The isolates included a large number of different genera and species, but at the phylum level all except one of the were members of only four bacterial phyla: Proteobacteria 82 isolates , Firmicutes 61 , Actinobacteria 29 and Bacteroidetes 4. In fact, it is a challenge to obtain isolates that do not belong to the big four, and these four phyla therefore dominate our present understanding of microbiology.
A logical question to ask is how many prokaryotic phyla there are altogether, in order to estimate how biased a sampling of four may be. Pie charts showing the phylum-level distribution of prokaryotic isolates a in the Australian Collection of Microorganisms  and b in the prokaryote genome sequences completed or in progress as of 20 August .
In the mid s, Norman Pace and colleagues outlined a molecular approach that bypassed the need to cultivate a microorganism in order to determine the sequence of its 16S rRNA gene 16S rDNA [ 12 ]. Essentially, bulk nucleic acids are extracted directly from environmental samples, 16S rDNA sequences are isolated from the bulk DNA, typically via PCR using primers broadly targeting 16S rDNAs and cloning, and these sequences are compared with known sequences Figure 2.
Gene sequences obtained in this manner 'environmental clone sequences' can then be assigned a location in a phylogenetic tree and can thus act as a marker for the organism from which they were obtained. The approach can be brought full circle by applying 16S rRNA-targeted nucleic-acid probes specific for the organisms of interest to visualize and quantify the target group in the environmental sample using techniques such as whole-cell fluorescence in situ hybridization FISH and membrane hybridization [ 13 ] Figure 2. Access to whole genomes of uncultivated organisms is also possible using the same basic approach but with large-insert cloning vectors, such as BACs, which remove the need for PCR.
Many researchers have applied the rRNA approach to a wide variety of environmental samples over the past decade and, perhaps not surprisingly given the great plate-count anomaly, the number of recognized bacterial phyla has exploded from the original estimate of 11 in [ 5 ] to 36 in [ 14 ]. This increase is due not only to environmental sequences that have filled out the tree, but also to a steady trickle of sequences from 'exotic' cultured organisms, particularly thermophiles, that highlight new lineages.
Figure 3a presents a recent conservative estimate of bacterial diversity at the phylum level; it is conservative because it includes only phyla for which at least four near-full-length 16S rDNA sequences over 1, nucleotides are known. These lineages include cultivated bacteria such as Chrysiogenes and Dictyoglomus , which are recognized as representing independent phyla in the taxonomic outline of Bergey's Manual of Systematic Bacteriology [ 8 ].
The latest tally of bacterial phyla is therefore probably nearer Evolutionary distance dendrograms of a bacterial and b archaeal diversity derived from comparative analysis of 16S rRNA gene sequences. This modified database will be available from the Ribosomal Database Project  user-submitted alignments download site . Major lineages phyla are shown as wedges with horizontal dimensions reflecting the known degree of divergence within that lineage. Phyla with cultivated representatives are in gray and, where possible, named according to the taxonomic outline of Bergey's Manual .
Phyla known only from environmental sequences are in white; because they are not formally recognized as taxonomic groups, they are usually named after the first clones found from within the group [14,20]. Note that environmental groups E2 and E3 defined in  are part of the Thermoplasmata phylum in the archaeal tree in b.
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The number of genome sequences completed or in progress for each phylum is given in brackets after the phylum name, with the exception of Methanopyrus kandleri , which is not included in the tree because it is represented by a single sequence. The scale bar represents 0.enter site
As more 16S rDNA sequences accumulate from both cultured and uncultured prokaryotes, the boundaries of existing phyla are being challenged and need to be re-evaluated. For example, the bacterial phylum Firmicutes, as currently defined [ 8 ], may not be monophyletic and may comprise at least four distinct phylum-level lineages that include the Haloanaerobiales, Thermoanerobacteriales, and Sulfobacillus groups [ 9 ]. Higher-level associations between bacterial phyla have not been resolved in 16S rDNA trees, with the exceptions of the sister-group affiliations of the Bacteroidetes and Chlorobi, and of the Chlamydiae and Verrucomicrobia [ 14 ].
Recently, trees based on concatenated ribosomal proteins obtained from complete genome sequences have suggested higher-order associations between Chlamydiae and Spirochaetes, between Thermotogae and Aquificae, and between Actinobacteria, Deinococcus-Thermus and Cyanobacteria [ 15 ]. The phylum Verrucomicrobia is also likely to be a member of the same group as Chlamydiae and Spirochaetes, given that it is a sister group to Chlamydiae; this prediction can be tested when a completed genome sequence becomes available for the Verrucomicrobia.
Several 'candidate' phyla [ 16 ], comprising only environmental clone sequences, have developed into large groups with sequence divergences similar to or greater than those within the big four phyla examples include OP11 [ 14 ] and WS6 [ 16 ] , and yet we know nothing about these lineages beyond a crude outline of their environmental distribution. Most have not even been knowingly observed under the microscope.
In a preliminary investigation of one candidate phylum, TM7, we determined that representatives of the group had typical Gram-positive cell envelopes and that they may have Archaea-like streptomycin resistance [ 17 ]. Detailed study of lineages like this one may yield insights into the evolutionary history of Gram-positive bacteria including, perhaps, a radical proposal that Gram-positive bacteria are related to Archaea [ 18 ] , which so far appear to have a restricted phylum-level distribution within the bacterial domain Actinobacteria and Firmicutes.
