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.

FIG. 1.

Phylogenetic relationship of ParA proteins from 20 actinobacterial plasmids. ParAs and their predicted homologs are designated by the name of the plasmid on which they reside. The subscript identifies the genus of the bacterium in which the plasmid resides (first letter) and either the first two letters of the species or the strain number. Linear plasmids are underscored. Bootstrap values of major, stable nodes are indicated as percentages. ParA sequences from the following plasmids were included in the analysis: pGKT1, Gordonia sp. KTR9; pGKT2, Gordonia sp. KTR9; pGKT3, Gordonia sp. KTR9; pKB1, G. westfalica strain DSM 44215^T (gi|40217320); pGBRO01, Gordonia bronchialis DSM 43247 (gi|262118119); pSCP1_a2, Streptomyces coelicolor A3(2) (gi|21234217); pCLP, Mycobacterium celatum, (gi|32455743); pMUM001, Mycobacterium ulcerans, (gi|42414723); pNF1, Nocardia farcinica IFM 10152 (gi|54027649); p103, Rhodococcus equi 103 (gi|10657915); pFAJ2600, Rhodococcus erythropolis NI86/21 (gi|2460009); pPBD2, R. erythropolis BD2 (gi|33867067); pREC1, R. erythropolis PR4 (gi|77404542); pREL1, R. erythropolis PR4 (gi|77454578); pRHL1, R. jostii RHA1, (gi|111025387); pRHL2, R. jostii RHA1, (gi|111026081); pRHL3, R. jostii RHA1, (gi|111027090); pSLA2-L, Streptomyces rochei 7434-AN4 (gi|30795070); pFP11, Streptomyces sp. F11 (gi|62184581); pFP1, Streptomyces sp. FQ1 (gi|62184597); pSV2, Streptomyces violaceoruber ANK95570 (gi|32455643). The predicted ParAs of pGKT1 and pKB1 have identical sequences.

FIG. 2.

The xpl gene cluster. Putative promoter regions (−10 and −35 regions) were found upstream of the xplR ORF (bp 156809 to 157548) at positions 156741 to 156761 and of the cyp151C ORF (bp 158328 to 159527 bp) at positions 158272 to 158290. A GntR binding site (*) (TnGTnnnACnA) was found 5 bp upstream of the promoter region for cyp151C. Regions targeted by qPCR are shown by barbell lines about the gene boxes.

FIG. 3.

Degradation of RDX by Rhodococcus jostii RHA1 containing XplA. Exponentially growing cells containing pTpXplA (▴) or an empty pTip vector (▪) were resuspended to an OD₆₀₀ of 12 in 0.1 M sodium phosphate, pH 7.5, containing 60 ppm RDX. The fitted curve represents a first-order decay with a half-life (t_1/2) of 0.61 ± 0.05 h.

FIG. 4.

PFGE (A) and Southern analysis (B) of KTR9 and derived mutant strains. A biotin-labeled 683-bp DNA fragment, specific for pGKT2, was used to probe a PFGE gel of uncut and XbaI-digested DNA samples from wild-type and mutant KTR9 strains. Lanes: 2, uncut KTR9; 3, uncut deletion mutant 1; 4, uncut deletion mutant 2; 5, cut KTR9; 6, cut deletion mutant 1; 7, cut deletion mutant 1. Lane L shows molecular mass markers, with sizes in kilodaltons at the left.

FIG. 5.

(A) Effects of various inorganic nitrogen sources on the RDX degradation activity (closed symbols) and growth (open symbols) of KTR9. A 4 mM concentration of (NH₄)₂SO₄ (triangles), KNO₃ (squares), or KNO₂ (diamonds) was included as a competing nitrogen source compared to RDX alone (circles). Error bars are the standard errors of three samples. (B) Quantitative real-time PCR expression analysis using primers for xplR, cyp151C, glnA-xplB, xplA, and a 16S rRNA. RNA was harvested from KTR9 cells grown for 24 h on media containing the indicated nitrogen sources: RDX alone (diagonally striped bars), RDX plus (NH₄)₂SO₄ (white bars), RDX plus KNO₂ (black bars), and RDX plus KNO₃ (horizontally striped bars). Relative expression of gene targets was normalized to corresponding gene expression levels for cultures in which RDX was the sole nitrogen source.