Organotrophic bacteria of the Baikal Rift Zone hot springs

Pyrosequencing of the 16S rRNA gene fragments was used to investigate the bacterial community of an Alla hot spring microbial mat. The mat community was mainly represented by the members of five phyla: Deinococcus-Thermus, Nitrospirae, Atribacteria (OP9), Proteobacteria, Chloroflexi, and Firmicutes, with other groups responsible for not more than 2% of the total number. From hot springs of the Baikal Rift Zone (Buryatia, Russia), 13 strains of aerobic alkaliphilic thermophilic organotrophic bacteria were isolated, and their morphology, ecology, physiology, and phylogenetic position were studied. Based on analysis of their 16S rRNA gene sequences, the isolates were identified as members of the family Bacillaceae. Strains Al-9-1, Se-1, Ga-1-1, Ga-9-2, and Se-1-10 were assigned to the genus Anoxybacillus; strains Ur-6, Br-2-2, А2, and Um-09m, to the genus Bacillus; strains Gor-10s and Gа-35, to the genus Paenabacillus. Secreted endopeptidases of the isolates were shown to have relatively narrow substrate specificity. The investigated enzymes were characterized by high pH (6.3–11.4) and temperature stability (23–70°C), which makes it possible to carry out organic matter degradation in the environment under variable ecological conditions.     

Microbial diversity of the Soldhar hot spring,India, assessed by analyzing 16S rRNA and protein-coding genes

Bacterial diversity of the Soldhar hot spring, located in the Chamoli district of Uttarakhand, India, was investigated using a clone library, denaturing gradient gel electrophoresis (DGGE) and functional genes. Physicochemical analysis of sediment samples indicated an oligotrophic environment with very low sulfur content. Based on the 16S rRNA gene studies Proteobacteria was the most predominant group in all the three approaches. Other dominant phyla were Deinococcus-Thermus and Aquificae. Pyrobaculum was the only archaeal genus detected by DGGE. In the functional gene analysis, the nifH library showed a single operational taxonomic unit (OTU) related to the genus Paenibacillus whereas the aoxB library showed three OTUs related to Acidovorax, Aminobacter and Agrobacterium. Our results demonstrate for the first time both the bacterial and archaeal diversity in the Soldhar hot spring by culture-independent techniques, thereby providing important information that will increase our understanding of the microbial ecology of the Soldhar hot spring.     

Diversity and phylogenetic profiling of niche-specific Bacilli from extreme environments of India

The diversity of culturable, aerobic and heterotrophic Bacillus and Bacillus-derived genera (BBDG) was investigated in various extreme environments (including thermal springs, cold deserts, mangroves, salt lakes, arid regions, salt pans and acidic soils) of India. Heat treatment followed by enrichment in different media led to a total of 893 bacterial isolates. Amplified ribosomal DNA restriction analysis (ARDRA) using three restriction enzymes AluI, MspI and HaeIII led to the clustering of these isolates into 12–74 groups for the different sites at 75 % similarity index, adding up to 559 groups. Phylogenetic analysis based on 16S rRNA gene sequencing led to the identification of 392 bacilli, grouped in two families, Bacillaceae (89.03 %) and Paenibacillaceae (10.97 %), and included 13 different genera with 75 distinct species. It was found that among the thirteen genera, nine (Bacillus, Halobacillus, Lysinibacillus, Oceanobacillus, Pontibacillus, Salinibacillus, Sediminibacillus, Thalassobacillus and Virgibacillus) belonged to Bacillaceae and four (Ammoniphilus, Aneurinibacillus, Brevibacillus and Paenibacillus) belonged to Paenibacillaceae. Novel isolates tolerant to low and high pH and temperature, salt and low moisture were identified. The major outcome of the present investigation was the identification of niche-specific species and also the ubiquitous presence of selected species of BBDG, which illustrate the diversity and pervasive nature of BBDG in extreme environments.     

Water-related environments: a multistep procedure to assess the diversity and enzymatic properties of cultivable bacteria

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.     

