Analysis of Ammonia-Oxidizing Bacteria from Hypersaline Mono Lake, California, on the Basis of 16S rRNA Sequences

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the β subdivision of the division Proteobacteria (β-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different β-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the γ subdivision of the Proteobacteria, did not amplify target from any samples.     

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.     

Wide distribution and diversity of members of the bacterial kingdom Acidobacterium in the environment

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.     

Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system

Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira-like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary.     

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.     

Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine

Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses. Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5 degrees C, pH 6.19) and XW-site (85.0 degrees C, pH 6.80) at a depth of 350 mbls (meters below land surface). Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria. The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.     

Diversity and distribution of DNA sequences with affinity to ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Arctic Ocean

The spatial distribution and diversity of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria (hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine Nitrosomonas-like sequences. Our results suggest that the Arctic Ocean beta-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.     

Grassland management regimens reduce small-scale heterogeneity and species diversity of beta-proteobacterial ammonia pxidizer populations.

The impact of soil management practices on ammonia oxidizer diversity and spatial heterogeneity was determined in improved (addition of N fertilizer), unimproved (no additions), and semi-improved (intermediate management) grassland pastures at the Sourhope Research Station in Scotland. Ammonia oxidizer diversity within each grassland soil was assessed by PCR amplification of microbial community DNA with both ammonia oxidizer-specific, 16S rRNA gene (rDNA) and functional, amoA, gene primers. PCR products were analysed by denaturing gradient gel electrophoresis, phylogenetic analysis of partial 16S rDNA and amoA sequences, and hybridization with ammonia oxidizer-specific oligonucleotide probes. Ammonia oxidizer populations in unimproved soils were more diverse than those in improved soils and were dominated by organisms representing Nitrosospira clusters 1 and 3 and Nitrosomonas cluster 7 (closely related phylogenetically to Nitrosomonas europaea). Improved soils were only dominated by Nitrosospira cluster 3 and Nitrosomonas cluster 7. These differences were also reflected in functional gene (amoA) diversity, with amoA gene sequences of both Nitrosomonas and Nitrosospira species detected. Replicate 0.5-g samples of unimproved soil demonstrated significant spatial heterogeneity in 16S rDNA-defined ammonia oxidizer clusters, which was reflected in heterogeneity in ammonium concentration and pH. Heterogeneity in soil characteristics and ammonia oxidizer diversity were lower in improved soils. The results therefore demonstrate significant effects of soil management on diversity and heterogeneity of ammonia oxidizer populations that are related to similar changes in relevant soil characteristics.     

Phylogenetic Diversity of Natural Populations of Ammonia Oxidizers Investigated by Specific PCR Amplification

Abstract The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels. Received: 25 September 1995; Revised: 15 January 1996; Accepted: 20 February 1996     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing bacteria

The presence of a copper-containing dissimilatory nitrite reductase gene (nirK) was discovered in several isolates of beta-subdivision ammonia-oxidizing bacteria using PCR and DNA sequencing. PCR primers Cunir3 and Cunir4 were designed based on published nirK sequences from denitrifying bacteria and used to amplify a 540-bp fragment of the nirK gene from Nitrosomonas marina and five additional isolates of ammonia-oxidizing bacteria. Amplification products of the expected size were cloned and sequenced. Alignment of the nucleic acid and deduced amino acid (AA) sequences shows significant similarity (62 to 75% DNA, 58 to 76% AA) between nitrite reductases present in these nitrifiers and the copper-containing nitrite reductase found in classic heterotrophic denitrifiers. While the presence of a nitrite reductase in Nitrosomonas europaea is known from early biochemical work, preliminary sequence data from its genome indicate a rather low similarity to the denitrifier nirKs. Phylogenetic analysis of the partial nitrifier nirK sequences indicates that the topology of the nirK tree corresponds to the 16S rRNA and amoA trees. While the role of nitrite reduction in the metabolism of nitrifying bacteria is still uncertain, these data show that the nirK gene is present in closely related nitrifying isolates from many oceanographic regions and suggest that nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Phylogenetic analysis of rhizosphere-associated beta-subclass proteobacterial ammonia oxidizers in a municipal wastewater treatment plant based on rhizoremediation technology

In wastewater treatment plants based on the rhizosphere zone (rhizoremediation technology), ammonia-oxidizing bacteria (AOB) play an important role in the removal of fixed nitrogen. However, the diversity of these bacteria in rhizoremediation wastewater treatment plants is largely unknown. We employed direct PCR amplification and cloning of 16S rRNA genes to determine the phylogenetic affiliation of AOB occurring in root and soil samples of a wastewater treatment plant (Merzdorf plant, Brandenburg, Germany). 16S rDNA clone libraries were screened by hybridization using an oligonucleotide probe specific for AOB of the beta subclass of proteobacteria. Comparative sequence analysis of all hybridization-positive clones revealed that the majority of rDNA sequences was affiliated to members of the genus Nitrosospira and formed a novel subcluster (SM cluster), whereas only three sequences were most closely related to Nitrosomonas species. Affiliation of the novel Nitrosospira-like sequences with those of isolates from soil and rhizosphere suggests that phylogenetic clusters reflect physiological differences between members of this genus.     

