A galactose-specific sugar: phosphotransferase permease is prevalent in the non-core genome of Streptococcus mutans.

Three genes predicted to encode the A, B and C domains of a sugar : phosphotransferase system (PTS) permease specific for galactose\(EII(Gal) ) were identified in the genomes of 35 of 57 recently sequenced isolates of Streptococcus mutans, the primary etiological agent of human dental caries. Mutants defective in the EII(Gal) complex were constructed in six of the isolates and showed markedly reduced growth rates on galactose-based medium relative to the parental strains. An EII(Gal) -deficient strain constructed using the invasive serotype f strain OMZ175 (OMZ/IIGal) expressed significantly lower PTS activity when galactose was present as the substrate. Galactose was shown to be an effective inducer of catabolite repression in OMZ175, but not in the EII(Gal) -deficient strain. In a mixed-species competition assay with galactose as the sole carbohydrate source, OMZ/IIGal was less effective than the parental strain at competing with the oral commensal bacterium Streptococcus gordonii, which has a high-affinity galactose transporter. Hence, a significant proportion of S. mutans strains encode a galactose PTS permease that could enhance the ability of these isolates to compete more effectively with commensal streptococci for galactose in salivary constituents and the diet.

Outcome of infection of C57BL/6 IL-10(-/-) mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up- and down-regulated in vivo.

Human Campylobacter jejuni infection can result in an asymptomatic carrier state, watery or bloody diarrhea, bacteremia, meningitis, or autoimmune neurological sequelae. Infection outcomes of C57BL/6 IL-10(-/-) mice orally infected with twenty-two phylogenetically diverse C. jejuni strains were evaluated to correlate colonization and disease phenotypes with genetic composition of the strains. Variation between strains was observed in colonization, timing of development of clinical signs, and occurrence of enteric lesions. Five pathotypes of C. jejuni in C57BL/6 IL-10(-/-) mice were delineated: little or no colonization, colonization without disease, colonization with enteritis, colonization with hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. Virulence gene content of ten sequenced strains was compared in silico; virulence gene content of twelve additional strains was compared using a C. jejuni pan-genome microarray. Neither total nor virulence gene content predicted pathotype; nor was pathotype correlated with multilocus sequence type. Each strain was unique with regard to absences of known virulence-related loci and/or possession of point mutations and indels, including phase variation, in virulence-related genes. An experiment in C. jejuni 11168-infected germ-free mice showed that expression levels of ninety open reading frames (ORFs) were significantly up- or down-regulated in the mouse cecum at least two-fold compared to in vitro growth. Genomic content of these ninety C. jejuni 11168 ORFs was significantly correlated with the capacity to colonize and cause enteritis in C57BL/6 IL-10(-/-) mice. Differences in gene expression levels and patterns are thus an important determinant of pathotype in C. jejuni strains in this mouse model.

Progress dissecting the oral microbiome in caries and health.

Recent rapid advances in "-omics" technologies have yielded new insights into the interaction of the oral microbiome with its host. Associations of species that are usually considered to be acid-tolerant with caries have been confirmed, while some recognized as health-associated are often present in greater proportions in the absence of caries. In addition, some newly identified bacteria have been suggested as potential contributors to the caries process. In spite of this progress, two major challenges remain. The first is that there is a great deal of heterogeneity in the phenotypic capabilities of individual species of oral bacteria. The second is that the most abundant taxa in oral biofilms display remarkable phenotypic plasticity, i.e., the bacteria associated most strongly with health or with caries can morph rapidly in response to alterations in environmental pH, carbohydrate availability and source, and oxygen tension and redox environment. However, new technologic advances coupled with "old-fashioned microbiology" are starting to erode the barriers to a more complete understanding of oral biofilm physiology and ecology, and in doing so are beginning to provide insights for the creation of novel cost-effective caries control therapies.

Resolution of the early placental mammal radiation using Bayesian phylogenetics.

Molecular phylogenetic studies have resolved placental mammals into four major groups, but have not established the full hierarchy of interordinal relationships, including the position of the root. The latter is critical for understanding the early biogeographic history of placentals. We investigated placental phylogeny using Bayesian and maximum-likelihood methods and a 16.4-kilobase molecular data set. Interordinal relationships are almost entirely resolved. The basal split is between Afrotheria and other placentals, at about 103 million years, and may be accounted for by the separation of South America and Africa in the Cretaceous. Crown-group Eutheria may have their most recent common ancestry in the Southern Hemisphere (Gondwana).