Investigation of the methanotrophic communities of the hot springs of the Uzon caldera,Kamchatka, by molecular ecological techniques

The methane-oxidizing microbial communities inhabiting the bottom sediments of 36 hot springs of the Uzon caldera (Kamchatka, Russia) located in the thermal fields Vostochnoe, Oranzhevoe, and Severnoe, as well as near the lakes Fumarol’noe and Khloridnoe and the Izvilistyi stream, were studied. Methanotrophic bacteria were detected by PCR and FISH in only 8 hot springs. The highest numbers of copies of the pmoA gene (molecular marker of methanotrophy) (2.8 × 10⁷ and 1.1 × 10⁷ copies/mL sediment) were detected in the Kul’turnyi and Kvadrat springs; however, in other springs, the numbers of the pmoA gene copies were significantly lower (5.4 × 10³–2.8 × 10⁶ copies/mL sediment). By using the FISH method, only type I methanotrophs were detected in these springs, with their percentage ranging from 0.3 to 20.5% of the total number of eubacteria. PCR-DGGE analysis of the pmoA gene showed that the diversity of methanotrophs was extremely low (no more that two components). Analysis of the deduced PmoA amino acid sequences demonstrated that methanotrophic bacteria of the genus Methylothermus, closely related to representatives of two valid species, widely occurred in the thermal springs near Lake Fumarol’noe. Other bacteria differing considerably from the detected Methylothermus species were detected as well. In the springs with low pH values (2.6–3.8), methanotrophic Gammaproteobacteria most closely related to the genera Methylomonas and Methylobacter were detected for the first time.     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.     

Bacterial Diversity and Functional Activity of Microbial Communities in Hot Springs of the Baikal Rift Zone

In this study the bacterial diversity of thermophilic microbial mats (40 to 65°C) in three alkaline hot springs of the Baikal Rift Zone (BRZ) was determined through pyrosequencing of 16S rRNA gene libraries. Significant diversity of bacterial species was found in the biomats of the hot springs with total number of detected phylotypes of 607. The highest share of the microbial community was represented by the phyla Chloroflexi (Seya Spring, 76.4%), Deinococcus-Thermus (Alla Spring, 45.1%), Nitrospira (Alla Spring, 36.1%), Cyanobacteria (Tsenkher Spring, 33.1%), and Proteobacteria (Tsenkher Spring, 22.6%), but their ratio varied significantly in different springs. A comparison of the biodiversity and composition of microbial communities between hot springs showed a decrease in biodiversity with increasing temperature. A large number of sequences showed a low degree of similarity with cultivated representatives in public databases. Microbial communities showed intensive rates of production and destruction of organic compounds, as revealed by the quantitative assessment of their functional activity.     

Diversity of methanogenic archaea from the 2012 terrestrial hot spring (Valley of Geysers,Kamchatka)

Archaeal diversity in the 2012 terrestrial hot spring (Valley of Geysers, Kronotsky Nature Reserve, Kamchatka, Russia) was investigated using molecular and cultivation-based approaches. Analysis of the 16S rRNA gene sequences revealed predominance among archaea of uncultured microorganisms of the pSL12 and THSCG clusters. Analysis of the mcrA genes revealed that members of the order Methanomassiliicoccales were predominant (68%) among methanogens; the latter constituted 0.15% of the total number of archaea. Five stable thermophilic methanogenic associations utilizing hydrogen, formate, acetate, or methanol as substrates were obtained from the sediments of spring 2012. The diversity of cultured methanogens was limited to members of the genera Methanothermobacter, Methanothrix, and Methanomethylovorans. The association growing at 65°C and producing methane from methanol contained two components, which probably formed a syntrophic relationship: a Methanothermobacter methanogenic archaeon and a bacterium representing an separate cluster within the Firmicutes phylum, which was phylogenetically related to the genera Thermacetogenium and Syntrophaceticus. These data indicate high diversity of methanogens, notwithstanding their low abundance among archaea. The group of thermophilic Methanomassiliicoccales, which predominated among methanogens, is of special interest.     

Phylogenetic diversity of microbial communities of the Posolsk Bank bottom sediments,Lake Baikal

Massive parallel sequencing (the Roche 454 platform) of the 16S rRNA gene fragments was used to investigate microbial diversity in the sediments of the Posolsk Bank cold methane seep. Bacterial communities from all sediment horizons were found to contain members of the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Firmicutes, Nitrospirae, Chloroflexi, Proteobacteria, and the candidate phyla Aminicenantes (OP8) and Atribacteria (OP9). Among Bacteria, members of the Chloroflexi and Proteobacteria were the most numerous (42 and 46%, respectively). Among archaea, the Thaumarchaeota predominated in the upper sediment layer (40.1%), while Bathyarchaeota (54.2%) and Euryarchaeota (95%) were predominant at 70 and 140 cm, respectively. Specific migration pathways of fluid flows circulating in the zone of gas hydrate stability (400 m) may be responsible for considerable numbers of the sequences of Chloroflexi, Acidobacteria, and the candidate phyla Aminicenantes and Atribacteria in the upper sediment layers and of the Deinococcus-Thermus phylum in deep bottom sediments.     

