兼性甲烷氧化菌的发现历程

兼性甲烷氧化菌在新陈代谢上具有独一无二的特性:它们能够利用甲烷或一些含碳碳键的有机物作为唯一碳源和能源.甲基细胞菌属(Methylocella)、甲基孢囊菌属(Methylocystis)和甲基帽菌属(Methylocapsa)的一些菌株已经被确定为兼性甲烷氧化菌.它们都属于a-变形菌纲,能够像利用甲烷一样在大分子有机酸或乙醇里生长.本文全面系统地总结了兼性甲烷氧化菌的研究发展历史,推断出兼性甲烷氧化菌易在酸性环境富集生长;介绍了与之有相近功能的兼性甲烷氧化生物;浅析了其对多碳化合物的代谢机理;最后讨论了兼性甲烷氧化菌研究的现存问题和工程应用前景.     

甲烷氧化菌素的产生和铜捕获作用

甲烷氧化菌素(mb)是由甲烷氧化菌产生的与铜捕获和吸收有关的生物螯合剂。采用NMS/CAS-Cu劈半平板法检测发现甲基弯菌 Methylosinus trichosporium IMV3011在以甲烷和甲醇为碳源时均具有向外界分泌mb的能力。甲基弯菌 Methylosinus trichosporium IMV3011在限铜胁迫下能够在培养介质中积累mb,甲醇为碳源时mb的积累量明显高于甲烷为碳源时mb的积累量,推测mb的合成与细胞中NADH含量有关。mb的专一性研究发现从甲基弯菌 Methylosinus trichosporium IMV3011发酵上清液中分离到的mb能够提高其它3种甲烷氧化菌由无铜培养基向含铜培养基转移时的pMMO活性的表达,并明显缩短它们由无铜培养基转移到含铜培养基时生长的延滞期,提高生长速度。表明其它3种甲烷氧化菌同样能够吸收来自于甲基弯菌Methylosinus trichosporium IMV3011的mb捕获的铜。     

低氧生境中好氧甲烷氧化菌的缺氧耐受机理及种群结构研究进展

甲烷生物氧化在全球大气甲烷平衡和温室气体的控制中起着重要作用.氧气是甲烷生物氧化过程中的重要影响因素之一.生境中氧浓度不仅影响好氧甲烷氧化菌的种群结构、活性及甲烷碳的分配,而且好氧甲烷氧化菌在不同氧浓度下具有不同的代谢途径.理解低氧生境中好氧甲烷氧化菌的缺氧耐受机理和甲烷生物氧化过程,对甲烷驱动型生态系统的碳循环和生物多样性有着重要意义.本文以好氧甲烷氧化菌为对象,综述了低氧生境中好氧甲烷氧化菌的活性及其种群结构、好氧甲烷氧化菌的缺氧耐受机理以及低氧生境中甲烷氧化菌与非甲烷氧化菌的关系,并对今后的研究方向进行了展望.     

甲烷氧化过程中铜的作用研究进展

甲烷生物氧化在全球甲烷平衡和温室效应控制中扮演着重要的角色,而铜是甲烷生物氧化过程中的重要影响因子.一方面,铜是调控不同类型甲烷单加氧酶表达的主要影响因子,是组成颗粒性甲烷单加氧酶的必需金属元素;另一方面,在自然环境体系中,铜含量及其形态的变化对甲烷氧化菌的分布、代谢甲烷和非甲烷类有机化合物的能力以及甲烷氧化菌的特异性铜捕获系统也会产生较大影响.准确把握铜在甲烷生物氧化过程中发挥的作用将有助于全面了解甲烷生物氧化过程,进而更好地指导甲烷氧化微生物在温室气体减排及非甲烷有机物污染修复中的应用.本文主要从铜的角度,概述了铜对甲烷氧化菌的分布和活性的影响,介绍了铜在调控甲烷单加氧酶的表达和活性以及调节甲烷氧化菌铜捕获系统方面的作用,并展望了其研究方向.     

