Diversity of Bacterial Communities in Container Habitats of Mosquitoes

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n = 12), cemetery urns (n = 23), and miscellaneous containers that included two tree holes (n = 19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Diversity and composition of sulfate- and sulfite-reducing prokaryotes as affected by marine-freshwater gradient and sulfate availability

Sulfate- and sulfite-reducing prokaryotes (SSRP) communities play a key role in both sulfur and carbon cycles. In estuarine ecosystems, sulfate concentrations change with tides and could be limited in tidal freshwater reach or deep sediments. In a subtropical estuary of northern Taiwan in December 2007, we examined the compositional changes of SSRP communities. We examined three sites: from the lower estuarine brackish-water reach (site GR and mangrove vegetation site, GM) to the upper estuarine tidal freshwater reach (site HR), as well as from surface to a 50-cm depth. The partial sequence of sulfite reductase (dsrB) genes was used as a molecular marker of SSRP, linked to polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE) techniques. SSRP communities of the DGGE profiles varied with sites according to one-way analyses of similarities (Global R = 0.69, P = 0.001). Using cluster analysis, the DGGE profile was found to show site-specific clusters and a distinct depth zonation (five, six, and two SSRP communities at the GM, GR, and HR sites, respectively). SSRP composition was highly correlated to the combination of salinity, reduced sulfur, and total organic carbon contents (BIO-ENV analysis, r _s  = 0.56). After analyzing a total of 35 dsrB sequences in the DGGE gel, six groups with 15 phylotypes were found, which were closely related to marine-freshwater gradient. Moreover, sequences neighboring sulfite-reducing prokaryotes were observed, in addition to those affiliated to sulfate-reducing prokaryotes. Four phylotypes harvested in HR resembled the genus Desulfitobacterium, a sulfite-reducing prokaryote, which failed to use sulfate as an electron acceptor and were active in freshwater and sulfate-limited habitat. The other five phylotypes in the HR reach belonged to the sulfate-reducing prokaryotes of the genera Desulfatiferula, Desulfosarcina, Desulfovibrio, and Desulfotomaculum, which appeared to tolerate low salinity and low sulfate supply. SSRP phylotypes at the mangrove-vegetated GM site (five phylotypes in two groups) were phylogenetically less diverse, when compared with those at the non-mangrove-vegetated GR site (three phylotypes in three groups) and the tidally influenced freshwater HR site (nine phylotypes in five groups). Phylotypes found at GR and GM were all affiliated to marine sulfate-reducing prokaryote strains of the genera Desulfofaba, Desulfobotulus, Desulfatiferula, Desulfosarcina, and Desulfotomaculum. Notably, a phylotype recorded in the surface sediment at GR resembled the genus Desulfobulbus, which was recorded from freshwater environment consisting of the freshwater input at GR during ebb tides.     

Morphological,Bacterial, and Secondary Metabolite Changes of <Emphasis Type="Italic">Aplysina aerophoba</Emphasis> upon Long-Term Maintenance Under Artificial Conditions

The aim of this study was to analyze successional changes in the bacterial community over a period of 6 months of cultivation of Aplysina aerophoba sponges under different artificial cultivation conditions by use of denaturing gradient gel electrophoresis (DGGE). The cultivation conditions varied concerning the water temperature (20 ± 2 °C and 25 ± 2 °C) of the aquaria, additional illumination of one aquarium, and feeding of the sponges. Amplicons from DGGE separation of dominant colonizing or variably appearing bacteria were sequenced and aligned for taxonomical identification. In addition, secondary metabolites typically found in A. aerophoba were analyzed to investigate changes in the natural product profile during cultivation. The cultivation of sponges under any given condition did not lead to a depletion of their bacterial community in the course of the experiment. On the contrary, the distinctive set of associated bacteria was maintained in spite of a dramatic loss of biomass and morphological degradation during the cultivation period. Generally, all sequences obtained from the DGGE gels were related to bacteria of five phyla: Actinobacteria, Cyanobacteria, α-Proteobacteria, γ-Proteobacteria, and Chloroflexi. Despite the overall stability of the bacterial community in A. aerophoba, an unambiguous variability was detected for the Cyanobacteria “A. aerophoba clone TK09”. This variability was ascribed to the predominant light conditions. The analysis of the metabolic pattern revealed that the concentration of a class of characteristic-brominated compounds typically found in A. aerophoba, like aeroplysinin-1, aerophobin-1, aerophobin-2, and isofistularin-3, increased over the 6 months of cultivation.     

Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.     

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the division Proteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).     

Assessing the Viability of Bacterial Species in Drinking Water by Combined Cellular and Molecular Analyses

The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining (“live/dead staining” indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotpes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water.     

Dissolved organic carbon concentrations in four Norway spruce stands of different ages

Boreal forests are increasing in age partly due to reduced logging and efficient wildfire control. As a result, they also stock more carbon. Whether increased forest C stock causes greater production of dissolved organic carbon (DOC) is uncertain. DOC in bulk precipitation, throughfall and soil water was studied in 10-, 30-, 60- and 120-year-old stands of Norway spruce (Picea abies (L.) Karst.) DOC concentrations in throughfall and O horizon soil water followed the order 10 < 30 < 60 = 120 and 10 = 30 < 120 < 60, respectively. DOC fluxes followed the order 10 = 30 < 60 = 120 in throughfall, while no significant difference between stands was found for O horizon soil water. Above-ground tree litter varied according to 10 < 30 < 60 = 120, a pattern identical to that for DOC concentrations in throughfall and resembling but not identical to that for DOC concentrations in O horizon soil water. This indicates additional sources for DOC in soil water. Seasonality in DOC concentrations was observed at the base of the O horizon, and seasonality in DOC fluxes in both throughfall and O horizon soil water. Our results suggest differences in the polarity of DOC between the 10-year stand and the others, which we interpret as reflecting the lack of grown trees and possibly the different vegetation on the 10-year stand.     

Community Structure of Planktonic Fungi and the Impact of Parasitic Chytrids on Phytoplankton in Lake Inba,Japan

Freshwater fungi have received little attention by scientific research in recent years, especially fungi of the pelagic zone. Recently, parasitic fungi, termed chytrids, have been found to play important roles in aquatic food webs. Yet, the diversity and community structure of planktonic fungi including chytrids are not well studied. In this study, we examined the temporal fluctuations of freshwater fungi, including chytrids, in Lake Inba by using molecular techniques of denaturing gradient gel electrophoresis (DGGE). DGGE profiles, and associated sequence analysis, indicated that chytrids were present on all sampling dates from May to October (n = 12). In addition, analysis showed that a large proportion of the sequences belonged to chytrids of both parasitic and saprotrophic species. This finding was supported by microscopic observations using Calcofluor white to stain chytrids infecting various phytoplankton species. The percentages of infection by chytrids on two dominant diatom species, Aulacoseira granulata and Aulacoseira ambigua, showed a similar seasonal pattern in the DGGE band profiles. From the phylogenetic analysis and microscopic identification, the chytrids infecting the two diatoms are likely to be affiliated to Chytriomyces sp. and Zygorhizidium sp.. This is the first study to show that DGGE is a useful preliminary approach for examining the diversity of planktonic fungi including chytrids. Our results indicate both parasitic and saprotrophic chytrids are a significant component of freshwater fungi inhabiting the pelagic zone of Lake Inba, Japan. Further modification of DGGE, together with new molecular techniques and microscopic observation, would reveal the hidden diversity and ecological significance of planktonic fungi in aquatic ecosystems.     

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C₂₉, C₃₁ and C₃₃ alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4–5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.     

Will rising atmospheric CO2 affect leaf litter quality and in situ decomposition rates in native plant communities?