Dragonflies (Odonata) of thermal springs in Central Asia

Sixteen species of dragonflies were recorded in 12 thermal springs of Central Asia. Of these, Ischnura forcipata, I. pumilio, Orthetrum anceps, O. brunneum, and Sympetrum haritonovi were widely distributed in hydrothermal waters. In the high mountains of Pamir, dragonflies can develop only in hot springs. The maximum altitude recorded for the habitat of O. brunneum was 3950 m a.s.l.     

Diversity,molecular phylogeny and fingerprint profiles of airborne <Emphasis Type="Italic">Aspergillus</Emphasis> species using random amplified polymorphic DNA

In the present study, diversity and phylogenetic relationship of Aspergillus species isolated from Tehran air was studied using random amplified polymorphic DNA (RAPD)–polymerase chain reaction (RAPD-PCR). Thirty-eight Aspergillus isolates belonging to 12 species i.e. A. niger (28.94 %, 11 isolates), A. flavus (18.42 %, 7 isolates), A. tubingensis (13.15 %, 5 isolates), A. japonicus (10.52 %, 4 isolates), A. ochraceus (10.52 %, 4 isolates), and 2.63 %, 1 isolate from each A. nidulans, A. amstelodami, A. oryzae, A. terreus, A. versicolor, A. flavipes and A. fumigatus were obtained by settle plate method which they were distributed in 18 out of 22 sampling sites examined. Fungal DNA was extracted from cultured mycelia of all Aspergillus isolates on Sabouraud Dextrose Agar and used for amplification of gene fragments in RAPD-PCR using 11 primers. RAPD-PCR data was analyzed using UPGMA software. Resulting dendrogram of combined selected primers including PM1, OPW-04, OPW-05, P160, P54, P10 and OPA14 indicated the distribution of 12 Aspergillus species in 8 major clusters. The similarity coefficient of all 38 Aspergillus isolates ranged from 0.02 to 0.40 indicating a wide degree of similarities and differences within and between species. Taken together, our results showed that various Aspergillus species including some important human pathogenic ones exist in the outdoor air of Tehran by different extents in distribution and diversity and suggested inter- and intra-species genetic diversity among Aspergillus species by RAPD-PCR as a rapid, sensitive and reproducible method.     

Distribution of sediment bacterial and archaeal communities in plateau freshwater lakes

Both Bacteria and Archaea might be involved in various biogeochemical processes in lacustrine sediment ecosystems. However, the factors governing the intra-lake distribution of sediment bacterial and archaeal communities in various freshwater lakes remain unclear. The present study investigated the sediment bacterial and archaeal communities in 13 freshwater lakes on the Yunnan Plateau. Quantitative PCR assay showed a large variation in bacterial and archaeal abundances. Illumina MiSeq sequencing illustrated high bacterial and archaeal diversities. Bacterial abundance was regulated by sediment total organic carbon and total nitrogen, and water depth, while nitrate nitrogen was an important determinant of bacterial diversity. Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, and Verrucomicrobia were the major components of sediment bacterial communities. Proteobacteria was the largest phylum, but its major classes and their proportions varied greatly among different lakes, affected by sediment nitrate nitrogen. In addition, both Euryarchaeota and Crenarchaeota were important members in sediment archaeal communities, while unclassified Archaea usually showed the dominance.     

Phylogeny and bioactivity of epiphytic Gram-positive bacteria isolated from three co-occurring antarctic macroalgae

