Demethylation of aromatic compounds by strain B10 and complete degradation of 3-methoxybenzoate in co-culture with Desulfosarcina strains

Summary A pure culture presumably of an Acetobacterium sp. from a waste water pond, strain B10, was able to grow with several methoxylated aromatic compounds by demethylation (or demethoxylation) to the corresponding hydroxilated substances. Acetate was formed from the eliminated methyl or methoxy groups and from CO₂. Demethylation of 3-methoxybenzoate occurred simultaneously with glucose or lactate fermentation if induced, methanol-grown cells of strain B10 were used as an inoculum. If 2-vanillin or 2,3-dimethoxybenzaldehyde were supplied as the only carbon sources, these substances were first oxidized to the corresponding benzoic acid derivatives and subsequently demethylated. In mixed cultures of strain B10 and Desulfosarcina variabilis or Desulfosarcina strain DSU3 the 3-hydroxybenzoate formed by strain B10 from 3-methoxybenzoate was completely degraded to acetate and presumably CO₂ by the sulphate reducers. Acetate could be oxidized to CO₂ upon extended incubation. The complete degradation of 3-methoxybenzoate to CO₂ by co-cultures of strain B10 and Desulfosarcina strains seemed to proceed via a commensalistic, rather than via a syntrophic interaction of the participating organisms. Offprint requests to: J. Winter     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

Proposal of Desulfosarcina ovata subsp. sediminis subsp. nov., a novel toluene-degrading sulfate-reducing bacterium isolated from tidal flat sediment of Tokyo Bay

Strain 28bB2T^T is a sulfate-reducing bacterium isolated in a previous study, obtained from a p-xylene-degrading enrichment culture. Physiological, phylogenetic and genomic characterizations of strain 28bB2T^T were performed to establish the taxonomic status of the strain. Cells of strain 28bB2T^T were short oval-shaped (0.8–1.2 × 1.2–2.7 μm), motile, and Gram-negative. For growth, the optimum pH was pH 6.5–7.0 and the optimum temperature was 28–32 °C. Strain 28bB2T^T oxidized toluene but could not utilize p-xylene. Sulfate and thiosulfate were used as electron acceptors. The G + C content of the genomic DNA was 53.8 mol%. The genome consisted of an approximately 8.3 Mb of chromosome and two extrachromosomal elements. On the basis of 16S rRNA gene analysis, strain 28bB2T^T was revealed to belong to the genus Desulfosarcina, with high sequence identities to Desulfosarcina ovata oXyS1^T (99.5%) and Desulfosarcina cetonica DSM 7267^T (98.7%). Results of Average Nucleotide Identity (ANI) calculation and digital DNA–DNA hybridization (dDDH) analysis showed that the strain 28bB2T^T should be classified as a subspecies under D. ovata. Based on physiological and phylogenetic data, strain 28bB2T^T (=NBRC 106234 =DSM 23484) is proposed as the type strain of a novel species in genus Desulfosarcina, Desulfosarcina ovata subsp. sediminis subsp. nov.     

Identification of active gaseous-alkane degraders at natural gas seeps

Natural gas seeps release significant amounts of methane and other gases including ethane and propane contributing to global climate change. In this study, bacterial actively consuming short-chain alkanes were identified by cultivation, whole-genome sequencing, and stable-isotope probing (SIP)-metagenomics using ¹³C-propane and ¹³C-ethane from two different natural gas seeps, Pipe Creek and Andreiasu Everlasting Fire. Nearly 100 metagenome-assembled genomes (MAGs) (completeness 70–99%) were recovered from both sites. Among these, 16 MAGs had genes encoding the soluble di-iron monooxygenase (SDIMO). The MAGs were affiliated to Actinobacteria (two MAGs), Alphaproteobacteria (ten MAGs), and Gammaproteobacteria (four MAGs). Additionally, three gaseous-alkane degraders were isolated in pure culture, all of which could grow on ethane, propane, and butane and possessed SDIMO-related genes. Two Rhodoblastus strains (PC2 and PC3) were from Pipe Creek and a Mycolicibacterium strain (ANDR5) from Andreiasu. Strains PC2 and PC3 encoded putative butane monooxygenases (MOs) and strain ANDR5 contained a propane MO. Mycolicibacterium strain ANDR5 and MAG19a, highly abundant in incubations with ¹³C-ethane, share an amino acid identity (AAI) of 99.3%. We show using a combination of enrichment and isolation, and cultivation-independent techniques, that these natural gas seeps contain a diverse community of active bacteria oxidising gaseous-alkanes, which play an important role in biogeochemical cycling of natural gas.Subject terms: Metagenomics, Environmental microbiology     

A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea

Recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium, Methylococcus capsulatus (Bath) has revealed a highly versatile metabolic potential. In parallel, environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea, further supporting the hypothesis of reverse methanogenesis.     