The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua.

The mammalian order Xenarthra (armadillos, anteaters and sloths) is one of the four major clades of placentals, but it remains poorly studied from the molecular phylogenetics perspective. We present here a study encompassing most of the order's diversity in order to establish xenarthrans' intra-ordinal relationships, discuss the evolution of their morphological characters, search for their extant sister group and specify the timing of their radiation with special emphasis on the status of the controversial fossil Eurotamandua. Sequences of three genes (nuclear exon 28 of the Von Willebrand factor and mitochondrial 12S and 16S rRNAs) are compared for eight of the 13 living genera. Phylogenetic analyses confirm the order's monophyly and that of its three major lineages: armadillos (Cingulata), anteaters (Vermilingua) and sloths ('Tardigrada', renamed in 'Folivora'), and our results strongly support the grouping of hairy xenarthrans (anteaters and sloths) into Pilosa. Within placentals, Afrotheria might be the first lineage to branch off, followed by Xenarthra. The morphological adaptative convergence between New World xenarthrans and Old World pangolins is confirmed. Molecular datings place the early emergence of armadillos around the Cretaceous/Tertiary boundary, followed by the divergence between anteaters and sloths in the Early Eocene era. These Tertiary dates contradict the concept of a very ancient origin of modern xenarthran lineages. They also question the placement of the purported fossil anteater (Eurotamandua) from the Middle Eocene period of Europe with the Vermilingua and instead suggest the independent and convergent evolution of this enigmatic taxon.

Phylogenetic analyses do not support horizontal gene transfers from bacteria to vertebrates.

Horizontal gene transfer (HGT) has long been recognized as a principal force in the evolution of genomes. Genome sequences of Archaea and Bacteria have revealed the existence of genes whose similarity to loci in distantly related organisms is explained most parsimoniously by HGT events. In most multicellular organisms, such genetic fixation can occur only in the germ line. Therefore, it is notable that the publication of the human genome reports 113 incidents of direct HGT between bacteria and vertebrates, without any apparent occurrence in evolutionary intermediates, that is, non-vertebrate eukaryotes. Phylogenetic analysis arguably provides the most objective approach for determining the occurrence and directionality of HGT. Here we report a phylogenetic analysis of 28 proposed HGT genes, whose presence in the human genome had been confirmed by polymerase chain reaction (PCR). The results indicate that most putative HGT genes are present in more anciently derived eukaryotes (many such sequences available in non-vertebrate EST databases) and can be explained in terms of descent through common ancestry. They are, therefore, unlikely to be examples of direct HGT from bacteria to vertebrates.

Universal trees based on large combined protein sequence data sets.

Universal trees of life based on small-subunit (SSU) ribosomal RNA (rRNA) support the separate mono/holophyly of the domains Archaea (archaebacteria), Bacteria (eubacteria) and Eucarya (eukaryotes) and the placement of extreme thermophiles at the base of the Bacteria. The concept of universal tree reconstruction recently has been upset by protein trees that show intermixing of species from different domains. Such tree topologies have been attributed to either extensive horizontal gene transfer or degradation of phylogenetic signals because of saturation for amino acid substitutions. Here we use large combined alignments of 23 orthologous proteins conserved across 45 species from all domains to construct highly robust universal trees. Although individual protein trees are variable in their support of domain integrity, trees based on combined protein data sets strongly support separate monophyletic domains. Within the Bacteria, we placed spirochaetes as the earliest derived bacterial group. However, elimination from the combined protein alignment of nine protein data sets, which were likely candidates for horizontal gene transfer, resulted in trees showing thermophiles as the earliest evolved bacterial lineage. Thus, combined protein universal trees are highly congruent with SSU rRNA trees in their strong support for the separate monophyly of domains as well as the early evolution of thermophilic Bacteria.

Integrated fossil and molecular data reconstruct bat echolocation.

