA binding. Different regulators from protein families like AraC/XylS, LysR, XylR/NtrC, IclR, etc. have been identified, that are involved in aromatic hydrocarbon regulation. These regulatory proteins have different structures and have different mechanisms of regulation. The proteins of the XylS/AraC family have two domains structure: a highly conserved C-terminus that contains two HTH motifs and the N-terminus end containing the regulatory domain. The LysR type regulatory proteins (LTTRs) act as tetramers that have a helix-turn-helix (HTH) domain at the N terminus and a regulatory binding domain at the C terminus. The IclR regulatory proteins also have a helix-turn-helix DNA binding motif in the N-terminus domain-like LTTRs but include an effector binding motif in the C-terminus domain that is also involved in subunit multimerization. In contrast, the XylR-like regulatory proteins have three domain structures; one for effector sensing, another for ATP binding and hydrolysis, and a domain for DNA binding which contains an HTH motif. This review describes in depth and critical assessment of the aerobic bacterial degradation pathways of aromatic hydrocarbon pollutants with state of art information, underscores areas that are viable and others that require further development, with particular reference to metabolic engineering and synthetic biology applications.

Complete Genome Sequence of Caballeronia sp. Strain NK8 (MAFF311271), a Chlorobenzoate-Degrading Bacterium.

Caballeronia sp. strain NK8 grows on 3-chlorobenzoate and shows chemotaxis toward 3-chlorobenzoate and its degradation products, such as chlorocatechols. Complete genome sequencing revealed a 9.2-Mb genome consisting of three chromosomes and four plasmids. The genes for degradation of 3-chlorobenzoate and chlorocatechols were located on plasmids pNK81 and pNK84, respectively.

Transcriptome differences between Cupriavidus necator NH9 grown with 3-chlorobenzoate and that grown with benzoate.

RNA-seq analysis of Cupriavidus necator NH9, a 3-chlorobenzoate degradative bacterium, cultured with 3-chlorobenzaote and benzoate, revealed strong induction of genes encoding enzymes in degradation pathways of the respective compound, including the genes to convert 3-chlorobenzaote and benzoate to chlorocatechol and catechol, respectively, and the genes of chlorocatechol ortho-cleavage pathway for conversion to central metabolites. The genes encoding transporters, components of the stress response, flagellar proteins, and chemotaxis proteins showed altered expression patterns between 3-chlorobenzoate and benzoate. Gene Ontology enrichment analysis revealed that chemotaxis-related terms were significantly upregulated by benzoate compared with 3-chlorobenzoate. Consistent with this, in semisolid agar plate assays, NH9 cells showed stronger chemotaxis to benzoate than to 3-chlorobenzoate. These results, combined with the absence of genes related to uptake/chemotaxis for 3-chlorobenzoate located closely to the degradation genes of 3-chlorobenzoate, suggested that NH9 has not fully adapted to the utilization of chlorinated benzoate, unlike benzoate, in nature.

Crystal structure of the full-length LysR-type transcription regulator CbnR in complex with promoter DNA.

LysR-type transcription regulators (LTTRs) comprise one of the largest families of transcriptional regulators in bacteria. They are typically homo-tetrameric proteins and interact with promoter DNA of ~ 50-60 bp. Earlier biochemical studies have suggested that LTTR binding to promoter DNA bends the DNA and, upon inducer binding, the bend angle of the DNA is reduced through a quaternary structure change of the tetrameric LTTR, leading to the activation of transcription. To date, crystal structures of full-length LTTRs, DNA-binding domains (DBD) with their target DNAs, and the regulatory domains with and without inducer molecules have been reported. However, these crystal structures have not provided direct evidence of the quaternary structure changes of LTTRs or of the molecular mechanism underlying these changes. Here, we report the first crystal structure of a full-length LTTR, CbnR, in complex with its promoter DNA. The crystal structure showed that, in the absence of bound inducer molecules, the four DBDs of the tetrameric CbnR interact with the promoter DNA, bending the DNA by ~ 70°. Structural comparison between the DNA-free and DNA-bound forms demonstrates that the quaternary structure change of the tetrameric CbnR required for promoter region-binding arises from relative orientation changes of the three domains in each subunit. The mechanism of the quaternary structure change caused by inducer binding is also discussed based on the present crystal structure, affinity analysis between CbnR and the promoter DNA, and earlier mutational studies on CbnR. DATABASE: Atomic coordinates and structure factors for the full-length Cupriavidus necator NH9 CbnR in complex with promoter DNA are available in the Protein Data Bank under the accession code 7D98.

