Bacterial community structure and activity in different Cd-treated forest soils

In this study we compared indicators of Cd bioavailability (water extracts, Lakanen extracts, free ions) and ecotoxicity in forest soils with contrasting physico-chemical characteristics. Soil samples were treated with CdCl(2) solutions (0, 0.1, 1, 10 and 100 mM) and incubated for 30 days. Microbial activity indexes (acid phosphatase, beta-glucosidase, basal respiration) and changes in bacterial community structure using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting were investigated. The Cd concentrations measured ranged from 1% to 37% of the total additions in water extracts, to higher levels in Lakanen extracts. Effects of Cd were observed at bioavailable concentrations exceeding United Nations/European Economic Commission UN/ECE guidelines for total Cd in the soil solution. Basal respiration was the most affected index, while enzymatic activities showed variable responses to the Cd treatments. We also noticed that soils with pH higher than 6.7 and clay content higher than 50% showed inhibition of basal respiration but no marked shift in bacterial community structure. Soils with lower pH (pH <5.8) with less clay content (<50%) showed in addition strong changes in the bacterial community structure. Our results provide evidence for the importance of relating the effects of Cd on the soil communities to soil properties and to bioavailability.     

Characterization of the bacterial community associated with the surface and mucus layer of whiting (Merlangius merlangus)

The bacterial community inhabiting the mucus layer and surface of whiting was examined to determine whether the bacteria present are a reflection of the surrounding water or an indigenous bacterial flora is present. The outer mucus, mouth mucus and gut of four whiting harvested from a site in the Irish Sea and the surrounding water were examined by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA gene and clone library construction. The water community was the most diverse, with only a small number of shared water-mucus phylotypes present. The bacterial flora associated with the outer mucus layer were more diverse than that of the mouth mucus and gut. All three mucus layers were characterized by the presence of a dominant phylotype, identified as clone wom-1, highly similar to Photobacterium iliopiscarium. In addition to other Photobacterium phylotypes, members of the CFB and Clostridia groups were also detected. Subsequently, whiting from 11 different sites along the east and south coast of Ireland were compared by tRFLP analysis. Strikingly, the mucus layer of whiting at all sites was characterized by the presence and dominance of a TRF corresponding to the clone wom-1 which was virtually absent from the water column.     

非培养方法在土壤微生物生态学研究中的应用

由于有相当数量的土壤微生物是目前不可培养的,因此利用传统培养技术来研究土壤微生物,不仅费时费力,所得到的结果可能和真实的情况相差甚远。近年来发展了三类不需培养的方法来研究土壤微生物的种类和数量,这些方法大体上分为生物化学、生理学和分子生物学三类。生物化学方法主要根据细胞膜磷脂酸(PLFA)的种类和数量来判定微生物的多样性;BIOLOG微量板分析系统是生理学方法的代表,它主要是根据土样细胞悬液对95种单一碳源的利用模式来说明群落结构的变化;分子生物学方法是发展应用最广的方法。基本步骤是提取土壤的总DNA,然后用通用引物或选择性高的引物来扩增16SrRNA基因。由于对扩增产物分析方法的不同,该方法又可分为PCR-DGGE,PCR-RFLP等。最近在PCR-RFLP基础上发展起来的T—RFLP分析方法,将微生物的多样性分析工作同RDP(ribosomal database project)数据库结合,充分利用了Internet的数据资源共享的优势,具有分辨率高,可实现自动化等优点。是未来土壤微生物生态学研究的有力工具。     

Phylogenetic Analysis of the Gut Bacterial Microflora of the Fungus-Growing Termite Odontotermes formosanus

We constructed a bacterial 16S rRNA gene clone library from the gut microbial community of O. formosanus and phylogenetically analyzed it in order to contribute to the evolutional study of digestive symbiosis and method development for termite control. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 out of 280 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. The representative phylotypes were affiliated to four phylogenetic groups, Firmicutes, the Bacteroidetes/Chlorobi group, Proteobacteria, and Actinobacteria of the domain Bacteira. No one clone affiliated with the phylum Spirochaetes was identified, in contrast to the case of wood-feeding termites. The phylogenetic analysis revealed that nearly half of the representative clones (25 phylotypes) formed monophyletic clusters with clones obtained from other termite species, especially with the sequences retrieved from fungus-growing termites. These results indicate that the presence of termite-specific bacterial lineages implies a coevolutional relationship of gut microbes and host termites.     

