Microbial Transformation of Terpenoids. I. Indentification of Metabolites Produced by a Pseudomonad from Citronellal and Citral

A bacterium capable of utilizing citronellal or citral as the sole source of carbon and energy has been isolated from soil by the enrichment culture technique. It metabolizes citronellal to citronellic acid (65%), citronellol (0.6%), dihydrocitronellol (0.6%), menthol (0.75%), and 3,7-dimethyl-1,7-octane diol (1.7%). The metabolites of citral were geranic acid (62%), 6-methyl-5-heptanoic acid (0.5%), 3-methyl-2-butenoic acid (1%), and 1-hydroxy-3, 7-dimethyl-6-octen-2-one (0.75%).     

Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27

A Pseudomonas sp. strain, HH69, and a mixed culture, designated HH27, were isolated by selective enrichment from soil samples. The pure strain and the mixed culture grew aerobically on dibenzofuran as the sole source of carbon and energy. Degradation proceeded via salicylic acid which was branched into the gentisic acid and the catechol pathway. Both salicylic acid and gentisic acid accumulated in the culture medium of strain HH69. The acids were slowly metabolized after growth ceased. The enzymes responsible for their metabolism showed relatively low activities. Besides the above-mentioned acids, 2-hydroxyacetophenone, benzopyran-4-one (chromone), several 2-substituted chroman-4-ones, and traces of the four isomeric monohydroxydiben-zofurans were identified in the culture medium. 2,2′,3-Trihydroxybiphenyl was isolated from the medium of a dibenzofuran-converting mutant derived from parent strain HH69, which can no longer grow on dibenzofuran. This gives evidence for a novel type of dioxygenases responsible for the attack on the biarylether structure of the dibenzofuran molecule. A meta-fission mechanism for cleavage of the dihydroxylated aromatic nucleus of 2,2′,3-trihydroxybiphenyl is suggested as the next enzymatic step in the degradative pathway.     

Selection in chemostat culture of a mutant strain of Clostridium tyrobutyricum improved in its reduction of ketones

Summary Growth of Clostridium tyrobutyricum on mannitol was possible only when an ancillary oxidant was additionally supplied. Hexacyanoferrate(III) in the presence of methylviologen could fulfil this role, which is normally better accomplished by acetate (and some other organic electron acceptors including acetoin, crotonate and 3-hydroxybutyrate). Several ketones, including pentan-2-one, although slowly reducible by the organism were unable to support its batch culture growth on mannitol. However, when a chemostat culture supplied with excess mannitol but limiting acetate was supplemented with pentan-2-one, a spontaneous mutant strain was selected that was much improved in its specific rate of reduction of this and other ketones, and which was capable of batch growth on mannitol plus pentan-2-one. This procedure may more generally be employed to select strains of anaerobic bacteria improved in their bioreductive abilities. Offprint requests to: J. G. Morris     

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.     

Hydrogen-using bacteria in a methanogenic acetate enrichment culture

A rcher , D.B. 1984. Hydrogen-using bacteria in a methanogenic acetate enrichment culture. Journal of Applied Bacteriology 56 , 125–129.
In a study of the anaerobic utilization of acetate, an enrichment culture of sewage sludge organisms was initiated with calcium acetate as the sole carbon and energy source. A mixed bacterial population became established from which 14 anaerobic species were isolated. Two of the isolates were methanogenic bacteria but only one of these, Methanosarcina barkeri , utilised acetate as an energy source in axenic culture. The other methanogenic isolate, a Methanobacterium sp., utilised H₂/CO₂ but not acetate. A third methanogen, which was morphologically identical to Methanothrix soehngenii , was detected in the enrichment but was not obtained in monoculture. 2-Bromoethanesulphonate, a specific inhibitor of methanogenesis. completely inhibited the enrichment at a concentration of 10 μmol/1. Addition of H₂ formate or methanol to the enrichment did not affect the rate of methanogenesis. An H₂-utilizing Desulfovibrio sp. was also isolated from the enrichment.     

