Combined Use of Cultivation-Dependent and Cultivation-Independent Methods Indicates that Members of Most Haloarchaeal Groups in an Australian Crystallizer Pond Are Cultivable

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10⁶ CFU/ml) were obtained on solid media. Long incubation times (≥8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.     

Phylogenetic relationships of the endemic Antarctic benthic hydroids (Cnidaria, Hydrozoa): what does the mitochondrial 16S rRNA tell us about it?

Hydroidolinan hydrozoans are widely represented in the benthic Antarctic ecosystem, mainly by some endemic and putative monophyletic groups, never included in molecular phylogenetic analyses. 38 partial sequences of the mitochondrial 16S rRNA gene were obtained for 38 species belonging to 14 families (six anthoathecates and eight leptothecates) and 20 genera (7 anthoathecates and 13 leptothecates). These sequences were combined with 108 additional sequences retrieved from the GenBank to investigate both the hypothetical monophyletism and the phylogenetic relationships of those endemic Antarctic groups; the potential use of the marker for barcoding was also investigated. Our results uphold the monophyly of some important hydroidolinan groups, such as the superfamily Plumularioidea (together with all its families, including Schizotrichidae, fam. nov.) and the Aplanulata. Concerning the Antarctic endemic groups, most results as monophyletic (Oswaldella, Schizotricha and Staurotheca), some genera form part of the expectable groups (e.g. Abietinella, located into the monophyletic Zygophylacinae clade) and, finally, others have shown a surprising position (e.g. Stegella, closely related to Lafoeinae, or Billardia and Stegopoma, allied with Hebellidae). Finally, our study has shown the utility of the marker to recognize the Antarctic species considered, but the low genetic divergence in some of the most important Antarctic groups suggests being careful when using it for DNA barcoding in the case of the original Antarctic hydroid fauna.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

An Outbreak of Nonflocculating Catabolic Populations Caused the Breakdown of a Phenol-Digesting Activated-Sludge Process

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter⁻¹ day⁻¹ and then to 1.5 g liter⁻¹ day⁻¹. After the loading rate was increased to 1.5 g liter⁻¹ day⁻¹, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter⁻¹ day⁻¹. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter⁻¹ day⁻¹. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Bacillus thermophilum sp. nov., isolated from a microbial fuel cell

A novel thermophilic, Gram-staining positive bacterium, designated DX-2^T, was isolated from the anode biofilm of a microbial fuel cell. Cells of the strain were oxidase positive, catalase positive, facultative anaerobic, motile rods. The isolate grew at 30–60 °C (optimum 50 °C) and pH 5–9 (optimum pH 8–8.5). The pairwise 16S rRNA gene sequence similarities showed that strain DX-2^T was most closely related to Bacillus fumarioli LMG 17489^T (96.2 %), B. firmus JCM 2512^T (96.0 %) and B. foraminis DSM 19613^T (95.7 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DX-2^T formed a cluster with B. smithii (95.5 %) and B. infernus (94.9 %). The genomic G+C content of DX-2^T was 43.7 mol%. The predominant respiratory quinone was MK-7. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and unknown phospholipids. The major cellular fatty acid was iso-C_16:0. Based on its phenotypic characteristics, chemotaxonomic features, and results of phylogenetic analysis, the strain was identified to represent a distinct novel species in the genus Bacillus, and the name proposed is B. thermophilum sp. nov. The type strain is DX-2^T (=CCTCC AB2012194^T = KCTC 33128^T).     

