Atrazine-degrading Genes Research Articles

Atrazine degradation by stable mixed cultures enriched from agricultural soil and their characterization

The aim of this work was to enrich stable mixed cultures from atrazine-contaminated soil. The cultures were examined for their atrazine biodegradation efficiencies in comparison with J14a, a known atrazine-degrading strain of Agrobacterium radiobacter. The cultures were also characterized to identify community structure and bacterial species present. The cultures were enriched and then stabilized in bacterial media. The stable mixed cultures and J14a were tested in a medium containing 100 microg l(-1) of atrazine. For all cultures, atrazine was removed 33-51% within 7 days and the cell optical density increased from 0.05 to between 0.50 and 0.70. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence analysis of the 16 S rRNA gene as Alcaligenes faecalis, Klebsiella ornithinolytica, Bacillus megaterium and Agrobacterium tumefaciens, respectively. An atrazine-degrading gene, atzA, was present in ND2 and ND4. The stable mixed cultures obtained could degrade atrazine. Klebsiella ornithinolytica ND2 and Ag. tumefaciens ND4 are atrazine degraders. The novel stable mixed cultures could be used for bioremediating crop fields contaminated with atrazine. This is the first report of the atzA gene in Kl. ornithinolytica.

Fitness drift of an atrazine‐degrading population under atrazine selection pressure

Pseudomonas sp. ADP harbouring the atrazine catabolic plasmid ADP1 was subcultured in liquid medium containing atrazine as sole source of nitrogen. After approximately 320 generations, a new population evolved which replaced the initial population. This newly evolved population grew faster and degraded atrazine more rapidly than the initial population. Plasmid profiles and Southern blot analyses revealed that the evolved strain, unlike the ancestral strain, presented a tandem duplication of the atzB gene encoding the second enzyme of the atrazine catabolic pathway responsible for the transformation of hydroxyatrazine to N-isopropylammelide. This duplication resulted from a homologous recombination that occurred between two direct repeats of 6.2 kb flanking the atzB gene and constituted by the insertion sequences IS1071, ISPps1 and a pdhL homologous sequence. This study highlights the IS-mediated plasticity of atrazine-degrading potential and demonstrates that insertion sequences not only help to disperse the atrazine-degrading gene but also improve the fitness of the atrazine-degrading population.

Isolation and characterization of atrazine-degrading Arthrobacter sp. AD26 and use of this strain in bioremediation of contaminated soil

A bacterial strain (AD26) capable of utilizing atrazine as a sole nitrogen source for growth was isolated from an industrial wastewater sample by enrichment culture. The 16S rRNA gene sequencing identified AD26 as an Arthrobacter sp. PCR assays indicated that AD26 contained atrazine-degrading genes trzN and atzBC. The trzN gene of AD26 only differs from the trzN of Arthrobacter aurescens TC1 by one base (A-->T at 907) and one amino acid (Met-->Leu at 303). The specific activity of trzN of AD26 in crude atrazine-containing minimal media than two well characterized atrazine-degrading bacteria, Pseudomonas sp. ADP and Arthrobacter aurescens TC1. After incubating for 48 h at 30 degrees C, the OD(600) of AD26 reached 2.6 compared with 1.33 of ADP. AD26 was capable of degrading 500 mg/L of atrazine in minimal medium at 95% in 72 h, while the degradative rates by TC1 and ADP were only 90% and 86%, respectively. A bioremediation trial of contaminated soil has indicated that AD26 can degrade as high as 98% of atrazine contained in soil (300 mg/kg) after incubating for 20 d at 26 degrees C, nominating this strain as a good candidate for use in bioremediation programs.

Study on the Atrazine-degrading Genes in Arthrobacter sp. AG1

Abstract Atrazine could be used as the sole carbon, nitrogen and energy source necessary for growth, by strain Arthrobacter sp. AG1, and the atrazine-degrading genes of AG1 were found to be a combination of trzN, atzB and atzC. The atrazine chloride hydrolysase gene trzN was cloned by PCR amplification, whose sequence shared 99% identity with that of Norcardioides sp. C190. Two large plasmids were found in AG1, trzN and atzB were confirmed to be localized on the larger plasmid pAG1 by the method of southern hybridization. Subculturing of AG1 in liquid LB for three generations resulted in the loss of degrading activity in 34% of the subsequent cells; however, none of the plasmid was lost. PCR amplification results showed that the mutants had only lost the trzN gene instead of atzB and atzC. It was deduced that mutation might be because of the trzN gene deletion from the plasmid pAG1. This study provided new evidence that atrazine metabolic genotypes resulted from horizontal gene transfer between different bacteria under selective environmental pressure.