Oxysterols from human bile induce apoptosis of canine gallbladder epithelial cells in monolayer culture

Oxysterols have been detected in various mammalian organs and blood. Biliary epithelium is exposed to high concentrations of cholesterol, and we have identified three keto-oxysterols (cholest-4-en-3-one, cholesta-4,6-dien-3-one, cholesta-3,5-dien-7-one) in human bile and gallstones. Because the effects of oxysterols on biliary physiology are not well defined, we investigated their biological effects on dog gallbladder epithelial cells. Enriched medium (culture medium containing taurocholate and lecithin and cholesterol +/- various oxysterols) was applied to confluent monolayers of dog gallbladder epithelial cells in culture. Cytotoxicity and apoptosis were studied by morphological analysis and flow cytometry. Oxysterols in the mitochondrial fraction were identified by gas chromatography/mass spectrometry, whereas release of cytochrome c from mitochondria was assayed by spectrophotometry and Western blot analysis. Compared with cells treated with culture medium or with enriched medium containing cholesterol, oxysterol-treated cells showed significantly increased apoptosis (P < 0.05). Exogenously applied oxysterols were recovered from the mitochondrial fraction. Cytochrome c release from mitochondria was increased significantly by cholest-4-en-3-one, cholesta-4,6-dien-3-one, and 5beta-cholestan-3-one (all P < 0.05). Thus oxysterols recovered from human bile and gallstones induce apoptosis of biliary epithelium via a mitochondrial-dependent pathway and may play a role in the pathogenesis of chronic inflammation and carcinogenesis in the gallbladder.     

Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.     

Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae)

An isolate of Curvularia sp. was obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado. The ethyl acetate extract from culture of this fungus afforded two benzopyran derivatives: (2'S)-2-(propan-2'-ol)-5-hydroxy-benzopyran-4-one (2) and 2,3-dihydro-2-methyl-benzopyran-4,5-diol (4); and two known benzopyrans: 2-methyl-5-methoxy-benzopyran-4-one (1) and (2R)-2,3-dihydro-2-methyl-5-methoxy-benzopyran-4-one (3). The structures of 2 and 4 were established on the basis of comprehensive spectroscopic analysis, mainly using 1D and 2D NMR experiments. The benzopyrans 1 and 2 showed weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides. Analyses of the biological activities were also carried out on HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cells, aiming to evaluate their potential effects on mammalian cell line proliferation. Results from both cell lines indicated that compound 2 was able to induce cell proliferation: 70% on HeLa cells and 25% on CHO cells.     

Rapid degradation of the triazinone herbicide metamitron by a Rhodococcus sp. isolated from treated soil

N.R. PAREKH, A. WALKER, S.J. ROBERTS AND S.J. WELCH. 1994. Seven bacterial isolates which degraded the herbicide metamitron (3-methyl-4-amino-6-phenyl-1,2,4-triazin-5-one) were obtained from field-enhanced soil by liquid enrichment culture. All isolates appeared to be identical and a representative, 0246b, was identified as a Rhodococcus sp. by cell wall and fatty acid analyses. This isolate degraded metamitron as the sole source of carbon within 24 h at 25C and this is the first report of a bacterium capable of growing with metamitron as the sole source of carbon. Metamitron was degraded less rapidly when it was the sole source of both carbon and nitrogen. The rate and extent of degradation was affected by the presence and type of additional sources of carbon and nitrogen in the culture medium. In studies with [¹⁴C]-phenyl-labelled metamitron Rhodococcus sp. 0246b partly mineralized the phenyl ring.     

Characterization of a novel biocatalyst system for sulfide oxidation

It has been demonstrated that an enrichment culture dominated by Thiomicrospira sp. CVO may be cultured on H2S(g) as an energy source under sulfide-limiting conditions in suspended culture with nitrate as the electron acceptor. Hydrogen sulfide (10,000 ppmv) was completely removed from the feed gas and oxidized to sulfate in <3 s of gas-liquid contacting time. Maximum loading of the biomass for sulfide oxidation was observed to be 5.8 mmol H2S/h-g biomass protein, comparable to that reported previously for Thiobacillus denitrificans under similar conditions. However, the enrichment culture was shown to be more tolerant of extremes in pH and elevated temperature than T. denitrificans. Coupled with a reported tolerance of CVO for up to 10% NaCl, these observations suggest that a CVO-based culture is potentially a more robust biocatalyst system for sulfide oxidation than cultures based on Thiobacilli.     