Though field data for naturally senesced leaf litter are rare, it is commonly assumed that rising atmospheric CO₂ concentrations will reduce leaf litter quality and decomposition rates in terrestrial ecosystems and that this will lead to decreased rates of nutrient cycling and increased carbon sequestration in native ecosystems. We generally found that the quality of␣naturally senesced leaf litter (i.e. concentrations of C, N and lignin; C:N, lignin:N) of a variety of native plant species produced in alpine, temperate and tropical communities maintained at elevated CO₂ (600–680 μl l⁻¹) was not significantly different from that produced in similar communities maintained at current ambient CO₂ concentrations (340–355 μl l⁻¹). When this litter was allowed to decompose in situ in a humid tropical forest in Panama (Cecropia peltata, Elettaria cardamomum, and Ficus benjamina, 130 days exposure) and in a lowland temperate calcareous grassland in Switzerland (Carex flacca and a graminoid species mixture; 261 days exposure), decomposition rates of litter produced under ambient and elevated CO₂ did not differ significantly. The one exception to this pattern occurred in the high alpine sedge, Carex curvula, growing in the Swiss Alps. Decomposition of litter produced in situ under elevated CO₂ was significantly slower than that of litter produced under ambient CO₂ (14% vs. 21% of the initial litter mass had decomposed over a 61-day exposure period, respectively). Overall, our results indicate that relatively little or no change in leaf litter quality can be expected in plant communities growing under soil fertilities common in many native ecosystems as atmospheric CO₂ concentrations continue to rise. Even in situations where small reductions in litter quality do occur, these may not necessarily lead to significantly slower rates of decomposition. Hence in many native species in situ litter decomposition rates, and the time course of decomposition, may remain relatively unaffected by rising CO₂. Received: 12 September 1996 / Accepted: 30 November 1996     

The Native Bacterioplankton Community in the Central Baltic Sea Is Influenced by Freshwater Bacterial Species

The Baltic Sea is one of the largest brackish environments on Earth. Despite extensive knowledge about food web interactions and pelagic ecosystem functioning, information about the bacterial community composition in the Baltic Sea is scarce. We hypothesized that due to the eutrophic low-salinity environment and the long water residence time (>5 years), the bacterioplankton community from the Baltic proper shows a native “brackish” composition influenced by both freshwater and marine phylotypes. The bacterial community composition in surface water (3-m depth) was examined at a single station throughout a full year. Denaturing gradient gel electrophoresis (DGGE) showed that the community composition changed over the year. Further, it indicated that at the four extensive samplings (16S rRNA gene clone libraries and bacterial isolates from low- and high-nutrient agar plates and seawater cultures), different bacterial assemblages associated with different environmental conditions were present. Overall, the sequencing of 26 DGGE bands, 160 clones, 209 plate isolates, and 9 dilution culture isolates showed that the bacterial assemblage in surface waters of the central Baltic Sea was dominated by Bacteroidetes but exhibited a pronounced influence of typical freshwater phylogenetic groups within Actinobacteria, Verrucomicrobia, and Betaproteobacteria and a lack of typical marine taxa. This first comprehensive analysis of bacterial community composition in the central Baltic Sea points to the existence of an autochthonous estuarine community uniquely adapted to the environmental conditions prevailing in this brackish environment.     

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of “species” during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.     

Spatial Heterogeneity of Bacterial Populations in Monomictic Lake Estanya (Huesca,Spain)

Bacterial population changes were investigated in the monomictic Lake Estanya by combining microscopic analysis and two molecular methods involving the amplification of 16S rDNA genes using primers for the domain Bacteria and subsequent restriction fragment length polymorphism (PCR–RFLP) and denaturing gradient gel electrophoresis (PCR–DGGE). Both approaches revealed the vertical distribution of predominant microbial morphotypes and phylotypes in both holomictic and stratified periods, respectively, and showed that variations in structure and composition of bacterial populations are occurring in this lake as a function of depth and time. Through principal component analysis (PCA), these shifts could be related to different physicochemical parameters with temperature, oxygen concentration, and the incident light being of paramount importance as structuring variables. Comparison of RFLP and DGGE profiles by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling map (MDS) showed equivalent results. Both techniques revealed that bacterial populations, present in the whole water column in the holomictic period, showed a high similarity with those located in the deeper part of the lake in the stratified period, evidencing that other factors, both biotic and abiotic, should also be considered as a force driving change in the composition of the bacterial community. Furthermore, DGGE analysis showed that sequences from prominent bands were affiliated to members of four major phyla of the domain Bacteria: Cyanobacteria, Bacteroidetes, Proteobacteria, and Actinobacteria, most of which corresponded to heterotrophic bacterial populations involved in carbon, sulfide, and nitrogen biogeochemical cycles, which were indistinguishable under the light microscope.     

