Progress 09/01/02 to 02/28/06
Outputs
In order to determine the regulation of atrazine catabolic genes (atzA-E) it us important to have an understanding of their structure and relationship to each other. This was done using a genomic DNA BAC sequencing approach in Arthrobacter TC1, a whole plasmid sequencing approach in Pseudomonas sp. strain ADP, and gene cloning and analysis studies done in Enterobacter cloacae strain 99 and other bacteria. In A. aurescens, which we have now completely sequenced, the TrzN gene shared 99% amino acid identity to TrzN from Nocardioides sp. strain C190 while the AtzB and AtzC proteins shared 100% and 99% amino acid identity, to those from Pseudomonas sp. strain ADP. The three genes were not organized in an operon-like structure, suggesting independent regulation. Plasmid analysis indicated that these genes are located on a 380 kb plasmid. In Pseudomonas ADP, the atzDEF genes, however, are localized in an operon structure downstream of a lysR transcriptional regulator. Studies
done using a atzD-Lux transcriptional fusion analysis indicated that the atzD gene is inducible by cyanuric acid, but not atrazine. Studies done by others have shown that the genes are regulated by cynauric acid and overall celll N status. We have now obtained data to show that allophanate, and not urea, is the intermediate in cyanuric acid metabolism by a wide variety of bacteria, including Enterobacter cloacae strain 99. Genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were present in several cyanuric acid-metabolizing bacteria and the. TrzF gene from Enterobacter cloacae strain 99 encodes allophanate hydrolase activity. Taken together these results indicate that s-triazine degradation appears to follow a common pathway in bacteria and that cyanuric acid and the cells nitrogen status influence regulation of these genes.
Impacts
Results of these studies will be used to determine how plasmids containing herbicide degradation genes evolve and how atrazine degradation genes are regulated. In addition, these studies will be used to build biosensors to determine if triazine compounds are in soil or water.
Publications
- Sajjaphan, K., L. P. Wackett, M. Palmer, B. Blackmon , J. Tomkins, and M. J. Sadowsky. 2004. Sequence and analysis of a 161 kb atrazine catabolic gene region in Arthrobacter aurescens strain TC1 indicates a plasmid origin. Appl. Environ. Microbiol. 70:4402-4407.
- Cheng, G., Shapir, N., Sadowsky, M.J., and Wackett, L.P. 2005. Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism. 2005. Source: Appl. Environ. Microbiol. 71(8): 4437-4445.
- Shapir, N., C. Rosendahl, G. Johnson, M. Andreina, M. J. Sadowsky, and L. P. Wackett. 2005. Substrate specificity and colorimetric assay for recombinant TrzN derived from Arthrobacter aurescens TC1. Appl. Environ. Microbiol. 71:2214-2220.
- Shapir, N., M. J. Sadowsky, and L. P. Wackett. 2005. Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP. J. Bacteriol. 187:3731-3738.
- Shapir, N., C. Pedersen, O. Gil, L. Strong, J. Seffernick, M. J. Sadowsky, and L. P. Wackett. 2006. TrzN from Arthrobacter aurescens TC1 is a zinc amidohydrolase. J. Bacteriol. In press.
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Progress 01/01/05 to 12/31/05
Outputs
Herbicides containing an s-triazine ring are metabolized by bacteria, through the intermediate cyanuric acid, to liberate 3 moles of ammonia. Over the last 25 years research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. We have now obtained data to show that allophanate, and not urea, is the intermediate in cyanuric acid metabolism by a wide variety of bacteria. This intermediate was confirmed by studies showing that synthetic allophanate readily decarboxylates to form urea under the acidic extraction and chromatography conditions used in previous studies and the kinetics of allophanate formation is consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments. We also showed that extracts from cells grown on cyanuric acid contained allophanate hydrolase activity, that genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze
allophanate, respectively, were present in several cyanuric acid-metabolizing bacteria, Moreover, we cloned a new gene TrzF from Enterobacter cloacae strain 99, we showed that it encodes allophanate hydrolase activity. Taken together, these results indicate that s-triazine metabolism in bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease.
Impacts
Results from these studies are useful in determining how s-triazines, including atrazine, degrade in the environment. Results from these studies will allow researchers to determine if breakdown products from triazine herbicides are found in soils and water.
Publications
- Cheng, G., Shapir, N., Sadowsky, M.J., and Wackett, L.P. 2005. Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism. 2005. Source: Appl. Environ. Microbiol. 71(8): 4437-4445.