JTP-109192, a novel G protein-coupled receptor 119 agonist, prevents atherosclerosis by improving hypercholesterolaemia in congenic spontaneously hyperlipidaemic mice.

G protein-coupled receptor 119 (GPR119) expression in pancreatic ß-cells and intestinal L-cells is a potential therapeutic target for the treatment of type 2 diabetes. Previously, we have reported that the GPR119 agonist JTP-109192 improves glucose metabolism with single and repeated administration. Conversely, overexpression of the Gpr119 gene reportedly regulates cholesterol transporter expression in animal models, and a natural GPR119 agonist, oleoylethanolamide (OEA), improves atherosclerosis. Therefore, improving dyslipidaemia is considered a possible feature of GPR119 agonists. In the present study, the lipid-lowering effect of JTP-109192 was examined in BALB/c background spontaneously hyperlipidaemic (SHL) mice with repeated administration, once daily for 12 weeks. On repeated administration, JTP-109192 revealed a cholesterol-lowering effect and improved atherosclerosis following histopathological examination. With further investigation, the cholesterol-lowering effect and subsequent antiatherosclerotic effect of JTP-109192 was attributed to changes in intestinal cholesterol metabolism gene expression. Based on these results, JTP-109192 represents a new potential antihypercholesterolaemic agent for the treatment of dyslipidaemia.

Hyperglycemia contributes to the development of Leydig cell hyperplasia in male Spontaneously Diabetic Torii rats.

Spontaneously Diabetic Torii (SDT) rats are a well-known animal model of non-obese type 2 diabetes mellitus. Although this animal model has been studied extensively over the last decade, the incidence rates of Leydig cell hyperplasia and tumors in this model have not been reported. In this study, pathophysiological analyses of the testes were performed on male SDT rats, to understand the effect of insulin treatment on the development of Leydig cell hyperplasia and tumors and the expression of integrins and extracellular matrix proteins. Testicular Leydig cell hyperplasia and tumors were observed in SDT rats at 64 weeks of age but were rarely identified in Sprague-Dawley (SD) rats of the same age. Insulin treatment decreased plasma glucose and HbA1c levels, and interestingly, decreased the number of hyperplastic Leydig cell foci and Leydig cell tumors in treated animals. A similar reduction in the expression of Ki67 in these Leydig cell foci was also observed. In addition, insulin treatment decreased the expression of integrin α5, integrin ß1, integrin αvß3, fibronectin, and vitronectin in hyperplastic Leydig cell foci. These results suggest that insulin might decrease the incidence of Leydig cell hyperplasia by reducing Leydig cell proliferation and the expression of integrins and extracellular matrix proteins through the reduction of serum glucose concentrations in these animals.

Complete Genome Sequence of 3-Chlorobenzoate-Degrading Bacterium Cupriavidus necator NH9 and Reclassification of the Strains of the Genera Cupriavidus and Ralstonia Based on Phylogenetic and Whole-Genome Sequence Analyses.

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain's ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C. necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia. As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

Crystal structure of the DNA-binding domain of the LysR-type transcriptional regulator CbnR in complex with a DNA fragment of the recognition-binding site in the promoter region.

