Mixed-population DNA Research Articles

Culture-independent identification of gut bacteria in fourth-instar red imported fire ant, Solenopsis invicta Buren, larvae

Red imported fire ants (RIFA), Solenopsis invicta Buren, are medical, urban, and agricultural pests from South America. They are successful invaders due to their preference for disturbed habitats, high reproductive rates, and the ability to feed on a wide variety of food items (omnivorous). Fourth-instar larvae are used by the colony to digest solid food and then regurgitate it for consumption by workers and queens. Larvae are an ideal source of investigations of endosymbiotic bacteria possibly involved in nutrient distributions. Our study utilized 16S rDNA sequencing to describe the composition of the bacterial community in fourth-instar ant larvae in order to identify possible endosymbiotic bacteria present therein. The 16S rRNA gene was directly amplified from mixed-population DNA of whole fire ant larval guts and cloned into Escherichia coli. Bacterial communities from three geographically separated RIFA colonies were examined. Sequenced bacterial clones from guts were determined to be predominantly from the phylum Proteobacteria and the family Enterobacteriaceae. Our results did not detect the presence of endosymbiotic bacteria in the guts of RIFA larvae among the colonies. In addition, minimal species overlap was found when bacterial inventories were compared among colonies. Thus, bacteria coadapted with red imported fire ant larvae were not detected. Identified bacteria were not closely affiliated with endosymbiotic bacteria common in other insect species. Bacteria communities appeared to be unique to each geographical location and were determined by the foods consumed by the ants.

A molecular view of archaeal diversity in marine and terrestrial hot water environments

Molecular phylogenetic survey of naturally occurring archaeal diversities in hot water environments was carried out by using the PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. Mixed population DNA was directly extracted from the effluent hot water or sediment of a shallow marine hydrothermal vent at Tachibana Bay, or the acidic hot water of hot spring pools at Mt. Unzen, in Nagasaki Prefecture, Japan. Based on the partial rDNA sequences amplified with an Archaea-specific primer set, the archaeal populations of hot water environments consisted of phylogenetically and physiologically diverse group of microorganisms. The archaeal populations were varied in each sample and subject to its environmental conditions. In addition, the large number of archaeal rDNA sequences recovered from hot water environments revealed the distant relationship not only to the rDNA sequences of the cultivated thermophilic archaea, but also to the sequences of unidentified archaeal rDNA clones found in other hot water environments. The findings extend our view of archaeal diversity in hot water environments and phylogenetic organization of these organisms.

A molecular view of archaeal diversity in marine and terrestrial hot water environments

Molecular phylogenetic survey of naturally occurring archaeal diversities in hot water environments was carried out by using the PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. Mixed population DNA was directly extracted from the effluent hot water or sediment of a shallow marine hydrothermal vent at Tachibana Bay, or the acidic hot water of hot spring pools at Mt. Unzen, in Nagasaki Prefecture, Japan. Based on the partial rDNA sequences amplified with an Archaea-specific primer set, the archaeal populations of hot water environments consisted of phylogenetically and physiologically diverse group of microorganisms. The archaeal populations were varied in each sample and subject to its environmental conditions. In addition, the large number of archaeal rDNA sequences recovered from hot water environments revealed the distant relationship not only to the rDNA sequences of the cultivated thermophilic archaea, but also to the sequences of unidentified archaeal rDNA clones found in other hot water environments. The findings extend our view of archaeal diversity in hot water environments and phylogenetic organization of these organisms.

Molecular phylogenetic identification of the intestinal anaerobic microbial community in the hindgut of the termite, Reticulitermes speratus, without cultivation.

A termite maintains an anaerobic microbial community in its hindgut, which seems to be the minimum size of an anaerobic habitat. This microbial community consists of bacteria and various anaerobic flagellates, and it is established that termites are totally dependent on the microbes for the utilization of their food. The molecular phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy that does not rely on cultivation of the resident microorganisms. Small subunit ribosomal RNA (ssrRNA) genes were directly amplified from the mixed-population DNA of the termite gut by polymerase chain reaction (PCR) and clonally isolated. Most sequenced clones were phylogenetically affiliated with the four major groups of the domain Bacteria: the Proteobacteria group, the Spirochete group, the Bacteroides group, and the Low G + C gram-positive bacteria. The 16S rRNA sequence data show that the majority of the intestinal microflora of the termite consists of new species that are yet to be cultured. The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite (R. speratus) was analyzed without cultivation. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order. The diversity of nitrogen-fixing organisms was also investigated without culturing the resident microorganisms. Fragments of the nifH gene, which encodes the dinitrogenase reductase, were directly amplified from the mixed-population DNA of the termite gut and were clonally isolated. The phylogenetic analysis of the nifH amino acid sequences showed that there was a remarkable diversity of nitrogenase genes in the termite gut. The molecular phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of R. speratus was also examined by the same strategy. The whole-cell hybridization experiments indicated that the sequence originated from a large hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that the hypermastigote and trichomonads shared a recent common ancestry.