Cohnella humi sp. nov., Isolated from Russian Soil

A Gram-positive, catalase and oxidase positive, rod-shaped bacteria, and spore-forming, designated as J20-3^T was isolated from a peat soil, collected near a coal mine at Prokopyevsk, (GPS; N53°52′51″, E86°43′39″) Kemerovo Oblast, Russia. A polyphasic taxonomy study using phenotypic, phylogenetic, and genotypic method was performed to characterize strain J20-3^T. Comparative 16S rRNA gene sequence analysis indicated that strain J20-3^T represented a novel subline within the genus Cohnella in the family Paenibacillaceae. According to 16S rRNA gene sequence, strain J20-3^T showed 93.7–97.2 % similarity levels with other Cohnella species. Strain J20-3^T exhibited relatively low level of DNA–DNA hybridization value with type strains KACC 11643^T (40 %), KACC 11771^T (37.5 %), and KACC 15372^T (30.5 %). The strain showed typical chemotaxonomic characteristic of the genus Cohnella, with the presence of predominant respiratory quinone MK-7; major fatty acids are C_15:0, C_16:0, iso, and C_16:0. The DNA G+C content of the strain J20-3^T was 56.3 mol%. The polar lipid profile of the strain J20-3^T included major amount of diphosphatidylglycerol, phosphatidylglycerol, and phosphoatidylethanolamine. On the basis of its phenotypic and genotypic properties, and its phylogenetic distinctiveness, strain J20-3^T should be classified as a novel species in the genus Cohnella, for which the name Cohnella humi sp. nov. is proposed.     

Phylogenetic and functional gene structure shifts of the oral microbiomes in periodontitis patients

Determining the composition and function of subgingival dental plaque is crucial to understanding human periodontal health and disease, but it is challenging because of the complexity of the interactions between human microbiomes and human body. Here, we examined the phylogenetic and functional gene differences between periodontal and healthy individuals using MiSeq sequencing of 16S rRNA gene amplicons and a specific functional gene array (a combination of GeoChip 4.0 for biogeochemical processes and HuMiChip 1.0 for human microbiomes). Our analyses indicated that the phylogenetic and functional gene structure of the oral microbiomes were distinctly different between periodontal and healthy groups. Also, 16S rRNA gene sequencing analysis indicated that 39 genera were significantly different between healthy and periodontitis groups, and Fusobacterium, Porphyromonas, Treponema, Filifactor, Eubacterium, Tannerella, Hallella, Parvimonas, Peptostreptococcus and Catonella showed higher relative abundances in the periodontitis group. In addition, functional gene array data showed that a lower gene number but higher signal intensity of major genes existed in periodontitis, and a variety of genes involved in virulence factors, amino acid metabolism and glycosaminoglycan and pyrimidine degradation were enriched in periodontitis, suggesting their potential importance in periodontal pathogenesis. However, the genes involved in amino acid synthesis and pyrimidine synthesis exhibited a significantly lower relative abundance compared with healthy group. Overall, this study provides new insights into our understanding of phylogenetic and functional gene structure of subgingival microbial communities of periodontal patients and their importance in pathogenesis of periodontitis.     

Sunlight effects on the DMSP-sulfur and leucine assimilation activities of polar heterotrophic bacterioplankton

The influence of solar ultraviolet radiation and photosynthetically active radiation (PAR) on summertime marine bacterial uptake and assimilation of sulfur from radiolabeled dimethlysulfoniopropionate (³⁵S-DMSP) was studied at four Arctic and two Antarctic stations. Incubations with ³H-leucine were also conducted for comparative purposes as a measurement of bacterial activity. Arctic waters were characterized by large numbers of colonial Phaeocystis pouchetii and higher DMSP concentrations than in the two diatom-dominated Antarctic samples. Exposure to full sunlight radiation (280–700?nm), and to a lesser extent to PAR?+?UVA (320–700?nm), generally decreased the bacterial assimilation of ³H-leucine with respect to darkness, and caused variable effects on ³⁵S-DMSP assimilation. By using a single-cell approach involving microautoradiography we found high percentages of sulfur assimilating cells within the bacterial groups Gammaproteobacteria, Bacteroidetes, SAR11 and Roseobacter despite the varying DMSP concentrations between Arctic and Antarctic samples. The dominant SAR11 clade contributed 50–70% of the cells assimilating both substrates in the Arctic stations, whereas either Gammaproteobacteria or SAR11 were the largest contributors to ³H-leucine uptake in samples from the two Antarctic stations. Only one station was analyzed for single-cell ³⁵S-DMSP assimilation in Antarctica, and Gammaproteobacteria were major contributors to its uptake, providing the first evidence for Antarctic bacteria actively taking up ³⁵S-DMSP. PAR?+?UVA repeatedly increased the number of SAR11 cells assimilating ³H-leucine. This pattern also occurred with other ³⁵S-DMSP assimilating groups, though not so consistently. Our results support a widespread capability of polar bacteria to assimilate DMSP-sulfur during the season of maximum DMSP concentrations, and show for the first time that all major polar taxa can be highly active at this assimilation under the appropriate circumstances. Our findings further confirm the role of sunlight as a modulator of heterotrophic carbon and sulfur fluxes in the surface ocean.     

