Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase (trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.     

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.     

一株阿特拉津降解菌的分离鉴定及降解特性

从农药厂废水处理池的活性污泥中分离到一株阿特拉津降解菌X-4, 根据其生理生化特性和16S rRNA基因序列相似性分析, 将其初步鉴定为节杆菌属(Arthrobacter sp.)。该菌能以阿特拉津为唯一碳氮源生长, 42 h内对100 mg/L的阿特拉津降解效果为95.7%, 降解阿特拉津的最适温度为30 °C, pH为7.0。该菌对多种重金属离子都存在抗性, 显示了其在去除阿特拉津和重金属复合污染方面的应用潜力。对其降解基因的初步研究显示, 该菌含有trzN、atzB和atzC 3个阿特拉津降解相关基因。     

Isolation and characterisation of an isoproturon-mineralising Methylopila sp. TES from French agricultural soil

Using enrichment culture three isoproturon (IPU) mineralising bacterial isolates were isolated from a French agricultural soil mineralising up to 50% of the initially added 14C-ring labelled IPU within only eight days. These isolates showed similar metabolic (BIOLOG GN) and amplified rDNA restriction (ARDRA) profiles. Partial 16S rDNA sequencing revealed that they were identical and identified as Methylopila sp TES. This strain harbours a large plasmid (220 kb) putatively bearing essential IPU-degrading genes as demonstrated by a curing experiment. Methylopila sp. TES transformed IPU and its known metabolites to CO2 and biomass but did not degrade chlorotoluron, monolinuron, diuron and linuron.     

Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea

A strictly aerobic, Gram-positive, motile, coccoid-shaped, halotolerant actinobacterium (10% NaCl, w/v), designated MSL-23^T, was isolated from a soil sample on Bigeum Island, Korea. Results of 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Nocardioides, with the highest sequence similarity (95.63%) being to Nocardioides kribbensis KCTC 19038^T. The major menaquinone was MK-8(H₄), and the predominant cellular fatty acids were i-C_16:0, ai-C_17:0, C_18:1 ω9c and 10-methyl-C_16:0. The DNA G+C content was 69.7 mol%. The 16S rRNA gene sequence of strain MSL 23^T and its chemotaxonomic properties showed it to be unique in the genus Nocardioides. Phenotypic characteristics distinguished strain MSL-23^T from other Nocardioides species. On the basis of the phenotypic, chemotaxonomic and phylogenetic data strain MSL-23^T represents a novel species, for which the name Nocardioides halotolerans sp. nov. is proposed, with MSL-23^T (=KCTC 19274^T=DSM 19273^T) as the type strain.     

Isolation and characterization of alkane-utilizing Nocardioides sp. strain CF8

A butane-utilizing bacterial strain CF8 was isolated and identified as a member of the genus Nocardioides from chemotaxonomic and 16S rDNA sequence analysis. Strain CF8 grew on alkanes ranging from C(2) to C(16) in addition to butane and various other substrates including primary alcohols, carboxylic acids, and phenol. Butane degradation by strain CF8 was inactivated by light, a specific inactivator of copper-containing monooxygenases. The unique thermal aggregation phenomenon of acetylene-binding polypeptides was also observed for strain CF8. These results suggest that butane monooxygenase in strain CF8 is a third example of the copper-containing monooxygenases previously described in ammonia oxidizers and methanotrophs.     

Evidence of atrazine mineralization in a soil from the Nile Delta: Isolation of Arthrobacter sp. TES6, an atrazine-degrading strain

