Three species of Pseudomonas capable of utilizing atrazine as a sole source of carbon were isolated by enrichment from soil with a long history of atrazine application. Atrazine was metabolized via N-dealkylation with preferential formation of deisopropylatrazine over deethylatrazine. Two of the species were able to carry out the dechlorination of both deisopropylatrazine and deethylatrazine following incubation in glucose-supplemented mineral salts medium. The dehalogenation of atrazine and its metabolites as a bacterial degradation process is shown.

Rhodococcus strains were screened for their ability to degrade the herbicide atrazine. Only rhodococci that degrade the herbicide EPTC (s-ethyl-dipropylthiocarbamate) metabolized atrazine. Rhodococcus strain TE1 metabolized atrazine under aerobic conditions to produce deethyl-and deisopropylatrazine, which were not degraded further and which accumulated in the incubation medium. The bacterium also metabolized the other s-triazine herbicides propazine, simazine, and cyanazine. The N dealkylation of triazine herbicides by Rhodococcus strain TE1 was associated with a 77-kb plasmid previously shown to be required for EPTC degradation.

Arthrobacter sp. strain TE1 isolated from s-ethyl-N,N-dipropylthiocarbamate (EPTC)-exposed soil degraded this herbicide effectively and could grow on EPTC as the sole carbon source. TE1 harboured four plasmids of 65.5, 60, 50.5, and 2.5 megadaltons. Spontaneous mutants unable to degrade EPTC arose at a high frequency, and this was further increased by treatment of the culture with acridine orange or incubation at high temperature. All EPTC degradation-deficient (E-) mutants 'lacked the 50.5-megadalton plasmid. This plasmid could be transferred from TE1 to Emutants by conjugation, resulting in the restoration of EPTC-degrading ability to the mutants.

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We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA-and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.The s-triazine herbicides have been used in a variety of weed control programs with major crops. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is one of the most heavily used herbicides in North America (21). The detection of atrazine in groundwater and surface water has prompted some environmental concerns (1, 14). There has been considerable interest in finding microbial activities that might be used to degrade atrazine and other s-triazine compounds in herbicide wastes and contaminated soils (6,7,12,16,24). Several microorganisms that can metabolize atrazine have been isolated during the last few years (2,3,17,18,25,33). We recently reported that Rhodococcus sp. strain TE1 can degrade atrazine efficiently to produce the dealkylated metabolites deisopropylatrazine (2-chloro-4-ethylamino-6-amino-s-triazine [CEAT]) and deethylatrazine (2-chloro-4-amino-6-isopropylamino-s-triazine [CIAT]) and that this catabolic function was associated with an indigenous 77-kb plasmid in this bacterium (4, 28). Two recently reported Pseudomonas isolates (18, 33) degrade atrazine efficiently but by different pathways.Cook and Hutter (5, 8) isolated and characterized an isolate of Rhodococcus corallinus that dechlorinates and deaminates the dealkylated s-triazine compounds CEAT and CIAT but not atrazine. The enzyme, named s-triazine hydrolase and encoded by the trzA gene, has been purified (23). The combination of the catabolic activities of both Rhodococcus sp. strain TE1 and R. corallinus would result in dealkylation and dechlorination of atrazine.We have been involved in...

The genetic studies of metabolically diverse Rhodococcus spp. have been hampered by the lack of a system of introducing exogenous DNA. The authors improved an existing Escherichia coli-Rhodococcus shuttle vector (pMVS301) by removing much of the DNA not needed for replication and adding a multicloning site. This improved vector (pBS305) is 7.9 kb in length. Its ability to transform Rhodococcus was tested using electroporation parameters optimized for introduction of pMVS301 into Rhodococcus. Transformation efficiencies as high as 10(5) cfu micrograms-1 DNA were obtained although efficiencies varied depending on the Rhodococcus strain tested. The improved vector pBS305 offers great utility for genetic studies of Rhodococcus because its small size enables movement of large inserts of DNA into Rhodococcus, it has multicloning sites, contains a highly selective thiostrepton marker, and can be replicated in both E. coli and Rhodococcus.

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The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T-) was examined. Within 7 min of the addition of myxin at 5 ,ug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 ,g/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of "4C-uracil incorporation was found in the presence of myxin in 15Tcells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 ,Ag of myxin per ml and with 0.8 X 108 cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 u/Ag/ml) of the myxin was retained in the cells.