The Evolution of Insertion Sequences within Enteric Bacteria

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.     

Phylogenetic analysis of ticks (Acari: Ixodida) using mitochondrial genomes and nuclear rRNA genes indicates that the genus Amblyomma is polyphyletic

Our understanding of the phylogenetic relationships among tick lineages has been limited by the lack of resolution provided by the most commonly used phylogenetic markers. Mitochondrial genomes are increasingly used to address controversial phylogenetic relationships. To date, the complete mitochondrial genomes of eleven tick species have been sequenced; however, only three of these species are metastriate ticks, the most speciose lineage of ticks. In this study, we present the nucleotide sequences of the complete mitochondrial genomes of five more species of metastriate ticks: Amblyomma elaphense, Amblyomma fimbriatum, Amblyomma sphenodonti, Bothriocroton concolor and Bothriocroton undatum. We use complete mitochondrial genome sequences to address the phylogenetic placement of two morphologically 'primitive' species -Am. elaphense and Am. sphenodonti - with respect to the genus Amblyomma. Our analysis of these five mitochondrial genomes with the other eleven tick mitochondrial genomes, as well as analysis of nuclear rRNA genes, provides strong evidence that the genus Amblyomma is polyphyletic with the inclusion of Am. sphenodonti and Am. elaphense. A new genus or two new genera may be required to describe Am. sphenodonti and Am. elaphense. It is also possible that these two species are sisters to two established genera, Bothriocroton in the case of Am. sphenodonti, and Haemaphysalis in the case of Am. elaphense. However, other arrangements of these taxa cannot be excluded with the current data. Thus, while Am. sphenodonti and Am. elaphense do not belong in the genus Amblyomma, the phylogenetic placement of these two species cannot be resolved without more data from metastriate ticks, either greater sampling of mitochondrial genomes, or a large data set of nuclear genes.     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene ( rpoB  ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

Phylogenetic analysis of gamma-proteobacteria inferred from nucleotide sequence comparisons of the house-keeping genes adk, aroE and gdh: comparisons with phylogeny inferred from 16S rRNA gene sequences

Nucleotide sequence comparisons of three house-keeping genes, adenylate kinase (adk), shikimate dehydrogenase (aroE), and glucose-6-phosphate dehydrogenase (gdh), were used to infer the phylogeny of 33 gamma-proteobacteria. Phylogenetic trees inferred from each gene, and from the concatenated sequences of all three genes, are, in general, similar to a 16S rRNA gene-inferred tree. Similar grouping of bacteria are revealed at the family, genus, species and strain levels in all five trees. The house-keeping genes, however, show a higher rate of nucleotide sequence substitutions. Consequently, they can possibly probe deeper branches of a phylogenetic tree than the 16S rRNA gene. However, because their nucleotide sequences are not as highly conserved among gamma-proteobacteria, family- or genus-specific primers would need to be designed for the amplification of any of these three house-keeping genes. Since these genes are used in multilocus sequence typing, it is expected that the number of sequences publicly available for many taxa will increase over time proving them very useful either at complementing 16S rRNA-inferred phylogenies or for specific, targeted, phylogenetic analysis.     

What is the phylogenetic placement of Dipteronia dyerana Henry? An example of plant species placement based on nucleotide sequences