Molecular and morphological data have important roles in illuminating evolutionary history. DNA data often yield well resolved phylogenies for living taxa, but are generally unattainable for fossils. A distinct advantage of morphology is that some types of morphological data may be collected for extinct and extant taxa. Fossils provide a unique window on evolutionary history and may preserve combinations of primitive and derived characters that are not found in extant taxa. Given their unique character complexes, fossils are critical in documenting sequences of character transformation over geologic time and may elucidate otherwise ambiguous patterns of evolution that are not revealed by molecular data alone. Here, we employ a methodological approach that allows for the integration of molecular and paleontological data in deciphering one of the most innovative features in the evolutionary history of mammals-laryngeal echolocation in bats. Molecular data alone, including an expanded data set that includes new sequences for the A2AB gene, suggest that microbats are paraphyletic but do not resolve whether laryngeal echolocation evolved independently in different microbat lineages or evolved in the common ancestor of bats and was subsequently lost in megabats. When scaffolds from molecular phylogenies are incorporated into parsimony analyses of morphological characters, including morphological characters for the Eocene taxa Icaronycteris, Archaeonycteris, Hassianycteris, and Palaeochiropteryx, the resulting trees suggest that laryngeal echolocation evolved in the common ancestor of fossil and extant bats and was subsequently lost in megabats. Molecular dating suggests that crown-group bats last shared a common ancestor 52 to 54 million years ago.

rbcL sequences reveal multiple cryptic introductions of the Japanese red alga Polysiphonia harveyi.

In Europe, the last 20 years have seen a spectacular increase in accidental introductions of marine species, but it has recently been suggested that both the actual number of invaders and their impacts have been seriously underestimated because of the prevalence of sibling species in marine habitats. The red alga Polysiphonia harveyi is regarded as an alien in the British Isles and Atlantic Europe, having appeared in various locations there during the past 170 years. Similar or conspecific populations are known from Atlantic North America and Japan. To choose between three competing hypotheses concerning the origin of P. harveyi in Europe, we employed rbcL sequence analysis in conjunction with karyological and interbreeding data for samples and isolates of P. harveyi and various congeners from the Pacific and North Atlantic Oceans. All cultured isolates of P. harveyi were completely interfertile, and there was no evidence of polyploidy or aneuploidy. Thus, this biological species is both morphologically and genetically variable: intraspecific rbcL divergences of up to 2.1% are high even for red algae. Seven rbcL haplotypes were identified. The four most divergent haplotypes were observed in Japanese samples from Hokkaido and south-central Honshu, which are linked by hypothetical 'missing' haplotypes that may be located in northern Honshu. These data are consistent with Japan being the centre of diversity and origin for P. harveyi. Two non-Japanese lineages were linked to Hokkaido and Honshu, respectively. A single haplotype was found in all North Atlantic and Mediterranean accessions, except for North Carolina, where the haplotype found was the same as that invading in New Zealand and California. The introduction of P. harveyi into New Zealand has gone unnoticed because P. strictissima is a morphologically indistinguishable native sibling species. The sequence divergence between them is 4-5%, greater than between some morphologically distinct red algal species. Two different types of cryptic invasions of P. harveyi have therefore occurred. In addition to its introduction as a cryptic sibling species in New Zealand, P. harveyi has been introduced at least twice into the North Atlantic from presumed different source populations. These two introductions are genetically and probably also physiologically divergent but completely interfertile.

Systematic identification of selective essential genes in Helicobacter pylori by genome prioritization and allelic replacement mutagenesis.

A comparative genomic approach was used to identify Helicobacter pylori 26695 open reading frames (ORFs) which are conserved in H. pylori J99 but highly diverged in other eubacteria. A survey of selected pathways of central intermediary metabolism was also carried out, and genes with a potentially selective role in H. pylori were identified. Forty-five ORFs identified in these two analyses were screened using a rapid vector-free allelic replacement mutagenesis technique, and 33 were shown to be essential in vitro. Notably, 13 ORFs gave essentiality results which are unexpected in view of their known or proposed functions, and phylogenetic analysis was used to investigate the annotation of 7 such ORFs which are highly diverged. We propose that the products of a number of these H. pylori-specific essential genes may be suitable targets for novel anti-H. pylori therapies.