Relationships between the Arctic and the Antarctic cyanobacteria; three Phormidium-like strains evaluated by a polyphasic approach

Selected strains of filamentous Phormidium-like cyanobacteria isolated from two Arctic regions (Ellesmere Island, High Canadian Arctic and Svalbard) and from Antarctica (Antarctic peninsula, South Shetland Islands and South Orkney Islands) were studied. The polyphasic approach used included phenotypic observations of morphological features and genotypic analyses (restriction fragment length polymorphism of 16S rRNA gene, internal transcribed space, 16S rRNA gene sequence analysis). Although genotypes generally correspond to observed morphotypes, the genetic analyses revealed a high degree of biodiversity that could not be unveiled using solely morphological evaluations. According to the phylogenetic analysis, the three clones were divided into two major clades, indicating that the phylogenetic distance between Arct-Ph5/Ant-Ph68 and Ant-Ph58 was so large they belonged to different genera. The polyphyletic position of strains of the genus Phormidium was confirmed by this study, attesting the need to entirely revise classification in this taxon in the future.     

Diversity of microbial communities colonizing the walls of a Karstic cave in Slovenia

Karstic cave systems in Slovenia receive substantial amounts of organic input from adjacent forest and freshwater systems. These caves host microbial communities that consist of distinct small colonies differing in colour and shape. Visible to the naked eye, the colonies cover cave walls and are strewn with light-reflecting water droplets. In this study, the diversity of prokaryotes constituting these unusual microbial communities in Pajsarjeva jama cave was examined. A molecular survey based on small subunit rRNA diversity showed a high diversity within the Bacteria , while members of Archaea were not recovered. A total of eight bacterial phyla were detected. The application of various species richness estimators confirmed the diverse nature of the microbial community sample. Members of Gammaproteobacteria were most abundant in the clone libraries constructed and were followed in abundance by members of Actinobacteria and Nitrospira . In addition, members of Alphaproteobacteria, Betaproteobacteria and Deltaproteobacteria as well as Acidobacteria, Verrucomicrobia, Planctomycetes, Chloroflexi and Gemmatimonadetes were identified in clone libraries. The high number of clones most closely related to environmental 16S rRNA gene clones showed the broad spectrum of unknown and yet to be cultivated microorganisms inhabiting these cave systems.     

Detection of methanotrophs with highly divergent pmoA genes from Arctic soils

Tundra soil samples from the Canadian Arctic community, Kuujjuaq, were analyzed for the presence of the soluble (sMMO) and particulate (pMMO) methane monooxygenase genes. Total genomic DNA extracted from these soils was used as template for PCR using sMMO- and pMMO-specific primers, mmoX1-mmoX2 and A189-A682, respectively. pMMO and sMMO genes were detected in the Kuujjuaq soil samples. Isolation of sMMO-possessing methanotrophic microorganisms from the three soils, as determined by the colony naphthalene oxidation assay, was carried out using direct plating (5 degrees C) and methane enrichment studies (5 degrees C and 25 degrees C). Direct plating did not yield sMMO-possessing methanotrophic bacteria, whereas methane enrichments yielded isolates possessing and expressing sMMO activity. Analysis of derived amino acid sequences of pmoA genes and partial 16S rRNA genes obtained by PCR, using DNA isolated directly from this environment and from isolates, revealed the presence of highly divergent PmoA/AmoA sequences and 16S rRNA sequences that cluster closely with but are distinct from the genes from the genera Methylosinus and Methylocystis.     

Succession of bacterial community composition over two consecutive years in two aquatic systems: a natural lake and a lake-reservoir

The succession in bacterial community composition was studied over two years in the epilimnion and hypolimnion of two freshwater systems: a natural lake (Pavin Lake) and a lake-reservoir (Sep Reservoir). The bacterial community composition was determined by cloning-sequencing of 16S rRNA and by terminal restriction fragment length polymorphism. Despite large hydrogeological differences, in the Sep Reservoir and Pavin Lake the dominant bacteria were from the same taxonomic divisions, particularly Actinobacteria and Betaproteobacteria. In both ecosystems, these major bacterial divisions showed temporal fluctuations that were much less marked than those occurring at a finer phylogenetic scale. Nutrient availability and mortality factors, the nature of which differed from one lake to another, covaried with the temporal variations in the bacterial community composition at all sampling depths, whereas factors related to seasonal forces (temperature and outflow for Sep Reservoir) seemed to account only for the variation of the hypolimnion bacterial community composition. No seasonal reproducibility in temporal evolution of bacterial community from one year to the next was observed.     