Phylogenetic Diversity and Community Structure of the Symbionts Associated with the Coralline Sponge Astrosclera willeyana of the Great Barrier Reef

The coralline sponge Astrosclera willeyana, considered to be a living representative of the reef-building stromatoporoids of the Mesozoic and the Paleozoic periods, occurs widely throughout the Indo-Pacific oceans. We aimed to examine, for the first time, the phylogenetic diversity of the microbial symbionts associated with A. willeyana using molecular methods and to investigate the spatial variability in the sponge-derived microbial communities of A. willeyana from diverse sites along the Great Barrier Reef (GBR). Both denaturing gradient gel electrophoresis (DGGE) analyses of 12 Astrosclera specimens and sequencing of a 16S rRNA gene clone library, constructed using a specimen of A. willeyana from the Yonge Reef (380 clones), revealed the presence of a complex microbial community with high diversity. An assessment of the 16S rRNA gene sequences to the particular phylogenetic groups showed domination of the Chloroflexi (42 %), followed by the Gammaproteobacteria (14 %), Actinobacteria (11 %), Acidobacteria (8 %), and the Deferribacteres (7 %). Of the microbes that were identified, a further 15 % belonged to the Deltaproteobacteria, Alphaproteobacteria, and Nitrospirae genera. The minor phylogenetic groups Gemmatimonadetes, Spirochaetes, Cyanobacteria, Poribacteria, and the Archaea composed 3 % of the community. Over 94 % of the sequences obtained from A. willeyana grouped together with other sponge- or coral-derived sequences, and of these, 72 % formed, with nearest relatives, 46 sponge-specific or sponge–coral clusters, highlighting the uniqueness of the microbial consortia in sponges. The DGGE results showed clear divisions according to the geographical origin of the samples, indicating closer relationships between the microbial communities with respect to their geographic origin (northern vs. southern GBR).     

Planktonic microbial community composition across steep physical/chemical gradients in permanently ice-covered Lake Bonney, Antarctica

Lake Bonney is a chemically stratified, permanently ice‐covered Antarctic lake that is unusual because anomalous nutrient concentrations in the east lobe suggest that denitrification occurs in the deep suboxic waters of the west lobe but not the east lobe, resulting in high concentrations of nitrate and nitrite below the east lobe chemocline. Environmental factors that usually control denitrification rates (e.g. organic carbon, nitrate, oxygen) do not appear to explain the nitrate distribution in the east lobe, suggesting that other factors (e.g. trace metals, salts, microbial community structure, etc.) may be involved. In order to explore the potential importance of microbial community composition, samples collected from multiple depths in both lobes were compared on the basis of 16S rRNA gene diversity. 16S rRNA polymerase chain reaction (PCR) clone libraries generated from five depths were subjected to restriction fragment length polymorphism (RFLP), rarefaction, statistical and phylogenetic analyses. Bacterial and archaeal 16S rRNA gene sequences were determined for clones corresponding to unique RFLP patterns. The bacterial community below the chemocline (at 25 m) in the east lobe was the least diverse of the five depths analysed and was compositionally distinct from the communities of the overlying waters. The greatest compositional overlap was observed between 16 and 19 m in the east lobe, while the east lobe at 25 m and the west lobe at 13 and 16 m had relatively distinct communities. Despite very little compositional overlap between the suboxic, hypersaline depths of the east and west lobes (25 m and 16 m, respectively), sequences closely related to the denitrifying Marinobacter strain ELB17 previously isolated from the east lobe were found in both libraries. Most of the Lake Bonney sequences are fairly distinct from those reported from other Antarctic environments. Archaeal 16S rRNA genes were only successfully amplified from the two hypersaline depths analysed, with only one identical halophilic sequence type occurring in both libraries, indicating extremely low archaeal diversity. Overall, microbial community composition varies both between lobes and across depths within lobes in Lake Bonney, reflecting the steep gradients in physical/chemical parameters across the chemocline, as well as the anomalous nutrient chemistry of the system.     

