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PubMed2004

Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome.

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.

Cell Nucleus↗
PubMed1999

In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting nonpathogenic Sterne strain Bacillus anthracis spores. A simplified affinity separation approach was employed, in which autoclaved anthrax spores were used as the separation matrix. An aptamer-magnetic bead-electrochemiluminescence (AM-ECL) sandwich assay scheme was devised for detecting anthrax spores. Using a low SELEX DNA to spore ratio (154 ng DNA/10(6) spores), at least three distinct populations of single-stranded DNA aptamers, having varied affinities for anthrax spores, were noted by the AM-ECL assay. Results reflect detection of spore components with a dynamic range equivalent to < 10- > 6 x 10(6) anthrax spores. In the low DNA to spore ratio experiments, aptamers could be liberated from spore pellets by heating at 96 degrees C for 5 min after each round of SELEX. When a much higher DNA to spore ratio (10,256 ng DNA/10(6) spores) was used for SELEX development, a higher affinity set of aptamers was selected that could not be heat-eluted even at 99 degrees C for 5 min following round four of SELEX. However, high affinity spore surface bound aptamers were detectable via their 5'-biotinylated tails using labeled avidin and could be eluted in deionized water. Aptamers have potential for use as inexpensive, in vitro-generated receptors for biosensors in biological warfare detection and other areas.

Bacillus anthracis↗
PubMed2003

Phylogenetic relationships and genotyping of the genus Streptococcus by sequence determination of the RNase P RNA gene, rnpB.

The rnpB gene is universally present in bacterial species and encodes the RNA subunit of endoribonuclease P. In this study, rnpB was sequenced in 50 type strains and 29 additional strains of the genus Streptococcus. Putative secondary-structure models and possible interactions in RNase P RNA molecules are discussed. Phylogenetic relationships were studied and Bayesian, maximum-parsimony and minimum-evolution analyses supported six main clades that comprised 22 of the 50 species analysed. Phylogenetic inference was also studied for the 16S rRNA gene; it indicated a similar tree topology, but with weaker support values than for rnpB. Combined analysis of rnpB and 16S resulted in a phylogeny with significantly better support. Variability in the rnpB and 16S genes among all type strains, calculated as Shannon-Wiener information index values, was 0.45 for rnpB and 0.15 for 16S. Intraspecies proximity was assessed by principal coordinate analysis of rnpB for 32 strains of six closely related species (two clades) and showed species-specific clusters, but heterogeneity occurred in two species. It can be concluded that the rnpB gene is suitable for phylogenetic analysis of closely related taxa and has potential as a tool for species discrimination.

Base Sequence↗
PubMed2005

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases.

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.

Animals↗
PubMed2001

Mechanisms of evolution in Rickettsia conorii and R. prowazekii.

Rickettsia conorii is an obligate intracellular bacterium that causes Mediterranean spotted fever in humans. We determined the 1,268,755-nucleotide complete genome sequence of R. conorii, containing 1374 open reading frames. This genome exhibits 804 of the 834 genes of the previously determined R. prowazekii genome plus 552 supplementary open reading frames and a 10-fold increase in the number of repetitive elements. Despite these differences, the two genomes exhibit a nearly perfect colinearity that allowed the clear identification of different stages of gene alterations with gene remnants and 37 genes split in 105 fragments, of which 59 are transcribed. A 38-kilobase sequence inversion was dated shortly after the divergence of the genus.

Adaptation, Physiological↗
PubMed2003

Plasmid-mediated antimicrobial resistance in Salmonella enterica.

The selective pressure imposed by the use of antimicrobials in both human and veterinary medicine promotes the spread of multiple antimicrobial resistance. The dissemination of antimicrobial resistance in Salmonella enterica strains, causing severe enteritis in human, has been reported worldwide and is largely attributed to conjugative DNA exchange. In the present review, the relevance of plasmids to the dissemination of antimicrobial resistance in S. enterica is discussed. Recent examples of plasmid-mediated resistance to expanded-spectrum cephalosporins are reported to illustrate the severity of current situation in enteric pathogens. The exchanges between plasmid(s) and the bacterial chromosome and the integration of resistance genes into specialised genetic elements, called integrons, play a major role in acquisition and dissemination of resistance genes. The evolution of a plasmid through the acquisition of integrons is reported, describing novel mechanisms for short-term accumulation of resistance determinants in plasmids circulating in Salmonella.

Animals↗
PubMed1992

Evolution of secondary metabolite production: potential roles for antibiotics as prebiotic effectors of catalytic RNA reactions.

It has been proposed that organic molecules related to known secondary metabolites have existed since the beginning of biochemical evolution and were present in primordial soups. Under primitive earth conditions certain of these molecules may have played roles as effectors in prebiotic reactions, especially those involving catalytic RNA (ribozymes). We demonstrate that a number of antibiotic-related secondary metabolites bind to group I introns and either inhibit splicing reactions or promote the formation of intron oligomers. This is consistent with the functional co-evolution of catalytic RNA and secondary metabolites as antibiotic inhibitors of translation, and supports the notion of an evolutionary relationship between group I introns and ribosomal RNA.

Anti-Bacterial Agents↗
PubMed1992

On the evolution of functional secondary metabolites (natural products).

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.

Anti-Bacterial Agents↗
PubMed2006

Evolution of bacteriophages infecting encapsulated bacteria: lessons from Escherichia coli K1-specific phages.

Bacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and host-range extension but may also facilitate the on purpose design of therapeutic phages based on well-characterized template phages.

Bacterial Capsules↗
PubMed2006

Evolution of mutation rates in bacteria.