Mitochondrial versus nuclear gene sequences in deep-level mammalian phylogeny reconstruction.

Both mitochondrial and nuclear gene sequences have been employed in efforts to reconstruct deep-level phylogenetic relationships. A fundamental question in molecular systematics concerns the efficacy of different types of sequences in recovering clades at different taxonomic levels. We compared the performance of four mitochondrial data sets (cytochrome b, cytochrome oxidase II, NADH dehydrogenase subunit I, 12S rRNA-tRNA-16S rRNA) and eight nuclear data sets (exonic regions of alpha-2B adrenergic receptor, aquaporin, ss-casein, gamma-fibrinogen, interphotoreceptor retinoid binding protein, kappa-casein, protamine, von Willebrand Factor) in recovering deep-level mammalian clades. We employed parsimony and minimum-evolution with a variety of distance corrections for superimposed substitutions. In 32 different pairwise comparisons between these mitochondrial and nuclear data sets, we used the maximum set of overlapping taxa. In each case, the variable-length bootstrap was used to resample at the size of the smaller data set. The nuclear exons consistently performed better than mitochondrial protein and rRNA-tRNA coding genes on a per-residue basis in recovering benchmark clades. We also concatenated nuclear genes for overlapping taxa and made comparisons with concatenated mitochondrial protein-coding genes from complete mitochondrial genomes. The variable-length bootstrap was used to score the recovery of benchmark clades as a function of the number of resampled base pairs. In every case, the nuclear concatenations were more efficient than the mitochondrial concatenations in recovering benchmark clades. Among genes included in our study, the nuclear genes were much less affected by superimposed substitutions. Nuclear genes having appropriate rates of substitution should receive strong consideration in efforts to reconstruct deep-level phylogenetic relationships.

Parallel adaptive radiations in two major clades of placental mammals.

Higher level relationships among placental mammals, as well as the historical biogeography and morphological diversification of this group, remain unclear. Here we analyse independent molecular data sets, having aligned lengths of DNA of 5,708 and 2,947 base pairs, respectively, for all orders of placental mammals. Phylogenetic analyses resolve placental orders into four groups: Xenarthra, Afrotheria, Laurasiatheria, and Euarchonta plus Glires. The first three groups are consistently monophyletic with different methods of analysis. Euarchonta plus Glires is monophyletic or paraphyletic depending on the phylogenetic method. A unique nine-base-pair deletion in exon 11 of the BRCA1 gene provides additional support for the monophyly of Afrotheria, which includes proboscideans, sirenians, hyracoids, tubulidentates, macroscelideans, chrysochlorids and tenrecids. Laurasiatheria contains cetartiodactyls, perissodactyls, carnivores, pangolins, bats and eulipotyphlan insectivores. Parallel adaptive radiations have occurred within Laurasiatheria and Afrotheria. In each group, there are aquatic, ungulate and insectivore-like forms.

Protein sequence signatures support the African clade of mammals.

DNA sequence evidence supports a superordinal clade of mammals that comprises elephants, sea cows, hyraxes, aardvarks, elephant shrews, golden moles, and tenrecs, which all have their origins in Africa, and therefore are dubbed Afrotheria. Morphologically, this appears an unlikely assemblage, which challenges-by including golden moles and tenrecs-the monophyly of the order Lipotyphla (Insectivora). We here identify in three proteins unique combinations of apomorphous amino acid replacements that support this clade. The statistical support for such "sequence signatures" as unambiguous synapomorphic evidence for the naturalness of the Afrotherian clade is reported. Using likelihood, combinatorial, and Bayesian methods we show that the posterior probability of the mammalian tree containing the Afrotherian clade is effectively 1.0, based on conservative assumptions. Presenting sequence data for another African insectivore, the otter shrew Micropotamogale lamottei, we demonstrate that such signatures are diagnostic for including newly investigated species in the Afrotheria. Sequence signatures provide "protein-morphological" synapomorphies that may aid in visualizing monophyletic groupings.

Independent adaptation to riverine habitats allowed survival of ancient cetacean lineages.

