Ribosomal DNA (rDNA) sequences have been aligned and compared in a number of living organisms, and this approach has provided a wealth of information about phylogenetic relationships. Studies of rDNA sequences have been used to infer phylogenetic history across a very broad spectrum, from studies among the basal lineages of life to relationships among closely related species and populations. The reasons for the systematic versatility of rDNA include the numerous rates of evolution among different regions of rDNA (both among and within genes), the presence of many copies of most rDNA sequences per genome, and the pattern of concerted evolution that occurs among repeated copies. These features facilitate the analysis of rDNA by direct RNA sequencing, DNA sequencing (either by cloning or amplification), and restriction enzyme methodologies. Constraints imposed by secondary structure of rRNA and concerted evolution need to be considered in phylogenetic analyses, but these constraints do not appear to impede seriously the usefulness of rDNA. An analysis of aligned sequences of the four nuclear and two mitochondrial rRNA genes identified regions of these genes that are likely to be useful to address phylogenetic problems over a wide range of levels of divergence. In general, the small subunit nuclear sequences appear to be best for elucidating Precambrian divergences, the large subunit nuclear sequences for Paleozoic and Mesozoic divergences, and the organellar sequences of both subunits for Cenozoic divergences. Primer sequences were designed for use in amplifying the entire nuclear rDNA array in 15 sections by use of the polymerase chain reaction; these "universal" primers complement previously described primers for the mitochondrial rRNA genes. Pairs of primers can be selected in conjunction with the analysis of divergence of the rRNA genes to address systematic problems throughout the hierarchy of life.

Ribosomal DNA: molecular evolution and phylogenetic inference.

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

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Listeria pathogenesis and molecular virulence determinants.

BACTERIOPLANKTON are recognized as important agents of biogeochemical change in marine ecosystems, yet relatively little is known about the species that make up these communities. Uncertainties about the genetic structure and diversity of natural bacterioplankton populations stem from the traditional difficulties associated with microbial cultivation techniques. Discrepancies between direct counts and plate counts are typically several orders of magnitude, raising doubts as to whether cultivated marine bacteria are actually representative of dominant planktonic species1–3. We have phylogenetically analysed clone libraries of eubacterial 16S ribosomal RNA genes amplified from natural populations of Sargasso Sea picoplankton by the polymerase chain reaction4. The analysis indicates the presence of a novel microbial group, the SAR 11 cluster, which appears to be a significant component of this oligotrophic bacterioplankton community. A second cluster of lineages related to the oxygenic phototrophs—cyanobacteria, prochlorophytes and chloroplasts—was also observed. However, none of the genes matched the small subunit rRNA sequences of cultivated marine cyanobacteria from similar habitats. The diversity of 16S rRNA genes observed within the clusters suggests that these bacterioplankton may be consortia of independent lineages sharing surprisingly distant common ancestors.

Genetic diversity in Sargasso Sea bacterioplankton.

A new hierarchic classification structure for the taxa between the taxonomic levels of genus and class is Proposed for the actinomycete line of descent as defined by analysis of small subunit (16S) rRNA and genes coding for this molecule (rDNA). While the traditional circumscription of a genus of the actinomycete subphylum is by and large in accord with the 16S rRNA/rDNA-based phylogenetic clustering of these organisms. most of the higher taxa proposed in the past do not take into account the phylogenetic clustering of genera. The rich chemical, morphological and physiological diversity of phylogenetically closely related genera makes the description of families and higher taxa so broad that they become meaningless for the description of the enclosed taxa. Here we present a classification system in which phylogenetically neighboring taxa at the genus level are clustered into families, suborders, orders, subclasses, and a class irrespective of those phenotypec characteristics on which the delineation of taxa has been based in the past. Rather than being based on a listing of a wide array of chemotaxonomic, morphological, and physiological properties, the delineation is based solely on 16S rDNA/rRNA sequence-based phylogenetic clustering and the presence of taxon-specific 16S rDNA RNA signature nucleotides.

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Proposal for a New Hierarchic Classification System, Actinobacteria classis nov.

Actinobacteria are Gram-positive bacteria with high G+C DNA content that constitute one of the largest bacterial phyla, and they are ubiquitously distributed in both aquatic and terrestrial ecosystems. Many Actinobacteria have a mycelial lifestyle and undergo complex morphological differentiation. They also have an extensive secondary metabolism and produce about two-thirds of all naturally derived antibiotics in current clinical use, as well as many anticancer, anthelmintic, and antifungal compounds. Consequently, these bacteria are of major importance for biotechnology, medicine, and agriculture. Actinobacteria play diverse roles in their associations with various higher organisms, since their members have adopted different lifestyles, and the phylum includes pathogens (notably, species of Corynebacterium, Mycobacterium, Nocardia, Propionibacterium, and Tropheryma), soil inhabitants (e.g., Micromonospora and Streptomyces species), plant commensals (e.g., Frankia spp.), and gastrointestinal commensals (Bifidobacterium spp.). Actinobacteria also play an important role as symbionts and as pathogens in plant-associated microbial communities. This review presents an update on the biology of this important bacterial phylum.