TM7 bacteria have also been implicated in human subgingival gum disease, which might promote their study [ 19 ]. The Archaea are formally divided into two phyla, Crenarchaeota and Euryarchaeota, from 16S rRNA phylogeny [ 8 ], but these groupings may be artifacts because analysis of concatenated ribosomal protein sequences suggests that Euryarchaeota, at least, is not a monophyletic group [ 15 ]. Figure 3b presents a current estimate of the major lineages in the archaeal 16S rDNA tree below the level of the Crenarchaeota and Euryarchaeota indicated to the right of the tree , using the same criteria and annotation used for the bacterial tree Figure 3a.
A higher tally of 23 phyla is arrived at if lineages not meeting the selection criteria are included in the estimate. These include Methanopyri [ 8 ], currently represented by a single sequence, and environmental group C3 [ 20 ], which has no full-length representatives. Most archaeal research has concentrated on the cultivated methanogenic such as Methanococci and thermophilic such as Thermoprotei and Thermococci lineages Figure 3b.
As is the case with the Bacteria, most candidate archeal phyla are completely uncharacterized at this point. A notable exception is candidate phylum C1 Figure 3b , which contains Cenarchaeum symbiosum , an uncultured archaeon that has been amenable to detailed study, including partial genome sequencing, because it exists as a near monoculture in a marine sponge [ 21 ]. Members of the C1 group are particularly prevalent in marine habitats [ 22 ]. The advent of large-scale DNA sequencing has provided unprecedented access to molecular data for inferring the tree of life. Currently, complete genome sequences of prokaryotes have been obtained only from pure cultures and hence, at the phylum level, microbial genomics reflects the bias towards the big four phyla Figures 1b , 3.
Increasing efforts are being made to select phylogenetically diverse prokaryotes Archaea for example for genome sequencing, using the 16S rRNA phylogeny as a guide [ 24 ].
Bergey's Manual of Systematic Bacteriology
But is selection solely on the basis of an exotic location in a 16S rRNA tree justified? The implicit assumption is that the evolutionary history of 16S rRNA represents the evolutionary history of the whole organism the whole genome , but the concept of a unified organismal phylogeny has been significantly compromised by the finding of widespread lateral gene transfer LGT between organisms [ 25 ].
LGT appears to affect the informational genes those involved in transcription and translation to a lesser extent than metabolic and other operational genes, leading to the hypothesis that a core set of vertically transmitted informational genes define organismal phylogeny [ 26 ]. Recent evidence suggests that this may not be the case for the Euryarchaeota, however; here, informational genes are apparently no less subject to LGT than operational genes [ 27 ]. Reliable detection of LGT by comparison of gene trees is complicated by gene duplication and loss [ 23 ], and different methods for detecting LGT are not particularly consistent [ 28 ].
The extent to which LGT blurs organismal phylogenies is therefore unclear at this point. At one extreme, if genomes are largely chimeric assemblages of genes with different histories, then any random sampling of organisms should provide a representative 'window' into genome space. On the other hand, if a core of vertically transmitted genes which includes 16S rDNA defines the organism, then striving to obtain genome sequences from all major lineages in the 16S rRNA tree [ 24 ] seems justified.
Either way, a more complete sampling of phyla defined using 16S rRNA should help to resolve the issue. The number of prokaryote genome-sequencing projects completed or in progress as of 20 August [ 29 ] is shown for each phylum-level lineage in the bacterial Figure 3a and archaeal Figure 3b domains. Several bacterial phyla that have cultivated representatives have no sequenced genomes Table 1.
Exploring prokaryotic diversity in the genomic era | Genome Biology | Full Text
These should provide compelling targets for future genome-sequencing projects. Phylum-level lineages comprising only environmental clone sequences Figure 3 also need to be sampled for genome sequences; this could best be achieved by obtaining one or more representatives of each phylum in pure culture. The classical approach to cultivating microorganisms is to prepare a solid or liquid growth medium containing an appropriate carbon source, energy source and electron acceptor depending on the physiological type of organism being isolated. The medium is then inoculated with a suitable source of microorganisms and left to incubate at a desired growth temperature until organisms multiply to the point at which we become aware of their presence by colony formation or increased turbidity.
This approach is not phylogenetically directed, however, and, as discussed above, typically ends up collecting fast-multiplying microbial weeds.
To isolate representatives of novel environmental lineages, a directed form of cultivation is required. In one such approach, the first step is to select a target group and design group-specific oligonucleotide probes [ 30 ] to detect or visualize the target organisms in environmental samples Figure 2. Bacteroidetes 5. Chlamydiae 6.
Chlorobi 7. Chloroflexi 8. Chrysiogenetes 9. Crenarchaeota Cyanobacteria Deferribacteres Deinococcus-Thermus Dictyoglomi Euryarchaeota Fibrobacteres Firmicutes Fusobacteria Gemmatimonadetes Lentisphaerae Nitrospirae Planctomycetes Proteobacteria Spirochaetes Synergistetes Tenericutes Thaumarchaeota Thermodesulfobacteria Thermomicrobia Thermotogae Verrucomicrobia 28 Acidithiobacillales 2 2. Aeromonadales 2 3.
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Alteromonadales 1 4. Cardiobacteriales 1 5. Chromatiales 3 6. Enterobacteriales 1 7.