LysR-type transcriptional regulators (LTTRs) are among the most abundant transcriptional regulators in bacteria. CbnR is an LTTR derived from Cupriavidus necator (formerly Alcaligenes eutrophus or Ralstonia eutropha) NH9 and is involved in transcriptional activation of the cbnABCD genes encoding chlorocatechol degradative enzymes. CbnR interacts with a cbnA promoter region of approximately 60 bp in length that contains the recognition-binding site (RBS) and activation-binding site (ABS). Upon inducer binding, CbnR seems to undergo conformational changes, leading to the activation of the transcription. Since the interaction of an LTTR with RBS is considered to be the first step of the transcriptional activation, the CbnR-RBS interaction is responsible for the selectivity of the promoter to be activated. To understand the sequence selectivity of CbnR, we determined the crystal structure of the DNA-binding domain of CbnR in complex with RBS of the cbnA promoter at 2.55 Å resolution. The crystal structure revealed details of the interactions between the DNA-binding domain and the promoter DNA. A comparison with the previously reported crystal structure of the DNA-binding domain of BenM in complex with its cognate RBS showed several differences in the DNA interactions, despite the structural similarity between CbnR and BenM. These differences explain the observed promoter sequence selectivity between CbnR and BenM. Particularly, the difference between Thr33 in CbnR and Ser33 in BenM appears to affect the conformations of neighboring residues, leading to the selective interactions with DNA. DATABASE: Atomic coordinates and structure factors for the DNA-binding domain of Cupriavidus necatorNH9 CbnR in complex with RBS are available in the Protein Data Bank under the accession code 5XXP.

Amino acid residues critical for DNA binding and inducer recognition in CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9.

CbnR, a LysR-type transcriptional regulator from Cupriavidus necator NH9, activates the transcription of chlorocatechol-degradative enzymes. To activate the transcription, CbnR needs to bind not only to the cbnA promoter but also to the inducer. In this study, the transcriptional activity and DNA-binding activity of twenty-five mutants of CbnR were analyzed. Of the 17 mutants of the DNA-binding domain, 11 mutants lost their ability to activate transcription. While most mutants without transcriptional activation did not show DNA-binding activity, Asn17Ala, Gln29Ala, and Pro30Ala retained DNA-binding activity, suggesting that transcriptional activation by CbnR requires more than its binding to promoter DNA. Of the 8 mutants of the regulatory domain, 6 mutants changed their responses to the inducer, when compared with wild-type CbnR. Interestingly, Arg199Ala and Val246Ala induced constitutive expression of the cbnA promoter without the inducer, suggesting that these mutations brought about a conformational change mimicking that induced by the inducer molecule.

JTT-130, a novel intestine-specific inhibitor of microsomal triglyceride transfer protein, ameliorates lipid metabolism and attenuates atherosclerosis in hyperlipidemic animal models.

JTT-130 was developed as an intestine-specific MTP inhibitor designed to rapidly catabolize after absorption to avoid causing hepatotoxicity due to hepatic MTP inhibition. In previous reports, we have demonstrated that JTT-130 suppresses dietary lipid absorption in the small intestine without inducing hepatic steatosis. Thus, in this report, JTT-130 was administered to hyperlipidemic animals fed a Western diet to investigate the effect of intestinal MTP inhibition on lipid metabolism and progression of atherosclerosis. JTT-130 potently lowered plasma non-high density lipoprotein-cholesterol, and elevated plasma high density lipoprotein-cholesterol (HDL-C), indicating improvement in atherogenic index in hamsters. HDL fractions obtained after two weeks treatment with JTT-130 significantly increased the efflux of cholesterol from macrophages, as an index parameter of HDL function. Furthermore, long-term treatment with JTT-130 also improved the plasma lipid profile without inducing hepatic steatosis in rabbits, resulting in the suppression of atherosclerosis formation in aortas. From these results, JTT-130 ameliorates lipid metabolism accompanied with the enhancement of the anti-atherosclerotic function of HDL, and attenuates the progression of atherosclerosis in hyperlipidemic animals. These findings indicate that intestinal MTP inhibition may be atherogenic in vivo and that JTT-130 may be a useful compound for the treatment of dyslipidemia and a potential anti-atherogenic drug.

A putative porin gene of Burkholderia sp. NK8 involved in chemotaxis toward ß-ketoadipate.