Characterisation of the bacterial community associated with early stages of Great Scallop (Pecten maximus), using denaturing gradient gel electrophoresis (DGGE)

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA was used to characterise and compare bacterial communities associated with scallop larvae (Pecten maximus), in different production units in a shellfish hatchery. Water and larvae samples were collected from three different aquaculture systems; stagnant, flow-through and a flow- through system with seawater treated with ozone. Samples were also collected from different algal cultures, inlet tanks and water pipes leading to the different aquaculture systems. Clear differences were seen between the bacterial community associated with the larvae and in the water from the different aquaculture systems. However, there were high similarities in the community composition between different water samples and between larvae samples collected at different time periods, indicating a high stability in the bacterial communities. Fifty three percent of the sequences from these samples were similar to 16S rRNA gene sequences of members of the gamma-subclass of the Proteobacteria. The different algal cultures had different bacterial communities, however 73 percent of the sequences were similar to 16S rRNA gene sequences of members of the alpha-subclass of the Proteobacteria. Differences in the DGGE profiles were also seen between the samples taken from the inlet tanks and water pipes, indicating a change in the bacterial community composition as the water passed through the pipes. To our knowledge this is the first study investigating bacterial communities associated with Great Scallop larvae in different aquaculture systems including noncultured components.     

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type.     

Wide Distribution and Diversity of Members of the Bacterial Kingdom Acidobacterium in the Environment

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms.     

Identification of bacteria responsible for ammonia oxidation in freshwater aquaria.

Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the beta subdivision of the class Proteobacteria. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established. Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established.     

Detection of diverse new Francisella-like bacteria in environmental samples

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.     

Detection of Proteobacteria from the rumen by PCR using methanotroph-specific primers

AIMS: To detect Proteobacteria, including methanotrophs, from the rumen fluid and the bacteria inhabiting the rumen epithelium. METHODS AND RESULTS: Proteobacteria inhabiting the rumen were detected by PCR using methanotroph-specific primers. The detected Proteobacteria were divided into clusters A, B, and C in addition to one clone, which was distinct from the clusters and closely related to Nitrosomonas sp. The clusters A, B, and C were close to Succinivibrio dextrinosolvens, Enterobacter cloacae, and Actinobacillus minor, respectively. The clones obtained from the rumen fluid each belonged to cluster A or B. The clones obtained from the rumen epithelium belonged to cluster B or C or to Nitrosomonas sp. CONCLUSIONS: It has been assumed that the rumen fluid and the rumen epithelium host different populations of Proteobacteria. Moreover, detection of Nitrosomonas from the rumen epithelium would indicate the possibility that the bacterium oxidizes ammonia and methane on the rumen surface. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the rumen fluid and the epithelium support different microbial populations, which would play specific roles in rumen function. Future study should focus on the relationship between these communities and physiological functions in the rumen.     

温州市境内城市河流底泥氨氧化菌富集培养物微生物群落结构分析

【背景】城市河流底泥含有丰富的微生物资源,底泥表面更是硝化作用的主要位点之一,其表面微生物在河流生态系统氮的转化过程中发挥着重要作用。【目的】以温州市境内的城市河流水系温瑞塘河茶山段舜岙河和横江河的4条河道作为采样点,比较分析4种不同环境下城市河流表层底泥氨氧化菌富集培养物的微生物群落结构。【方法】通过野外采样及室内培养对底泥中氨氧化功能菌进行富集培养,采用高通量测序技术分析微生物群落的组成、丰度和多样性。【结果】富集培养后主要优势类群为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。4个样品共涉及氨氧化细菌3个属,分别为亚硝化单胞菌属(Nitrosomonas)、亚硝化螺菌属(Nitrosospira)、亚硝化球菌属(Nitrosococcus),涉及氨氧化古菌1个属为Nitrososphaera,其中所有样品均以Nitrosomonas为主。不同底泥富集样品氨氧化微生物可操作分类单元(Operational taxonomic unit,OTU)组成存在明显差异,栽种有水生植物的河道底泥样品DA2具有最高的氨氧化细菌OTU数量和相对丰度,而存在生活餐饮污染的河道底泥样品DA4具有最高的氨氧化古菌OTU数量和相对丰度;相较于滞留水体,采自相对流动水体的富集样品DA2、DA4具有更高的氨氧化微生物OTU数量和相对丰度。【结论】阐述了4种不同环境下城市河流底泥氨氧化菌富集培养物微生物群落结构的多样性,确定了富集培养之后的优势类群,为氨氧化微生物培养源的选择提供了参考,也为城市河流底泥中氨氧化菌进一步的筛选分离及其生理生态特征的研究提供了科学依据。