Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

Abstract

Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain Gordonia sp. KTR9. This plasmid carries xplA, encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with xplA are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator. Rhodococcus jostii RHA1 expressing xplA from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an Escherichia coli strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of glnA-xplB, xplA, and xplR showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.

Role of Nitrogen Limitation in Transformation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9

“…Conversely, the lack of GlnR cis-acting elements upstream of xplAB may suggest that GlnR does not directly regulate xplAB expression. Real-time PCR analyses as described previously (20) confirmed the relative changes in transcript levels for genes listed in Table 2.…”

“…We identified a regulator located upstream of the xplAB gene cluster, designated xplR, which is transcriptionally upregulated in the presence of RDX and subject to nitrogen repression (20). We hypothesized that XplR may repress xplAB in the presence of preferred nitrogen sources such as (NH 4 ) 2 SO 4 .…”

“…RDX degradation by a Gordonia sp. strain KTR9 isolate was inhibited in the presence of competing inorganic nitrogen sources, and reduction in RDX degradation correlated to expression of xplA (20). Recently, a role for the global nitrogen regulator, GlnR, in RDX degradation has been suggested for KTR9 since glnR mutants of KTR9 are unable to grow on RDX as a sole nitrogen source (21).…”

“…To further investigate nitrogen limitation in the catabolism of RDX, gene expression analyses of KTR9 and a glnR KTR9 deletion mutant were conducted under excess and limiting nitrogen growth conditions. KTR9 was grown as described previously (20), and triplicate cultures in the late exponential phase of growth were used to inoculate media containing the following nitrogen-containing compounds: (i) 4 mM (NH 4 ) 2 SO 4 , (ii) 0.9 mM (NH 4 ) 2 SO 4 , (iii) 40 mg liter Ϫ1 (180 M) RDX, and (iv) 40 mg liter Ϫ1 RDX plus 4 mM (NH 4 ) 2 SO 4 . RDX concentrations were determined by high-performance liquid chromatography (HPLC) analysis (14), and ammonium concentrations were verified using the AQUANAL-plus test kit for ammonium (Sigma, St. Louis, MO).…”

Genomic and Transcriptomic Studies of an RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine)-Degrading Actinobacterium

“…strain KTR9 was isolated from soil at an explosives testing facility (19). Although the strain was reported to utilize hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a sole source of carbon and nitrogen under aerobic growth conditions, subsequent studies have replicated only its ability to utilize RDX as a sole source of nitrogen (9). KTR9 is similar to other mycolic acid-containing actinomycetes that degrade RDX (4,14,18) in that it utilizes XplA, a cytochrome P450, to catalyze the denitration of RDX (11,17,18).…”