Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Degradation of p-nitrophenol and pentachlorophenol mixtures by Sphingomonas sp. UG30 in soil perfusion bioreactors

The degradation of mixtures of pentachlorophenol (PCP) and p-nitrophenol (PNP) were evaluated in pure cultures of Sphingomonas sp. UG30, statically incubated soils (60% water-holding capacity) and soil perfusion bioreactors where encapsulated cells of UG30 were used as a soil inoculant. In pure-culture studies, conditions were optimized for mineralization of PCP and PNP mixtures at concentrations of 30 mg l⁻¹ each. Optimum in vitro mineralization of PCP and PNP mixtures by UG30 was facilitated using ammonium phosphate as a nitrogen source, while inhibition was observed with ammonium nitrate. The bioreactor system used columns containing soil treated with mixtures of 100, 225 or 500 mg kg⁻¹ of PCP and PNP. Rapid dissipation of both substrates was observed at the 100 mg kg⁻¹ level. Inoculation with UG30 enhanced PCP degradation at the 100 mg kg⁻¹ level in bioreactors but not in static soil microcosms. At higher PCP and PNP concentrations (225 mg kg⁻¹), occasional complete degradation of PNP was observed, and PCP degradation was about 80% compared to about 25% in statically incubated soils after 20 days at 22°C. There was no additional degradation of the PCP and PNP mixtures attributable to inoculation with encapsulated cells of UG30 in either soil system at concentrations of 225 or 500 mg kg⁻¹. Journal of Industrial Microbiology & Biotechnology (2000) 25, 93–99. Received 25 February 2000/ Accepted in revised form 07 June 2000     

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.     

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of ¹⁴C-labeled isoproturon to ¹⁴CO₂ and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Dehydration of the off-flavor chemical 2-methylisoborneol by the R-limonene-degrading bacteria Pseudomonas sp. strain 19-rlim and Sphingomonas sp. strain BIR2-rlima

The terpene 2-methylisoborneol (MIB), a major cause of off-flavor in farm-raised catfish and drinking water, is transformed by various different terpene-degrading bacteria. Two of these, the R-limonene-degrading strains Pseudomonas sp. 19-rlim and Sphingomonas sp. BIR2-rlima, dehydrated MIB with the formation of odorless metabolites 2-methylenebornane and 4-methylcamphene. These metabolites which have a structural resemblance to camphor, could be further transformed by camphor-degrading bacteria to more oxidized products. The bacterial dehydrations demonstrated here may have application in removing MIB where it is a problem.     

The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil

Aims: To study how repeated applications of an herbicide bromoxynil to a soil, mimicking the regime used in the field, affected the degradation of the compound and whether such affects were reflected by changes in the indigenous bacterial community present. Methods and Results: Bromoxynil degradation was monitored in soil microcosms using HPLC. Its impact on the bacterial community was determined using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of five bacterial taxa (Pseudomonads, Actinobacteria, α‐Proteobacteria, Acidobacteria and nitrifying bacteria). Three applications of 10 mg kg^?1 of bromoxynil at 28‐day intervals resulted in rapid degradation, the time for removal of 50% of the compound decreasing from 6·4 days on the first application to 4·9 days by the third. Bacterial population profiles showed significant similarity throughout the experiment. With the addition of 50 mg kg^?1 bromoxynil to soil, the degradation was preceded by a lag phase and the time for 50% of the compound to be degraded increased from 7 days to 28 days by the third application. The bacterial population showed significant differences 7 days after the final application of bromoxynil that correlated with an inhibition of degradation during the same period. Conclusions: These analyses highlighted that the addition of bromoxynil gave rise to significant shifts in the community diversity and its structure as measured by four abundant taxa, when compared with the control microcosm. These changes persisted even after bromoxynil had been degraded. Significance and Impact of the Study: Here we show that bromoxynil can exert an inhibitory effect on the bacterial population that results in decreased rates of degradation and increased persistence of the compound. In addition, we demonstrate that molecular approaches can identify statistically significant changes in microbial communities that occur in conjunction with changes in the rate of degradation of the compound in the soil.     

Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1

The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined. Received: 12 November 1996 / Received revision: 21 February 1997 / Accepted: 22 February 1997     

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.     