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Taxonomy, Physiology, and Natural Products of Actinobacteria

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

Phylogenetic Analysis of the Genus Listeria Based on Reverse Transcriptase Sequencing of 16S rRNA

Reverse transcriptase sequencing of 16S ribosomal ribonucleic acids of some representatives of the genus Rhodococcus were performed in an attempt to clarify their intra- and inter-generic relationships. The results indicate that the genus Rhodococcus as presently constituted is phylogenetically heterogeneous. On the basis of the 16S ribosomal-RNA sequence and earlier chemical data it is proposed that the genus Gordona (Tsukamura) be revived to accommodate those rhodococcal species containing relatively long-chain mycolic acids (48 to 66 carbon atoms) and MK-9(H2) as the predominant menaquinone type.

Evidence of phylogenetic heterogeneity within the genus rhodococcus: revival of the genus gordona (tsukamura)

The Phylogeny of Mycolate-less Wall Chemotype IV Actinomycetes and Description of Pseudonocardiaceae fam. nov.

Reverse transcriptase sequencing of long regions of 16S ribosomal ribonucleic acids of Corynebacterium paurometabolum and Rhodococcus aurantiacus was performed to determine the relationship of these organisms to other mycolic acid-containing actinomycetes These two species were highly related to each other (more than 99% sequence homology in a large stretch of 1,184 nucleotides) but were distinct from the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus On the basis of our findings and previous findings we propose that C paurometabolum and R aurantiacus be reduced to a single species and reclassified in a new genus, Tsukamurella The type species is Tsukamurella paurometabolum comb nov

Tsukamurella gen. nov. Harboring Corynebacterium paurometabolum and Rhodococcus aurantiacus

The phylogenetic position of various budding and/or or prosthecate Gram-negative eubacteria was determined by different methods. Members of the genera Hyphomicrobium, Filomicrobium, Pedomicrobium were investigated by 16S rRNA cataloguing, a 1373 nucleotide long portion of the 16S rRNA was sequenced from Hyphomicrobium vulgare and the 5S rRNAs were analyzed from two Hyphomicrobium strains, Hyphomonas polymorpha and Caulobacter crescentus. Comparison with published sequences indicated a membership of all of these organisms to the alpha subdivision of purple bacteria. While C. crescentus and Hyphomonas polymorpha constitute separate individual lines of descent, the position of a coherent cluster embracing Hyphomicrobium, Pedomicrobium and Filomicrobium is not yet settled. 16S rRNA cataloguing indicate the presence of a distinct line equivalent to other subgroups in its phylogenetic depth. 5S rRNA analysis, on the other hand, groups Hyphomicrobium vulgare and strain IFAM 1761 with members of subgroup alpha-2 (Rhodopseudomonas palustris, Nitrobacter winogradskyi and relatives). In contrast to the present classification, Pedomicrobium ferrugineum and Filimicrobium fusiforme are more closely related to certain Hyphomicrobium strains than these are related among each other. Budding mode of reproduction and prosthecate morphology are dominating morphological features of members of the alpha subdivision. These characteristics may gain diagnostic significance in a future formal description of this subdivision and its subgroups as a higher rank.

A phylogenetic survey of budding, and/or prosthecate, non-phototrophic eubacteria: membership of Hyphomicrobium, Hyphomonas, Pedomicrobium, Filomicrobium, Caulobacter and "dichotomicrobium" to the alpha-subdivision of purple non-sulfur bacteria.

Jan Smida

Papers

Phylogenetic Analysis of the Genus Listeria Based on Reverse Transcriptase Sequencing of 16S rRNA

Evidence of phylogenetic heterogeneity within the genus rhodococcus: revival of the genus gordona (tsukamura)

The Phylogeny of Mycolate-less Wall Chemotype IV Actinomycetes and Description of Pseudonocardiaceae fam. nov.

Tsukamurella gen. nov. Harboring Corynebacterium paurometabolum and Rhodococcus aurantiacus

A phylogenetic survey of budding, and/or prosthecate, non-phototrophic eubacteria: membership of Hyphomicrobium, Hyphomonas, Pedomicrobium, Filomicrobium, Caulobacter and "dichotomicrobium" to the alpha-subdivision of purple non-sulfur bacteria.