Burkholderia sp. NK8 can utilize 3-chlorobenzoate (3CB) as a sole source of carbon because it has a megaplasmid (pNK8) that carries the gene cluster (tfdT-CDEF) encoding chlorocatechol-degrading enzymes. The expression of tfdT-CDEF is induced by 3CB. In this study, we found that NK8 cells were attracted to 3CB and its degradation products, 3- and 4-chlorocatechol, and ß-ketoadipate. Capillary assays revealed that a pNK8-eliminated strain (NK82) was defective in chemotaxis toward ß-ketoadipate. The introduction of a plasmid carrying a putative outer membrane porin gene, which we name ompNK8, into strain NK82 restored chemotaxis toward ß-ketoadipate. RT-PCR analyses demonstrated that the transcription of the ompNK8 gene was enhanced in the presence of 3CB.

Regional distribution of styrene analogues generated from polystyrene degradation along the coastlines of the North-East Pacific Ocean and Hawaii.

Beach sand and seawater taken from the coastlines of the North-East Pacific Ocean and Hawaii State were investigated to determine the causes of global chemical contamination from polystyrene (PS). All samples were found to contain styrene monomer (SM), styrene dimers (SD), and styrene trimers (ST) with a concentration distribution of styrene analogues in the order of ST > SD > SM. The contamination by styrene analogues along the West Coast proved more severe than in Alaska and other regions. The Western Coastlines of the USA seem be affected by both land- and ocean-based pollution sources, which might result from it being a heavily populated area as the data suggest a possible proportional relationship between PS pollution and population. Our results suggest the presence of new global chemical contaminants derived from PS in the ocean, and along coasts.

New analytical method for the determination of styrene oligomers formed from polystyrene decomposition and its application at the coastlines of the North-West Pacific ocean.

The pollution caused by plastic debris is an environmental problem with increasing concern in the oceans. Among the plastic polymers, polystyrene (PS) is one of the most problematic plastics due to the direct public health risk associated with their dispersion, as well as the numerous adverse environmental impacts which arise both directly from the plastics and from their degradation products. Little is known about their potential distribution characteristics throughout the oceans. For the first time, we report here on the regional distribution of styrene monomer (SM), styrene dimers (SD; 2,4-diphenyl-1-butene, SD1; 1,3-diphenyl propane, SD2), and styrene trimer (2,4,6-triphenyl-1-hexene: ST1), as products of PS decomposition determined from samples of sand and seawater from the shorelines of the North-West Pacific ocean. In order to quantitatively determine SM, SD (=SD1+SD2), and ST1, a new analytical method was developed. The detection limit was 3.3 µg L(-1), based on a signal-to-noise ratio of three, which was well-suited to quantify levels of SM, SD, and ST1 in samples. Surprisingly, the concentrations of SM, SD, and ST1 in sand samples from the shorelines were consistently greater than those in seawater samples from the same location. The results of this study suggest that SM, SD, and ST1 can be widely dispersed throughout the North-West Pacific oceans.

A 2,4-dichlorophenoxyacetic acid degradation plasmid pM7012 discloses distribution of an unclassified megaplasmid group across bacterial species.

Analysis of the complete nucleotide sequence of plasmid pM7012 from 2,4-dichlorophenoxyacetic-acid (2,4-D)-degrading bacterium Burkholderia sp. M701 revealed that the plasmid had 582 142 bp, with 541 putative protein-coding sequences and 39 putative tRNA genes for the transport of the standard 20 aa. pM7012 contains sequences homologous to the regions involved in conjugal transfer and plasmid maintenance found in plasmids byi_2p from Burkholderia sp. YI23 and pBVIE01 from Burkholderia sp. G4. No relaxase gene was found in any of these plasmids, although genes for a type IV secretion system and type IV coupling proteins were identified. Plasmids with no relaxase gene have been classified as non-mobile plasmids. However, nucleotide sequences with a high level of similarity to the genes for plasmid transfer, plasmid maintenance, 2,4-D degradation and arsenic resistance contained on pM7012 were also detected in eight other megaplasmids (~600 or 900 kb) found in seven Burkholderia strains and a strain of Cupriavidus, which were isolated as 2,4-D-degrading bacteria in Japan and the United States. These results suggested that the 2,4-D degradation megaplasmids related to pM7012 are mobile and distributed across various bacterial species worldwide, and that the plasmid group could be distinguished from known mobile plasmid groups.