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR-denaturing gradient gel electrophoresis

Culture-independent PCR–denaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for nifH PCR amplification, which simplified the current procedure and resulted in good DGGE profiles. The results revealed a novel nitrogen-fixing bacterial profile and fundamental diazotrophic biodiversity in mangrove sediments, as reflected by the numerous bands present DGGE patterns. Canonical correspondence analysis (CCA) revealed that the sediments organic carbon concentration and available soil potassium accounted for a significant amount of the variability in the nitrogen-fixing bacterial community composition. The predominant DGGE bands were sequenced, yielding 31 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from Proteobacteria, e.g. α, γ, β, δ-subdivisions, and characterized by sequences of members of genera Azotobacter, Desulfuromonas, Sphingomonas, Geobacter, Pseudomonas, Bradyrhizobium and Derxia. These results significantly expand our knowledge of the nitrogen-fixing bacterial diversity of the mangrove environment.     

Distribution of dissolved organic carbon in Lake Baikal and its watershed

 Concentrations of dissolved organic carbon (DOC) in Lake Baikal ranged from 90 to 110 μM C, considerably higher than those in oceanic environments. The DOC concentrations in the epilimnion were higher than those in the hypolimnion. Since particulate organic carbon (POC) concentrations in the pelagic waters of Lake Baikal were <10–40 μM C in the epilimnion and 2–5 μM C in the hypolimnion, DOC constitutes a major component of the organic carbon pool in Lake Baikal, especially in the deep layers. The DOC concentrations downstream of the Barguzin and Selenga Rivers were quite high (400–500 μM C). Probably because of the high concentrations of DOC in these rivers, the DOC levels in Barguzin Bay and offshore at the mouth of the Selenga River were higher than those in the pelagic regions of the central and south basins of Lake Baikal. The relationship between DOC and electric conductivity revealed the transport of DOC from rivers to the pelagic area in Lake Baikal. The spatial distribution of DOC suggested that a major part of DOC in the lake was allochthonous (land-derived). Received: July 26, 2002 / Accepted: September 16, 2002 Present address:Research Institute for Humanity and Nature, 335 Takashima-cho, Marutamachi Kawaramachi, Kamigyo-ku, Kyoto 602-0878, Japan Tel. +81-75-229-6167; Fax +81-75-229-6150 e-mail: yos@chikyu.ac.jp Acknowldgments The authors wish to thank Director Prof. M. Grachev and Dr. O. Timoshkin of the Limnological Institute, Siberian Branch, Academy of Science, Russia, for arranging the cruise on Lake Baikal. We are also indebted to Drs. V. Sinyukovich, I. Khanaev, and A. Zhdanov for their kind assistance during the expeditions. We wish to thank Ms. Y. Ito for measuring the DOC concentrations. This work was supported and financed by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (No. 09041159) and for Scientific Research of Priority Area B (No. 11213101) and by the International Geosphere-Biosphere Programme (IGBP) at Nagoya University. Correspondence to:T. Yoshioka     

Endophytic Bacteria Associated with Growing Shoot Tips of Banana (Musa sp.) cv. Grand Naine and the Affinity of Endophytes to the Host

