Sphingomonas sp. strain KA1, carrying a carbazole dioxygenase gene homologue,degrades chlorinated dibenzo-p-dioxins in soil

Hybridization analysis showed that a newly isolated carbazole (CAR)-degrading bacterium Sphingomonas sp. strain KA1 did not possess the gene encoding the terminal oxygenase component (carAa) of CAR 1,9a-dioxygenase at high homology (more than 90% identity) to that of another CAR-degrader, Pseudomonas resinovorans strain CA10. However, PCR experiments using the primers for amplifying the internal fragment of the carAa gene (810 bp for strain CA10) showed that a PCR product of unexpected size (1100 bp) was amplified. Sequence analysis revealed that this DNA region contained the portion of two possible ORFs, which showed moderate homology to CarAa and CarBa from strain CA10 (61% and 40% identities at the amino acid level, respectively). Inoculation of strain KA1 into dioxin-contaminated model soil resulted in 96% and 70% degradation of 2-mono- and 2,3-dichlorinated dibenzo-p-dioxin, respectively, after 7-day incubation.     

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10⁻⁵ to 10⁻⁸ per recipient. A total of 12 distinct plasmids, designated pB1–pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10⁻¹ to 10⁻² per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPβ subgroup, whereas two plasmids were identified as IncPα plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPβ plasmids at the DNA sequence level. In order to characterize the gene “load” of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified. Received: 11 October 1999 / Accepted: 11 January 2000     

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.     

Isolation and characterization of an atrazine-degrading Rhodococcus sp. strain MB-P1 from contaminated soil

Aims:  The aim of this study is to isolate and characterize organisms capable of utilizing high concentration atrazine from the contaminated sites.
Methods and Results:  A selective enrichment was used for isolating atrazine-degrading organisms from the contaminated sites resulting in isolation of an efficient atrazine-degrading organism designated as strain MB-P1. On the basis of 16S rRNA gene sequencing, total cellular fatty acid analysis and physiological and biochemical tests, strain MB-P1 was identified as a member of genus Rhodococcus . High performance liquid chromatography was performed to identify the atrazine degradation intermediates demonstrating that the degradation proceeds via formation of 'de-ethylatrazine' and 'de-isopropylatrazine'. Further, plasmid curing by SDS method showed atrazine-degrading gene(s) to be plasmid-encoded.
Conclusions:  We have successfully isolated a Rhodococcus sp. strain MB-P1 which is capable of utilizing atrazine as sole source of carbon and energy at very high concentrations of 1000 ppm. The pathway for degradation of atrazine has also been determined. The metabolic gene(s) responsible for atrazine degradation was found to be plasmid-encoded.
Significance and Impact of the Study:  Rhodococcus sp. strain MB-P1 could be used as an ideal model system for in-situ degradation and restoration of ecological niches which are heavily contaminated with atrazine.     

Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid,a platform chemical from inedible biomass

Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80 g/L LA by some isolated strains, Brevibacterium epidermidis LA39–2 consumed 62.6 g/L LA following 8 days incubation. The strain also utilized both 90 and 100 g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10 days incubation.     

Characterization of Thiobacillus thioparus isolated from an activated sludge bioreactor used for hydrogen sulfide treatment

AIMS: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H(2)S) degradation. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H(2)S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H(2)S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H(2)S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. CONCLUSIONS: It was concluded that the use of AS process for H(2)S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. SIGNIFICANCE AND IMPACT OF THE STUDY: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H(2)S.     

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

AIMS: Isolation of the genes relative to PCB biodegradation and identification of the bph gene function in Rhodococcus sp. R04. METHODS AND RESULTS: A 8.7-kb fragment carrying the biphenyl catabolic genes bphABCD was isolated from the gene library in Rhodococcus sp. R04. Based on the deduced amino acid sequence homology, seven bph genes, bphA1A2A3A4, bphB, bphC and bphD, were thought to be responsible for the initial four steps of biphenyl degradation. In Escherichia coli, BphA exhibited poor activity for biphenyl transformation, and BphB, BphC and BphD were found to be catalytically active towards 2,3-dihydro-2,3-dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, respectively (activities of 50, 8.1 and 2.4 micromol l(-1) min(-1) mg(-1)). SDS-PAGE analysis indicated that the sizes of bphA1A2A3A4, bphB, bphC and bphD gene products were 49, 19, 14, 47, 32, 30 and 31 kDa, respectively. After disruption of bph genes, the bphA1 mutants lost the ability to grow on biphenyl, the bphB and bphD mutants were able to transform a little of biphenyl, but hardly grew on biphenyl. CONCLUSION: The cloned bph genes indeed play an important role in the biphenyl catabolism in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This bph gene organization in Rhodococcus sp. R04 differs from that of other biphenyl degraders reported previously, indicating it is a novel type of bph gene cluster. Analysis of the phylogenetic tree suggested that BphA1 and BphA2 in Rhodococcus sp. R04 had a different evolutionary relationship with those in the other PCB degraders.     