A novel pathway for mineralization of the thiocarbamate herbicide molinate by a defined bacterial mixed culture

A bacterial mixed culture able to mineralize molinate was established, through enrichment, using mineral medium with molinate as the only carbon, nitrogen and energy source. The combination of five cultivable isolates, purified from the enrichment culture, permitted the reconstitution of a degrading consortium. Both enrichment and defined cultures were able to mineralize molinate without accumulation of degradation products by the end of the growth. Among the five isolates constituting the defined mixed culture, an actinomycete, strain ON4, was essential for biodegradation, being involved in the cleavage of the thioester bond of molinate, the initial step of the degradation pathway. Isolate ON4 was able to grow on molinate at concentrations below 2 mM, with the accumulation of ethanethiol and diethyl disulphide. These sulphur compounds were toxic to strain ON4 when accumulating at higher concentrations. However, this inhibitory effect was avoided by the presence of other members of the mixed culture, out of which isolates ON1 and ON2 were observed to consume ethanethiol and diethyl disulphide. In this way, interactions among defined mixed culture members involve metabolic and detoxifying association.     

Microbial remediation of NTO in aqueous industrial wastes

The bioremediation of aqueous wastes containing 5-nitro-1,2,4-triazol-3-one (NTO) was investigated. The microorganism used is a Bacillus licheniformis strain, isolated from the contaminated solutions by enrichment techniques. The biodegradation was carried out in the waste (15 g l-1 NTO) and proceeded through the nitroreduction of NTO, followed by the ring cleavage of the formed primary amine 5-amino-1,2,4-triazol-3-one (ATO). Both steps were optimized and according to the optimal conditions, the nitroreduction of NTO is total in 24 h, while the degradation of ATO requires 2 weeks of incubation. The end products of the biodegradation were carbon dioxide (40%), urea and a polar compound, assumed to be hydroxyurea. A mechanism of ATO ring cleavage was postulated in the light of experimental data, and led us to propose an overall degradation sequence for NTO.     

Characterization of a highly enriched dehalococcoides-containing culture that grows on vinyl chloride and trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 +/- 1.4) x 10(8) 16S rRNA gene copies/micromol of Cl(-) when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 +/- 1.3) x 10(8) 16S rRNA gene copies/micromol of Cl(-). The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H(2) enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

一株高效氧化胆固醇的简单节杆菌构建与转化条件优化

为实现胆固醇的高效生物氧化,利用基因工程手段将编码简单节杆菌胆固醇氧化酶的DNA片段克隆到质粒pTY2中,构建pTY2-5332插入表达载体。该载体以简单节杆菌基因组中的16S rDNA位点为整合点,提高了胆固醇氧化酶在基因组中的拷贝数,实现了简单节杆菌胆固醇氧化酶的过表达。重组菌的生长实验分析表明,插入到16S rDNA位点的胆固醇氧化酶没有影响简单节杆菌的生长。重组菌可在20h将2g/L的胆固醇完全转化为4-胆甾烯-3酮,比原始菌的转化时间缩短了4h,提高了胆固醇的转化效率。经过转化条件优化确定了该重组菌以2%的接种量后继续培养16h,然后胆固醇经120目过筛后投料量为2g/L,并添加2%（体积比）二甲基甲酰胺作为促溶剂;胆固醇添加18h后可完全转化为4-胆甾烯-3-酮,是优化前的1.11倍。     

Methyltransferase activity in Allanthm altissima cell suspension cultures

Enzymes for the methylation of 1hydroxycanthin-6-one and a series of coumarins have been isolated from Ailanthus altissima cell suspension cultures. The coumarin methyltransferases methylate aesculetin to scopoletin and isoscopoletin, but not scopoletin, to scoparone. Fraxetin was methylated to isofraxidine but not to fraxidine and only fraxidine was methylated to 6,7,8-trimethoxycoumarin. These enzymes were studied throughout the culture growth cycle with two cell lines: 1, which produced 1-methoxycanthin-6-one as the major alkaloid and 2, in which canthin-6-one was the major alkaloid.Abbreviations UV ultraviolet - DEAE diethylaminoethyl - dw dry weight - MS mass spectrometry - NMR nuclear magnetic resonance - TLC thin layer chromatography     

Comparison of methods for isolating Salmonella bacteria from faeces of naturally infected pigs