Restriction fragment length polymorphisms of 16S rRNA genes in the differentiation of fast-growing mycobacterial species

Abstract DNA from several species of fast growing mycobacteria displayed a characteristic restriction fragment length polymorphism (RFLP) pattern when hybridizated to a Mycobacterium fortuitum 16S rRNA gene fragment. The resulting patterns were identical when comparing different strains belonging to the same species. The RFLP results were consistent with those obtained by DNA-DNA hybridization studies. Using this approach, we have been able to identify the number of copies for 16S rRNA genes in several fast-growing mycobacteria.     

Bacterial diversity in rock varnish of extreme arid region of Turpan Basin

The bacterial community composition and diversity in rock varnish of Turpan Basin were investigated by restriction fragment length polymorphism (RFLP) and clone library of the 16S rRNA gene. 114 positive clones were screened, which could be grouped into 28 phylotypes and then further divided into 23 different operational taxonomic units (OTUs). These were affiliated into 5 phyla (Acidobacteria, Proteobacteria, Chloroflexi, Firmicutes and Cyanobacteria). Clones from actinobacteria were the dominant, accounting for 67.5% of total clones in the library, followed by Proteobacteria (15.8%), Chloroflexi (13.2%), Firmicutes (2.6%) and Cyanobacteria (0.9%). Rubrobacter (accounts for 35%) in the phylum Actinobacteria was the dominant genus and contained many species which might be resistant to gamma radiation. A 70% of the library clone sequences showed less 97% similarity to 16S rRNA gene sequences of standard strains obtained by pure culture. Shannon–Wiener index value of this study is 2.52 and is lower than deep-sea sediments, soils, lakes and other environments. Results of this study showed that bacterial diversity in rock varnishes of Turpan Basin was low, but maybe exist a large number of new unknown taxons, especially species that could well adapted to drought and resist radiation.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Identification of dominant bacterial phylotypes in a cadmium-treated forest soil

The presence of heavy metals in soils can lead to changes in microbial community structure, characterized by the dominance of groups that are able to tolerate contamination. Such groups may provide good microbial indicators of heavy-metal pollution in soil. Through terminal restriction fragment length polymorphism (T-RFLP) profiling, changes in the bacterial community structure of an acidic forest soil that had been incubated with cadmium (Cd) for 30 days were investigated. T-RFLP revealed, in particular, three operational taxonomic units (OTUs) strongly dominating in relative abundance in the contaminated soil. By cloning of the amplified 16S rRNA genes and partial sequencing of 25 clones, these three dominant OTUs were phylogenetically characterized. One dominant OTU in the cadmium-contaminated soil was derived from Betaproteobacteria, genus Burkholderia, and the other two were from uncultured members of the class Actinobacteria, closely related to the genus Streptomyces. To confirm T-RFLP data, four primers were designed on the basis of this study's dominant sequences, targeting the OTUs corresponding to Burkholderia or Actinobacteria. Real-time PCR showed that Burkholderia target sequences were more abundant in cadmium-treated soil (7.8 x 10(7)+/- 3.0 x 10(7) targets g(-1) soil) than in untreated soil (4.0 x 10(6)+/- 8.9 x 10(5) targets g(-1) soil). It was concluded that the genus Burkholderia includes species that may be particularly dominant under cadmium contamination.     

利用小亚基核糖体RNA 技术分析温室黄瓜近根土壤古菌和真菌多样性

土壤古菌和真菌在温室生态系统是仅次于细菌的微生物,具有类似于细菌的重要生态功能。通过构建古菌16S rRNA和真菌18S rRNA基因克隆文库,分析温室黄瓜近根土壤古菌和真菌群落结构组成,为开发利用温室这一特殊的生态环境中丰富的微生物资源以及理解微生物与植物间的互作提供参考依据。采用研磨-冻融-溶菌酶-蛋白酶K-SDS热处理以及CTAB处理等理化方法,提取和纯化微生物总DNA,构建古菌16S rRNA和真菌18S rRNA基因克隆文库。利用DOTUR软件将古菌和真菌序列按照相似性97％的标准分成若干个可操作分类单元 (OTUs)。土壤古菌克隆文库主要包括泉古菌门和未分类的古菌两大类,并有少部分广域古菌类群,所有泉古菌均属于热变形菌纲,共45个OTUs;真菌克隆文库包括真菌门的大多数亚门真菌,共24个OTUs,未发现担子菌亚门真菌。古菌多样性比较丰富,且发现少量的广域古菌 (甲烷菌),这一情况可能与温室长期高温高湿,高有机质含量,土壤处于缺氧环境有关;土壤真菌的优势种群为子囊菌,占到土壤真菌的80％以上,这可能与绝大多数植物真菌性病害属于土传病害,通过菌丝体、菌核或子囊壳在土壤病残体中越冬有一定的关系。     