Detection and organization of atrazine-degrading genetic potential of seventeen bacterial isolates belonging to divergent taxa indicate a recent common origin of their catabolic functions

A collection of 17 atrazine-degrading bacteria isolated from soils was studied to determine the composition of the atrazine-degrading genetic potential (i.e. trzN, trzD and atz) and the presence of IS1071. The characterization of seven new atrazine-degrading bacteria revealed for the first time the trzN-atzBC gene composition in Gram-negative bacteria such as Sinorhizobium sp. or Polaromonas sp. Three main atrazine-degrading gene combinations (i) trzN-atzBC, (ii) atzABC-trzD and (iii) atzABCDEF were observed. The atz and trz genes were often located on plasmids, suggesting that plasmid conjugation could play an important role in their dispersion. In addition, the observation of these genes (i) on the chromosome, (ii) on the same DNA fragment but on different plasmids and (iii) on DNA fragments also hybridizing with IS1071 suggests that transposition may also contribute to disperse the atrazine-degrading genes.

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.

Combined metabolic activity within an atrazine-mineralizing community enriched from agrochemical factory soil

The main objective of this work was to characterize an atrazine-mineralizing community originating from agrochemical factory soil, especially to elucidate the catabolic pathway and individual metabolic and genetic potentials of culturable members. A stable four-member bacterial community, characterized by colony morphology and 16S rDNA sequencing, was rapidly able to mineralize atrazine to CO 2 and NH 3 . Two primary organisms were identified as Arthrobacter species (ATZ1 and ATZ2) and two secondary organisms (CA1 and CA2) belonged to the genera Ochrobactrum and Pseudomonas, respectively. PCR assessment of atrazine-degrading genetic potential of the community, revealed the presence of trzN, trzD, atzB and atzC genes. Isolates ATZ1 and ATZ2 were capable of dechlorinating atrazine to hydroxyatrazine and contained the trzN gene. ATZ2 further degraded hydroxyatrazine to cyanuric acid and contained atzB and atzC genes whereas ATZ1 contained atzC but not atzB. Isolates CA1 and CA2 grew on cyanuric acid and contained the trzD gene. Complete atrazine degradation was a result of the combined metabolic attack on the atrazine molecule, and complex interactions may exist between the community members sharing carbon and nitrogen from atrazine mineralization. Scientific relevance: Despite numerous reports on atrazine degradation by pure bacterial cultures, the pathways and the atrazine-degrading gene combinations harboured by bacterial communities are only poorly described. In this work, we characterized a four-member atrazine-mineralizing community enriched from an agrochemical factory soil, which was capable of rapidly metabolizing atrazine to CO 2 . This study will contribute towards better understanding of the genetic potential and metabolic activities of atrazine-degrading communities, which are generally considered to be responsible for atrazine mineralization in the natural environment.

Genetic rearrangement of the atzAB atrazine-degrading gene cassette from pADP1::Tn 5 to the chromosome of Variovorax sp. MD1 and MD2

We report the characterization of the rearrangement phenomena responsible for the movement of the atrazine-degrading atzA and B genes from pADP1::Tn 5 to the chromosome of Variovorax sp. MD1 and MD2. Long PCRs and Southern blot analyses revealed that the two genes forming a gene cassette moved in a unique rearrangement event. It also revealed that the boundaries of the plasmid sequence inserted in the chromosome correspond to IS 1071or to sequences close to IS 1071. It suggests that this genetic rearrangement could result from the transposition of the composite transposon delimited by IS 1071 insertion sequences and containing atzA and atzB genes. In addition, for MD1 and MD2 strains the sequencing of the remaining sequence on pADP1::Tn 5 indicated that the deletion of the atzA and B genes from the plasmid might be the result of a recombination event that occurred between the IS 1071 insertion sequences surrounding the atzAB gene cassette.