A cultivation-based assessment of endophytic bacteria present in deep-seated shoot tips of banana suckers was made with a view to generate information on the associated organisms, potential endophytic contaminants in tissue-cultured bananas and to assess if the endophytes shared a beneficial relationship with the host. Plating the tissue homogenate from the central core of suckers showed colony growth on nutrient agar from just 75% and 42% of the 12 stocks during May and November, respectively (average 58%; 6 × 10³ colony-forming units per gram), yielding diverse organisms belonging to firmicutes (Bacillus, Brevibacillus, Paenibacillus, Virgibacillus, Staphylococcus spp.), actinobacteria (Cellulomonas, Micrococcus, Corynebacterium, Kocuria spp.), α-proteobacteria (Paracoccus sp.), and γ-proteobacteria (Pseudomonas, Acinetobacter spp.). Each shoot tip showed one to three different organisms and no specific organism appeared common to different sucker tips. Tissue homogenate from shoot tips including the ones that did not yield culturable bacteria displayed abundant bacterial cells during microscopic examination suggesting that a high proportion of cells were in viable-but-nonculturable state, or their cultivation requirements were not met. Direct application of cultivation-independent approach to study endophytic bacterial community using bacterial 16S ribosomal RNA universal primers resulted in high interference from chloroplast and mitochondrial genome sequences. Dislodging the bacterial cells from shoot tips that did not show cultivable bacteria and incubating the tissue crush in dilute-nutrient broth led to the activation of four organisms (Klebsiella, Agrobacterium, Pseudacidovorax spp., and an unidentified isolate). The endophytic organisms in general showed better growth at 30–37 °C compared with 25 °C, and the growth of endophytes as well as pathogenic Erwinia carotovora were promoted with the supply of host tissue extract (HTE) while that of the isolates from nonplant sources were inhibited or unaffected by HTE, suggesting an affinity or dependence of the endophytes on the host and the prospect of an HTE-based assay for discriminating the nonendophytes from endophytes.     

Microbial Succession in a Compost-packed Biofilter Treating Benzene-contaminated Air

Air artificially contaminated with increasing concentrations of benzene was treated in a laboratory scale compost-packed biofilter for 240 days with a removal efficiency of 81–100%. The bacterial community in the packing material (PM) at different heights of the biofilter was analysed every 60 days. Bacterial plate counts and ribosomal intergenic spacer analysis (RISA) of the isolated strains showed that the number of cultivable aerobic heterotrophic bacteria and the species diversity increased with benzene availability. Identification of the isolated species and the main bands in denaturing gradient gel electrophoresis (DGGE) profiles from total compost DNA during the treatment revealed that, at a relatively low volumetric benzene load (1.2≤VBL≤6.4 g m⁻³ _PM h⁻¹), besides low G+C Gram positive bacteria, originally present in the packing compost, bacteroidetes and β- and γ-proteobacteria became detectable in the colonising population. At the VBL value (24.8 g m⁻³ _PM h⁻¹) ensuring the maximum elimination capacity of the biofilter (20.1 g m⁻³ _PM h⁻¹), strains affiliated to the genus Rhodococcus dominated the microflora, followed by β-proteobacteria comprising the genera Bordetella and Neisseria. Under these conditions, more than 35% of the isolated strains were able to grow on benzene as the sole carbon source. Comparison of DGGE and automated RISA profiles of the total community and isolated strains showed that a complex bacterial succession occurred in the reactor in response to the increasing concentrations of the pollutant and that cultivable bacteria played a major role in benzene degradation under the adopted conditions.     

Pyrosequencing Reveals a Highly Diverse and Cultivar-Specific Bacterial Endophyte Community in Potato Roots

In this study, we examined the bacterial endophyte community of potato (Solanum tuberosum) cultivar/clones using two different molecular-based techniques (bacterial automated ribosomal intergenic spacer analysis (B-ARISA) and pyrosequencing). B-ARISA profiles revealed a significant difference in the endophytic community between cultivars (perMANOVA, p < 0.001), and canonical correspondence analysis showed a significant correlation between the community structure and plant biomass (p = 0.001). Pyrosequencing detected, on average, 477 ± 71 bacterial operational taxonomic units (OTUs, 97% genetic similarity) residing within the roots of each cultivar, with a Chao estimated total OTU richness of 1,265 ± 313. Across all cultivars, a total of 238 known genera from 15 phyla were identified. Interestingly, five of the ten most common genera (Rheinheimera, Dyadobacter, Devosia, Pedobacter, and Pseudoxanthomonas) have not, to our knowledge, been previously reported as endophytes of potato. Like the B-ARISA analysis, the endophytic communities differed between cultivar/clones (∫-libshuff, p < 0.001) and exhibited low similarities on both a presence/absence (0.145 ± 0.019) and abundance (0.420 ± 0.081) basis. Seventeen OTUs showed a strong positive (r > 0.600) or negative (r < −0.600) correlation with plant biomass, suggesting a possible link between plant production and endophyte abundance. This study represents one of the most comprehensive assessments of the bacterial endophytic communities to date, and similar analyses in other plant species, cultivars, or tissues could be utilized to further elucidate the potential contribution(s) of endophytic communities to plant physiology and production.     