A series of experiments was conducted using faecal samples collected from commercial swine farms to evaluate the effects of variation in methods used for the detection of Salmonella bacteria. The primary objective of the studies was to compare the protocols routinely used in two laboratories in the USA. The studies included five experiments comparing the enrichment protocols used routinely in the respective laboratories (Method 1: 10 g faeces--buffered peptone water (BPW) pre-enrichment--selective enrichment in Rappaport/Vassiliadis (RV) broth; Method 2: approximately 1g faeces--primary enrichments in tetrathionate and Hajna GN broths--secondary enrichment in RV broth). The effects of enrichment temperatures (37 vs 42 degrees C) using RV broth (two experiments) and delayed secondary enrichment (four experiments) were also evaluated. Direct comparison of Method 1 and Method 2 indicated comparable results. However, when compared using faecal samples of equal weight, the Method 2 enrichment protocol was more sensitive for detecting Salmonella bacteria than the Method 1 protocol. Enrichment in RV at 42 degrees C was superior to 37 degrees C, particularly for samples that were pre-enriched in BPW. Delayed secondary enrichment increased detection of Salmonella bacteria in swine faeces. These results highlight the imperfect sensitivity of culture methods, and the need for researchers to consider the sensitivity of bacteriological methods in the design and interpretation of the results of epidemiologic studies based on faecal culture.     

Microbiological Hydroxylation of Cinerone to Cinerolone

Cinerone [2-(2′-cis-butenyl)-3-methyl-2-cyclopenten-1-one] is hydroxylated to cinerolone [2-(2′-cis-butenyl)-3-methyl-4-hydroxy-2-cyclopenten-1-one] by a number of streptomycetes, bacteria, and fungi. Aspergillus niger ATCC 9,142 and Streptomyces aureofaciens ATCC 10,762 were found to be the most effective hydroxylators. The optical activity of the product was found to range from [α]_D²⁵ 0° to -8.6°, depending on the organism and conditions of culture. Two additional hydroxylated products of cinerone have been isolated and identified as 2-n-butyl-4-hydroxy-3-methyl-2-cyclopenten-1-one and 2-(2′-cis-butenyl-4′-hydroxy)-3-methyl-2-cyclopenten-1-one, respectively.     

On the origin of the cholestenoic acids in human circulation

3 Beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid are metabolites of cholesterol present at significant concentrations (40-80 ng/ml) in human circulation. The 7 alpha-hydroxylated acids may be formed from cholesterol via two major pathways initiated by oxidations at either the 7 alpha- or 27-positions. In an attempt to clarify the origin and possible precursor-product relationships between these cholestenoic acids, we measured their deuterium enrichment in a unique experiment, after infusion of 10 g of [2H(6)]-cholesterol to a healthy volunteer. The observed extent and time-course of deuterium enrichment of circulating 3 beta-hydroxy-5-cholestenoic and 3 beta,7 alpha-dihydroxy-5-cholestenoic acid were almost identical, while different from that of cholesterol and 7 alpha-hydroxycholesterol. Notably, the deuterium enrichment of 7 alpha-hydroxy-3-oxo-4-cholestenoic acid was similar to that of 7 alpha-hydroxycholesterol (and its metabolite 7 alpha-hydroxy-4-cholesten-3-one), though distinct from the other cholestenoic acids. Finally, the enrichment of unesterified 27-hydroxycholesterol followed a similar, though less pronounced, time curve to the delta(5)-cholestenoic acids. In conclusion, these results suggest that plasma 3 beta-hydroxy-5-cholestenoic acid is formed from a pool of cholesterol distinct from that used for the formation of the bulk of 27-hydroxycholesterol. The results are also in accordance with a formation of 3 beta,7 alpha-dihydroxy-5-cholestenoic acid directly from 3 beta-hydroxy-5-cholestenoic acid, and a formation of most of the circulating 7 alpha-hydroxy-4-cholesten-3-one from 7 alpha-hydroxycholesterol. These results are consistent with a flux of 7 alpha-hydroxycholesterol from the liver into the circulation, and an extrahepatic metabolism of this steroid into 7 alpha-hydroxy-3-oxo-4-cholestenoic acid.     

Acidophilic degradation of methanol by a methanogenic enrichment culture

Abstract An acidophilic methanogenic enrichment culture was obtained in a continuous up-flow anaerobic sludge blanket reactor operated at pH 4.2 with methanol as the sole carbon source. The specific methylotrophic methanogenic activity of the enriched reactor sludge at pH 5 was 3.57 g COD g⁻¹ volatile suspended solids day⁻¹ and the apparent doubling time of the biomass was 15.8 h. Acidic conditions were obligatory, since the enrichment culture was not able to produce methane or to grow at pH 7. Based on morphological characteristics, the dominant methanogenic species in the enrichment culture was a Methanosarcina .     

Continuous enrichment cultures: insights into prokaryotic diversity and metabolic interactions in deep-sea vent chimneys

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.