The s-triazine herbicide atrazine was rapidly mineralized (i.e., about 60% of ¹⁴C-ring-labelled atrazine released as ¹⁴CO₂ within 21 days) by an agricultural soil from the Nile Delta (Egypt) that had been cropped with corn and periodically treated with this herbicide. Seven strains able to degrade atrazine were isolated by enrichment cultures of this soil. DNA fingerprint and phylogenetic studies based on 16S rRNA analysis showed that the seven strains were identical and belonged to the phylogeny of the genus Arthrobacter (99% similarity with Arthrobacter sp. AD38, EU710554). One strain, designated Arthrobacter sp. strain TES6, degraded atrazine and mineralized the ¹⁴C-chain-labelled atrazine. However, it was unable to mineralize the ¹⁴C-ring-labelled atrazine. Atrazine biodegradation ended in a metabolite that co-eluted with cyanuric acid in HPLC. This was consistent with its atrazine-degrading genetic potential, shown to be dependent on the trzN, atzB, and atzC gene combination. Southern blot analysis revealed that the three genes were located on a large plasmid of about 175 kb and clustered on a 22-kb SmaI fragment. These results reveal for the first time the adaptation of a North African agricultural soil to atrazine mineralization and raise interesting questions about the pandemic dispersion of the trzN, atzBC genes among atrazine-degrading bacteria worldwide.     

阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

Promoting effect of Fusarium oxysporum [f.sp. strigae] on abundance of nitrifying prokaryotes in a maize rhizosphere across soil types

The integrated application of resistant crop varieties with biological control agents (BCAs) such as the Fusarium oxysporum [f.sp. strigae] strain “Foxy-2” has shown to be effective in fighting off the weed Striga hermonthica which is parasitic to several cereals cultivated in Sub-Saharan Africa (Schaub et al., 2006; Venne et al., 2009). “Foxy-2” proliferates in the rhizosphere and has been mainly studied for its virulence and mode of action. Contrary, no understanding is available regarding its interactions with key rhizosphere microorganisms steering relevant nutrient cycles in soils including nitrogen (N). In this study, we tested the hypothesis that “Foxy-2” displaces indigenous prokaryotic, N cycling communities in the maize rhizosphere due to competition for organic resources. Consequently, we evaluated if the application of an N-rich organic residue (i.e., Tithonia diversifolia with C/N ratio = 13, lignin content = 8.9%, polyphenol content = 1.7%) compensates these presumed competition effects. In a rhizobox experiment, quantitative polymerase chain reaction was used to follow the response of rhizosphere ammonia-oxidizing archaea (AOA) and bacteria (AOB) as well as total bacteria and archaea following “Foxy-2” inoculation in two physico-chemically contrasting soils (sandy Ferric Alisol versus clayey Humic Nitisol). Soils were treated with or without “Foxy-2”, S. hermonthica seeds, and T. diversifolia residues. Contrary to our expectations, we observed a distinct soil texture dependent, promoting effect of “Foxy-2” on rhizosphere prokaryotes. Abundance of AOA and total prokaryotic communities increased in response to “Foxy-2” in the sandy soil, while AOB remained unaffected. This effect on AOA was accelerated when T. diversifolia residues were incorporated. Further, in the clayey soil, AOA abundance was promoted when exposed to S. hermonthica infestation of maize. This suggested their capability to adapt to this biotic stress situation. It was concluded that “Foxy-2” did not pose a negative effect on targeted indigenous microorganisms, but the underlying mechanisms for the observed promoting effect of AOA abundance by “Foxy-2” inoculation are yet to be understood.     

Molecular taxonomic studies on some ll-diaminopimelic acid-containing coryneforms from herbage: Description of Nocardioides fastidiosa sp. nov.

Molecular taxonomic studies were performed on two LL-diaminopimelic acid-containing coryneform isolates from herbage. The results indicate that the herbage strains represent a new species of the genus Nocardioides for which the name Nocardioides fastidiosa sp. nov. is proposed. The type strain is NCIB 12713.     

Metabolic ability and gene characteristics of Arthrobacter sp. strain DNS10, the sole atrazine-degrading strain in a consortium isolated from black soil

Strain DNS10 was the only member that could utilize atrazine as the sole nitrogen source for growth in an atrazine-degrading consortium which was isolated from black soil previously in our laboratory. It belongs to the genus Arthrobacter according to the sequence of 16S rRNA gene and is designated as Arthrobacter sp. DNS10. 16S rRNA gene phylogenetic analysis showed that strain DNS10 was located in a different evolutionary branch comparing with other Arthrobacter sp. atrazine-degrading strains. The degrading genes such as trzN, atzB and atzC harbored in strain DNS10 revealed high sequence similarity with those in Arthrobacter aurescens TC1 and Pseudomonas sp. ADP. These genes enabled the strain DNS10 to decompose atrazine to cyanuric acid. This was further proved by the results that the strain DNS10 (10⁸ CFU mL⁻¹) could degrade the whole atrazine (100 mg L⁻¹) in the medium within 24 h at 30 °C and there was 66.13 ± 2.11 mg L⁻¹ cyanuric acid accumulated at 24 h. These results imply that the strain DNS10 seems to be an excellent atrazine-degrading strain. Furthermore, this paper helps us in the better understanding of the strain evolution by comparing the metabolic ability and gene characteristics of strain DNS10 with other geographically distinct atrazine-degrading strains.     