Molecular detection and diversity of medium-chain-length polyhydroxyalkanoates-producing bacteria enriched from activated sludge

AIMS: Knowledge of the species composition of complex bacterial communities is still very limited. The main objectives of this study were to identify medium-chain-length polyhydroxyalkanoates (mcl-PHAs)-producing bacteria from activated sludge fed with methanol as well as to characterize their PHA operon. METHODS AND RESULTS: The identification was based on PCR amplification of mcl-PHA synthase gene fragments. In the analysed sample, four isolates possessing mcl-PHA synthesis systems were distinguished. The results of a 16S rDNA sequence analysis revealed that three strains belonged to Pseudomonas species and the fourth one was characterized as Comamonas testosteroni. CONCLUSIONS: The results of this study indicate that the PCR-RFLP approach is an excellent way to identify mcl-PHA-synthesizing micro-organisms. The discovery of 4 genetic variants, among the 20 analysed, demonstrates that microbial diversity of activated sludge is high and thus offers a great opportunity for the discovery of novel gene forms. SIGNIFICANCE AND IMPACT OF THE STUDY: An important discovery of this study is that C. testosteroni could harbour mcl-PHA operon. Moreover, the results obtained indicate that PHAs synthesis ability can be spread by horizontal gene transfer. The results of a comparative phylogenetic analysis revealed that mcl-PHA-synthesizing bacteria can be divided into Pseudomonas fluorescens and Pseudomonas aeruginosa groups.     

The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Rhodococcus cercidiphylli sp. nov., a new endophytic actinobacterium isolated from a Cercidiphyllum japonicum leaf

An endophytic actinobacterium, designated YIM 65003(T), was isolated from a surface sterilized leaf sample of Cercidiphyllum japonicum collected from Yunnan province, south-west China. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. Analysis of the 16S rRNA gene sequence revealed that the isolate was most closely related to Rhodococcus fascians DSM 20669(T) (99.6%) and Rhodococcus yunnanensis YIM 70056(T) (99.0%). DNA-DNA hybridization with the above microorganisms (46.3% and 48.8%, respectively), in combination with differences in the biochemical and physiological properties, suggested that strain YIM 65003(T) should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus cercidiphylli sp. nov. is proposed, with YIM 65003(T) (=CCTCC AB 207160(T)=DSM 45141(T)) as the type strain. The 16S rRNA gene sequence of strain YIM 65003(T) has been deposited in GenBank under the accession number EU325542.     

Genetic and antigenic characterization of swine H1N2 influenza viruses isolated from Korean pigs

H1N2 influenza viruses are circulating in pigs worldwide and cause considerable economic losses to the pig industry. We genetically analyzed the genes of our isolates from Korean pigs, and compared the antigenicity of our isolates with swine H1N2 viruses isolated from pigs in the U.S.A. In addition, we serologically surveyed the infection rate of swine H1N2 viruses in pigs. We found that H1N2 isolates from Korean pigs are genetically more related to swine H1N2 viruses isolated from pigs in the U.S.A. than those in European countries. When antigenicity was compared, our isolates were weakly reacted to antibodies against swine H1N2 viruses isolated from pigs in the U.S.A. The serological surveillance using sera from pigs in Korea showed that about 46% was positive for H1N2 viruses. Our results suggest that swine H1N2 viruses are widespread in Korean pigs, and the development of a vaccine against H1N2 viruses may help to control their infection in pigs.     

