Zhao Jian-Shen, Manno Dominic, Hawari Jalal
Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada.
Microbiology (Reading). 2008 Apr;154(Pt 4):1026-1037. doi: 10.1099/mic.0.2007/013409-0.
Shewanella halifaxensis HAW-EB4 was previously isolated for its potential to mineralize hexahydro-1,3,5-trinitro-1,3,5-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 alpha (551 nm) or gamma (418-420 nm) bands during anaerobic incubation with RDX. In both cases we found that RDX biotransformation was accompanied by oxidation of reduced cytochrome. Furthermore, O(2), 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.
哈利法克斯希瓦氏菌HAW-EB4最初是从哈利法克斯港附近一个受未爆炸弹药（UXO）污染的海洋沉积物地点分离出来的，因其具有使六氢-1,3,5-三硝基-1,3,5-三嗪（RDX）矿化的潜力。本研究旨在确定末端电子受体（TEA）对菌株HAW-EB4生长以及RDX代谢相关酶促过程的影响。结果表明，需氧条件最有利于细菌生长，但在三甲胺N-氧化物（TMAO）存在或不存在TEA的厌氧条件下，有利于RDX代谢。RDX作为底物既不刺激呼吸生长，也不诱导其自身的生物转化。菌株HAW-EB4利用周质蛋白将RDX转化为亚硝基化合物[六氢-1-亚硝基-3,5-二硝基-1,3,5-三嗪（MNX）、六氢-1,3-二亚硝基-5-硝基-1,3,5-三嗪（DNX）和六氢-1,3,5-三亚硝基-1,3,5-三嗪（TNX）]和开环产物（如亚甲基二硝胺），在以TMAO为生长底物或在不存在TEA的条件下预培养的细胞中形成更多的亚硝基化合物。通过光谱学、SDS-PAGE和血红素染色分析发现，菌株HAW-EB4在不同的TEA条件下生长时会产生不同的c型细胞色素，在以TMAO为生长底物的细胞中产生的细胞色素更多。来自以TMAO为生长底物的细胞总周质蛋白的粗细胞色素将RDX代谢为与使用总周质蛋白和全细胞进行测定时发现的产物相似的产物。为了证明细胞色素参与RDX代谢，我们在与RDX厌氧孵育期间通过它们在α（551 nm）或γ（418 - 420 nm）波段的吸光度监测连二亚硫酸盐或NADH还原的细胞色素。在这两种情况下，我们都发现RDX生物转化伴随着还原型细胞色素的氧化。此外，还原型细胞色素的氧化剂O₂抑制RDX转化。目前的结果表明，哈利法克斯希瓦氏菌HAW-EB4在TMAO还原条件下能最佳地代谢RDX，并且c型细胞色素参与其中。