Abstract

DNA sequence data have been widely used to evaluate species delimitations and examine infraspecific relationships. However, species placements inferred from different nucleotide sequences are frequently in conflict. As an example of plant species placement based on nucleotide sequences, the phylogenetic placement of Dipteronia dyerana Henry (Aceraceae) was analyzed in the present study. The study species included eight Acer species (from different sections of Acer), two Dipteronia species, and two outgroup taxa. Phylogenetic trees based on five datasets (ITS, trnL‐F, trnD‐trnT, psbM‐trnD, and rpl16 regions) as well as their combined datasets were generated by using maximum parsimony (MP) and maximum likelihood (ML) analyses. Further analyses were conducted to compare the strict consensus trees based on single regions and the combination of different regions. The results revealed a significant discrepancy among the phylogenetic placements of D. dyerana, inferred from various sequences. Phylogenetic trees using MP analysis based on trnD‐trnT, rpl16, and the four chloroplast combined sequences supported the genus Dipteronia as a monophyletic group, while in the other trees D. dyerana was positioned either in parallel with D. sinensis and Acer species or within the genus Acer. In ML analysis, only rpl16 and the four chloroplast combined sequence datasets supported the genus Dipteronia as a monophyletic group. We concluded that, although significant genetic differentiation occurred between D. dyerana and D. sinensis, D. dyerana was more advanced than D. sinensis. However, whether Dipteronia is monophyletic remains to be further investigated, e.g., by using more closely related taxa and more sequences. Furthermore, in addition to internal transcribed spacer sequences, more chloroplast gene sequences should be used for phylogenetic analyses of species.     

Phylogeny of Cryptocercus species (Blattodea: Cryptocercidae) inferred from nuclear ribosomal DNA

The wood-feeding cockroaches of the genus Cryptocercus occur in temperate forests. Of the seven known species, five occur in the United States and two in Eurasia. Until 1997, all populations in the United States were considered a single species. Populations in the western United States were elevated to a species status based on variation in DNA sequence and morphology. In 1999, three new species were described from the eastern United States based on variation in chromosome number and mitochondrial DNA, bringing the number of species in the United States to five. The objective of this study was to determine if the DNA sequence of nuclear rRNA also signals the existence of four species in the eastern United States and to compare the inferred relationships with those proposed based on mitochondrial sequences. We obtained the DNA sequence from a portion of the 5.8S and 28S rRNA genes and the entire ITS2 region from 38 individuals and 30 additional clones to assess intraindividual, intraspecific, and interspecific variation. We found extensive sequence variation among the various species and little or no intraindividual and intraspecific variation. Phylogenetic analysis indicated the existence of monophyletic lineages among the eastern United States samples, which largely corresponded to the four species previously described. The inferred relationships were well-supported by bootstrap analysis and decay indices. Although the nuclear rRNA sequences resulted in a coherent phylogenetic tree, the ITS2 region contained many insertions and deletions, which may introduce homoplasy and ambiguity in alignment as more taxa are added to the data set.     

Phylogenetic Placement of Pentatomid Stink Bug Gut Symbionts

Insect bacterial symbionts are ubiquitous, however, only a few groups of host families have been well studied in relation to their associations with microbes. The determination of the phylogenetic relationships among bacteria associated with different species within an insect family can provide insights into the biology and evolution of these interactions. We studied the phylogenetic placement of vertically transmitted bacterial symbionts associated with the posterior midgut (crypt-bearing) region of pentatomid stink bugs (Hemiptera, Pentatomidae). Our results demonstrate that different host species carried one major bacterium in their midgut. Phylogenetic analyses of the 16S rRNA gene sequences obtained from the midgut of stink bugs placed all symbionts in a clade with Erwinia and Pantoea species, both plant-associated bacteria. Results indicate that symbiont monophyly occurs among recently diverged taxa (e.g., within a genus) but does not occur in the Pentatomidae. Results suggest that these vertically transmitted symbionts are occasionally replaced by other taxonomically similar bacteria over evolutionary time. Our findings highlight how the evolutionary history of hemipteran symbionts in unexplored host families may have unpredictable levels of complexity.     

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.     

Evolution of Coenzyme B(12) Synthesis among Enteric Bacteria: Evidence for Loss and Reacquisition of a Multigene Complex

We have examined the distribution of cobalamin (coenzyme B(12)) synthetic ability and cobalamin-dependent metabolism among enteric bacteria. Most species of enteric bacteria tested synthesize cobalamin under both aerobic and anaerobic conditions and ferment glycerol in a cobalamin-dependent fashion. The group of species including Escherichia coli and Salmonella typhimurium cannot ferment glycerol. E. coli strains cannot synthesize cobalamin de novo, and Salmonella spp. synthesize cobalamin only under anaerobic conditions. In addition, the cobalamin synthetic genes of Salmonella spp. (cob) show a regulatory pattern different from that of other enteric taxa tested. We propose that the cobalamin synthetic genes, as well as genes providing cobalamin-dependent diol dehydratase, were lost by a common ancestor of E. coli and Salmonella spp. and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source. Consistent with this hypothesis, the S. typhimurium cob genes do not hybridize with the genomes of other enteric species. The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes. This process may be an important aspect of bacterial population genetics and evolution.     

Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico

PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale. Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates. Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups. Two isolates were closely related to M. loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M. amorphae, M. plurifarium and M. huakuii. However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences. Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene. A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene. These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.     

Enterobacterial repetitive intergenic consensus (ERIC) sequences in Escherichia coli: Evolution and implications for ERIC-PCR

Enterobacterial repetitive intergenic consensus (ERIC) sequences are 127-bp imperfect palindromes that occur in multiple copies in the genomes of enteric bacteria and vibrios. Here we investigate the distribution of these elements in the complete genome sequences of nine Escherichia coli (including Shigella species) strains. There is a significant tendency for copies to be adjacent to more highly expressed genes. There is considerable variation among strains with respect to the presence of an element in any particular intergenic region, but some copies appear to have been conserved since before the divergence of E. coli and Salmonella enterica. In comparisons of orthologous copies between these species, ERIC sequences are surprisingly conserved, implying that they have acquired some function, perhaps related to mRNA stability. The relationships among copies within E. coli are consistent with a master copy mode of generation. Insertion of new copies seems to occur at, and involve duplication of, the dinucleotide TA. Two classes of inserts of about 70 bp each occur at different specific sites within ERIC sequences; these inserts evolve independently of the ERIC sequences. The small number of ERIC sequences in E. coli genomes indicates that a widely used bacterial fingerprinting method using primers based on ERIC sequences (ERIC-PCR) does not rely on the presence of ERIC sequences.     

Nonhomogeneous model of sequence evolution indicates independent origins of primary endosymbionts within the enterobacteriales (gamma-Proteobacteria)

Standard methods of phylogenetic reconstruction are based on models that assume homogeneity of nucleotide composition among taxa. However, this assumption is often violated in biological data sets. In this study, we examine possible effects of nucleotide heterogeneity among lineages on the phylogenetic reconstruction of a bacterial group that spans a wide range of genomic nucleotide contents: obligately endosymbiotic bacteria and free-living or commensal species in the gamma-Proteobacteria. We focus on AT-rich primary endosymbionts to better understand the origins of obligately intracellular lifestyles. Previous phylogenetic analyses of this bacterial group point to the importance of accounting for base compositional variation in estimating relationships, particularly between endosymbiotic and free-living taxa. Here, we develop an approach to compare susceptibility of various phylogenetic reconstruction methods to the effects of nucleotide heterogeneity. First, we identify candidate trees of gamma-Proteobacteria groEL and 16S rRNA using approaches that assume homogeneous and stationary base composition, including Bayesian, maximum likelihood, parsimony, and distance methods. We then create permutations of the resulting candidate trees by varying the placement of the AT-rich endosymbiont Buchnera. These permutations are evaluated under the nonhomogeneous and nonstationary maximum likelihood model of Galtier and Gouy, which allows equilibrium base content to vary among examined lineages. Our results show that commonly used phylogenetic methods produce incongruent trees of the Enterobacteriales, and that the placement of Buchnera is especially unstable. However, under a nonhomogeneous model, various groEL and 16S rRNA phylogenies that separate Buchnera from other AT-rich endosymbionts (Blochmannia and Wigglesworthia) have consistently and significantly higher likelihood scores. Blochmannia and Wigglesworthia appear to have evolved from secondary endosymbionts, and represent an origin of primary endosymbiosis that is independent from Buchnera. This application of a nonhomogeneous model offers a computationally feasible way to test specific phylogenetic hypotheses for taxa with heterogeneous and nonstationary base composition.     

Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes

Butterflies of the genus Papilio have served as the basis for numerous studies in insect physiology, genetics, and ecology. However, phylogenetic work on relationships among major lineages in the genus has been limited and inconclusive. We have sequenced 2.3 kb of DNA from the mitochondrial cytochrome oxidase I and II genes (COI and COII) for 23 Papilio taxa and two outgroups, Pachliopta neptunus and Eurytides marcellus, in order to assess the potential of these genes for use in Papilio phylogenetics and to examine patterns of gene evolution across a broad taxonomic range. Nucleotide and amino acid variation is distributed heterogeneously, both within and between genes. Structural features of the proteins are not always reliable predictors of variation. In a combined analysis, these sequences support a nearly fully resolved topology within subgenera and species groups, though higher level relationships among species groups require additional study. The most noteworthy findings are that neither Papilio alexanor nor P. xuthus belongs in the machaon group and that the subgenus Pterourus is paraphyletic with respect to the subgenus Pyrrhosticta. We leave relationships among members of the phorcas species group as a trichotomy. These two protein coding genes, particularly COI, show excellent performance in resolving relationships at the level of species and species groups among Papilionidae. We strongly endorse a similar approach for future studies aimed at these levels.     

The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli.

The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide. Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined. The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin. The cob operon of E. coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase. The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes. All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E. coli genetic map. Although the nucleotide sequences of the Salmonella cob genes and the E. coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor. On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source. The cob operon of E. coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.     

Phylogenetic relationships among taxa of the liverwort Conocephalum conicum (Conocephalaceae) revealed by psbA sequence

The 5' flanking and coding regions of the psbA gene were sequenced to clarify relationships among several taxa discovered recently in thalloid liverwort Conocephalum conicum. Twenty six samples included in this study represent five "cryptic species" detected in worldwide collection, five groups discovered from Japan and finally three "chemo-types" with different dominant volatile component. Pairwise differences in nucleotide sequences of coding region between samples of Conocephalum conicum varied greatly depending on the combination, ranging from 0.001 to 0.018 per site. In total, seven amino acid substitutions were found among all samples. NJ and parsimony trees among the taxa were constructed for the first time. The phylogenetic relationships among taxa were basically consistent with those inferred from isozyme and morphological studies. Conocephalum conicum -species FS and T taxon found in Japan have identical sequence of psbA gene, suggesting that they are conspecific. Similarly, YFS and KYT taxa cluster together with FS cryptic species, and "chemo-types" I and II with "cryptic species" J. A chemotype III was very different from any other taxa of C. conicum. These results suggest that morphological species, Conocephalum conicum is highly differentiated at the molecular level and some of the taxa may in fact represents different species as previous studies suggested.     

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

Phylogenetic relationships of bryophytes inferred from nuclear-encoded rRNA gene sequences

We investigate phylogenetic relationships among hornworts, liverworts and mosses, and their relationships to other green plant groups, by analysis of nucleotide variation in complete 18s rRNA gene sequences of three green algae, two hornworts, seven liverworts, nine mosses, and six tracheophytes. Parsimony and maximum-likelihood analyses yield a single optimal tree in which the hornworts are resolved as the basal group among land plants, and the liverworts and mosses are sister taxa that together form the sister clade to the tracheophytes. This phylogeny is internally robust as indicated by decay indices and by comparison (using both parsimony and likelihood criteria) to topologies representing five alternative hypotheses of bryophyte relationships. We discuss some possible reasons for differences between the phylogeny inferred from the rRNA data and those inferred from other character sets.     

New gammaproteobacteria associated with blood-feeding leeches and a broad phylogenetic analysis of leech endosymbionts

Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech, Haementeria ghilianii, and two others from Placobdelloides species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class Gammaproteobacteria. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus Placobdella as well as Aeromonas veronii from the medicinal leech, Hirudo medicinalis, and a Rickettsia sp. detected in Hemiclepsis marginata. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.     

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.