The four species of "river dolphins" are associated with six separate great river systems on three subcontinents and have been grouped for more than a century into a single taxon based on their similar appearance. However, several morphologists recently questioned the monophyly of that group. By using phylogenetic analyses of nucleotide sequences from three mitochondrial and two nuclear genes, we demonstrate with statistical significance that extant river dolphins are not monophyletic and suggest that they are relict species whose adaptation to riverine habitats incidentally insured their survival against major environmental changes in the marine ecosystem or the emergence of Delphinidae.

Molecular phylogenetic examination of the delphinoidea trichotomy: congruent evidence from three nuclear loci indicates that porpoises (Phocoenidae) share a more recent common ancestry with white whales (Monodontidae) than they do with true dolphins (Delphinidae).

Porpoises (Phocoenidae), dolphins (Delphinidae), and the two species of Monodontidae (beluga and narwhal) together constitute the superfamily Delphinoidea. Although there is extensive evidence supporting the monophyly of this superfamily, previous studies involving morphology, as well as sequence analysis of mitochondrial genes, have failed to yield a clear picture of the relative relationships within the group. Here we present the first examination of this issue from the perspective of single-copy nuclear genes at the DNA sequence level. The data involve three such loci: von Willebrand factor (vWF), interphotoreceptor retinoid binding protein (IRBP), and lactalbumin. The vWF and IRBP data sets consist of protein-coding fragments, whereas the sequenced lactalbumin fragment is predominately intronic. All phylogenetic analyses involving at least one representative from each of the three Delphinoidea families congruently support a beluga/porpoise clade. The levels of sequence divergence for most of these data appear to roughly concur with a paleontological date for the radiation of the Delphinoidea at 11-15 MYA but, in agreement with mitochondrial DNA sequence analyses, suggest that the extant major groups of cetaceans radiated approximately 25 MYA, 10 million years later than inferred from paleontological data.

Species-specific oligonucleotide probes for macroalgae: molecular discrimination of two marine fouling species of Enteromorpha (Ulvophyceae).

The green seaweeds Enteromorpha intestinalis and E. compressa are important fouling organisms commonly found in polluted and nutrient-enriched marine and brackish water habitats, where they are used in environmental monitoring. Discrimination of the two species is extremely difficult because of overlapping morphological characters. In this study a quick molecular method for species identification was developed based on the nuclear rDNA ITS2 sequence data of 54 E. intestinalis samples and 20 E. compressa samples from a wide geographical range. Oligonucleotide probes were designed for species-specific hybridization to dot-blots of the PCR-amplified ITS1, 5.8S gene and ITS2 fragment of both E. intestinalis and E. compressa. Specificity of the oligonucleotide probes was confirmed by tests with taxonomically diverse species that could morphologically be confused with E. intestinalis or E. compressa. This is the first use of species-specific probes for macroalgae. The restriction endonuclease NruI digested specifically the amplified PCR product from E. compressa into two fragments detectable on agarose gels, but no suitable restriction sites were identifiable in the PCR product of E. intestinalis.

Molecular evidence regarding the origin of echolocation and flight in bats.

Bats (order Chiroptera) are one of the few orders of mammals that echolocate and the only group with the capacity for powered flight. The order is subdivided into Microchiroptera and Megachiroptera, with an array of characteristics defining each group, including complex laryngeal echolocation systems in microbats and enhanced visual acuity in megabats. The respective monophylies of the two suborders have been tacitly assumed, although microbat monophyly is uncorroborated by molecular data. Here we present a phylogenetic analysis of bat relationships using DNA sequence data from four nuclear genes and three mitochondrial genes (total of 8,230 base pairs), indicating that microbat families in the superfamily Rhinolophoidea are more closely related to megabats than they are to other microbats. This implies that echolocation systems either evolved independently in rhinolophoids and other microbats or were lost in the evolution of megabats. Our data also reject flying lemur (order Dermoptera) as the bat sister group, indicating that presumed shared derived characters for flying lemurs and bats are convergent features that evolved in association with gliding and flight, respectively.

Molecular phylogenetic evidence for a reversible morphogenetic switch controlling the gross morphology of two common genera of green seaweeds, Ulva and Enteromorpha.

