Genetic and phenotypic diversity of carbofuran-degrading bacteria isolated from agricultural soils

Thirty-seven carbofuran-degrading bacteria were isolated from agricultural soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize carbofuran as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that the isolates were related to members of the genera Rhodococcus, Sphingomonas, and Sphingobium, including new types of carbofuran-degrading bacteria, Bosea and Microbacterium. Among the 37 isolates, 15 different chromosomal DNA patterns were obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences. Five of the 15 representative isolates were able to degrade carbofuran phenol, fenoxycarb, and carbaryl, in addition to carbofuran. Ten of the 15 representative isolates had 1 to 8 plasmids. Among the 10 plasmid-containing isolates, plasmid-cured strains were obtained from 5 strains. The cured strains could not degrade carbofuran and other pesticides anymore, suggesting that the carbofuran degradative genes were on the plasmid DNAs in these strains. When analyzed with PCR amplification and dot-blot hybridization using the primers targeting for the previously reported carbofuran hydrolase gene (mcd), all of the isolates did not show any positive signals, suggesting that their carbofuran hydrolase genes had no significant sequence homology with the mcd gene.     

Bacterial metabolism of carbofuran.

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.     

Bacterial metabolism of carbofuran

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.     

Genetic and phenotypic diversity of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid]-degrading bacteria isolated from soils

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.     

Aromatic hydrocarbon-degrading bacteria from soil near Scott Base, Antarctica

Hydrocarbons persist in Antarctic soils when fuel oils such as JP8 jet fuel are spilled. For clean-up of hydrocarbon-contaminated soils in Antarctica, bioremediation has been proposed using hydrocarbon-degrading microbes indigenous to Antarctic soils. A number of alkane-degrading bacteria have been isolated previously from Antarctic soils. In this paper we describe the direct isolation of aromatic hydrocarbon-degrading bacteria from oil-contaminated Antarctic soil. Isolates that grew on JP8 jet fuel were characterised for their ability to degrade aromatic and aliphatic hydrocarbons and for growth at a range of temperatures. All isolates were gram-negative, oxidase-positive, rod-shaped bacteria. Representative strains were identified using 16S rDNA sequence analysis as either Sphingomonas spp. or Pseudomonas spp. Aromatic-degrading bacteria from Antarctic soils were psychrotolerant and appear similar to those found worldwide. Accepted: 27 September 1999     

Diversity of carbofuran-degrading soil bacteria and detection of plasmid-encoded sequences homologous to the mcd gene

Abstract: Fifty-five bacterial isolates, from English and French soils with different histories of carbofuran field treatment, which hydrolysed the N -methylcarbamate insecticide carbofuran to carbofuran 7-phenol were characterised phenotypically and genetically. The isolates were compared by using 125 physiological tests and morphological features, plasmid profiles and restriction fragment length polymorphism (RFLP) patterns of total DNA using the rRNA operon of Escherichia coli as a DNA probe. Cluster analysis of both phenotypic characters and RFLP patterns showed a high degree of diversity amongst the bacteria. Ten distinct plasmid profiles with 2–4 plasmids ranging in size from 84 to about 438 kb were visualised in 50 isolates. The majority of isolates had one of two types of plasmid profiles. Plasmid profiles and Eco RI restricted total DNA patterns were hybridised with an internal fragment of the carbofuran hydrolase ( mcd ) gene and 22 diverse soil isolates exhibited sequence homology with this gene probe. Our results indicate that sequences homologous to the mcd gene are located on a conserved Eco RI fragment (12 or 14 kb) of a plasmid (100, 105, 115 or 124 kb) found in diverse soil isolates from geographically distant areas. Thirty-three isolates did not exhibit detectable homology to the mcd gene probe and the hydrolase enzymes and genes in these isolates need further investigation.     

Two unidentified aerobic bacterial strains that transform 2,4,6-trinitrotoluene

Several strains of aerobic bacteria tolerant to some chemical toxins were isolated from the water of man-made Yerevan Lake and from the soil and air of different regions of the city of Yerevan. Some of these bacteria were capable of degrading 2,4,6-trinitrotoluene (TNT) during shake-flask fermentation in liquid medium. Two bacterial strains with good ability to degrade TNT were isolated. These strains showed visible morphological and physiological differences during growth on numerous elective media. The comparative ability of these strains to transform TNT was investigated.     