Identification and Characterization of the Butane-Utilizing Bacterium,Arthrobacter sp. PG-3-2, Harboring a Novel bmoX Gene

A bacterium, PG-3-2, capable of butane-utilization as a sole carbon source was isolated from Puguang oilfield in Sichuan Province, China and identified as Arthrobacter sp. by 16S rRNA gene sequence and morphology characteristics. Butane-saturated medium was defined as optimal for the growth of PG-3-2. Proliferation of PG-3-2 was enhanced at low butanol concentrations (≤50 mM) and repressed at high concentrations (≥100 mM). Growth of strain PG-3-2 was supported by alkanes from C₂ to C₁₀ (except pentane) and various carbon substrates including primary alcohols, secondary alcohols, carboxylic acids, aldehydes, ketones, but not methane or its oxidation products. The rate of butane degradation by PG-3-2 was relatively high during the lag phase and prophase of the exponential phase. A bmoX gene, which encodes the alpha hydroxylase subunit of butane monooxygenase, was amplified from the genome of this bacterium. Sequence analysis revealed a high level of homology with alkane monooxygenase, thus indicating the existence of a novel bmoX gene involved in the butane degradation pathway in this Arthrobacter strain.     

Insight from the draft genome of Dietzia cinnamea P4 reveals mechanisms of survival in complex tropical soil habitats and biotechnology potential

The draft genome of Dietzia cinnamea strain P4 was determined using pyrosequencing. In total, 428 supercontigs were obtained and analyzed. We here describe and interpret the main features of the draft genome. The genome contained a total of 3,555,295 bp, arranged in a single replicon with an average G+C percentage of 70.9%. It revealed the presence of complete pathways for basically all central metabolic routes. Also present were complete sets of genes for the glyoxalate and reductive carboxylate cycles. Autotrophic growth was suggested to occur by the presence of genes for aerobic CO oxidation, formate/formaldehyde oxidation, the reverse tricarboxylic acid cycle and the 3-hydropropionate cycle for CO₂ fixation. Secondary metabolism was evidenced by the presence of genes for the biosynthesis of terpene compounds, frenolicin, nanaomycin and avilamycin A antibiotics. Furthermore, a probable role in azinomycin B synthesis, an important product with antitumor activity, was indicated. The complete alk operon for the degradation of n-alkanes was found to be present, as were clusters of genes for biphenyl ring dihydroxylation. This study brings new insights in the genetics and physiology of D. cinnamea P4, which is useful in biotechnology and bioremediation.     

Metabolism of Propane, n-Propylamine, and Propionate by Hydrocarbon-Utilizing Bacteria

Studies were conducted on the oxidation and assimilation of various three-carbon compounds by a gram-positive rod isolated from soil and designated strain R-22. This organism can utilize propane, propionate, or n-propylamine as sole source of carbon and energy. Respiration rates, enzyme assays, and ¹⁴CO₂ incorporation experiments suggest that propane is metabolized via methyl ketone formation; propionate and n-propylamine are metabolized via the methylmalonyl-succinate pathway. Isocitrate lyase activity was found in cells grown on acetate and was not present in cells grown on propionate or n-propylamine. ¹⁴CO₂ was incorporated into pyruvate when propionate and n-propylamine were oxidized in the presence of NaAsO₂, but insignificant radioactivity was found in pyruvate produced during the oxidation of propane and acetone. The n-propylamine dissimilatory mechanism was inducible in strain R-22, and amine dehydrogenase activity was detected in cells grown on n-propylamine. Radiorespirometer and ¹⁴CO₂ incorporation studies with several propane-utilizing organisms indicate that the methylmalonyl-succinate pathway is the predominant one for the metabolism of propionate.     

Genome‐scale metabolic reconstruction and constraint‐based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold‐adapted bacteria and is currently exploited as a new alternative expression host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of –omics data. A genome‐scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold‐dependent changes in gene expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome‐scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system‐level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.     

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with ¹³C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood–Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.     

Anaerobic degradation of phenylacetic acid by mixed and pure cultures

Summary A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.     

Integrated genomics and phenotype microarray analysis of Saccharomyces cerevisiae industrial strains for rice wine fermentation and recombinant protein production

The industrial potential of Saccharomyces cerevisiae has extended beyond its traditional use in fermentation to various applications, including recombinant protein production. Herein, comparative genomics was performed with three industrial S. cerevisiae strains and revealed a heterozygous diploid genome for the 98-5 and KSD-YC strains (exploited for rice wine fermentation) and a haploid genome for strain Y2805 (used for recombinant protein production). Phylogenomic analysis indicated that Y2805 was closely associated with the reference strain S288C, whereas KSD-YC and 98-5 were grouped with Asian and European wine strains, respectively. Particularly, a single nucleotide polymorphism (SNP) in FDC1, involved in the biosynthesis of 4-vinylguaiacol (4-VG, a phenolic compound with a clove-like aroma), was found in KSD-YC, consistent with its lack of 4-VG production. Phenotype microarray (PM) analysis showed that KSD-YC and 98-5 displayed broader substrate utilization than S288C and Y2805. The SNPs detected by genome comparison were mapped to the genes responsible for the observed phenotypic differences. In addition, detailed information on the structural organization of Y2805 selection markers was validated by Sanger sequencing. Integrated genomics and PM analysis elucidated the evolutionary history and genetic diversity of industrial S. cerevisiae strains, providing a platform to improve fermentation processes and genetic manipulation.     