Geomesophilobacter sediminis gen. nov., sp. nov., Geomonas propionica sp. nov. and Geomonas anaerohicana sp. nov., three novel members in the family Geobacterecace isolated from river sediment and paddy soil

Bacteria in the family Geobacteraceae have been proven to fill important niches in a diversity of anaerobic environments and global biogeochemical processes. Here, three bacterial strains in this family, designated Red875^T, Red259^T, and Red421^T were isolated from river sediment and paddy soils in Japan. All of them are Gram-staining-negative, strictly anaerobic, motile, flagellum-harboring cells that form red colonies on agar plates and are capable of utilizing Fe(III)-NTA, Fe(III) citrate, ferrihydrite, MnO₂, fumarate, and nitrate as electron acceptors with acetate, propionate, pyruvate, and glucose as electron donors. Phylogenetic analysis based on the 16S rRNA gene and 92 concatenated core proteins sequences revealed that strains Red259^T and Red421^T clustered with the type strains of Geomonas species, whereas strain Red875^T formed an independent lineage within the family Geobacteraceae. Genome comparison based on  average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values clearly distinguished these three strains from other Geobacteraceae members, with lower values than the thresholds for species delineation. Moreover, strain Red875^T also shared low average amino acid identity (AAI) and percentage of conserved proteins (POCP) values with the type species of the family Geobacteraceae. Based on these physiological, chemotaxonomic, and phylogenetic distinctions, we propose that strain Red875^T (=NBRC 114290^T = MCCC 1K04407^T) represents a novel genus in the family Geobacteraceae, namely, Geomesophilobacter sediminis gen. nov., sp. nov., and strains Red259^T (=NBRC 114288^T = MCCC 1K05016^T) and Red421^T (=NBRC 114289^T = MCCC 1K06216^T) represent two novel independent species in the genus Geomonas, namely, Geomonas propionica sp. nov. and Geomonas anaerohicana sp. nov., respectively.     

普洱地区茶叶内生细菌与根际土壤细菌群落结构分析

[目的]茶叶内生细菌、根际土壤细菌在普洱茶的发酵中起着重要的作用,还可以促进茶树生长,诱导茶树抗病性.研究其群落结构组成及相互关系可为微生物资源开发利用提供理论依据.[方法]本研究以普洱地区茶树叶片和根际土壤为材料,采用高通量测序技术,对茶叶及根际土壤细菌的16S核糖体RNA基因(16S rRNA)进行测序,比较分析茶...     

黄顶菊入侵对土壤中主要功能细菌的影响

以我国北方大面积发生的入侵植物黄顶菊为研究对象,对黄顶菊根际土壤中可培养的主要功能细菌进行了分离筛选,通过rep-PCR聚类和多样性分析研究了其群落结构的变化,并利用16S rRNA序列比对,对主要优势菌群进行鉴定.结果表明: 相对于本地植物万寿菊和空白对照,黄顶菊显著增加了根际土壤中固氮菌、有机磷细菌、无机磷细菌和钾细菌的数量.rep-PCR分析显示,黄顶菊根际4种功能细菌的种群结构与本地植物和对照相比有显著差异,3种土壤中相同的聚类群极少.多样性分析表明,黄顶菊根际微生物物种多样性更加丰富,群落结构更加复杂,优势种群比较明显,生态多样性较高.对从3种土壤中分离得到的主要优势菌群的鉴定结果也进一步证明了这一结论.研究结果为阐明黄顶菊入侵的微生态机制提供了理论基础.     

Transformation of metolachlor in soil inoculated with a Streptomyces sp.

Microbial detoxication of pesticides may offer a promising alternative to existing physical-chemical treatment methods. We investigated a strain of Streptomyces sp. which can transform metolachlor in a liquid medium for its ability to decontaminate herbicide-treated soil. A cell suspension of Streptomyces sp. was added to a silt loam soil (Hagerstown, pH 6.1) which was amended with 10 g of metolachlor containing 5 nCi ring-UL-¹⁴C metolachlor per gram of soil, and the mixture was incubated at 28°C. Inoculation of the sterile soil resulted in the rapid transformation of metolachlor. Analyses of one-week-old samples indicated that approximately 70% of the added radioactivity was recovered in the ethyl acetate and water fractions as products from the inoculated reaction mixture, whereas in the uninoculated control less than 8% of the ¹⁴C was found as products and about 80% was recovered in the form of unchanged metolachlor. In native soil, however, the rate of metolachlor disappearance was not enhanced by Streptomyces inoculation. In inoculated sterile soil the yields of products were affected by inoculum size, inoculation temperature and substrate concentration, but these variables had no effect on product formation in the inoculated native soil. Addition of Na₂CO₃ (200 g/g soil) into native soil significantly promoted growth of Streptomyces due to the higher pH (7.8) and also stimulated transformation of metolachlor by 30%. Our results suggest that proliferation of the inoculated organisms under favorable conditions is essential for their function as metolachlor degraders in native soil.     