Genetic transfer of the mcd gene in soil

AIMS: To investigate the role of horizontal gene transfer of mcd (methylcarbamate-degrading) gene in high genetic diversity of carbofuran-degrading bacteria. METHODS AND RESULTS: The actuality of genetic transfer from degraders to an Agrobacterium tumefaciens strain was determined in liquid medium. The mcd gene was chosen for transfer experiments. Transconjugants were obtained irrespective of the type of the donor strain (Gram-positive or Gram-negative), size of the inoculum, or nature and concentration of the pesticide in the medium. Soil microcosms, inoculated with or without the donor and/or recipient strains were used. The size of the initial degrading population (treated or untreated soil) and the nature of the inoculated donor strains were considered. More transconjugants were isolated in the previously treated soil than in the untreated soil. Agrobacterium transconjugants were isolated even when the donor strain was not inoculated, probably as a result of gene transfer from indigenous degrading population to the recipient strain. Moreover, potential transconjugants belonging to the Pseudomonas genus were isolated. CONCLUSIONS: Our results seem to demonstrate that the mcd gene is transferable in soil among bacterial populations. SIGNIFICANCE AND IMPACTS OF THE STUDY: The transfer of the mcd gene is partly responsible for the high genetic diversity of micro-organisms able to catabolize carbofuran.     

Carbofuran degradation mediated by three related plasmid systems

Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements.     

Isolation and characterization of agar-degrading Paenibacillus spp. associated with the rhizosphere of spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.     

Isolation and characterization of 23 carbofuran-degrading bacteria from soils from distant geographical areas

The aim of this work was to isolate, identify and type carbofuran-degrading bacteria from two geographically distant soils. Restriction Fragment Length Polymorphism (RFLP) patterns of the 16S rRNA gene and partial 16S rRNA sequence analysis were used to classify the 23 isolates obtained. Nine of them showed high similarity to Pseudomonas strains, seven showed similarity to the Flexibacter/Cytophaga/Bacteroides group and the remainder showed similarity to other bacterial genera. Isolates within the same group were sub-typed by comparing partial 16S rRNA sequences and SDS-PAGE analysis of their total protein profiles. Many of the UK isolates showed similarity to the Pseudomonas genera, while most of the Greek isolates showed similarity to the Flexibacter/Cytophaga/Bacteroides group. Only two Chrysobacterium strains isolated from both the UK and Greek soils were identical.     

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3% to 2.5% of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75-160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.     

Isolation and characterization of fenobucarb-degrading bacteria from rice paddy soils

Forty-five fenobucarb-degrading bacteria were isolated from rice paddy soils, and their genetic and phenotypic characteristics were investigated. The isolates were able to utilize fenobucarb as a sole source of carbon and energy. Analysis of the 16S rRNA gene sequence indicated that all the isolates were related to members of the genera Sphingobium and Novosphingobium. Among 45 isolates, 21 different chromosomal DNA fingerprinting patterns were obtained. All these strains exhibited similar growth and degradation patterns on fenobucarb. 2-sec-butylphenol was identified as an intermediate during fenobucarb degradation by HPLC analysis. All of the isolates were able to degrade another carbamate insecticide, carbaryl, and 2-sec-butylphenol, but not other fenobucarb related compounds such as aldicarb and fenoxycarb. Representative strains of the different repetitive extragenic palindromic sequence PCR fingerprint types had one to six plasmids. The plasmid-cured strains lost their degradation abilities, suggesting that fenobucarb degradative genes were on their plasmid DNAs in these strains. When analyzed with PCR amplification using the primers targeting for the previously reported carbamate hydrolase genes, most of the isolates did not exhibit any positive signals for different genes involved in carbamate degradation such as mcd, cahA and cehA genes. This is the first report that microorganisms involved in the degradation of fenobucarb have been isolated and the intermediate of fenobucarb biodegradation was identified.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

Species variation and plasmid incidence among fluorescent Pseudomonas strains isolated from agricultural and industrial soils

Abstract: A total of 132 different fluorescent Pseudomonas strains were isolated from several agricultural and industrial soils. The bacteria from the two different soil environments were compared for species and biotype variation, antibiotic and heavy metal resistance profiles, ability to degrade polyaromatic hydrocarbons, and plasmid incidence. Irrespective of the soil type, the isolates belonged to Pseudomonas fluorescens biotypes I–VI and Pseudomonas putida biotype B. Except for a streptomycin resistant isolate from one of the industrial soils, all the strains had the same antibiotic resistance profile. However, there was a higher incidence of heavy metal resistance and polyaromatic hydrocarbon degradation phenotypes in the isolates from industrial soils than from the agricultural soils. Only 2 out of 68 strains from agricultural soil were found to carry plasmids, while 28 out of 64 strains from industrial soil had plasmids. A majority of the plasmids (56%) were estimated to be larger than 50 kb, indicating that they could encode transfer functions. However, transferability as indicated by the ability to mobilize an IncQ plasmid (tra⁻, mob⁺), was observed with only one plasmid. None of the plasmid(s) containing isolates hybridized to a ³²P-labelled repP probe suggesting that none of the indigenous plasmids in the soil fluorescent Pseudomonas strains was related to the IncP group of conjugative plasmids commonly associated with resistance and catabolic genes.     

