Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine

Abstract

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...

Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species

“…5. None of the known genes for s-triazine-degrading enzymes, including the melamine-degrading genes trzA and triA (Shao et al 1995;Seffernick et al 2001), were detected in strain ATD6. The melamine-deaminating activity of the strain was detected in more than 50% saturation of ammonium sulfate fraction of the cell extract (data not shown).…”

“…The sequence of the gene triA was 98% identical to atzA (Seffernick et al 2001), so the primer pairs for atzA are also available to detect triA. To detect the trzA gene (Shao et al 1995), a pair of primers, trzA1f (5′-GACCAGAATCGCAATCACC-3′) and trzA1r (5′-TACAGCAGTGTTCCGACTC-3′), are used. The primer pairs used for amplifying trzC-encoding ammelide aminohydrolase (TrzC) (Eaton and Karns 1991), trzD-encoding cyanuric acid amidohydrolase (TrzD) (Karns 1999), and trzF-encoding allophanate hydrolase (TrzF) (Shapir et al 2006) were trzCf (5′-GGTAGAGCCTCACATCCATC-3′) and trzCr (5′-GAGCCAAACATTACGGAGAC-3′), trzDf (5′-CCTCGCGTTCAAGGTCTACT-3′) and trzDr (5′-TCGAAGCGATAACTGCATTG-3′), and trzFf (5′-GAAAAGCCAGAGGTGAGGAG-3′) and trzFr (5′-ACCTGCCGTGAGAATGAGTC-3′), respectively.…”

“…R. corallinus was originally considered to be a junior synonym of Rhodococcus rubropertinctus in a DNA homology study (Mordarski et al 1980), and has since been reclassified as Gordonia rubripertincta (Stackebrandt et al 1988;Euzeby 1998). Genes encoding enzymes for melamine deamination, trzA for s-triazine hydrolase (TrzA) and triA for melamine deaminase (TriA), were cloned from G. rubripertincta strain NRRL B-15444R and A. citrulli strain NRRL B-12227, respectively (Shao et al 1995;Seffernick et al 2001). Shelton et al (1997) determined the melamine metabolism pathway used by Klebsiella terragena strain DRS-1, isolated from sewage sludge enrichments (Hapeman et al 1995).…”

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“…This includes upper pathway and lower pathway. The reaction pathways shown are combined from the various reaction pathways provided (Behki et al, 1993;Nagy et al, 1995;Shao et al, 1995). Unlike both bacteria (Pseudomonas sp.…”

“…Respective encoding genes (Table 5) have been reported (Nagy et al, 1995;Shao and Behki, 1996). AtrA gene in Rhodococcus strain TE1 involved in N-dealkylation of atrazine and forms DIA and DEA (Shao et al, 1995). AtzB (hydroxyatrazine ethylaminohydrolase) catalyzes hydroxyatrazine and form Nisopropylammelide which further catalyzed by AtzC (N-isopropylammelide isopropylamino hydrolase), forming a cyanuric acid (Martinez et al, 2001;Shapir et al, 2007).…”