Halocafeteria seosinensis gen. et sp. nov. (Bicosoecida), a halophilic bacterivorous nanoflagellate isolated from a solar saltern

Recently, heterotrophic nanoflagellates (HNF) have been reported to actively ingest prokaryotes in high salinity waters. We report the isolation and culture of an HNF from a Korean saltern pond of 300‰ salinity. The organism is biflagellated with an acronematic anterior flagellum and never glides on surfaces. The mitochondria have tubular cristae. Neither transitional helix nor spiral fiber were observed in the transition zones of the flagella. The cell has a cytostome supported by an arc of eight microtubules, suggesting that our isolate is a bicosoecid. Our isolate had neither mastigonemes, lorica, body scales, nor cytopharynx and thus could not be placed in any of the presently described bicosoecid genera. Phylogenetic analysis of 18S rRNA gene sequences from stramenopiles confirmed the bicosoecid affinities of our isolate, but did not place it within any established genus or family. Its closest relatives include Caecitellus and Cafeteria. The optimal range of growth temperature was 30–35°C. The isolated HNF grew optimally at 150‰ salinity and tolerated up to 363‰ salinity, but it failed to grow below 75‰ salinity, indicating that it could be a borderline extreme halophile. On the basis of its morphological features and position in 18S rRNA trees we propose a novel genus for our isolate; Halocafeteria, n. gen. The species name Halocafeteria seosinensis sp. nov. is proposed.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.     

Molecular diversity of 16S rRNA and gyrB genes in copper mines

The molecular diversities of the microbial communities from four sites impacted by acid mine drainage (AMD) at Dexing Copper Mine in Jiangxi province of China were studied using 16S rRNA sequences and gyrB sequences. Of the four sampled sites, each habitat exhibited distinct geochemical characteristics and the sites were linked geographically allowing us to correlate microbial community structure to geochemical characteristics. In the present study, we examined the molecular diversity of 16S rRNA and gyrB genes from water at these sites using a PCR-based cloning approach. We found that the microbial community appears to be composed primarily of Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicutes, Chlorella and unknown phylotypes. Of clones affiliated with Nitrospira, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III were all detected. Principal-component analysis (PCA) revealed that the distribution of the microbial communities was influenced greatly by geochemical characteristics. The overall PCA profiles showed that the sites with similar geochemical characteristics had more similar microbial community structures. Moreover, our results also indicated that gyrB sequence analysis may be very useful for differentiating very closely related species in the study of microbial communities. H. Yin and L. Cao contributed equally to this work.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Molecular methods of analysing bacterial diversity: the case of Rhizobia

The distribution of rhizobia, bacteria which nodulate the roots of leguminous plants, was surveyed for 91 species of the familyLeguminosae. A total of 117 strains of rhizobia were isolated, and 28 strains were obtained from culture collections. The sample total of 145 strains was discriminated by rapid ribosomal RNA (rRNA) sequencing. The partial sequences (157 bases from position (inEscherichia coli) 1220 to 1377 from 5 end) of 16S rRNA revealed the presence of 16 groups in these rhizobia. Further, DNA-DNA homology studies suggested that the differences of the 16 groups were enough to justify establishing at least 16 species.     

Different <Emphasis Type="Italic">Planctomycetes</Emphasis> diversity patterns in latitudinal surface seawater of the open sea and in sediment

The 16S rRNA gene approach was applied to investigate the diversity of Planctomycetes in latitudinal surface seawater of the Western Pacific Ocean. The results revealed that the Pirellula-Rhodopirellula-Blastopirellula clade dominated the Planctomycetes community at all surface seawater sites while the minority genera Gemmata and Planctomyces were only found at sites H5 and H2 respectively. Although the clone frequency of the PRB clade seemed stable (between 83.3% and 94.1%) for all surface seawater sites, the retrieved Pirellula-Rhodopirellula-Blastopirellula clade presented unexpected diversity. Interestingly, low latitude seawater appeared to have higher diversity than mid-latitudes. integral-LIBSHUFF software analysis revealed significantly different diversity patterns between in latitudinal surface seawater and in the sediment of South China Sea station M2896. Our data suggested that different hydrological and geographic features contributed to the shift of Planctomycetes diversity in marine environments. This is, to our knowledge, the first systematic assessment of Planctomycetes in latitudinal surface seawater of the open sea and the first comparison of diversity pattern between surface seawater and sediments and has broadened our understanding of Planctomycetes diversity in marine environments.     