Characterization of a carbofuran-degrading bacterium and investigation of the role of plasmids in catabolism of the insecticide carbofuran

A bacterium capable of using the carbamate insecticide carbofuran as a sole source of carbon and energy, was isolated from soil. The ability to catabolise carbofuran phenol, produced by cleavage of the carbamate ester linkage of the insecticide, was lost at very high frequency when the bacterium was grown in the absence of carbofuran. Plasmid analyses together with curing and mating experiments indicated that the presence of a large plasmid (pIH3, >199 kb) was required for the degradation of carbofuran phenol.Abbreviations Rif^r Rifampicin resistant - Rif^s Rifampicin sensitive - CFH⁺ Carbofuran hydrolase activity present - CFH^- Carbofuran hydrolase activity absent - CFP⁺ ability to degrade carbofuran phenol present - CFP^- ability to degrade carbofuran phenol absent - MS mineral salts medium. MSCF minimal mineral salts medium containing 0.25 mM carbofuran as sole source of carbon and energy - YP MS medium containing 5 g/l yeast extract and 5 g/l Bactopeptone. YPCF as above but with the addition of 1 mM carbofuran - EPTC S-ethyl-N,N-dipropylthiocarbamate - 2,4-D 2,4-dichlorophenoxyacetic acid - NAG N-acetylglucosamine - 3-HB 3-hydroxybutyrate     

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.     

Cellulolytic bacteria from soils in harsh environments

It is believed that the exposure of organisms to harsh climate conditions may select for differential enzymatic activities, making the surviving organisms a very promising source for bioprospecting. Soil bacteria play an important role in degradation of organic matter, which is mostly due to their ability to decompose cellulose-based materials. This work focuses on the isolation and identification of cellulolytic bacteria from soil found in two environments with stressful climate conditions (Antarctica and the Brazilian semi-arid caatinga). Cellulolytic bacteria were selected using enrichments at high and low temperatures (4 or 60°C) in liquid media (trypic soy broth—TSB and minimum salt medium—MM) supplemented with cellulose (1%). Many of the isolates (119 out of 254—46.9%) displayed the ability to degrade carboxymethyl-cellulose, indicating the presence of endoglucolytic activity, while only a minority of these isolates (23 out of 254—9.1%) showed exoglucolytic activity (degradation of avicel). The obtained isolates revealed a preferential endoglucolytic activity according to the temperature of enrichments. Also, the identification of some isolates by partial sequencing of the 16S rRNA gene indicated that the Bacteroidetes (e.g., Pedobacter, Chryseobacterium and Flavobacterium) were the main phylum of cellulolytic bacteria isolated from soil in Antarctica; the Firmicutes (e.g., Bacillus) were more commonly isolated from samples from the caatinga; and Actinobacteria were found in both types of soil (e.g., Microbacterium and Arthrobacter). In conclusion, this work reports the isolation of bacteria able to degrade cellulose-based material from soil at very low or very high temperatures, a finding that should be further explored in the search for cellulolytic enzymes to be used in the bioenergy industry.     

Streptomycin as a selective agent to facilitate recovery and isolation of introduced and indigenous Sphingomonas from environmental samples

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 microg ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minority of the Sphingomonas isolates, which all originated from soils containing high phenanthrene concentrations.     

塔里木盆地荒漠盐碱生境嗜盐碱细菌的初步研究

为了探索塔里木盆地荒漠盐碱生境嗜（耐）盐碱细菌的分离方法,采用纯培养技术探讨了不同土壤预处理方法、盐度及不同分离培养基对不同盐度土壤中嗜（耐）盐碱细菌分离效果的影响。结果表明：高盐土壤嗜（耐）盐碱细菌的多样性高于中度盐分和低度盐分的土壤,而总菌落数则相反;半量的Horikoshi I（NaCl 10％～15％）为3种土样最佳的分离培养基,碱性复合培养基和高盐碱培养基A次之;分离嗜（耐）盐碱细菌以获得资源为主要目的时,富集培养法最佳。以反映土壤嗜（耐）盐碱细菌生态分布而言,用土壤悬液法;塔里木盆地嗜（耐）盐碱细菌生长盐浓度及pH值范围较宽,最适生长盐浓度为10％左右,pH值多为8—10左右。分离到的120株嗜（耐）盐碱细菌中,有33株为嗜盐碱细菌,占分离菌株的27．5％。