一株分离自网箱养殖区沉积物的硫氧化菌B1-1的鉴定及其硫氧化特性

【背景】海水网箱养殖是一种高密度、高投饵的人工养殖方式,其产生的硫化物污染严重影响了海水养殖业的经济效益及可持续发展。【目的】鉴定从网箱养殖区沉积物中分离得到的硫氧化菌B1-1并研究其硫氧化特性,为利用生物技术修复网箱养殖环境提供理论依据。【方法】通过形态观察、生理生化特征及16S rRNA基因序列分析对硫氧化菌B1-1进行鉴定;通过摇床培养对该菌株氧化硫代硫酸盐的最适培养条件(pH、温度、底物浓度、外加碳源、外加氮源、金属离子)进行研究。【结果】该菌株被鉴定为HalothiobacillushydrothermalisB1-1。菌株B1-1生物氧化硫代硫酸盐的最适pH为8.0,最适生长温度为30°C,最适底物浓度为5.0g/L;外加碳源对该菌株氧化能力无显著影响,外加铵盐(硫酸铵或草酸铵)可以将延滞期缩短至12 h,添加Mg~(2+)可以显著提高菌株的氧化能力,氧化率达100%。【结论】Halothiobacillus hydrothermalisB1-1耐盐性较强,对硫代硫酸盐氧化率高,可作为修复受硫化物污染网箱养殖环境的潜在菌株资源。     

lambda-Transducing phages carrying transfer genes isolated from an abnormal prophage insertion into the traY gene of F

A set of lambda-transducing phages carrying transfer (tra) genes has been isolated from an abnormal lysogen in which a lambda prophage was inserted into the traY gene of Flac. These have been characterized genetically for complementation of Flac tra and finP point mutants and for the presence of oriT. Studies of tra gene expression during lambda repression showed that tra genes on the transducing phages were expressed from the lambda PL promoter as well as from the transfer promoters when these were present. The molecular weights of the traM (14,000) and traJ (23,500) proteins were measured after infection of ultraviolet-irradiated cells with one of the phages, ED lambda 102, and overproduction of the traJ protein upon induction of an ED lambda 102 lysogen was demonstrated. A proportion of this traJ protein was located in the inner membrane and cytoplasmic fractions of the cell, the majority being in the outer membrane. Physical analysis of the DNA carried by the lambda tra phages by determination of the phage buoyant densities in CsCl, by restriction enzyme digestion and by electron microscope heteroduplex analysis, was used to define the DNA segments encoding the tra functions. Correlation of the physical and genetical data improved the positioning of the tra genes within the transfer region. These results were combined with new restriction enzyme cleavage data to construct an improved map of this region.     

Biodegradation of high concentrations of formaldehyde using Escherichia coli expressing the formaldehyde dismutase gene of Methylobacterium sp. FD1

In the present study, formaldehyde dismutase from Methylobacterium sp. FD1 was partially purified and analyzed by nanoLC–MS/MS; it was then cloned from the genomic DNA of FD1 by PCR. The open reading frame of the formaldehyde dismutase gene of FD1 was estimated to be 1203 bp in length. The molecular weight and pI of formaldehyde dismutase (401 aa), as deduced from the FD1 gene, were calculated at 42,877.32 and 6.56, respectively. NAD(H)-binding residues and zinc-binding residues were found in the amino acid sequence of the deduced formaldehyde dismutase of FD1 by BLAST search. The resting Escherichia coli cells that were transformed with the FD1 formaldehyde dismutase gene degraded high concentrations of formaldehyde and produced formic acid and methanol that were molar equivalents of one-half of the degraded formaldehyde. The lyophilized cells of the recombinant E. coli also degraded high concentrations of formaldehyde.     

Clostridium quinii sp. nov., a new saccharolytic anaerobic bacterium isolated from granular sludge

A new species of sporulating saccharolytic anaerobe, designated as Clostridium quinii sp. nov., is described. A gram-positive strain BS1, was isolated from the granular metanogenic sludge (UASB) from a waste-water treatment plant at a sugar refinery. The strain exhibits a series of morphological stages, developing from a spore to a small rod to a motile rod (peritrichous flagella) in the exponential growth phase, and then swelling to form cigar-shaped cells, exhibiting tumbling movements, in the late exponential growth phase before finally becoming large nonmotile ovoid cells in the stationary phase. Swelling occurs as a result of glucose being taken up and stored as a glycogen-like substance. The main fermentation products when growing on glucose is H₂, CO₂, formate, acetate and ethanol as well as small amounts of butyrate during exponential growth. Lactate is formed during the stationary phase, when glucose is abundant. Optimal conditions for growth is 40–45°C and pH of around 7.4. The type strain BS1 contains 28.0% mol G+C.     