Evolutionary success of bacteria relies on the constant fine-tuning of their mutation rates, which optimizes their adaptability to constantly changing environmental conditions. When adaptation is limited by the mutation supply rate, under some conditions, natural selection favours increased mutation rates by acting on allelic variation of the genetic systems that control fidelity of DNA replication and repair. Mutator alleles are carried to high frequency through hitchhiking with the adaptive mutations they generate. However, when fitness gain no longer counterbalances the fitness loss due to continuous generation of deleterious mutations, natural selection favours reduction of mutation rates. Selection and counter-selection of high mutation rates depends on many factors: the number of mutations required for adaptation, the strength of mutator alleles, bacterial population size, competition with other strains, migration, and spatial and temporal environmental heterogeneity. Such modulations of mutation rates may also play a role in the evolution of antibiotic resistance.

Bacteria↗
PubMed2006

Analysis of bifidobacterial evolution using a multilocus approach.

Bifidobacteria represent one of the most numerous groups of bacteria found in the gastrointestinal tract of humans and animals. In man, gastrointestinal bifidobacteria are associated with health effects and for this reason they are often used as functional ingredients in food and pharmaceutical products. Such applications may benefit from or require a clear and reliable bifidobacterial species identification. The increasing number of available bacterial genome sequences has provided a large amount of housekeeping gene sequences that can be used both for identification of bifidobacterial species as well as for understanding bifidobacterial evolution. In order to assess their relative positions in the evolutionary process, fragments from seven conserved genes, clpC, dnaB, dnaG, dnaJ1, purF, rpoC and xfp, were sequenced from each of the currently described type strains of the genus Bifidobacterium. The results demonstrate that the concatenation of these seven gene sequences for phylogenetic purposes allows a significant increase in the discriminatory power between taxa.

Animals↗
PubMed2006

Genetic population structure of the soil bacterium Myxococcus xanthus at the centimeter scale.

Myxococcus xanthus is a gram-negative soil bacterium best known for its remarkable life history of social swarming, social predation, and multicellular fruiting body formation. Very little is known about genetic diversity within this species or how social strategies might vary among neighboring strains at small spatial scales. To investigate the small-scale population structure of M. xanthus, 78 clones were isolated from a patch of soil (16 by 16 cm) in Tübingen, Germany. Among these isolates, 21 genotypes could be distinguished from a concatemer of three gene fragments: csgA (developmental C signal), fibA (extracellular matrix-associated zinc metalloprotease), and pilA (the pilin subunit of type IV pili). Accumulation curves showed that most of the diversity present at this scale was sampled. The pilA gene contains both conserved and highly variable regions, and two frequency-distribution tests provide evidence for balancing selection on this gene. The functional domains in the csgA gene were found to be conserved. Three instances of lateral gene transfer could be inferred from a comparison of individual gene phylogenies, but no evidence was found for linkage equilibrium, supporting the view that M. xanthus evolution is largely clonal. This study shows that M. xanthus is surrounded by a variety of distinct conspecifics in its natural soil habitat at a spatial scale at which encounters among genotypes are likely.

Bacterial Proteins↗
PubMed1993

Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont.

As a result of the nucleotide sequence analysis of an aphid endosymbiont's operon homologous to the Escherichia coli groE, we noted that directional base substitutions tending toward an increase of A + T content represent an obvious evolutionary trend in this prokaryotic operon, housed for a long period by an eukaryotic cell. This result, when taken together with previous reports, raised the possibility that genomic DNA of prokaryotes residing in an eukaryotic cell is subject to A/T-biased directional mutation pressure and/or both negative and positive selection operating under conditions specific to the intracellular environments.

Adenine↗
PubMed1992

Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family.

SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli. We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus. The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments. Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes. Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains. It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.

Amino Acid Sequence↗
PubMed1977

On the cysteine and cystine content of proteins. Differences between intracellular and extracellular proteins.

Analysis of published data on the cysteine and half-cystine content of proteins indicates that most intracellular proteins may be classified as sulfhydryl proteins (those containing cysteine but little or no half-cystine) and that such sulfhydryl proteins have a low cysteine content. The mean systeine content found for 32 intracellular mammalian proteins was 1.6% and intracellular proteins of many bacteria have similar or lower values. Extracellular mammalian proteins are primarily disulfide proteins (those containing half-cystine but little or no cysteine) have a high half-cystine content, the mean value found for some 34 extracellular mammalian proteins being 4.1%. This is contrasted with many of the extracellular proteins from facultative bacteria which are cyst(e)ine-free proteins, being lacking in both cysteine and half-cystine. These and related observations are interpreted in terms of the evolution of life in a reducing atmosphere and the subsequent transition to an oxidizing environment. It is suggested that disulfide proteins evolved primarily after the accumulation of oxygen in the atmosphere.

Bacterial Proteins↗
PubMed1989

Nucleotide sequences from the colicin E5, E6 and E9 operons: presence of a degenerate transposon-like structure in the ColE9-J plasmid.

The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, their cis- or trans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys) present in the ColE9-J plasmid. The ColE6 gene organisation, in the order col-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%-94% homologous. In ColE9-J, the genes are organised as col-imm-lys-E5imm-lys. The E9 col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which is trans-acting. Neither the predicted structures of E5Imm[E9] nor the cis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conserved btuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene, lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Spinach chloroplast thioredoxins in evolutionary drift.

The amino acid sequences surrounding the active sites of spinach chloroplast thioredoxins m and f have been determined. Both types of thioredoxins share common ancestor genes with the E. coli one, demonstrated by invariant active site sequences. The m-type thioredoxins have closer homology with the E. coli one in the sequence analyzed as well as in enzymatic specificity, whereas the f-type is less homologous both in sequence and specificity. It suggests that the m-type gene represents a prototype conserved throughout evolutionary processes whereas the f-type has undergone mutations resulting in a modified specificity.

Amino Acid Sequence↗

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