Ulva and Enteromorpha are two of the most common, ubiquitous, and environmentally important genera of green seaweeds. They are widely regarded as easily distinguishable because of their dramatically different morphologies: Ulva species are flat, lettucelike blades two cell layers thick, and Enteromorpha species form hollow liquid- or gas-filled tubes one cell thick, which may also be highly branched. We present molecular phylogenetic analyses of nuclear ribosomal RNA ITS sequences from 39 samples representing 21 purported species within these two genera. The results clearly indicate that the two genera are not respectively monophyletic and that the characteristic Ulva and Enteromorpha morphologies have arisen independently several times throughout the evolutionary diversification of the group. The analyses demonstrate that this radical change in gross morphology can also happen within clades exhibiting sequence divergence typical of conspecific assemblages of this group. We suggest that this morphological flexibility is the result of some form of developmental switch that results in either blades or tubes, but that this putative switch must be activated relatively infrequently, since there is evidence that some lineages have retained their form for significant periods. This discovery suggests a possible new model system for study of the molecular mechanisms involved in the interplay between environmental stimuli and plant development.

Additional support for Afrotheria and Paenungulata, the performance of mitochondrial versus nuclear genes, and the impact of data partitions with heterogeneous base composition.

We concatenated sequences for four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes [aquaporin, alpha 2B adrenergic receptor (A2AB), interphotoreceptor retinoid-binding protein (IRBP), von Willebrand factor (vWF)] into a multigene data set representing 11 eutherian orders (Artiodactyla, Hyracoidea, Insectivora, Lagomorpha, Macroscelidea, Perissodactyla, Primates, Proboscidea, Rodentia, Sirenia, Tubulidentata). Within this data set, we recognized nine mitochondrial partitions (both stems and loops, for each of 12S rRNA, tRNA valine, and 16S rRNA; and first, second, and third codon positions of cytochrome b) and 12 nuclear partitions (first, second, and third codon positions, respectively, of each of the four nuclear genes). Four of the 21 partitions (third positions of cytochrome b, A2AB, IRBP, and vWF) showed significant heterogeneity in base composition across taxa. Phylogenetic analyses (parsimony, minimum evolution, maximum likelihood) based on sequences for all 21 partitions provide 99-100% bootstrap support for Afrotheria and Paenungulata. With the elimination of the four partitions exhibiting heterogeneity in base composition, there is also high bootstrap support (89-100%) for cow + horse. Statistical tests reject Altungulata, Anagalida, and Ungulata. Data set heterogeneity between mitochondrial and nuclear genes is most evident when all partitions are included in the phylogenetic analyses. Mitochondrial-gene trees associate cow with horse, whereas nuclear-gene trees associate cow with hedgehog and these two with horse. However, after eliminating third positions of A2AB, IRBP, and vWF, nuclear data agree with mitochondrial data in supporting cow + horse. Nuclear genes provide stronger support for both Afrotheria and Paenungulata. Removal of third positions of cytochrome b results in improved performance for the mitochondrial genes in recovering these clades.

Molecular evidence for multiple origins of Insectivora and for a new order of endemic African insectivore mammals.

The traditional views regarding the mammalian order Insectivora are that the group descended from a single common ancestor and that it is comprised of the following families: Soricidae (shrews), Tenrecidae (tenrecs), Solenodontidae (solenodons), Talpidae (moles), Erinaceidae (hedgehogs and gymnures), and Chrysochloridae (golden moles). Here we present a molecular analysis that includes representatives of all six families of insectivores, as well as 37 other taxa representing marsupials, monotremes, and all but two orders of placental mammals. These data come from complete sequences of the mitochondrial 12S rRNA, tRNA-Valine, and 16S rRNA genes (2.6 kb). A wide range of different methods of phylogenetic analysis groups the tenrecs and golden moles (both endemic to Africa) in an all-African superordinal clade comprised of elephants, sirenians, hyracoids, aardvark, and elephant shrews, to the exclusion of the other four remaining families of insectivores. Statistical analyses reject the idea of a monophyletic Insectivora as well as traditional concepts of the insectivore suborder Soricomorpha. These findings are supported by sequence analyses of several nuclear genes presented here: vWF, A2AB, and alpha-beta hemoglobin. These results require that the order Insectivora be partitioned and that the two African families (golden moles and tenrecs) be placed in a new order. The African superordinal clade now includes six orders of placental mammals.