Nutrient amendments in soil DNA stable isotope probing experiments reduce the observed methanotroph diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in non-amended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.     

Assimilation of acetate by the putative atmospheric methane oxidizers belonging to the USCα clade

Forest soils are a major biological sink for atmospheric methane, yet the identity and physiology of the microorganisms responsible for this process remain unclear. Although members of the upland soil cluster α (USCα) are assumed to represent methanotrophic bacteria adapted to the oxidation of the trace level of methane in the atmosphere and to be an important sink of this greenhouse gas, so far they have resisted isolation. In particular, the question of whether the atmospheric methane oxidizers are able to obtain all their energy and carbon solely from atmospheric methane still waits to be answered. In this study, we performed stable-isotope probing (SIP) of RNA and DNA to investigate the assimilation of (13) C-methane and (13) C-acetate by USCα in an acidic forest soil. RNA-SIP showed that pmoA mRNA of USCα was not labelled by (13) C of supplemented (13) C methane, although catalysed reporter deposition - fluorescence in situ hybridization (CARD-FISH) targeting pmoA mRNA of USCα detected its expression in the incubated soil. In contrast, incorporation of (13) C-acetate into USCαpmoA mRNA was observed. USCαpmoA genes were not labelled, indicating that they had not grown during the incubation. Our results indicate that the contribution of alternative carbon sources, such as acetate, to the metabolism of the putative atmospheric methane oxidizers in upland forest soils might be substantial.     

Methane assimilation and trophic interactions with marine Methylomicrobium in deep-water coral reef sediment off the coast of Norway

Deep-water coral reefs are seafloor environments with diverse biological communities surrounded by cold permanent darkness. Sources of energy and carbon for the nourishment of these reefs are presently unclear. We investigated one aspect of the food web using DNA stable-isotope probing (DNA-SIP). Sediment from beneath a Lophelia pertusa reef off the coast of Norway was incubated until assimilation of 5 micromol 13CH4 g(-1) wet weight occurred. Extracted DNA was separated into 'light' and 'heavy' fractions for analysis of labelling. Bacterial community fingerprinting of PCR-amplified 16S rRNA gene fragments revealed two predominant 13C-specific bands. Sequencing of these bands indicated that carbon from 13CH4 had been assimilated by a Methylomicrobium and an uncultivated member of the Gammaproteobacteria. Cloning and sequencing of 16S rRNA genes from the heavy DNA, in addition to genes encoding particulate methane monooxygenase and methanol dehydrogenase, all linked Methylomicrobium with methane metabolism. Putative cross-feeders were affiliated with Methylophaga (Gammaproteobacteria), Hyphomicrobium (Alphaproteobacteria) and previously unrecognized methylotrophs of the Gammaproteobacteria, Alphaproteobacteria, Deferribacteres and Bacteroidetes. This first marine methane SIP study provides evidence for the presence of methylotrophs that participate in sediment food webs associated with deep-water coral reefs.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.     

Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.     

Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing

Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.     

Biochemical and molecular characterization of methanotrophs in soil from a pristine New Zealand beech forest

Methane (CH4) oxidation and the methanotrophic community structure of a pristine New Zealand beech forest were investigated using biochemical and molecular methods. Phospholipid-fatty acid-stable-isotope probing (PLFA-SIP) was used to identify the active population of methanotrophs in soil beneath the forest floor, while terminal-restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the pmoA gene were used to characterize the methanotrophic community. PLFA-SIP suggested that type II methanotrophs were the predominant active group. T-RFLP and cloning and sequencing of the pmoA genes revealed that the methanotrophic community was diverse, and a slightly higher number of type II methanotrophs were detected in the clone library. Most of the clones from type II methanotrophs were related to uncultured pmoA genes obtained directly from environmental samples, while clones from type I were distantly related to Methylococcus capsulatus. A combined data analysis suggested that the type II methanotrophs may be mainly responsible for atmospheric CH4 consumption. Further sequence analysis suggested that most of the methanotrophs detected shared their phylogeny with methanotrophs reported from soils in the Northern Hemisphere. However, some of the pmoA sequences obtained from this forest had comparatively low similarity (<97%) to known sequences available in public databases, suggesting that they may belong to novel groups of methanotrophic bacteria. Different methods of methanotrophic community analysis were also compared, and it is suggested that a combination of molecular methods with PLFA-SIP can address several shortcomings of stable isotope probing.     