甲烷氧化细菌的一个新种

从沼气发酵装置中,分离出一株H型专性甲烷氧化细菌81Z菌株。它具有甲烷氧化细菌的一般典型性状。根据其极生单鞭毛和过氧化氢酶阴性的特征,该菌株明显地区别于已知的任何一种甲烷氧化细菌,被认为是一个新种并命名为沼气甲基弯菌(Methylosinus methanica)。本文还讨论了它在厌氧条件下的生命活动。     

甲烷氧化菌研究进展

甲烷氧化菌以甲烷为其唯一的碳源和能源 ,在全球大气甲烷平衡中起着重要的作用 ,它还可以降解卤代化合物 ,在污染治理方面具有潜在价值。本文从甲烷氧化菌的分类出发 ,对甲烷氧化菌氧化甲烷的机理及影响因素、甲烷氧化菌的生理、生态分布及检测方法、甲烷氧化菌降解有机污染物的潜在应用等作一综述 ,分析目前研究中存在的问题 ,并指出今后应加强研究的方面。     

填埋覆土甲烷氧化微生物及甲烷氧化作用机理研究进展

甲烷是一种长期存在于大气中的温室气体,它对温室效应的贡献率是二氧化碳的26倍.生活垃圾填埋场是大气甲烷的主要产生源之一,由其产生的甲烷约占全球甲烷排放总量的1.5%～15%.甲烷氧化微生物在调节全球甲烷平衡中起着重要作用.垃圾填埋场覆土具有相当强的甲烷氧化能力.填埋覆土甲烷氧化菌及其氧化作用机理的研究,已成为环境微生物学研究领域的热点之一.本文对生活垃圾填埋场填埋覆土中甲烷氧化微生物、甲烷氧化机理及动力学机制、甲烷与微量填埋气体的共氧化机制以及影响甲烷氧化的环境因子研究的最新进展进行综述,并对生活垃圾填埋场甲烷氧化微生物的研究进行展望.     

基于甲烷氧化菌的脱氮技术研究进展

甲烷氧化菌利用甲烷作为唯一碳源和能源,在氧化甲烷的过程中能有效地实现脱氮,该过程分为好氧甲烷氧化耦合反硝化(aerobic methane oxidation coupled to denitrification,AME-D)和厌氧甲烷氧化耦合反硝化(anaerobic methane oxidation coupled to denitrification,ANME-D),在碳循环和氮循环的研究中具有重要意义。本文通过总结近年来有关甲烷氧化菌的分类与分布,阐述AME-D和ANME-D的基本原理、影响因素和应用情况,提出相应的研究方向,以期为甲烷氧化菌在污水脱氮中的应用提供参考。     

垃圾填埋场甲烷氧化菌及甲烷减排的研究进展

垃圾填埋场是全球最重要的人为甲烷排放源之一,其全球年甲烷释放量为35-69 Tg,垃圾填埋场甲烷减排是目前全球温室气体研究的热点。甲烷氧化菌能够氧化分解甲烷,作为减少大气甲烷排放的重要生物汇,对保持大气中甲烷浓度的平衡具有重要意义。从甲烷氧化菌的类型及其特征、甲烷氧化机理着手,介绍了多样性研究方法、填埋场中甲烷氧化菌的活性影响因素及甲烷生物减排应用等最新研究进展。在综述前人研究的基础上,探讨了目前研究的不足,提出了利用甲烷氧化菌复合微生物菌剂等综合处理措施,旨为垃圾填埋场甲烷减排的研究和应用提供新的思路。     

土壤大气甲烷氧化菌研究进展

土壤微生物催化是大气中痕量甲烷(约1.8ppmv)氧化的唯一生物途径。目前的研究表明好氧土壤中存在专性和选择性大气甲烷氧化菌2种类型:前者(USCα和USCγ)广泛分布于各种好氧旱地土壤,其甲烷氧化酶对低浓度甲烷亲和力极高,属真正的寡营养型,但至今尚未获得该种类的纯培养菌株。后者属于传统甲烷氧化菌Methylocystis/Methylosinus属,广泛分布于各种周期性排放高浓度甲烷的土壤环境中。该属大部分菌株含有亲和力不同的2套甲烷单加氧酶系统,其中的高亲和力甲烷单加氧酶使这些菌株可以在相当长的时间内(3个月)保持大气浓度甲烷氧化活性,但其生长和繁殖还需依赖于土壤内部阶段性产生的高浓度甲烷。本文详细阐述了2类大气甲烷氧化菌的发现历程及其可能的生存策略,最后系统梳理了几种关键的环境因子(土壤温度及湿度、土壤pH、植被、土地利用及氮输入)对大气甲烷氧化菌群落结构和甲烷氧化活性的影响,提出并展望了土壤大气甲烷氧化菌研究的重要方向。     

Copper methanobactin: a molecule whose time has come

Copper plays a key role in the physiology of methanotrophs. One way that these bacteria meet their high copper requirement is by the biosynthesis and release of high affinity copper binding compounds called methanobactins. Recent advances in methanobactin characterization include the first crystal structure, detailed spectroscopic analyses, and studies of metal ion specificity. Methanobactin may function in copper uptake, regulation of methane monooxygenase expression, protection against copper toxicity, and particulate methane monooxygenase activity. Methanobactin can extract copper from insoluble minerals and could be important for mineral weathering. Many methanobactin properties are reminiscent of iron siderophores, suggesting a similar mechanism of handling. Methanobactin-like compounds have also been identified in yeast mitochondria, suggesting that these molecules are a more universal phenomenon.     

A TonB-Dependent Transporter Is Responsible for Methanobactin Uptake by Methylosinus trichosporium OB3b

Methanobactin, a small modified polypeptide synthesized by methanotrophs for copper uptake, has been found to be chromosomally encoded. The gene encoding the polypeptide precursor of methanobactin, mbnA, is part of a gene cluster that also includes several genes encoding proteins of unknown function (but speculated to be involved in methanobactin formation) as well as mbnT, which encodes a TonB-dependent transporter hypothesized to be responsible for methanobactin uptake. To determine if mbnT is truly responsible for methanobactin uptake, a knockout was constructed in Methylosinus trichosporium OB3b using marker exchange mutagenesis. The resulting M. trichosporium mbnT::Gm^r mutant was found to be able to produce methanobactin but was unable to internalize it. Further, if this mutant was grown in the presence of copper and exogenous methanobactin, copper uptake was significantly reduced. Expression of mmoX and pmoA, encoding polypeptides of the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), respectively, also changed significantly when methanobactin was added, which indicates that the mutant was unable to collect copper under these conditions. Copper uptake and gene expression, however, were not affected in wild-type M. trichosporium OB3b, indicating that the TonB-dependent transporter encoded by mbnT is responsible for methanobactin uptake and that methanobactin is a key mechanism used by methanotrophs for copper uptake. When the mbnT::Gm^r mutant was grown under a range of copper concentrations in the absence of methanobactin, however, the phenotype of the mutant was indistinguishable from that of wild-type M. trichosporium OB3b, indicating that this methanotroph has multiple mechanisms for copper uptake.     

Detoxification of Mercury by Methanobactin from Methylosinus trichosporium OB3b

Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl₂ and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b ΔmbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the ΔmbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.     

Purification and physical-chemical properties of methanobactin: a chalkophore from Methylosinus trichosporium OB3b

Methanobactin is an extracellular, copper-binding chromopeptide from the methane-oxidizing bacterium, Methylosinus trichosporium OB3b, believed to be involved in copper detoxification, sequestration, and uptake. Although small (1217.2 Da), methanobactin possesses a complex three-dimensional macrocyclic structure with several unusual moieties. The molecule binds one copper and has the N-2-isopropylester-(4-thionyl-5-hydroxyimidazolate)-Gly(1)-Ser(2)-Cys(3)-Tyr(4)-pyrrolidine-(4-hydroxy-5-thionylimidazolate)-Ser(5)-Cys(6)-Met(7) sequence [Kim, H. J., et al. (2004) Science 305, 1612-1615]. We report methods for purifying methanobactin from M. trichosporium OB3b and present initial evidence of its physiological function. MALDI-TOF MS was used to systematically monitor samples for optimizing purification conditions, and for detecting and analyzing specific metal-methanobactin complexes. Purification was performed by first stabilizing the extracted compound with copper followed by separation using reversed-phase HPLC in neutral pH buffers. Purified methanobactin exhibited UV-visible maxima at 342 nm, a shoulder at 388 nm, and a broad peak at 282 nm. These features were lost upon CuCl(2) titration with appearance of new features at 335, 356, 290, and 255 nm. Furthermore, methanobactin contains two fluorescent moieties, which exhibit broad emissions at 440-460 nm (lambda(max)(ex) at 388 nm) and 390-430 nm (lambda(max)(ex) = 342 nm), respectively. Finally, methanobactin eliminates the growth lag in M. trichosporium OB3b and substantially increases growth rates when cultures are exposed to elevated copper levels.     