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.     

<Emphasis Type="Italic">Nocardioides islandiensis</Emphasis> sp. nov., isolated from soil in Bigeum Island Korea

A novel actinobacterium designated as MSL-26^T was isolated from soil in Bigeum Island Korea. A polyphasic study was undertaken to establish the taxonomic position of isolate MSL-26^T. Strain MSL-26^T was found to have chemical and morphological characteristics similar to Nocardioides. The strain grew optimally at pH 7·5 and 28°C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain MSL-26^T forms a distinct line of descent within the radiation enclosed by the genus Nocardioides. The cell wall of strain MSL-26^T contained LL-2, 6-diaminopimelic acid. The principal menaquinone was MK-8 (H₄). The phospholipids detected were diphosphatidylglycerol, phosphatidylglycerol and some unidentified lipids. C_18:1 w7c (50.38%) was the major fatty acid. The DNA G + C content of strain MSL-26^T was 71.4 mol%. The 16S rRNA gene sequence of strain MSL-26^T shares the highest sequence similarity with Nocardioides kribbensis KCTC 19038^T (95.78%) and Nocardioides aquaticus DSM 11439^T (95.52%). Based on the morphological, physiological, biochemical and chemotaxonomical data presented in this study, strain MSL-26^T should be classified as a novel species, for which the name Nocardioides islandiensis sp. nov. is proposed. The type strain is MSL-26^T (=KCTC 19275^T =DSM 19321^T)     

Isolation and characterization of an atrazine-degrading bacterium from industrial wastewater in China

AIMS: To isolate and characterize atrazine-degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination. METHODS AND RESULTS: A high efficiency atrazine-degrading bacterium, strain AD1, which was capable of utilizing atrazine as a sole nitrogen source for growth, was isolated from industrial wastewater. 16S rDNA sequencing identified AD1 as an Arthrobacter sp. The atrazine chlorohydrolase gene (atzA) isolated from strain AD1 differed from that found in the Pseudomonas sp. ADP by only one nucleotide. However, it was found located on the bacterial chromosome rather than on plasmids as previously reported for other bacteria. CONCLUSIONS: Atrazine chlorohydrolase gene, atzA, either encoded by chromosome or plasmid, is highly conserved. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison analysis of atrazine degradation gene structure and arrangement in this and other bacteria provides insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.     

Biodegradaon of phenanthrene in soil microcosms stimulated by an introduced Alcaligenes sp.

Abstract A phenanthrene degrading strain of Alcaligenes sp. was isolated from oil polluted soil. Addition of Alcaligenes sp. to soil microcosms supplemented with phenanthrene (1 mg/g dry soil) resulted in degradation of the added phenanthrene within 11 days. The phenanthrene concentration declined only 12% in uninoculated soil during 42 days. The total phenanthrene degradation potential of Alcaligenes sp. was 2.3 mg/g dry soil during a period of 22 days. The amount of CO₂ evolved during 22 days corresponded to the conversion of 91% of the degraded phenanthrene to CO₂. The Alcaligenes sp. were not able to degrade phenanthrene in sterile soil.     

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.     

An unusual strain of Desulfovibrio sp. from an Antarctic lake

Abstract An unusual sulphate-reducing bacterium was isolated from Lake Fryxell, Antarctica. Designated strain FESu, it illustrates the difficulty in assigning some isolates of Desulfovibrio spp. to a described species. FESu was characterized by its relatively low mol% G + C (45%), its rod-shaped morphology, inability to grow on lactate, pyruvate, malate or choline in the absence of sulphate, and by its lack of desulfoviridin.