Acinetobacter apis sp. nov., isolated from the intestinal tract of a honey bee,Apis mellifera

A novel Gram-negative, obligate aerobic, non-motile, and both coccobacillus- and bacillus-shaped bacterium, designated strain HYN18^T, was isolated from the intestinal tract of a honey bee (Apis mellifera). The isolate was oxidasenegative and catalase-positive. Strain HYN18^T showed optimum growth at 25°C, pH 6–7, and in the presence of 1% (w/v) NaCl in trypticase soy broth medium. The isolate was negative for hydrolyses of starch, casein, gelatin and urea, indole production from tryptone and hemolysis on sheep blood agar. A phylogenetic analysis based on the 16S rRNA gene and rpoB gene sequence showed that strain HYN18^T was most closely related to Acinetobacter nectaris SAP 763.2^T and A. boissieri SAP 284.1^T with 98.3% and 98.1% similarity (16S rRNA gene), respectively, and 84.4% similarity with Acinetobacter nectaris SAP 763.2^T (rpoB gene). The major cellular fatty acids were summed features 3 (comprising C_16:1ω7c /C_16:1ω6c ), C_12:0 and C_16:0. The main isoprenoid quinone was ubiquinone-9 (Q-9). The polar lipids of strain HYN18^T were phosphatidylethanolamine, three unidentified lipids, an unidentified phospholipid and an unidentified glycolipid. The DNA G+C content was 40.6 mol%. DNADNA hybridization experiments indicated less than 33 ± 10% relatedness to the closest phylogenetic species, Acinetobacter nectaris SAP 763.2^T. Thus, the phenotypic, phylogenetic and genotypic analyses indicate that strain HYN18^T is a novel species within the genus Acinetobacter, for which the name Acinetobacter apis is proposed. The type strain is HYN18^T (=KACC 16906^T =JCM 18575^T).     

Community Structures and Distribution of Anaerobic Ammonium Oxidizing and nirS-Encoding Nitrite-Reducing Bacteria in Surface Sediments of the South China Sea

Anaerobic ammonium oxidation (anammox) and denitrification are two important processes responsible for nitrogen loss; monitoring of microbial communities carrying out these two processes offers a unique opportunity to understand the microbial nitrogen cycle. The aim of the current study was to characterize community structures and distribution of anammox and nirS-encoding nitrite-reducing bacteria in surface sediments of the northern South China Sea (SCS). The consistent phylogenetic results of three biomarkers of anammox bacteria, including 16S rRNA, hzo, and Scalindua-nirS genes, showed that Scalindua-like bacteria were the only anammox group presenting in surface sediments of the SCS. However, a relatively high micro-diversity was found within this group, including several SCS habitat-specific phylotypes, Candidatus “Scalindua zhenghei”. Comparing to 16S rRNA gene, hzo and Scalindua-nirS genes provided a relatively higher resolution to elucidate anammox bacteria. For the nirS-encoding nitrite-reducing bacteria, the detected nirS gene sequences were closely related to various marine nirS denitrifiers, especially those which originated from coastal and estuarine sediments with a much higher diversity than anammox bacteria. Anammox bacterial communities shifted along with the seawater depth, while nirS-encoding nitrite-reducing bacteria did not. Although nirS-encoding nitrite-reducing bacteria have a much higher abundance and diversity than anammox bacteria, they showed similar abundance variation patterns in research sites, suggesting the two microbial groups might be affected by the similar environmental factors. The significant correlations among the abundance of the two microbial groups with the molar ratio of NH₄ ⁺ to (NO₂ ^??+?NO₃ ^?), pH, and organic matters of sediments strongly supported this hypothesis.     