A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea

Recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium, Methylococcus capsulatus (Bath) has revealed a highly versatile metabolic potential. In parallel, environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea, further supporting the hypothesis of reverse methanogenesis.     

Chloroform degradation by butane-grown cells of Rhodococcus aetherovorans BCP1

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0–633 μM range (0–75.5 mg l⁻¹) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 μmol , respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 μM) did not inhibit butane utilization. Acetylene (1,720 μM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 μM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Methylketones from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Cell suspensions of methane-utilizing bacteria grown on methane oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). The product methylketones accumulated extracellularly. The rate of production of methylketones varied with the organism used for oxidation; however, the average rate of acetone, 2-butanone, 2-pentanone, and 2-hexanone production was 1.2, 1.0, 0.15, and 0.025 μmol/h per 5.0 mg of protein in cell suspensions. Primary alcohols and aldehydes were also detected in low amounts as products of n-alkane (propane and butane) oxidation, but were rapidly metabolized further by cell suspensions. The optimal conditions for in vivo methylketone formation from n-alkanes were compared in Methylococcus capsulatus (Texas strain), Methylosinus sp. (CRL-15), and Methylobacterium sp. (CRL-26). The rate of acetone and 2-butanone production was linear for the first 60 min of incubation and directly increased with cell concentration up to 10 mg of protein per ml for all three cultures tested. The optimal temperatures for the production of acetone and 2-butanone were 35°C for Methylosinus trichosporium sp. (CRL-15) and Methylobacterium sp. (CRL-26) and 40°C for Methylcoccus capsulatus (Texas). Metal-chelating agents inhibited the production of methylketones, suggesting the involvement of a metal-containing enzymatic system in the oxidation of n-alkanes to the corresponding methylketones. The soluble crude extracts derived from methane-utilizing bacteria contained an oxidized nicotinamide adenine dinucleotide-dependent dehydrogenase which catalyzed the oxidation of secondary alcohols.     

枯草芽孢杆菌N2-10的基因组测序和比较基因组分析

【背景】枯草芽孢杆菌N2-10是一株具有较强抑菌能力且能产纤维素酶等多种水解酶的革兰氏阳性菌,在发酵饲料中具有较大的应用潜力。【目的】通过获得枯草芽孢杆菌N2-10的全基因组序列信息,进一步解析菌株次级代谢产物合成基因信息,并通过比较基因组学分析菌株N2-10与模式菌株的差异性,为阐明N2-10抑菌和益生机制提供理论基础。【方法】通过二代Illumina NovaSeq联合三代PacBio Sequel测序平台,对菌株N2-10进行全基因组测序,将测序数据进行基因组组装、基因预测与功能注释,并利用比较基因组学分析N2-10与其他菌株的差异。【结果】菌株N2-10基因组大小为4 036 899 bp,GC含量为43.88%;共编码4 163个编码基因,所有编码基因总长度为3594369bp,编码区总长度占基因组总长度的89.04%;含有85个tRNA、10个5S rRNA、10个16S rRNA、10个23S rRNA,以及2个CRISPR-Cas、1个前噬菌体和6个基因岛;在GO (gene ontolog)、COG (clusters of orthologous groups of...     

Atlantic salmon (Salmo salar L.) genetics in the 21st century: taking leaps forward in aquaculture and biological understanding

Atlantic salmon (Salmo salar L.) is among the most iconic and economically important fish species and was the first member of Salmonidae to have a high‐quality reference genome assembly published. Advances in genomics have become increasingly central to the genetic improvement of farmed Atlantic salmon as well as conservation of wild salmon stocks. The salmon genome has also been pivotal in shaping our understanding of the evolutionary and functional consequences arising from an ancestral whole‐genome duplication event characterising all Salmonidae members. Here, we provide a review of the current status of Atlantic salmon genetics and genomics, focussed on progress made from genome‐wide research aimed at improving aquaculture production and enhancing understanding of salmonid ecology, physiology and evolution. We present our views on the future direction of salmon genomics, including the role of emerging technologies (e.g. genome editing) in elucidating genetic features that underpin functional variation in traits of commercial and evolutionary importance.     

Relationship between Stereoisomerism and Biological Activity of Pyrethroids

Higher homologous acids of chrysanthemic acid described in the previous papers were esterified with (±)-allethrolone. The toxicity of these esters and the related compounds were evaluated by the topical application method to Musca domestica vicina Macq. The allethronyl homochry-santhemate (entry Nos. 4, 5) was shown to be toxic and the dextrorotatory form was far more toxic than the laevorotatory one. Further elongation of the ester linkage resulted in a loss of toxicity. The cyclobutane carboxylic acid ester (entry No. 10) was shown to be toxic and so, the cyclopropane ring might be replaced to some extent by the cyclobutane ring, provided the other requirements were fulfiled. However, further elongation of the ester linkage also reduced the toxicity. The lactones (entry Nos. 12–16) obtained by the hot sulfuric acid treatment were non-toxic.     

Enzymes involved in phthalate degradation in sulphate-reducing bacteria

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the short-lived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology.