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Transposable elements and fitness of bacteria.

A stochastic model was designed to describe the evolution of bacterial cultures during 10,000 generations. It is based on a decreasing law for the generation of beneficial mutations as they become fixed in the genomes. Seven beneficial mutations on average were necessary to improve the relative fitness from 1.0 to 1.43 and the model was consistent with the population biology and the genetic data of 12 experimental lines. In one bacterial line, comparison between the model and the data suggests that pivotal mutations mediated by insertion sequences account for a large part of bacterial adaptation. In a more detailed analysis of one simulation, it was shown that only 0.01% of the mutations generated by a population over 10,000 generations can go to fixation as a consequence of their improved fitness. However in the model, the probability of being better fit than its parent should be set initially at ca. 10% to promote an evolution similar to the observed data.

Bacteria↗

Construction of a medium-density horse gene map.

A medium-density map of the horse genome (Equus caballus) was constructed using genes evenly distributed over the human genome. Three hundred and twenty-three exonic primer pairs were used to screen the INRA and the CHORI-241 equine BAC libraries by polymerase chain reaction and by filter hybridization respectively. Two hundred and thirty-seven BACs containing equine gene orthologues, confirmed by sequencing, were isolated. The BACs were localized to horse chromosomes by fluorescent in situ hybridization (FISH). Overall, 165 genes were assigned to the equine genomic map by radiation hybrid (RH) (using an equine RH(5000) panel) and/or by FISH mapping. A comparison of localizations of 713 genes mapped on the horse genome and on the human genome revealed 59 homologous segments and 131 conserved segments. Two of these homologies (ECA27/HSA8 and ECA12p/HSA11p) had not been previously identified. An enhanced resolution of conserved and rearranged chromosomal segments presented in this study provides clarification of chromosome evolution history.

Animals↗

Genomic duplication, fractionation and the origin of regulatory novelty.

Having diverged 50 MYA, rice remained diploid while the maize lineage became tetraploid and then fractionated by losing genes from one or the other duplicate region. We sequenced and annotated 13 maize genes (counting the duplicate gene as one gene) on one or the other of the pair of homeologous maize regions; 12 genes were present in one cluster in rice. Excellent maize-rice synteny was evident, but only after the fractionated maize regions were condensed onto a finished rice map. Excluding the gene we used to define homeologs, we found zero retention. Once retained, fractionation (loss of functioning DNA sequence) could occur within cis-acting gene space. We chose a retained duplicate basic leucine zipper transcription factor gene because it was well marked with big, exact phylogenetic footprints (CNSs). Detailed alignments of lg2 and retained duplicate lrs1 to their rice ortholog found that fractionation of conserved noncoding sequences (CNSs) was rare, as expected. Of 30 CNSs, 27 were conserved. The 3 unexpected, missing CNSs and a large insertion support subfunctionalization as a reflection of fractionation of cis-acting gene space and the recent evolution of lg2's novel maize leaf and shoot developmental functions. In general, the principles of fractionation and consolidation work well in making sense of maize gene and genomic sequence data.

Chromosomes, Artificial, Bacterial↗

X-ray crystallographic analysis of 6-aminohexanoate-dimer hydrolase: molecular basis for the birth of a nylon oligomer-degrading enzyme.

6-Aminohexanoate-dimer hydrolase (EII), responsible for the degradation of nylon-6 industry by-products, and its analogous enzyme (EII') that has only approximately 0.5% of the specific activity toward the 6-aminohexanoate-linear dimer, are encoded on plasmid pOAD2 of Arthrobacter sp. (formerly Flavobacterium sp.) KI72. Here, we report the three-dimensional structure of Hyb-24 (a hybrid between the EII and EII' proteins; EII'-level activity) by x-ray crystallography at 1.8 A resolution and refined to an R-factor and R-free of 18.5 and 20.3%, respectively. The fold adopted by the 392-amino acid polypeptide generated a two-domain structure that is similar to the folds of the penicillin-recognizing family of serine-reactive hydrolases, especially to those of d-alanyl-d-alanine-carboxypeptidase from Streptomyces and carboxylesterase from Burkholderia. Enzyme assay using purified enzymes revealed that EII and Hyb-24 possess hydrolytic activity for carboxyl esters with short acyl chains but no detectable activity for d-alanyl-d-alanine. In addition, on the basis of the spatial location and role of amino acid residues constituting the active sites of the nylon oligomer hydrolase, carboxylesterase, d-alanyl-d-alanine-peptidase, and beta-lactamases, we conclude that the nylon oligomer hydrolase utilizes nucleophilic Ser(112) as a common active site both for nylon oligomer-hydrolytic and esterolytic activities. However, it requires at least two additional amino acid residues (Asp(181) and Asn(266)) specific for nylon oligomer-hydrolytic activity. Here, we propose that amino acid replacements in the catalytic cleft of a preexisting esterase with the beta-lactamase fold resulted in the evolution of the nylon oligomer hydrolase.

Amidohydrolases↗

A virulence-associated gene microarray: a tool for investigation of the evolution and pathogenic potential of Staphylococcus aureus.

An oligonucleotide probe microarray for investigation of the evolution of epidemic Staphylococcus aureus strains has been constructed. The array comprises 383 probes based on virulence-associated genes present in four key strains. Twelve strains including seven for which the complete chromosomal nucleotide sequence was available were tested on the array. Twenty-six per cent of the probes were able to differentiate between strains to give a minimum of two gene differences between pairs. A gene difference distance tree based on the array data had approximately the same topology as one prepared using concatenated MLST sequences. Differences in the topologies of these trees were found to indicate that large-scale recombination events had occurred during the evolution of the species. One such occurrence appears to have been a key event in the genesis of the EMRSA-15 clone (ST22) that currently represents the most prevalent methicillin-resistant S. aureus (MRSA) in the UK.

Bacterial Proteins↗

Repetitive extragenic palindromic sequences in the Pseudomonas syringae pv. tomato DC3000 genome: extragenic signals for genome reannotation.

Repetitive extragenic palindromic (REPs) sequences were first described in enterobacteriacea and later in Pseudomonas putida. We have detected a new variant (51 base pairs) of REP sequences that appears to be disseminated in more than 300 copies in the Pseudomonas syringae DC3000 genome. The finding of REP sequences in P. syringae confirms the broad presence of this type of repetitive sequence in bacteria. We analyzed the distribution of REP sequences and the structure of the clusters, and we show that palindromy is conserved. REP sequences appear to be allocated to the extragenic space, with a special preference for the intergenic spaces limited by convergent genes, while their presence is scarce between divergent genes. Using REP sequences as markers of extragenicity we re-annotated a set of genes of the P. syringae DC3000 genome demonstrating that REP sequences can be used for refinement of annotation of a genome. The similarity detected between virulence genes from evolutionarily distant pathogenic bacteria suggests the acquisition of clusters of virulence genes by horizontal gene transfer. We did not detect the presence of P. syringae REP elements in the principal pathogenicity gene clusters. This absence suggests that genome fragments lacking REP sequences could point to regions recently acquired from other organisms, and REP sequences might be new tracers for gaining insight into key aspects of bacterial genome evolution, especially when studying pathogenicity acquisition. In addition, as the P. syringae REP sequence is species-specific with respect to the sequenced genomes, it is an exceptional candidate for use as a fingerprint in precise genotyping and epidemiological studies.

Base Sequence↗

An evaluation of bacterial resistance to chemo-antibiotics in a homogeneous area comprising East Veneto and South Friuli.

The experiment, now in progress, consists in the gathering and processing of data obtained from the Antibiotic Sensitivity Test done in all the hospital laboratories in the aforesaid area and within which there may well be some interchange of patients. The Antibiotic Sensitivity Tests are arrived at from the identification of all the microorganisms found in the various biological specimens by rigorously standardized methodology. The data, reported on identical modules by all the laboratories and computerized by the CED of Treviso Hospital for half-yearly evaluation, will furnish information on the evolution of bacterial resistance and the related activity of the chemo-antibiotics for each individual hospital, for hospital groups, and for all the hospitals taken together. Details of the program of this operation, of the difficulties encountered, and of the current state of the survey are discussed.

Anti-Bacterial Agents↗

[Neonatal bacterial infections at the CUH of Dakar].