Diversity and Structure of Bacterial Communities in Arctic versus Antarctic Pack Ice

A comprehensive assessment of bacterial diversity and community composition in arctic and antarctic pack ice was conducted through cultivation and cultivation-independent molecular techniques. We sequenced 16S rRNA genes from 115 and 87 pure cultures of bacteria isolated from arctic and antarctic pack ice, respectively. Most of the 33 arctic phylotypes were >97% identical to previously described antarctic species or to our own antarctic isolates. At both poles, the α- and γ-proteobacteria and the Cytophaga-Flavobacterium group were the dominant taxonomic bacterial groups identified by cultivation as well as by molecular methods. The analysis of 16S rRNA gene clone libraries from multiple arctic and antarctic pack ice samples revealed a high incidence of closely overlapping 16S rRNA gene clone and isolate sequences. Simultaneous analysis of environmental samples with fluorescence in situ hybridization (FISH) showed that ~95% of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells hybridized with the general bacterial probe EUB338. More than 90% of those were further assignable. Approximately 50 and 36% were identified as γ-proteobacteria in arctic and antarctic samples,respectively. Approximately 25% were identified as α-proteobacteria, and 25% were identified as belonging to the Cytophaga-Flavobacterium group. For the quantification of specific members of the sea ice community, new oligonucleotide probes were developed which target the genera Octadecabacter, Glaciecola, Psychrobacter, Marinobacter, Shewanella, and Polaribacter. High FISH detection rates of these groups as well as high viable counts corroborated the overlap of clone and isolate sequences. A terrestrial influence on the arctic pack ice community was suggested by the presence of limnic phylotypes.     

Aquatic Microbial Habitats Within a Neotropical Rainforest: Bromeliads and pH-Associated Trends in Bacterial Diversity and Composition

Tank-forming bromeliads, suspended in the rainforest canopy, possess foliage arranged in compact rosettes capable of long-term retention of rainwater. This large and unique aquatic habitat is inhabited by microorganisms involved in the important decomposition of impounded material. Moreover, these communities are likely influenced by environmental factors such as pH, oxygen, and light. Bacterial community composition and diversity was determined for the tanks of several bromeliad species (Aechmea and Werauhia) from northern Costa Rica, which span a range of parameters, including tank morphology and pH. These were compared with a nearby forest soil sample, an artificial tank (amber bottle), and a commercially available species (Aechmea). Bacterial community diversity, as measured by 16S rRNA analysis and tRFLP, showed a significant positive correlation with tank pH. A majority of 16S rRNA bacterial phylotypes found in association with acidic bromeliad tanks of pH < 5.1 were affiliated with the Alphaproteobacteria, Acidobacteria, Planctomycetes, and Bacteroidetes, and were similar to those found in acidic peat bogs, yet distinct from the underlying soil community. In contrast, bromeliads with tank pH > 5.3, including the commercial bromeliad with the highest pH (6.7), were dominated by Betaproteobacteria, Firmicutes, and Bacteroidetes. To empirically determine the effect of pH on bacterial community, the tank pH of a specimen of Aechmea was depressed, in the field, from 6.5 to 4.5, for 62 days. The resulting community changed predictably with decreased abundance of Betaproteobacteria and Firmicutes and a concomitant increase in Alphaproteobacteria and Acidobacteria. Collectively, these results suggest that bromeliad tanks provide important habitats for a diverse microbial community, distinct from the surrounding environment, which are influenced greatly by acid–base conditions. Additionally, total organic carbon (∼46%) and nitrogen (∼2%) of bromeliad-impounded sediment was elevated relative to soil and gene surveys confirmed the presence of both chitinases and nitrogenases, suggesting that bromeliad tanks may provide important habitats for microbes involved in the biological cycling of carbon and nitrogen in tropical forests.