Diverse populations of lake water bacteria exhibit chemotaxis towards inorganic nutrients

Chemotaxis allows microorganisms to rapidly respond to different environmental stimuli; however, understanding of this process is limited by conventional assays, which typically focus on the response of single axenic cultures to given compounds. In this study, we used a modified capillary assay coupled with flow cytometry and 16S rRNA gene amplicon pyrosequencing to enumerate and identify populations within a lake water microbial community that exhibited chemotaxis towards ammonium, nitrate and phosphate. All compounds elicited chemotactic responses from populations within the lake water, with members of Sphingobacteriales exhibiting the strongest responses to nitrate and phosphate, and representatives of the Variovorax, Actinobacteria ACK-M1 and Methylophilaceae exhibiting the strongest responses to ammonium. Our results suggest that chemotaxis towards inorganic substrates may influence the rates of biogeochemical processes.     

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

The Distribution of Phytoplasmas in Myanmar

Phytoplasma‐infected plants with symptoms of general yellowing, stunting, little leaves, white leaves, virescence, phyllody and witches’ broom growth of axillary shoots were collected from various plant species in Myanmar during 2010 and 2011. Restriction fragment length polymorphism (RFLP), sequence analysis of the PCR‐amplified 16S ribosomal RNA gene and phylogenetic analyses were used to identify and classify the phytoplasmas. Based on RFLP and sequence analyses, 13 isolates were identified and classified into one subgroup of 16SrI‐B, two subgroups of 16SrII‐A and 16SrII‐C, and one of 16SrXI group phytoplasmas. Phylogenetic analyses also supported the relationship of Myanmar isolates with the three 16Sr groups. This study showed that at least three 16Sr groups exist and 16SrII group phytoplasmas are widely distributed in Myanmar.     

Differences in vegetation composition and plant species identity lead to only minor changes in soil-borne microbial communities in a former arable field

To examine the relationship between plant species composition and microbial community diversity and structure, we carried out a molecular analysis of microbial community structure and diversity in two field experiments. In the first experiment, we examined bacterial community structure in bulk and rhizosphere soils in fields exposed to different plant diversity treatments, via a 16S rRNA gene clone library approach. Clear differences were observed between bacterial communities of the bulk soil and the rhizosphere, with the latter containing lower bacterial diversity. The second experiment focused on the influence of 12 different native grassland plant species on bacterial community size and structure in the rhizosphere, as well as the structure of Acidobacteria and Verrucomicrobia community structures. In general, bacterial and phylum-specific quantitative PCR and PCR-denaturing gradient gel electrophoresis revealed only weak influences of plant species on rhizosphere communities. Thus, although plants did exert an influence on microbial species composition and diversity, these interactions were not specific and selective enough to lead to major impacts of vegetation composition and plant species on below-ground microbial communities.     

Characterization of the depth-related changes in the microbial communities in Lake Hovsgol sediment by 16S rRNA gene-based approaches

The undisturbed sediment of Lake Hovsgol (Mongolia) is scientifically important because it represents a record of the environmental changes that took place between the Holocene (the present age) and Pleistocene (the last ice age; 12,000 14C years before present day). Here, we investigated how the current microbial communities change as the depth increases by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA genes of the microbial communities. The microbial diversity, as estimated by the Shannon index, decreased as the depth increased. In particular, significant changes in archaeal diversity were observed in the middle depth (at 39-42 cm depth of total 60 cm depth) that marks the border between the Holocene and Pleistocene. Phylotype belonging to Beta-and Gamma-Proteobacteria were the predominant bacteria and most of these persisted throughout the depth examined. However, as the depth increased, some bacteria (some genera belonging to Beta-Proteobacteria, Nitrospira, and OP8-9) were not detectable while others (some genera belonging to Alpha-, Beta-, Gamma-Proteobacteria) newly detected by DGGE. Crenarchaea were the predominant archaea and only one phylotype belonging to Euryarchaea was found. Both the archaeal and bacterial profiles revealed by the DGGE band patterns could be grouped into four and three subsets, respectively, subsets that were largely divided by the border between the Holocene and Pleistocene. Thus, the diversity of the current microbial communities in Lake Hovsgol sediments decreases with increasing depth. These changes probably relate to the environmental conditions in the sediments, which were shaped by the paleoclimatic events taking place between the Holocene and Pleistocene.