Competitive ligand exchange between Cu–humic acid complexes and methanobactin

Copper has been found to play a key role in the physiology of methanotrophic micro‐organisms, and methane oxidation may critically depend on the availability of Cu. In natural environments, such as soils, sediments, peat bogs, and surface waters, the presence of natural organic matter (NOM) can control the bioavailability of Cu by forming strong metal complexes. To promote Cu acquisition, methanotrophs exude methanobactin, a ligand known to have a high affinity for Cu. In this study, the capability of methanobactin for Cu acquisition from NOM was investigated using humic acid (HA) as a model substance. The kinetics of ligand exchange between Cu–HA and methanobactin was observed by UV–vis spectroscopy, and the speciation of Cu bound to methanobactin was determined by size‐exclusion chromatography coupled to an ICP‐MS. The results showed that Cu was mobilized from HA by a fast ligand exchange reaction following a second‐order rate law with first‐order kinetics for both methanobactin and Cu–HA complexes. The reaction rates decreased with decreasing temperature. Equilibrium experiments indicated that methanobactin was not sorbed to HA and proved that methanobactin is competitive with HA for Cu binding by forming strong 1:1 Cu–methanobactin complexes. Consequently, our results demonstrate that methanobactin can efficiently acquire Cu in organic‐rich environments.     

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl₂, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl₂, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl₂. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl₂. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin “piracy” may be commonplace.     

Genome sequence of the obligate methanotroph Methylosinus trichosporium strain OB3b

Methylosinus trichosporium OB3b (for "oddball" strain 3b) is an obligate aerobic methane-oxidizing alphaproteobacterium that was originally isolated in 1970 by Roger Whittenbury and colleagues. This strain has since been used extensively to elucidate the structure and function of several key enzymes of methane oxidation, including both particulate and soluble methane monooxygenase (sMMO) and the extracellular copper chelator methanobactin. In particular, the catalytic properties of soluble methane monooxygenase from M. trichosporium OB3b have been well characterized in context with biodegradation of recalcitrant hydrocarbons, such as trichloroethylene. The sequence of the M. trichosporium OB3b genome is the first reported from a member of the Methylocystaceae family in the order Rhizobiales.     

Methanobactin-promoted dissolution of Cu-substituted borosilicate glass

Mineral weathering plays an important role in the global cycling of carbon and metals and there is an increasing realization that subsurface microbial activity may be a key factor controlling specific biogeochemical reactions and their rates. Methanobactin (mb) is an extracellular copper-binding compound excreted by methanotrophs to acquire copper for the regulation of methane oxidation. Bioavailable Cu regulates the expression and activity of pMMO vs. sMMO (particulate vs. soluble methane monooxygenase, respectively), key enzymes responsible for bacterial methane oxidation. In this study, we investigate the effect of mb on the dissolution of Cu-substituted borosilicate glass at low temperature and near neutral pH conditions, using batch dissolution experiments. Methanobactin promotes the dissolution of Cu-substituted glasses at rates faster than control experiments. Glasses with lower concentrations of copper (80 p.p.m.) or no copper are dissolved more rapidly by mb than those with more abundant copper (800 p.p.m.). Within the first 2 h of reaction, mb sorption onto glass surfaces limits mass transfer of Cu into solution, and at higher concentrations (100 µmol) of the ligand, inhibits dissolution rates of all glass formulations. These results suggest that both the concentration of mb in solution and the solid phase Cu concentration impact silicate weathering rates and related cycling of carbon in near-surface geological settings.