茶园根际土壤细菌群落结构及多样性

【背景】茶园根际土壤的细菌群落结构与茶园生境土壤营养循环密切相关,其组成及多样性可以作为健康茶园的一个生物指标。【方法】采用PCR．变性梯度凝胶电泳（PCR—DGGE）分子指纹图谱技术,检测安溪铁观音种植区不同海拔茶园根际土壤样本的细菌群落结构,利用Shannon．Wiener多样性指数分析其多样性,采用非加权组平均法进行聚类分析得到其分布特征,利用蒙特卡罗检验和冗余分析分别揭示影响细菌群落分布的环境因子及细菌群落分布和环境变量之间的关系。【结果】茶园根际土壤细菌的DGGE结果显示,检测到的14种主要细菌中有11种细菌是不可培养的,3种细菌是可培养的,分别属于根瘤菌属、中华根瘤菌属和苍白杆菌属。聚类分析得到,同一海拔梯度茶园根际土壤细菌群落结构相似。Shan-non—Wiener多样性指数分析表明,400m海拔处茶园根际土壤细菌群落多样性最高。蒙特卡罗检验分析得到环境因子协同作用对茶园根际土壤细菌群落结构贡献率为59．6％。冗余分析显示,茶园根际土壤细菌群落结构与海拔密切相关。【结论与意义】茶园根际土壤细菌群落结构分布与海拔梯度密切相关,考虑不同海拔高度土壤细菌群落对茶园营养循环的影响,在铁观音的健康栽培和管理过程中具有重要意义。     

Colonization of contaminated soil by an introduced bacterium: effects of initial pentachlorophenol levels on the survival of Sphingomonas chlorophenolica strain RA2

The survival of a Sphingomonas species that was introduced into pentachlorophenol (PCP)-contaminated soil was monitored with two complementary methods, a respiration-based assay and a most probable number (MPN) technique. Sphingomonas chlorophenolicastrain RA2 is a PCP-mineralizing bacterium that was introduced into soil contaminated with a range of PCP concentrations (0–300 μg PCP g⁻¹ soil). The population of introduced microorganisms was followed for 170 days using a substrate-induced growth-response method and a MPN assay that specifically targets PCP-mineralizing bacteria. Varying the initial PCP concentration resulted in the emergence of three distinct patterns of survival. In soil contaminated with 300 μg PCP g⁻¹ the population of S. chlorophenolica strain RA2 immediately declined following introduction, increased by 200-fold and leveled off by the end of the 170-day incubation. In contrast, populations of S. chlorophenolica strain RA2 declined to levels below detection limits in uncontaminated soil by the end of the experiment. Intermediate PCP concentrations (10–100 μg PCP g⁻¹ soil) resulted in the establishment of S. chlorophenolica strain RA2 that slowly declined in numbers. These results indicate that Sphingomonas chlorophenolica strain RA2 is an effective colonizer of PCP-contaminated soil but will not persist in the absence of PCP. Received 14 April 1999/ Accepted in revised form 24 June 1999     

Monitoring by laser-flow-cytometry of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. strain 107 during biotreatment of a contaminated soil

A flow cytometric method (FCM) was used to detect and accurately enumerate a polycyclic aromatic hydrocarbon-degrading bacterial strain, Sphingomonas sp. 107, inoculated into a soil sample artificially contaminated with pyrene. To compare the FCM method with colony forming unit (CFU) assays, a rifampicin-resistant Sphingomonas sp. 107 was obtained which could be distinguished from the indigenous microflora, since there was no organism resistant to rifampicin in the soil that could transform indole to indigo (naphthalene dioxygenase activity). By combining light-scattering profiles (i.e., morphological properties), ethidium bromide influx (i.e., cell wall permeability), and fluorescence in situ hybridization against the 16S rRNA (i.e., detection specificity), we could enumerate the bacterial population of interest from the indigenous microflora and soil debris during the biotreatment. The FCM technique revealed that the number of inoculated Sphingomonas cells decreased gradually for 15 days of incubation before reaching a steady level of 7 to 12 x 10(5) cells.g-1 of soil. Similar values were obtained with the CFU assay. During this period, pyrene concentration decreased from 632 to 26 mg.kg-1 of dry soil. The FCM detection was improved by adding blocking reagent to the hybridization buffer to minimize the non-specific attachment of the fluorescent probe to soil particles. Combined with the improvements in probe technology, FCM detection was shown to be a good alternative to the conventional culture methods for the analysis of bacterial populations in environmental samples. This technique could be potentially useful for the detection of microorganisms that grow poorly in culture.     