Effects of oxygen exposure on respiratory activities of Desulfovibrio desulfuricans strain DvO1 isolated from activated sludge

The present study addresses the effects of oxygen exposure on the aerobic and anaerobic respiratory activity of Desulfovibrio desulfuricans strain DvO1. This strain was isolated from the highest sulfate-reduction positive most-probable-number dilution (10(6)) of an activated sludge sample, which had been subjected to 120 h of continuous aeration. Washed cell suspensions of strain DvO1 were aerated at 50% atmospheric oxygen saturation in sulfide-free media for a period of 33 h in the presence or absence of an external electron donor (10 mM lactate). During the aeration periods, samples were removed at intervals for determination of anaerobic INT [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride]-reducing activity, anaerobic sulfate-reducing activity, and oxygen-reducing activity. The cell suspension aerated in the absence of lactate showed negligible endogenous oxygen reduction rates and therefore did not consume oxygen during the aeration period. In contrast, the cell suspension aerated in the presence of lactate sustained significant rates of oxygen reduction during the entire 33 h aeration period. Despite this, no explicit differences in the potential INT-, oxygen-, or sulfate-reducing activities were evident between the two cell suspensions during the aeration periods. Strain DvO1 remained viable throughout the 33 h aeration periods irrespective of the presence or absence of lactate, however, the oxygen exposure resulted in a dose-dependent reversible metabolic inactivation. Notably, lactate-dependent anaerobic sulfate-reducing activity recovered quickly upon anaerobiosis, and was more oxygen tolerant than lactate-dependent oxygen-reducing activity.     

Degradation of 1,3-dichloropropene by a soil bacterial consortium and Rhodococcus sp. AS2C isolated from the consortium

A bacterial consortium capable of degrading the fumigant 1,3-D ((Z)- and (E)-1,3-dichloropropene) was enriched from an enhanced soil. This mixedculture degraded (Z)- and (E)-1,3-D only in the presence of a suitable biodegradable organic substrate, such as tryptone, tryptophan, or alanine. After 8 months of subculturing at 2- to 3-week intervals, a strain of Rhodococcus sp. (AS2C) that was capable of degrading 1,3-D cometabolically in the presenceof a suitable second substrate was isolated. (Z)-3-chloroallyl alcohol (3-CAA) and (Z)-3-chloroacrylic acid (3-CAAC), and (E)-3-CAA and (E)-3-CAAC were the metabolites of (Z)- and (E)-1,3-D, respectively. (E)-1,3-D was degraded faster than (Z)-1,3-D by the strain AS2C and the consortium. AS2C also degraded (E)-3-CAA faster than (Z)-3-CAA. Isomerization of (E)-1,3-D to (Z)-1,3-D orthe (Z) form to the (E) form did not occur.     

瓜类枯萎病菌遗传多样性和亲缘关系的SRAP分析

尖孢镰刀菌可造成不同瓜类的枯萎病.为明确不同寄主、不同地区的瓜类枯萎病菌菌株间的遗传多样性及亲缘关系,采用相关序列扩增多态性(SRAP)分子标记技术,对来源于不同地区、不同寄主的95株尖孢镰刀菌的基因组DNA进行多态性扩增.以筛选出的19对引物共扩增出238条带,多态性比率为100%,平均每对引物扩增出12.5个位点和12.5个多态性位点;尖孢镰刀菌苦瓜专化型共扩增出166条带,其中145条为多态性条带,多态性比率为87.4%,平均每对引物扩增出8.7个位点和7.7个多态性位点,说明尖孢镰刀菌的遗传变异较为广泛.瓜类枯萎病菌株间的遗传相似系数范围为0.68～0.99,样品间的平均Nei遗传多样性指数和Shannon指数分别为0.2390和0.3718.在遗传相似系数为0.74时,可将供试的95株尖孢镰刀菌划分为苦瓜、黄瓜、西瓜、甜瓜4个专化群.在SRAP聚类树中,同一寄主的尖孢镰刀菌聚在一个分支上,其中尖孢镰刀菌苦瓜专化型菌株间的遗传相似系数范围为0.78～0.99,Nei遗传多样性指数为0.1811,平均Shannon指数为0.2750,表明尖孢镰刀菌苦瓜专化型的遗传变异较大,且菌株的聚群与地理来源存在相关性.     

β-Methylmuconolactone, a key intermediate in the dissimilation of methylaromatic compounds by a modified 3-oxoadipate pathway evolved in nocardioform actinomycetes

Abstract p -Toluate-grown cells of Rhodococcus ruber N75, R. corallinus N657, R. rhodochrous N5 and Rhodococcus strains BCN1, BCN2 and 4PH1 metabolized 4-methylcatechol by a modified 3-oxoadipate pathway. Steps in the conversion of this compound to 4-methyl-3-oxoadipic acid were investigated. The conversion of 4-carboxymethyl-3-methylbut-2-en-1, 4-olide to 4-carboxymethyl-3-methylbut-2-en-1, 4-olide by a new enzyme is described.