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Shewanella halifaxensis HAW-EB4 was previously isolated for its potential to mineralize hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX)from a UXO (unexploded ordnance)-contaminated marine sediment site near Halifax Harbor. The present study was undertaken to determine the effect of terminal electron acceptors (TEA) on the growth of strain HAW-EB4 and on the enzymic processes involved in RDX metabolism. The results showed that aerobic conditions were optimal for bacterial growth, but that anaerobic conditions in the presence of trimethylamine N-oxide (TMAO) or in the absence of TEA favoured RDX metabolism. RDX as a substrate neither stimulated respiratory growth nor induced its own biotransformation. Strain HAW-EB4 used periplasmic proteins to transform RDX to both nitroso [hexahydro-1-nitroso-3,5-dinitro-1, 3, 5-triazine (MNX), hexahydro-1, 3-dinitroso-5-nitro-1,3, 5-triazine (DNX), and hexahydro-1,3, 5-trinitroso-1,3,5-triazine (TNX)] and ring cleavage products (such as methylenedinitramine), with more nitroso formation in cells grown on TMAO or pre-incubated in the absence of TEA. Using spectroscopy, SDS-PAGE and haem-staining analysis, strain HAW-EB4 was found to produce different sets of c-type cytochromes when grown on various TEA, with several more cytochromes produced in cells grown on TMAO. Crude cytochromes from total periplasmic proteins of TMAO-grown cells metabolized RDX to products similar to those found in assays using total periplasmic proteins and whole cells. To prove the involvement of cytochrome in RDX metabolism, we monitored dithionite- or NADH-reduced cytochromes by their absorbance at the (551 nm) or [gamma] (418-420 nm) bands during anaerobic incubation with RDX. In both caseswe found that RDX biotransformation was accompanied by oxidation of reduced cytochrome. Furthermore, 02, an oxidant of reduced cytochrome, inhibited RDX transformation. The present results demonstrate that S. halifaxensis HAW-EB4 metabolizes RDX optimally under TMAO-reducing conditions, and that c-type cytochromes are involved.

Shewanella halifaxensis and Shewanella sediminis were among a few aquatic γ-proteobacteria that were psychrophiles and the first anaerobic bacteria that degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although many mesophilic or psychrophilic strains of Shewanella and γ-proteobacteria were sequenced for their genomes, the genomic evolution pathways for temperature adaptation were poorly understood. On the other hand, the genes responsible for anaerobic RDX mineralization pathways remain unknown. To determine the unique genomic properties of bacteria responsible for both cold-adaptation and RDX degradation, the genomes of S. halifaxensis and S. sediminis were sequenced and compared with 108 other γ-proteobacteria including Shewanella that differ in temperature and NaP+ requirements, as well as RDX degradation capability. Results showed that for coping with marine environments their genomes had extensively exchanged with deep sea bacterial genomes. Many genes for NaP+-dependent nutrient transporters were recruited to use the high NaP+ content as an energy source. For coping with low temperatures, these two strains as well as other psychrophilic strains of Shewanella and γ-proteobacteria were found to decrease their genome G+C content and proteome alanine, proline and arginine content (p-value <0.01) to increase protein structural flexibility. Compared to poorer RDX-degrading strains, S. halifaxensis and S. sediminis have more number of genes for cytochromes and other enzymes related to RDX metabolic pathways. Experimentally, one cytochrome was found induced in S. halifaxensis by RDX when the chemical was the sole terminal electron acceptor. The isolated protein degraded RDX by mono-denitration and was identified as a multiheme 52 kDa cytochrome using a proteomic approach. The present analyses provided the first insight into divergent genomic evolution of bacterial strains for adaptation to the specific cold marine conditions and to the degradation of the pollutant RDX. The present study also provided the first evidence for the involvement of a specific c-type cytochrome in anaerobic RDX metabolism. [ABSTRACT FROM AUTHOR]

The article reports on the study of terminal electron acceptors (TEA). It aims to determine the effect of TEA on the development of strain Shewanella halefaxensis HAW-EB4. It also examines the role of TEA in the enzymic processes involved in the metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It shows that HAW-EB4 used periplasmic proteins in transforming RDX to nitroso and ring cleavage products. It also demonstrates that HAW-EB4 creates different sets of c-type chromosomes in different TEA and trimethylamine N-oxide (TMAO). The study also indicates that cytochrome and TMAO-reducing conditions are involved in RDX metabolism.