Description and functional testing of four species of the novel phototrophic genus Chioneia gen. nov., isolated from different East Antarctic environments

Seven Gram-negative, aerobic, non-sporulating, motile strains were isolated from terrestrial (R-67880^T, R-67883, R-36501 and R-36677^T) and aquatic (R-39604, R-39161^T and R-39594^T) East Antarctic environments (i.e. soil and aquatic microbial mats), between 2007 and 2014. Analysis of near-complete 16S rRNA gene sequences revealed that the strains potentially form a novel genus in the family Sphingomonadaceae (Alphaproteobacteria). DNA-DNA reassociation and average nucleotide identity values indicated distinction from close neighbors in the family Sphingomonadaceae and showed that the seven isolates form four different species. The main central pathways present in the strains are the glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The strains can use only a limited number of carbon sources and mainly depend on ammonia and sulfate as a nitrogen and sulfur source, respectively. The novel strains showed the potential of aerobic anoxygenic phototrophy, based on the presence of bacteriochlorophyll a pigments, which was corroborated by the presence of genes for all building blocks for a type 2 photosynthetic reaction center in the annotated genomes. Based on the results of phenotypic, genomic, phylogenetic and chemotaxonomic analyses, the strains could be assigned four new species in the novel genus Chioneia gen. nov. in the family Sphingomonadaceae, for which the names C. frigida sp. nov. (R-67880^T, R-67883 and R-36501), C. hiemis sp. nov. (R-36677^T), C. brumae sp. nov. (R-39161^T and R-39604) and C. algoris sp. nov. (R-39594^T) are proposed.     

Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-¹³C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of ¹³C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the ¹³C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.     

Analysis of the Attached Microbial Community on Mucilaginous Cyanobacterial Aggregates in the Eutrophic Lake Taihu Reveals the Importance of Planctomycetes

The phylogenetic diversity of the microbial community assemblage of the carpet-like mucilaginous cyanobacterial blooms in the eutrophic Lake Taihu was investigated. 16S ribosomal DNA clone libraries produced from the DNA of cyanobacterial assemblages that had been washed to remove unattached bacteria contained only cyanobacteria. However, a further treatment which included grinding the freeze-dried material to physically detach cells followed by the removal of larger cells by filtration allowed us to detect a large variety of bacteria within the cyanobacterial bloom community. Interestingly, the dominant members of the microbial community were Planctomycetes followed by Cytophaga–Flavobacterium–Bacteroides (CFB), Betaproteobacteria, and Gammaproteobacteria. The analysis of the 16S ribosomal DNA clone libraries made from enrichment culture revealed much higher phylogenetic diversity of bacteria. Dominant bacterial groups in the enrichment system were identified as members of the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria subdivisions, CFB group, and Planctomycetes. In addition, the clone libraries constructed from Planctomycetes-specific 16S ribosomal RNA primers also verified that the enrichment allowed a diversity of Planctomycetes to proliferate, although the community composition was altered after enrichment.     

POLYPHASIC STUDY OF ANTARCTIC CYANOBACTERIAL STRAINS1

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.     

Distribution of rDNA and polyploidy in <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> E. Desv. in Antarctic and Patagonic populations

Unlike the Arctic flora, with many flowering plant species offering opportunities to study evolutionary processes, the Antarctic flora offers only two. One of them is the Antarctic grass Deschampsia antarctica E. Desv., whose distribution spans from northern Patagonia (ca. 38°S) down to Alamode Island (ca. 68°S), in the west side of the Antarctic Peninsula. While some aspects of Antarctic plants have been extensively studied (e.g., anatomy, physiology, genetics), little is known about the related Patagonian populations. Particularly in cytogenetics, no single study has focused on continental populations and its relationships with the Antarctic plants. The combination of traditional fluorescent in situ hybridization (FISH) with a phylogenetic framework highlights the importance of cytogenetics in plant evolutionary studies, by allowing comparison of chromosome characters in phylogenetically related individuals. Most used characters for this purpose are the chromosome number, karyotype morphology and patterns of repetitive DNA. These were used to compare distant populations of D. antarctica in a phylogenetic framework, to obtain a first view of the cytogenetic structure of the species along its distribution. Patagonian populations have greater variability in the chromosomal and molecular characters, while Antarctic populations are very alike, hinting at a South American origin hypothesis. A polyploid population is reported for the first time, located on Central Patagonia populations, close to the northern limit of distribution range. Cytogenetic characteristics suggest that hybridization processes could have played an important role in the evolution of the genome of D. antarctica.     

Phylogeny and distribution of Bradyrhizobium symbionts nodulating cowpea (Vigna unguiculata L. Walp) and their association with the physicochemical properties of acidic African soils

In the N₂-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S–23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S–23S rRNA region yielded single polymorphic bands of varying lengths (573–1298 bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard’s similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.