好氧甲烷氧化菌生态学研究进展

好氧甲烷氧化菌是一群以甲烷为碳源和能源的细菌。好氧甲烷氧化菌在自然环境中分布广泛,人类已从土壤、淡水和海洋沉积、泥炭沼泽、热泉、海水和南极环境分离到甲烷氧化菌的纯培养。好氧甲烷氧化菌可分为14个属,包括研究较为深入的隶属于变形菌门Alpha和Gamma纲的细菌,以及属于疣微菌门的极端嗜热嗜酸甲烷氧化菌。最近,好氧甲烷氧化菌还被发现存在于苔藓类植物（尤其是泥炭苔藓）共生体中,兼性营养好氧甲烷氧化菌也被发现。本文通过对好氧甲烷氧化菌的分类、生理生化特征、分子生物学检测方法以及微生物生态学中的研究成果的总结与分析,以及对甲烷氧化菌研究所面临的问题进行讨论,以期为今后进一步开展好氧甲烷氧化菌及其在碳循环中的作用研究提供参考。     

水稻中一个NBS-LRR抗病同源基因家族的克隆和分析

利用克隆的抗病基因同源序列RS13作为探针,从水稻IR64的BAC文库中筛选到4个阳性克隆,其中一个克隆14E19能够覆盖其余3个克隆。对14E19进行全序列测定和分析,获得了73kb的全长DNA序列,基因预测显示其上有4个编码NBS-LRR结构域的基因(NL),分别命名为NL-A,B,C和D。对具有相同基因组背景的IRBB56同一染色体位置上跨度更大的BAC克隆106P13进行分析,发现其上有10个NL同源拷贝,其中4个同14E19上的NL一样。搜索日本晴、93—11、广陆矮4号的序列,发现三者有类似的同源序列。但与已知的NBS-LRR抗病基因同源性较低,说明NL是一个至少由10个成员(分别命名为NL-A至J)组成的新基因家族。对NL家族进行RT-PCR和cDNA库筛选分析,发现NL-B基因能够在抗白叶枯病品系IRBB4中表达,暗示该基因参与了抗病反应。     

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000-1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000-1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .     

Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.     

Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments

Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function.     

Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays

Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

First genome data from uncultured upland soil cluster alpha methanotrophs provide further evidence for a close phylogenetic relationship to Methylocapsa acidiphila B2 and for high-affinity methanotrophy involving particulate methane monooxygenase

Members of upland soil cluster alpha (USC alpha) are assumed to be methanotrophic bacteria (MB) adapted to the trace level of atmospheric methane. So far, these MB have eluded all cultivation attempts. While the 16S rRNA phylogeny of USC alpha members is still not known, phylogenies constructed for the active-site polypeptide (encoded by pmoA) of particulate methane monooxygenase (pMMO) placed USC alpha next to the alphaproteobacterial Methylocapsa acidiphila B2. To assess whether the pmoA tree reflects the evolutionary identity of USC alpha, a 42-kb genomic contig of a USC alpha representative was obtained from acidic forest soil by screening a metagenomic fosmid library of 250,000 clones using pmoA-targeted PCR. For comparison, a 101-kb genomic contig from M. acidiphila was analyzed, including the pmo operon. The following three lines of evidence confirmed a close phylogenetic relationship between USC alpha and M. acidiphila: (i) tetranucleotide frequency patterns of 5-kb genomic subfragments, (ii) annotation and comparative analysis of the genomic fragments against all completely sequenced genomes available in public domain databases, and (iii) three single gene phylogenies constructed using the deduced amino acid sequences of a putative prephenate dehydratase, a staphylococcal-like nuclease, and a putative zinc metalloprotease. A comparative analysis of the pmo operons of USC alpha and M. acidiphila corroborated previous reports that both the pmo operon structure and the predicted secondary structure of deduced pMMO are highly conserved among all MB.     

Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity

Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating ¹³C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient ¹³C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of ¹³CH₄ with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of ¹³CH₄ was slow and some cross-feeding of ¹³C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of ¹³CH₄, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of ¹³C into active methanotrophs while avoiding the cross-feeding of ¹³C.     

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA ( 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.     

苏云金芽胞杆菌大质粒pBMB165的克隆与分析

以pBeloBAC11为载体,成功构建了苏云金芽胞杆菌YBT-1765的基因组人工染色体(BAC)文库和质粒BAC文库.根据已克隆的包含复制子ori165在内的3.6kb片段中编码复制蛋白Rep165的核苷酸序列设计探针,通过染色体步移方式,对质粒文库和基因组文库进行筛选,得到13个覆盖YBT-1765菌株中质粒pBMB165不同区域的克隆子.通过Hind Ⅲ和BamH Ⅰ酶切分析,建立了质粒pBMB165的物理图谱和线状重叠连锁图,并测算出该质粒的大小为82kb.根据部分核苷酸序列初步统计了pBMB165上转座因子的存在机率.YBT-1765菌株基因组文库的构建和物理图谱的绘制为克隆苏云金芽胞杆菌大质粒提供了一套可行的方案,成功解决了大质粒难克隆的问题.