Diversity of halophilic archaea in the crystallizers of an Adriatic solar saltern

Haloarchaeal diversity in the crystallizers of Adriatic Secovlje salterns was investigated using gene fragments encoding 16S rRNA and bacteriorhodopsin as molecular markers. Screening of 180 clones from five gene libraries constructed for each gene targeted revealed 15 different 16S rRNA and 10 different bacteriorhodopsin phylotypes, indicating higher haloarchaeal diversity than previously reported in such hypersaline environments. Furthermore, results of rarefaction analysis indicated that analysis of an increasing number of clones would have revealed additional diversity. Finally, most sequences from the crystallizers grouped within the Halorubrum branch, whereas square-shaped 'Haloquadratum' relatives, repeatedly reported to dominate crystallizer communities, were rare. Presence of such special and diverse haloarchaeal community could be attributed to the Secovlje salterns rare continuous short-cycling salt production mechanism.     

Microbial diversity in production waters of a low-temperature biodegraded oil reservoir

Microbial communities in two production waters of a low-temperature and low-salinity petroleum reservoir in Canada were examined using cultural and molecular approaches. The predominant cultivated microorganisms were homoacetogens but sulfate-reducers, acetoclastic methanogens and denitrifiers also gave significant counts. The dominant members of the culturable population were affiliated with the Firmicutes, the "Deltaproteobacteria", the "Epsilonproteobacteria", the Spirochaetes and the Euryarchaeota. 16S rRNA gene clone libraries were also constructed from the total DNA collected from production waters. The bacterial library was entirely composed by a single phylotype related to Arcobacter. The archaeal phylotypes were generally very closely related to members of the orders Methanosarcinales and Methanomicrobiales. Consistent with earlier observations, our data suggest that methanogenesis is a dominant terminal process in the reservoir. Moreover, the cross-evaluation of culture-dependent and -independent techniques also indicates that, contrary to most studies, both acetoclastic and lithotrophic methanogens may be involved in this process. This first investigation of the microbial diversity in a non water-flooded low-temperature and low-salinity petroleum reservoir expands substantially our knowledge of the extent of microbial diversity and highlights the complexity of microbial communities involved in the oil field food chain.     

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

5S rRNA fingerprints of marine bacteria, halophilic archaea and natural prokaryotic assemblages along a salinity gradient

Natural prokaryotic assemblages from two multi-pond solar salterns and pure cultures of both marine bacteria and halophilic archaea were analyzed and compared by electrophoretic analysis of 5S rRNAs. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied. The culture-independent, PCR-free, fingerprinting analysis covered two objectives: (i) to compare natural assemblages among them and with results previously obtained through a PCR-dependent approach and (ii) to estimate the in situ relevance of those prokaryotic groups obtained with classical culture methodologies. Natural assemblages were analyzed through cluster analysis of quantitative 5S rRNA band patterns. The resulting groups were in accordance with environmental parameters (i.e., NaCl concentration) and with the clustering obtained after a PCR-dependent approach, showing the formation of three salinity-based groups of samples (<10%, 10-25% and >25% salinity). Similarities between the laboratory strains tested and dominant community members were studied by comparing 5S rRNA band patterns. The lack of match obtained after cluster analysis indicated that the prokaryotic populations relevant in the ponds below 25% salinity were neither Flavobacteria nor haloarchaeal strains belonging to the genera Halococcus, Haloarcula and Halobacterium. Members of Proteobacteria and Gram-positive bacteria were found to match bands in these samples. The 5S rRNA fingerprint from the dominant community members in the ponds above 30% salinity did not fit any of the cultured halophilic archaea studied, in agreement with earlier PCR results. This is consistent with a greater bias introduced by culture-dependent methods than by those based on PCR, especially for archaeal populations.     

