Diversity and abundance of uncultured cytophaga-like bacteria in the Delaware estuary.
ABSTRACT: The Cytophaga-Flavobacterium group is known to be abundant in aquatic ecosystems and to have a potentially unique role in the utilization of organic material. However, relatively little is known about the diversity and abundance of uncultured members of this bacterial group, in part because they are underrepresented in clone libraries of 16S rRNA genes. To circumvent a suspected bias in PCR, a primer set was designed to amplify 16S rRNA genes from the Cytophaga-Flavobacterium group and was used to construct a library of these genes from the Delaware Estuary. This library had several novel Cytophaga-like 16S rRNA genes, of which about 40% could be grouped together into two clusters (DE clusters 1 and 2) defined by sequences initially observed only in the Delaware library; the other 16S rRNA genes were classified into an additional four clades containing sequences from other environments. An oligonucleotide probe was designed for the cluster with the most clones (DE cluster 2) and was used in fluorescence in situ hybridization assays. Bacteria in DE cluster 2 accounted for about 10% of the total prokaryotic abundance in the Delaware Estuary and in a depth profile of the Chukchi Sea (Arctic Ocean). The presence of DE cluster 2 in the Arctic Ocean was confirmed by results from 16S rRNA clone libraries. The contribution of this cluster to the total bacterial biomass is probably larger than is indicated by the abundance of its members, because the average cell volume of bacteria in DE cluster 2 was larger than those of other bacteria and prokaryotes in the Delaware Estuary and Chukchi Sea. DE cluster 2 may be one of the more abundant bacterial groups in the Delaware Estuary and possibly other marine environments.
Project description:Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM). We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample. Forty-two different cultivatable strains were isolated from rich and poor solid media. In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector. Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group. The different methods gave similar distributions of taxa at the genus level and higher. However, the match at the species level among the methods was poor, and only one species was identified by all three methods. Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system. The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species. Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied. Culture-independent methods also suggested several not-yet-cultivated beta-proteobacteria to be RDOM consumers.
Project description:We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the gamma- and delta-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium -Bacteroides cluster, and the phyla Planctomyces and Holophaga-Acidobacterium. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the Planctomyces phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.
Project description:Temporal changes of the bacterioplankton from a meromictic lake (Lake Vilar, Banyoles, Spain) were analyzed with four culture-independent techniques: epifluorescence microscopy, PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting, fluorescence in situ whole-cell hybridization and flow cytometry sorting. Microscopically, blooms of one cyanobacterium (Synechococcus sp.-like), one green sulfur bacterium (Chlorobium phaeobacteroides-like), and one purple sulfur bacterium (Thiocystis minor-like) were observed at different depths and times. DGGE retrieved these populations and, additionally, populations related to the Cytophaga-Flavobacterium-Bacteroides phylum as predominant community members. The analyses of partial 16S ribosomal DNA sequences from the DGGE fingerprints (550 bp analyzed) revealed higher genetic diversity than expected from microscopic observation for most of these groups. Thus, the sequences of two Synechococcus spp. (both had a similarity of 97% to Synechococcus sp. strain PCC6307 in 16S rRNA), two Thiocystis spp. (similarities to Thiocystis minor of 93 and 94%, respectively), and three Cytophaga spp. (similarities to Cytophaga fermentans of 88 and 89% and to Cytophaga sp. of 93%, respectively) were obtained. The two populations of Synechococcus exhibited different pigment compositions and temporal distributions and their 16S rRNA sequences were 97.3% similar. The two Thiocystis populations differed neither in pigment composition nor in morphology, but their 16S rRNA sequences were only 92.3% similar and they also showed different distributions over time. Finally, two of the Cytophaga spp. showed 96.2% similarity between the 16S rRNA sequences, but one of them was found to be mostly attached to particles and only in winter. Thus, the identity of the main populations changed over time, but the function of the microbial guilds was maintained. Our data showed that temporal shifts in the identity of the predominant population is a new explanation for the environmental 16S rRNA microdiversity retrieved from microbial assemblages and support the hypothesis that clusters of closely related 16S rRNA environmental sequences may actually represent numerous closely related, yet ecologically distinct, populations.
Project description: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 beta- and gamma-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.
Project description: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 alpha- and gamma-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 approximately 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 gamma-proteobacteria in arctic and antarctic samples,respectively. Approximately 25% were identified as alpha-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.
Project description:The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.
Project description:Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the alpha, beta, and gamma Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.
Project description:Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.
Project description:The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.
Project description:Very little is known about growth rates of individual bacterial taxa and how they respond to environmental flux. Here, we characterized bacterial community diversity, structure and the relative abundance of 16S rRNA and 16S rRNA genes (rDNA) using pyrosequencing along the salinity gradient in the Delaware Bay. Indices of diversity, evenness, structure and growth rates of the surface bacterial community significantly varied along the transect, reflecting active mixing between the freshwater and marine ends of the estuary. There was no positive correlation between relative abundances of 16S rRNA and rDNA for the entire bacterial community, suggesting that abundance of bacteria does not necessarily reflect potential growth rate or activity. However, for almost half of the individual taxa, 16S rRNA positively correlated with rDNA, suggesting that activity did follow abundance in these cases. The positive relationship between 16S rRNA and rDNA was less in the whole water community than for free-living taxa, indicating that the two communities differed in activity. The 16S rRNA:rDNA ratios of some typically marine taxa reflected differences in light, nutrient concentrations and other environmental factors along the estuarine gradient. The ratios of individual freshwater taxa declined as salinity increased, whereas the 16S rRNA:rDNA ratios of only some typical marine bacteria increased as salinity increased. These data suggest that physical and other bottom-up factors differentially affect growth rates, but not necessarily abundance of individual taxa in this highly variable environment.