Isolation and structural determination of makinolide B from Streptomyces sp. MK-19.

A new 16-membered macrolide named makinolide B (1) was isolated from Streptomyces sp. MK-19. The structure of makinolide B (1) was determined on the basis of 2D NMR experiments involving DQF-COSY, TOCSY, HSQC, and HMBC methods. Application of the paper disk diffusion method to makinolide B (1) showed weak antibacterial activity against Staphylococcus aureus at the dose of 100 µg/disk.

Laboratory tests on sorption and transformation of the insecticide flubendiamide in Japanese tea field soil.

Flubendiamide belongs to the modern insecticides applied in Japanese tea cultivation to control smaller tea tortrix and tea leaf roller. Since fate and behavior in soil have been only monitored sparsely and fragmentarily until today, laboratory tests were performed on sorption, leaching, biotransformation and photo-induced biotransformation of flubendiamide in two different soils. In batch equilibrium tests, K(d) and K(OC) values were 15 and 298 L kg(-1) for the Japanese tea field soil as well as 16 and 1610 L kg(-1) for the German arable field soil classifying flubendiamide to be moderately mobile and slightly mobile, respectively. The affinity to the tea field soil was additionally confirmed by soil column tests where flubendiamide was predominantly retarded in the topsoil layers resulting in a percolate contamination of only 0.002 mg L(-1). In the aerobic biotransformation tests, flubendiamide did not substantially disappear within the 122-d incubation period. Due to DT(50)>122 d, flubendiamide was assessed very persistent. Supplementary, photo-induced impacts on biotransformation were studied in a special laboratory irradiation system. Despite a 14-d irradiation period, photo-induced biotransformation in the tea field soil was not identifiable, neither by HPLC/DAD nor by LC/MS/MS. 3-d irradiation tests in photosensibilizing acetone, however, showed that the primary photo-transformation product desiodo-flubendiamide was formed. How far this photochemical reaction may also occur in soil of perennial tea plant stands, however, has to be checked in field studies.

Specific gene responses of Rhodococcus jostii RHA1 during growth in soil.

Transcriptome analysis of Rhodococcus jostii RHA1 during growth in sterilized soil was performed. A total of 165 soil-specific genes were identified by subtracting genes upregulated in late growth phases and on solid medium from 264 genes commonly upregulated during growth on biphenyl or pyruvate in sterilized soil. Classification of the 165 genes into functional categories indicated that this soil-specific group is rich in genes for the metabolism of fatty acids, amino acids, carbohydrates, and nitrogen and relatively poor in those for cellular processes and signaling. The ro06365-ro06369 gene cluster, in which ro06365 to ro06368 were highly upregulated in transcriptome analysis, was characterized further. ro06365 and ro06366 show similarity to a nitrite/nitrate transporter and a nitrite reductase, respectively, suggesting their involvement in nitrogen metabolism. A strain with an ro06366 deletion, D6366, showed growth retardation when we used nitrate as the sole nitrogen source and no growth when we used nitrite. A strain with a deletion of ro06365 to ro06368, DNop, utilized neither nitrite nor nitrate and recovered growth using nitrite and nitrate by introduction of the deleted genes. Both of the mutants showed growth retardation in sterilized soil, and the growth retardation of DNop was more significant than that of D6366. When these mutants were cultivated in medium containing the same proportions of ammonium, nitrate, and nitrite ions as those in the sterilized soil, they showed growth retardation similar to that in the soil. These results suggest that the ro06365-ro06369 gene cluster has a significant role in nitrogen utilization in sterilized soil.