Speciation of two sympatric coastal fish species, Girella punctata and Girella leonina (Perciformes, Kyphosidae)

Girella punctata and Girella leonina are sympatric sister species showing extensive distributional overlap in shallow rocky reefs in the Pacific Ocean south of the Japanese Islands. Differences between the two species in external morphological characters, such as number of pored lateral line scales, colour of opercular flap and shape of caudal fin, are congruent with genetic divergence. Nucleotide identity between the two species in the 3.3 kbp region of partial mitochondrial DNA containing the D-loop region, in 12S and 16S ribosomal RNA (rRNA) and transfer RNA genes is 95%. To estimate divergence time, Bayesian analysis was conducted using a dataset comprising concatenated nucleotide sequences from the two rRNA genes of three girellid and nine other fish species. Using the Elopomorpha – Clupeocephala split (265 million years ago (mya)) as a calibration point, divergence between G. punctata and G. leonina is estimated as having occurred 6.0±1.4 mya. Speciation is suggested to have been caused by geographical isolation associated with formation of the Japanese Islands, which resulted in disjunction of Girella habitat.     

Molecular Characterization of Prokaryotic Communities Associated with Lonar Crater Basalts

The Lonar crater is an unusually well-preserved meteorite impact structure that is located in one of the largest volcanic provinces on Earth (i.e., the Deccan Traps in India). The diversity of endoliths in Lonar crater basalts or Deccan flood basalts is not known. Here, the phylogenetic diversity of endolithic Bacteria and Archaea inhabiting basalts retrieved from four discrete sampling sites on the Lonar crater walls and the lake-bed was assessed using culture-independent molecular methods. Taxonomic classification of 16S rRNA gene sequences from all four basalt samples revealed similarities as well as dissimilarities in the presence or absence of several prokaryotic taxa. Cluster analysis of Denaturing gradient gel electrophoresis fingerprints and UniFrac analysis of clone library sequences suggested substantial variations in bacterial and archaeal diversity between crater-wall and lake-bed sites. Although sequences affiliated to the bacterial phyla Actinobacteria, Acidobacteria and Chloroflexi were relatively more abundant in crater-wall basalts than in lake-bed basalts; the reverse was observed for sequences related to Proteobacteria, Firmicutes, Cyanobacteria and Bacteroidetes. Archaea in crater-wall and lake-bed basalt libraries were almost completely represented by Thaumarchaeota and Euryarchaeota, respectively. Diversity indices and richness estimates suggested the diversity of endolithic Bacteria to be higher than that of Archaea in the Lonar crater basalts. A substantial number of clone library sequences did not affiliate with extant Bacteria and Archaea. The detection of several putative lineages associated with C, N and S cycling suggests that the Lonar crater basalts are colonized by metabolically diverse prokaryotic communities involved in biogeochemical cycling of major elements.     

Characterization of a bacterium isolated from amber

A bacterium has been isolated from Baltic amber, after it was soaked in ethanol and flamed. The bacterium was a Gram-positive aerobic spore-forming rod whose 16S rDNA sequence had a 99.6% homology to that of Bacillus subtilis. Accordingly, the bacterium was identified as a strain in the species Bacillus subtilis. Considering the isolation procedure that was employed, the isolate should not be a contaminant of the contemporary Bacillus population; however, it may not be considered as a bacterium trapped when the amber was formed. These results suggest that amber might contain bacteria that were derived from the environments in which the amber had been located.     

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.     

