Analysis of the xplAB -Containing Gene Cluster Involved in the Bacterial Degradation of the Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine

Abstract

c Repeated use of the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) on military land has resulted in significant soil and groundwater pollution. Rates of degradation of RDX in the environment are low, and accumulated RDX, which the U.S. Environmental Protection Agency has determined is a possible human carcinogen, is now threatening drinking water supplies. RDX-degrading microorganisms have been isolated from RDX-contaminated land; however, despite the presence of these species in contaminated soils, RDX pollution persists. To further understand this problem, we studied RDX-degrading species belonging to four different genera (Rhodococcus, Microbacterium, Gordonia, and Williamsia) isolated from geographically distinct locations and established that the xplA and xplB (xplAB) genes, which encode a cytochrome P450 and a flavodoxin redox partner, respectively, are nearly identical in all these species. Together, the xplAB system catalyzes the reductive denitration of RDX and subsequent ring cleavage under aerobic and anaerobic conditions. In addition to xplAB, the Rhodococcus species studied here share a 14-kb region flanking xplAB; thus, it appears likely that the RDX-metabolizing ability was transferred as a genomic island within a transposable element. The conservation and transfer of xplAB-flanking genes suggest a role in RDX metabolism. We therefore independently knocked out genes within this cluster in the RDX-degrading species Rhodococcus rhodochrous 11Y. Analysis of the resulting mutants revealed that XplA is essential for RDX degradation and that XplB is not the sole contributor of reducing equivalents to XplA. While XplA expression is induced under nitrogen-limiting conditions and further enhanced by the presence of RDX, MarR is not regulated by RDX.

Iron-Dependent Enzyme Catalyzes the Initial Step in Biodegradation of N -Nitroglycine by Variovorax sp. Strain JS1663

“…These electron transfer proteins catalyze the reductive denitration of RDX, resulting in subsequent ring cleavage and the formation of nitrous oxide, ammonia, formaldehyde, and a linear nitramine 4-nitro-2,4-diazabutanal (18,33,48). The degradation of the nitramine ring cleavage product, nitramine 4-nitro-2,4-diazabutanal (18,28,49), is proposed to involve amidohydrolases from the purine catabolic pathway (49).…”

“…The degradation of the synthetic nitramine RDX is catalyzed by the cytochrome P450 XplA and its associated reductase, XplB (27,33). These electron transfer proteins catalyze the reductive denitration of RDX, resulting in subsequent ring cleavage and the formation of nitrous oxide, ammonia, formaldehyde, and a linear nitramine 4-nitro-2,4-diazabutanal (18,33,48).…”

“…The products of secondary nitramine degradation are often primary nitramines, similar to NNG. For example, a cytochrome P450 system catalyzes the initial reaction in bacteria that use RDX as the sole source of nitrogen (27)(28)(29)(30)(31)(32)(33). The enzyme cleaves nitro groups from RDX in a pathway that releases nitrite and yields linear nitramine products.…”

Effects of Perchlorate and Other Groundwater Inorganic Co-Contaminants on Aerobic RDX Degradation

“…Instead, the general availability of nitrogen repressed xplA expression [ 18 ]. Similarly, in Rhodococcus rhodochrous 11Y, xplA expression was induced under nitrogen-limiting conditions and further enhanced by the presence of RDX [ 19 ].…”

The Integrative Conjugative Element clc (ICEclc) of Pseudomonas aeruginosa JB2

“…Biodegradation of xenobiotic compounds is a key environmental service of bacterial communities, and MGE are well-established as playing a central role in the evolution of metabolic capacity essential for these activities ( Top et al, 2002 ; Top and Springael, 2003 ; Diaz, 2004 ; Shintani et al, 2010 ). While initial work exploring MGE centered largely on plasmids, the advent of genome sequencing has revealed the role of ICE and other types of genomic islands ( van der Meer and Sentchilo, 2003 ; Gaillard et al, 2006 ; Hickey et al, 2012 ; Chong et al, 2014 ; Pathak et al, 2016 ; Zamarro et al, 2016 ; Suenaga et al, 2017 ).…”