Phylogenetic analysis of the symbiotic intestinal microflora of the termite Cryptotermes domesticus

The phylogeny of microorganisms of the symbiotic community in the gut of a lower termite, Cryptotermes domesticus (order Isoptera, family Kalotermitidae), was investigated without culturing the resident microorganisms. Portions of the small-subunit rRNA genes (16S rRNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial sequences of 16S rRNA showed the existence of prokaryotic species related to the genera Methanobrevibacter, Leuconostoc, Bacteroides and Treponema, but most of the sequences were those of yet unknown species. Unique sequences showing very low sequence similarity to known 16S rRNA sequences were also found although they were significantly clustered with the High G+C Gram-positive bacteria. Comparisons of these sequences with those from the symbiotic microorganisms in other termite species revealed the existence of termite-specific groups of organisms.

Phylogenetic analysis of the symbiotic intestinal microflora of the termiteCryptotermes domesticus

The phylogeny of microorganisms of the symbiotic community in the gut of a lower termite, Cryptotermes domesticus (order Isoptera, family Kalotermitidae), was investigated without culturing the resident microorganisms. Portions of the small-subunit rRNA genes (16S rRNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial sequences of 16S rRNA showed the existence of prokaryotic species related to the genera Methanobrevibacter, Leuconostoc, Bacteroides and Treponema, but most of the sequences were those of yet unknown species. Unique sequences showing very low sequence similarity to known 16S rRNA sequences were also found although they were significantly clustered with the High G+C Gram-positive bacteria. Comparisons of these sequences with those from the symbiotic microorganisms in other termite species revealed the existence of termite-specific groups of organisms.

Phylogenetic identification of the symbiotic hypermastigote Trichonympha agilis in the hindgut of the termite Reticulitermes speratus based on small-subunit rRNA sequence.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Phylogenetic diversity of the intestinal bacterial community in the termite Reticulitermes speratus.

The phylogenetic diversity of the intestinal microflora of a lower termite, Reticulitermes speratus, was examined by a strategy which does not rely on cultivation of the resident microorganisms. Small-subunit rRNA genes (16S rDNAs) were directly amplified from the mixed-population DNA of the termite gut by the PCR and were clonally isolated. Analysis of partial 16S rDNA sequences showed the existence of well-characterized genera as well as the presence of bacterial species for which no 16S rDNA sequence data are available. Of 55 clones sequenced, 45 were phylogenetically affiliated with four of the major groups of the domain Bacteria: the Proteobacteria, the spirochete group, the Bacteroides group, and the low-G+C-content gram-positive bacteria. Within the Proteobacteria, the 16S rDNA clones showed a close relationship to those of cultivated species of enteric bacteria and sulfate-reducing bacteria, while the 16S rDNA clones in the remaining three groups showed only distant relationships to those of known organisms in these groups. Of the remaining 10 clones, among which 8 clones formed a cluster, there was only very low sequence similarity to known 16S rRNA sequences. None of these clones were affiliated with any of the major groups within the domain Bacteria. The 16S rDNA gene sequence data show that the majority of the intestinal microflora of R. speratus consists of new, uncultured species previously unknown to microbiologists.

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus.

The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite. Reticulitermes speratus, was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase (mcrA) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus, was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.

Phylogenetic analysis of the hyperthermophilic pink filament community in Octopus Spring, Yellowstone National Park.

The phylogenetic diversity of a well-known pink filament community associated with the 84 to 88 degrees C outflow from Octopus Spring, Yellowstone National Park, was examined. Three phylogenetic types ("phylotypes"), designated EM 3, EM 17, and EM 19, were identified by cloning and sequencing the small subunit rRNA genes (16S rDNA) obtained by PCR amplification of mixed-population DNA. All three phylotypes diverge deeply within the phylogenetic domain Bacteria sensu Woese (C. R. Woese, O. Kandler, and M. L. Wheelis, Proc. Natl. Acad. Sci. USA 87:4576-4579, 1990). No members of the Archaea or Eucarya were detected. EM 3 comprises a unique lineage within the Thermotogales group, and EM 17 and EM 19 are affiliated with the Aquificales. A total of 35 clones were examined, of which the majority (26 clones) were of a single sequence type (EM 17) closely related to Aquifex pyrophilus. In situ hybridization with clone-specific probes attributes the majority sequence, EM 17, to the pink filaments.

Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment.

Of the three primary phylogenetic domains--Archaea (archaebacteria), Bacteria (eubacteria), and Eucarya (eukaryotes)--Archaea is the least understood in terms of its diversity, physiologies, and ecological panorama. Although many species of Crenarchaeota (one of the two recognized archaeal kingdoms sensu Woese [Woese, C. R., Kandler, O. & Wheelis, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 4576-4579]) have been isolated, they constitute a relatively tight-knit cluster of lineages in phylogenetic analyses of rRNA sequences. It seemed possible that this limited diversity is merely apparent and reflects only a failure to culture organisms, not their absence. We report here phylogenetic characterization of many archaeal small subunit rRNA gene sequences obtained by polymerase chain reaction amplification of mixed population DNA extracted directly from sediment of a hot spring in Yellowstone National Park. This approach obviates the need for cultivation to identify organisms. The analyses document the existence not only of species belonging to well-characterized crenarchaeal genera or families but also of crenarchaeal species for which no close relatives have so far been found. The large number of distinct archaeal sequence types retrieved from this single hot spring was unexpected and demonstrates that Crenarchaeota is a much more diverse group than was previously suspected. The results have impact on our concepts of the phylogenetic organization of Archaea.

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.