牛源多杀性巴氏杆菌的分离与初步鉴定

【目的】本研究旨在对引起犊牛呼吸道综合征的多杀性巴氏杆菌进行分离鉴定,分析其亲缘关系和毒力基因的分布情况。【方法】收集2017年8月至2018年4月疑似患有犊牛呼吸道综合征的病牛鼻拭子进行细菌分离培养,对菌落形态和染色疑似巴氏杆菌的菌株进行16S rRNA测序和血清型鉴定,选择巴氏杆菌7类23种毒力基因,筛查临床分离株的毒力基因的分布。【结果】从8个省份的237份病料中分离出31株多杀性巴氏杆菌,分离率为13.1%。16S rRNA测序分析表明31株A型多杀性巴氏杆菌属于同一亚群,其序列同源性与中国分离株HB01以及国外分离株USDA-ARS-USMARC-60712、USDA-ARS-USMARC-60214、ATCC 43137以及36950亲缘关系较近。对分离出的31株A型多杀性巴氏杆菌的7类共23种毒力基因鉴定,结果显示31株多杀性巴氏杆菌所携带的毒力因子大多分布在17–19个,且集中度较高。【结论】A型多杀性巴氏杆菌为犊牛呼吸道综合症的主要流行血清型,通过对多杀性巴氏杆菌的临床分离株进化树和毒力基因分析,内蒙古、黑龙江、新疆、山西以及河北的7株分离株进化来源于同一分支,且均缺失毒力基因tadD和hgbA及携带毒力基因hsf-1,提示着其亲缘关系可能与其携带的特定毒力基因存在一定相关性。该研究为犊牛呼吸道综合征的病原学调查和多杀性巴氏杆菌流行病学调查提供了参考数据。     

Bacterial diversity in organically-enriched fish farm sediments

The bacterial diversity and community structure within both organically enriched and adjacent, unimpacted, near-shore marine sediments at two fish farms in southern Tasmania, Australia, was examined using 16S rRNA gene clone library construction and analysis. Sediments at both caged and reference sites at both farms showed a very high level of microbial diversity. Over 900 clones were analysed and grouped into 631 unique phylotypes. Reference sites were dominated by Delta- and Gammaproteobacteria and the Cytophaga-Flavobacteria-Bacteroides group. Cage site sediments were also dominated by these phylotypes, as well as members of the Alpha- and Epsilonproteobacteria. Diversity and coverage indices indicated that the actual diversity of the sediments was much greater than that detected, despite a large sampling effort. All libraries were shown to be statistically different from one another (P < 0.05). Many phylotypes did not group with cultured bacteria, but grouped with other environmental clones from a wide array of marine benthic environments. Diversity and evenness indices suggested that although both parameters changed after farming, diverse communities were present in all sediments. The response of the microbial community to organic load suggested that random, rather than predictable, succession events determine community composition and diversity, and that sediment type may influence bacterial community and sediment response to organic perturbation.     

Prokaryotic community composition and biogeochemical processes in deep subseafloor sediments from the Peru Margin

The community compositions of Bacteria and Archaea were investigated in deep, sub-seafloor sediments from the highly productive Peru Margin (ODP Leg 201, sites 1228 and 1229, c. 25 km apart) down to nearly 200 m below the seafloor using taxonomic (16S rRNA) and functional (mcrA and dsrA) gene markers. Bacterial and archaeal groups identified from clone libraries of 16S rRNA gene sequences at site 1229 agreed well with sequences amplified from bands excised from denaturing gradient gel electrophoresis (DGGE) depth profiles, with the exception of the Miscellaneous Crenarchaeotic Group (MCG). This suggested that the prokaryotic community at site 1228, obtained from DGGE profiling alone, was reliable. Sites were dominated by Bacteria in the Gammaproteobacteria, Chloroflexi (green non-sulphur bacteria) and Archaea in the MCG and South African Gold Mine Euryarchaeotic Group, although community composition changed with depth. The candidate division JS1 was present throughout both sites but was not dominant. The populations identified in the Peru Margin sediments consisted mainly of prokaryotes found in other deep subsurface sediments, and were more similar to communities from the Sea of Okhotsk (pelagic clays) than to those from the low organic carbon Nankai Trough sediments. Despite broad similarities in the prokaryotic community at the two sites, there were some differences, as well as differences in activity and geochemistry. Methanogens (mcrA) within the Methanosarcinales and Methanobacteriales were only found at site 1229 (4 depths analysed), whereas sulphate-reducing prokaryotes (dsrA) were only found at site 1228 (one depth), and these terminal-oxidizing prokaryotes may represent an active community component present at low abundance. This study clearly demonstrates that the deep subsurface sediments of the Peru Margin have a large diverse and metabolically active prokaryotic population.