Impact of the Maize Rhizosphere on the Genetic Structure, the Diversity and the Atrazine-degrading Gene Composition of Cultivable Atrazine-degrading Communities

Sixty-six atrazine-degrading bacterial communities utilizing atrazine as sole N source and citrate as principal C source were isolated from unplanted and maize planted soils treated with atrazine. Ribosomal intergenic spacer analysis (RISA) fingerprints revealed that the genetic structure of atrazine-degrading bacterial communities was modified in the maize rhizosphere. To assess the underlying microbial diversity, 16S rDNA sequences amplified from each bacterial community were cloned. Libraries containing 660 16S rDNA clones were screened by restriction fragment length polymorphism (RFLP) analysis. In all, 63 clone families were identified. Rarefaction curves did not reach a clear saturation, indicating that the analysis of a greater number of clones would have revealed further diversity. Recovered 16S rDNA sequences were related to Actinobacteria, Bacteroidetes and Proteobacteria. The four dominant RFLP families were highly similar to Variovorax paradoxus, Burkholderia cepacia, Arthrobacter sp. and Bosea sp. The composition of most of the atrazine-degrading bacterial communities consisted of 2–7 different bacterial species. Various atrazine-degrading gene compositions were observed, two of these atzABCDEF, trzND and atzBCDEF, trzN being largely dominant. The first was more frequently detected in bacterial communities isolated from the maize rhizosphere whereas the second was more frequently detected in communities isolated from bulk soil. Monitoring of atrazine-degrading activity showed that 76% of the bacterial communities degraded up to 80% of the initially added atrazine within 15 days of culture. Altogether our results indicate that the maize rhizosphere has an impact on the genetic structure, the diversity and atrazine-degrading gene composition of the atrazine-degrading communities.

Characterization of Arthrobacter nicotinovorans HIM, an atrazine-degrading bacterium, from agricultural soil New Zealand

Arthrobacter nicotinovorans HIM was isolated directly from an agricultural sandy dune soil 6 months after a single application of atrazine. It grew in minimal medium with atrazine as sole nitrogen source but was unable to mineralize 14C-ring-labelled atrazine. Atrazine was degraded to cyanuric acid. In addition to atrazine the bacterium degraded simazine, terbuthylazine, propazine, cyanazine and prometryn but was unable to grow on terbumeton. When added to soil, A. nicotinovorans HIM did enhance mineralization of 14C-ring-labelled atrazine and simazine, in combination with naturally occurring cyanuric acid degrading microbes resident in the soil. Using PCR, the atrazine-degradation genes atzABC were identified in A. nicotinovorans HIM. Cloning of the atzABC genes revealed significant homology (>99%) with the atrazine degradation genes of Pseudomonas sp. strain ADP. The atrazine degradation genes were held on a 96 kbp plasmid.

Plasmid localisation of atrazine-degrading genes in newly described Chelatobacter and Arthrobacter strains

Abstract In a previous study, we isolated a collection of atrazine-degrading bacteria from various soils. The aim of this study was to localise the atrazine-degrading genes in these 25 atrazine-degrading strains. In the case of the Gram-negative strains of Chelatobacter heintzii, six to seven plasmids were observed. The atzABC and trzD genes were located on two or three plasmids with variable molecular masses. For the Gram-positive strains of Arthrobacter crystallopoietes, the atzBC genes were located on a single plasmid of 117 kb. The organisation of atrazine-degrading genes seems to be highly variable between the strains studied. We have shown by a specific PCR the occurrence of IS1071-like sequences in all strains carrying atzBC genes, while these sequences seem to be absent in strains carrying only the atzA gene. Thus, we show a strong correlation between the presence of these insertion sequences and the presence of some genes involved in atrazine degradation.

Plasmid localisation of atrazine-degrading genes in newly described Chelatobacter and Arthrobacter strains

In a previous study, we isolated a collection of atrazine-degrading bacteria from various soils. The aim of this study was to localise the atrazine-degrading genes in these 25 atrazine-degrading strains. In the case of the Gram-negative strains of Chelatobacter heintzii, six to seven plasmids were observed. The atzABC and trzD genes were located on two or three plasmids with variable molecular masses. For the Gram-positive strains of Arthrobacter crystallopoietes, the atzBC genes were located on a single plasmid of 117 kb. The organisation of atrazine-degrading genes seems to be highly variable between the strains studied. We have shown by a specific PCR the occurrence of IS 1071-like sequences in all strains carrying atzBC genes, while these sequences seem to be absent in strains carrying only the atzA gene. Thus, we show a strong correlation between the presence of these insertion sequences and the presence of some genes involved in atrazine degradation.

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.