Marine macroalgae are emerging as an untapped source of novel microbial diversity and, therefore, of new bioactive secondary metabolites. This study was aimed at assessing the diversity and antimicrobial activity of the culturable Gram-positive bacteria associated with the surface of three co-occurring Antarctic macroalgae. Specimens of Adenocystis utricularis (brown alga), Iridaea cordata (red alga) and Monostroma hariotii (green alga) were collected from the intertidal zone of King George Island, Antarctica. Gram-positive bacteria were investigated by cultivation-based methods and 16S rRNA gene sequencing, and screened for antimicrobial activity against a panel of pathogenic microorganisms. Isolates were found to belong to 12 families, with a dominance of Microbacteriaceae and Micrococcaceae. Seventeen genera of Actinobacteria and 2 of Firmicutes were cultured from the three macroalgae, containing 29 phylotypes. Three phylotypes within Actinobacteria were regarded as potentially novel species. Sixteen isolates belonging to the genera Agrococcus, Arthrobacter, Micrococcus, Pseudarthrobacter, Pseudonocardia, Sanguibacter, Staphylococcus, Streptomyces and Tessaracoccus exhibited antibiotic activity against at least one of the indicator strains. The bacterial phylotype composition was distinct among the three macroalgae species, suggesting that these macroalgae host species-specific Gram-positive associates. The results highlight the importance of Antarctic macroalgae as a rich source of Gram-positive bacterial diversity and potentially novel species, and a reservoir of bacteria producing biologically active compounds with pharmacological potential.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

The high diversity of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> endosymbionts in soils of northwest Spain

The diversity of rhizobia that establish symbiosis with Lotus corniculatus has scarcely been studied. Several species of Mesorhizobium are endosymbionts of this legume, including Mesorhizobium loti, the type species of this genus. We analysed the genetic diversity of strains nodulating Lotus corniculatus in Northwest Spain and ten different RAPD patterns were identified among 22 isolates. The phylogenetic analysis of the 16S rRNA gene showed that the isolated strains belong to four divergent phylogenetic groups within the genus Mesorhizobium. These phylogenetic groups are widely distributed worldwide and the strains nodulate L. corniculatus in several countries of Europe, America and Asia. Three of the groups include the currently described Mesorhizobium species M. loti, M. erdmanii and M. jarvisii which are L. corniculatus endosymbionts. An analysis of the recA and atpD genes showed that our strains belong to several clusters, one of them very closely related to M. jarvisii and the remanining ones phylogenetically divergent from all currently described Mesorhizobium species. Some of these clusters include L. corniculatus nodulating strains isolated in Europe, America and Asia, although the recA and atpD genes have been sequenced in only a few L. corniculatus endosymbionts. The results of this study revealed great phylogenetic diversity of strains nodulating L. corniculatus, allowing us to predict that even more diversity will be discovered as further ecosystems are investigated.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

Comparative ecology of Ostracoda (Crustacea) in two rheocrene springs (Bolu,Turkey)

A total of ten taxa belonging to the class Ostracoda of the Crustacea (Darwinula stevensoni, Candona neglecta, Cypria ophthalmica, Ilyocypris bradyi, Prionocypris zenkeri, Herpetocypris chevreuxi, Psychrodromus olivaceus, Heterocypris incongruens, Scottia pseudobrowniana, Eucypris sp.) were collected from two rheocrene Darwinula stevensoni springs (Çetin Bey and Çaygökp?nar springs) on 15 separate occasions between November 2002 and November 2004. Almost all of the species identified exhibit cosmopolitan distributions – at least in the Holarctic region. The presence of Scottia pseudobrowniana represents the second recording of this species in the ostracod fauna of Turkey. The dominant taxa in both springs was Cy. ophthalmica, I. bradyi, Pr. zenkeri and Ca. neglecta. Correlation analyses suggested a significant positive relationship in relative abundance between I. bradyi, Pr. zenkeri and Cy. ophthalmica. Species composition differed significantly between the upper and lower study sites for each spring, but differences could not be detected between sites at the same elevation across sites. Environmental tolerance index (ETI) values suggest that species with high optima and tolerance ranges show cosmopolitan characteristics.     

Genetic Diversity of <Emphasis Type="Italic">Arcobacter</Emphasis> Species of Animal and Human Origin in Andhra Pradesh,India

Arcobacter is an emerging foodborne pathogen having zoonotic significance. Enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive sequence-based PCR (rep-PCR) analysis of a total of 41 Arcobacter isolates revealed a greater degree of genetic diversity. ERIC-PCR genotyping distinguished 14, 13 and 12 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. Rep-PCR genotyping distinguished 15, 12 and 11 genotypes among 16, 13 and 12 isolates of A. butzleri, A. cryaerophilus and A. skirrowii, respectively. The discriminatory power for ERIC and rep-PCR was found to be 0.997 and 0.996, respectively. Close clustering between isolates of animal and human origin are indicative of probable zoonotic significance.