Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

Because a natural entity “species” cannot be recognized as a group of strains that is genetically well separated from its phylogenetic neighbors, a pragmatic approach was taken to define a species by a polyphasic approach (L. G. Wayne, D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Truper, Int. J. Syst. Bacteriol. 37:463-464, 1987), in which a DNA reassociation value of about 70% plays a dominant role. With the establishment of rapid sequence analysis of 16S rRNA and the recognition of its potential to determine the phylogenetic position of any prokaryotic organism, the role of 16S rRNA similarities in the present species definition in bacteriology needs to be clarified. Comparative studies clearly reveal the limitations of the sequence analysis of this conserved gene and gene product in the determination of relationships at the strain level for which DNA-DNA reassociation experiments still constitute the superior method. Since today the primary structure of 16S rRNA is easier to determine than hybridization between DNA strands, the strength of the sequence analysis is to recognize the level at which DNA pairing studies need to be performed, which certainly applies to similarities of 97% and higher.

Taxonomic Note: A Place for DNA-DNA Reassociation and 16S rRNA Sequence Analysis in the Present Species Definition in Bacteriology

Interest in the role of the microbiome in human health has burgeoned over the past decade with the advent of new technologies for interrogating complex microbial communities. The large-scale dynamics of the microbiome can be described by many of the tools and observations used in the study of population ecology. Deciphering the metagenome and its aggregate genetic information can also be used to understand the functional properties of the microbial community. Both the microbiome and metagenome probably have important functions in health and disease; their exploration is a frontier in human genetics.

/pdf/the-human-microbiome-at-the-interface-of-health-and-disease-8umvuebp9h.pdf

The human microbiome: at the interface of health and disease

▪ Abstract Since the delineation of 12 bacterial phyla by comparative phylogenetic analyses of 16S ribosomal RNA in 1987 knowledge of microbial diversity has expanded dramatically owing to the sequencing of ribosomal RNA genes cloned from environmental DNA. Currently, only 26 of the approximately 52 identifiable major lineages, or phyla, within the domain Bacteria have cultivated representatives. Evidence from field studies indicates that many of the uncultivated phyla are found in diverse habitats, and some are extraordinarily abundant. In some important environments, including seawater, freshwater, and soil, many biologically and geochemically important organisms are at best only remotely related to any strain that has been characterized by phenotype or by genome sequencing. Genome sequence information that would allow ribosomal RNA gene trees to be related to broader patterns in microbial genome evolution is scant, and therefore microbial diversity remains largely unexplored territory.

The Uncultured Microbial Majority

Insect heritable symbionts have proven to be ubiquitous, based on molecular screening of various insect lineages. Recently, molecular and experimental approaches have yielded an immensely richer understanding of their diverse biological roles, resulting in a burgeoning research literature. Increasingly, commonalities and intermediates are being discovered between categories of symbionts once considered distinct: obligate mutualists that provision nutrients, facultative mutualists that provide protection against enemies or stress, and symbionts such as Wolbachia that manipulate reproductive systems. Among the most farreaching impacts of widespread heritable symbiosis is that it may promote speciation by increasing reproductive and ecological isolation of host populations, and it effectively provides a means for transfer of genetic information among host lineages. In addition, insect symbionts provide some of the extremes of cellular genomes, including the smallest and the fastest evolving, raising new questions about the limits of evolution of life.

Genomics and Evolution of Heritable Bacterial Symbionts

The primary endosymbionts of aphids are maternally inherited bacteria that live only within specialized host cells. Phylogenetic analysis of the 16S ribosomal DNA sequences of aphid endosymbionts reveals that they are a monophyletic group with a phylogeny completely concordant with that of their hosts, implying long-term cospeciation. Here we show that rates of base substitution are similar in the 16.S ribosomal DNA of different endosymbiont lineages. In addition, we calibrate these rates by assigning age estimates for ancestral aphid hosts to the corresponding endosymbionts. The resulting rate estimates (1—2% per 50 Ma) are among the most reliable available for prokaryotes. They are very near values previously conjectured by using more tenuous assumptions for dating divergence events in eubacteria. Rates calibrated using dates inferred from fossil aphids imply that Asian and American species of the aphid tribe Melaphidina diverged by the early Eocene; this result confirms an earlier hypothesis based on biogeographic evidence. Based on these rate estimates, the minimum age of this endosymbiotic association and the age of aphids as a whole is estimated at 160-280 Ma.

A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts

Aphids (superfamily Aphidoidea) contain eubacterial endosymbionts localized within specialized cells (mycetocytes). The endosymbionts are essential for the survival of the aphid hosts. Sequence analyses of the 16S rRNAs from endosymbionts of 11 aphid species from seven tribes and four families have indicated that the endosymbionts are monophyletic. Furthermore, phylogenetic relationships within the symbiont clade parallel the relationships of the corresponding aphid hosts. Our findings suggest that this endocytobiotic association was established in a common ancestor of the four aphid families with subsequent diversification into the present species of aphids and their endosymbionts.

Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families.

All aphids are thought to have an association with primary endosymbionts, which are located within specialized cells known as mycetocytes. The primary endosymbionts are gram-negative, spherical or slightly oval cells which have not been cultivated outside aphid hosts. Recently, it has been shown that the 16S rRNA sequences of the primary endosymbionts of 11 host species belonging to four aphid families are similar and thus that these organisms are related. Comparisons with the 16S rRNAs of other procaryotes indicated that the primary endosymbionts form a distinct lineage within the gamma-3 subgroup of the Proteobacteria. On the basis of these results we propose that the primary endosymbionts should be placed in Buchnera gen. nov. and Buchnera aphidicola sp. nov. The primary endosymbiont found in the mycetocytes of Schizaphis graminum (green bug) is designated the type strain of this species.

Buchnera gen. nov. and Buchnera aphidicola sp. nov., a Taxon Consisting of the Mycetocyte-Associated, Primary Endosymbionts of Aphids

Whiteflies (superfamily Aleyrodoidea) contain eubacterial endosymbionts localized within host cells known as mycetocytes. Sequence analysis of the genes for the 16S rRNA of the endosymbionts ofBemisia tabaci, Siphoninus phillyreae, andTrialeurodes vaporariorum indicates that these organisms are closely related and constitute a distinct lineage within the γ-subdivision of theProteobacteria. The endosymbionts of whiteflies are unrelated to the endosymbionts of aphids and mealybugs, which are in two separate lineages.

/pdf/the-eubacterial-endosymbionts-of-whiteflies-homoptera-3t15m3awh3.pdf

Mark A. Munson

Papers

A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts

Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families.

Buchnera gen. nov. and Buchnera aphidicola sp. nov., a Taxon Consisting of the Mycetocyte-Associated, Primary Endosymbionts of Aphids

The Eubacterial Endosymbionts of Whiteflies (Homoptera: Aleyrodoidea) Constitute a Lineage Distinct from the Endosymbionts of Aphids and Mealybugs

Phylogenetic relationships of the endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 165 rDNA sequences