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Progress 01/01/04 to 12/31/04
Outputs
In order to determine the regulation of atrazine catabolic genes it is important to have an understanding of their structure and the substrates that can act as inducers. The TrzN protein involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1 was cloned and expressed in Escherichia coli as a His-tagged protein. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds, 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan was reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. We constructed an atzD-Lux transcriptional fusion from the atzD gene from Pseudomonas
ADP and found that it is induced by cyanuric acid and other compounds upstream of this gene in the catabolic pathway.
Impacts
Results of these studies will be used to determine how plasmids containing herbicide degradation genes evolve and how atrazine degradation genes are regulated. In addition, these studies will be used to build biosensors to determine if triazine compounds are in soil or water.
Publications
- Shapir, N., C. Rosendahl1, G. Johnson, M. Andreina, M. J. Sadowsky, and L. P. Wackett. 2005. Substrate Specificity and Colorimetric Assay for Recombinant TrzN Derived from Arthrobacter aurescens TC1. Appl. Environ. Microbiol. In Press.
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Progress 01/01/03 to 12/31/03
Outputs
In order to determine the regulation of atrazine catabolic genes (atzA-E) it us important to have an understanding of their structure and relationship to each other. This was done using a genomic DNA BAC sequencing approach in Arthrobacter TC1 and a whole plasmid sequencing approach in Pseudomonas sp. strain ADP. In the former strain, high density BAC library filters, containing 3072 colonies, was screened by hybridization to radiolabeled single-stranded probes prepared from atzB, atzC, and trzN genes. The screening identified 89, 72, and 91 clones containing the trzN, atzB and atzC genes, respectively. One BAC clone hybridized to all three gene probes, which was used to create an approximately 8X coverage shotgun-sequencing library. ORF 175 was identified as TrzN, and shared 99% amino acid identity to TrzN from Nocardioides sp. strain C190 while ORFs 33 and 38 shared 100% and 99% amino acid identity, respectively, to the AtzB and AtzC proteins from Pseudomonas sp.
strain ADP. The three genes were not organized in an operon-like structure; the trzN gene was located 129 kb and 122.5 kb from the atzB and atzC genes, respectively. Plasmid analysis suggested that these genes are located on a 380 kb plasmid. In Pseudomonas ADP, the atzA-C genes are unlinked and localized to plasmid pADP-1. The atzDEF genes, however, are localized in an operon structure downstream of a lysR transcriptional regulator. Studies done using a atzD-Lux transcriptional fusion analysis indicated that the atzD gene is inducible by cyanuric acid, but not atrazine.
Impacts
Results of these studies will be used to determine how plasmids containing herbicide degradation genes evolve and how atrazine degradation genes are regulated. In addition, these studies will be used to build biosensors to determine if triazine compounds are in soil or water.
Publications
- Sajjaphan, K., L. P. Wackett, M. Palmer, B. Blackmon , J. Tomkins, and M. J. Sadowsky. 2004. Sequence and analysis of a 161 kb atrazine catabolic gene region in Arthrobacter aurescens strain TC1 indicates a plasmid origin. Appl. Environ. Microbiol. submitted
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Progress 01/01/02 to 12/31/02
Outputs
In Pseudomonas strain ADP, the genes for the biodegradation of the herbicide atrazine are localized to a large indigenous plasmid, pADP-1. We used a genomics approach to obtain the complete 108,845 nucleotide sequence of catabolic plasmid pADP-1. Functional analyses identified three new catabolic genes, atzDEF that reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. In these studies we will examine the genetic regulation of the atzDEF genes and use this knowledge to construct biosensors to detect atrazine in the environment. We have begun to sequence the analogous, but broader substrate genes from Arthrobacter aurescens strain TC1s in order to examine their regulation and produce biosensors with broad substrate specificity. TC1 was shown to metabolize 23 different s-triazine compounds, the most extensive and broad catabolism of s-triazine compounds observed to date. We constructed a Bacterial Artificial Chromosome (BAC) and
shotgun sequencing library from strain TC1. The genes for s-triazine degradation were localized to a 160 KB BAC and sequencing and assembly is currently being done to localize the genes and regulatory regions of this important gene region.
Impacts
Results from these studies will be used to construct biosensors and superior microorganisms that can be used to rapidly and cheaply identify triazine compounds in the environment and clean-up atrazine contamination of soil and water.
Publications
- No publications reported this period
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