Isolation and characterization of halotolerant phosphate solubilizing bacteria naturally colonizing the peanut rhizosphere in salt-affected soil

Abstract

In search of efficient salt-tolerant phosphate solubilizing bacteria (PSB), we conducted a survey of PSB that naturally colonize the rhizosphere of peanuts in Xinjiang, China, a typical inland saline area. A total of 23 PSB were isolated, which included 12 Bacillus strains, three Acinetobacter strains, two Pseudomonas strains, two Brevibacillus, and one strain of Gordonia terrae, Chryseobacterium lathyri, Ensifer sesbaniae, and Paenibacillus illinoisensis. All PSB have high potential for dissolving calcium phosphate [Ca₃(PO₄)₂] within the range of 65–496?mg·L^?1, but relatively weak ability to dissolve AlPO₄ in amounts ranging from 0.4 to 11.4?mg·L^?1, FePO₄ ranging from 0.1 to 5.5?mg·L^?1, and lecithin ranging from 2.1 to 10.3?mg·L^?1. The maximum tolerance for pH is 10, and the maximum tolerance for Na₂CO₃ is 50?mm. Six isolates that can grow under the condition of 1.5?M NaCl (YMX5, YMX11, TPMX5, TPMX16, TPMX18, and TPMX19) were selected to determine their ability to dissolve Ca₃(PO₄)₂ and tolerate pH changes. This study characterized salt-tolerant PSB isolates that can be used as bioinoculants to protect plants against salt stress. In addition, we describe the phosphate-solubilizing ability of Gordonia terrae at high NaCl concentrations.     

报告基因技术及其在土壤质量监测中的应用

报告基因包括lux、gfp、lacZ、inaZ、luc、cat、xylE及uidA等,其中土壤质量监测中最常用的报告基因有lux、gfp、lacZ和inaZ 4种.从不同角度比较了土壤质量监测中最常用的上述4种报告基因,简要阐述了应用于土壤质量监测中的微生物监测报告基因的定义、类别、性质及特点,展示了报告基因表达系统的构建方式与检测方法,总结了报告基因技术在监测土壤重金属、污染物质、营养物质等化学物质以及在土壤微生物及其活动、土壤根际微生物与土壤中原生动物、植物间的相互作用等方面的应用.讨论了目前报告基因技术应用的局限性及未来研究方向和重点.     

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 polycyclic aromatic hydrocarbons-degrading Sphingomonas sp. strain ZL5

A bacterial strain ZL5, capable of growing on phenanthrene as a sole carbon and energy source but not naphthalene, was isolated by selective enrichment from crude-oil-contaminated soil of Liaohe Oil Field in China. The isolate was identified as a Sphingomonas sp. strain on the basis of 16S ribosomal DNA analysis. Strain ZL5 grown on phenanthrene exhibited catechol 2,3-dioxygenase (C23O) activity but no catechol 1,2-dioxygenase, gentisate 1,2-dioxygenase, protocatechuate 3,4-dioxygenase and protocatechuate 4,5-dioxygenase activities. This suggests that the mode of cleavage of phenanthrene by strain ZL5 could be meta via the intermediate catechol, which is different from the protocatechuate way of other two bacteria, Alcaligenes faecelis AFK2 and Nocardioides sp. strain KP7, also capable of growing on phenanthrene but not naphthalene. A resident plasmid (approximately 60 kb in size), designated as pZL, was detected from strain ZL5. Curing the plasmid with mitomycin C and transferring the plasmid to E. coli revealed that pZL was responsible for polycyclic aromatic hydrocarbons degradation. The C23O gene located on plasmid pZL was cloned and overexpressed in E. coli JM109(DE3). The ring-fission activity of the purified C23O from the recombinant E. coli on dihydroxylated aromatics was in order of catechol > 4-methylcatechol > 3-methylcatechol > 4-chlorocatechol > 3,4-dihydroxyphenanthrene > 3-chlorocatechol.