OBJECTIVES: It's a retrospective study in order to determine the epidemiology of neonatal bacterial infection and to evaluate the efficiency of the antibiotic protocol in University Teaching Hospital in Dakar. MATERIAL AND METHODES: From January 1st 1997 to December 31st 1998 we have registered 7461 live births, samples of blood are taken from 2312 new-born baby and they received antibiotherapy (beta-lactamine + gentamycin) at the first day based on infections risk evaluated by anamnestic criterias. The treatment is seven to one days long, the antibiotic was adapted according to the antibiogram result. RESULTS: The neonatal infection diagnosis is confirmed in 246 cases, about 33 per 1000 live births or 10.6% of newborn babies having on antibiotherapy. Most current risk factors are premature rupture of membranes (85%) and neonatal suffering (87.8%). Isolated gerras are: Klebsiella pneumoniae (61.5%), Enterobacteria (11.5 Staphylococcus (8.7%), colibacille (6%), Streptococcus (5.5%), Enterococcus (4.1%) and Pseudomonas (2.7%). Most of these germs are resistant to antibiotics currently used in first intention (ampicillin, cefotaxim, gentamycin), in particularly 95% of Klebsiella. Most efficient antibiotics are amikacin, colistin, ceftriaxon and ciprofloxacine. Deaths occurs in 48 cases with 36 in early neonatal period, 79% of mortality rate related to infection by Klebsiella. CONCLUSION: First intention antibiotherapy must be always adapted to the bacterial ecology evolution and must be more selective by using major infections risk factors. We promote early infection diagnosis by using biologic markers which reference is represented by C Reactive Protein.

Anti-Bacterial Agents↗

Evolution of short sequence repeats in Mycobacterium tuberculosis.

Whole genome comparison has revealed the presence of short sequence repeats (also called mycobacterial interspersed repeat units and variable number tandem repeat units) used for genotyping schemes. In this study, we have used deletion analysis, single nucleotide polymorphism data and spoligotype taken from published data from others to investigate the evolution of selected repeats that form the common denominators of the majority of established schemes. Analysis of the number of repeats per locus from over 400 isolates revealed that the general trend globally appears to be loss of repeats in modern strains compared with ancestral strains.

DNA, Bacterial↗

Pitfalls of bacterial pan-genome analysis approaches: a case study of Mycobacterium tuberculosis and two less clonal bacterial species.

SUMMARY: Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety in methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations. AVAILABILITY AND IMPLEMENTATION: Panqc is freely available under an MIT license at https://github.com/maxgmarin/panqc.

Genome, Bacterial↗

Efficient differentiation of Mycobacterium tuberculosis strains of the W-Beijing family from Russia using highly polymorphic VNTR loci.

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen's University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter-Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a "gold standard" method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.

Bacterial Typing Techniques↗

The IStron CdISt1 of Clostridium difficile: molecular symbiosis of a group I intron and an insertion element.

The IStron CdISt1 was first discovered as an insertion into the tcdA gene of the clinical isolate C34. It combines structural and functional properties of a group I intron at its 5'-end with those of an insertion element at its 3'-end. Up to date four different types could be found, mainly differing in their IS-element portions. Contrasting classical group I introns, CdISt1 is always integrated in ORFs encoding bacterial protein. In case CdISt1 had only the IS-element function such insertion would inactivate the protein encoded by the host gene. It is only due to the self-splicing activity of the group I intron parts that CdISt1 integration does not abolish protein function. Both elements seem to exist in molecular symbiosis and CdISt1 could thus be a prototype of a novel class of genetic elements. Moreover, integration of the CdISt1 into the genome could be advantageous for the bacterium, a motor function for evolution of bacterial proteins is discussed. In clinical practice CdISt1 might well serve as a tool for epidemiological studies of C. difficile infections.

Journal Article↗

Relationship between blaSHV-12 and blaSHV-2a in Korea.

In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the prevalent extended-spectrum SHV enzymes, in Korea SHV-2a and SHV-12 are the most frequently identified extended-spectrum SHV enzymes. A 6.6 kb BamHI fragment containing the bla(SHV-12) gene of strain K7746 isolated from one university hospital in Korea was cloned into the pCRScriptCAM vector. Sequencing of the constructed recombinant plasmid pK7746-C1 revealed that the immediate upstream sequence of the bla(SHV-12) gene showed little similarity to the part of the prototype bla(SHV-1) gene due to the insertion of an IS26 element next to the -10 region. Instead, the upstream sequences of bla(SHV-12) retained 100% DNA identity with the part of plasmid pMPA2a from Klebsiella pneumoniae KPZU-3 carrying bla(SHV-2a). The restriction map of the inserted 6.6 kb DNA fragment of plasmid pK7746-C1 was also homologous to that of plasmid pMPA2a, suggesting a common lineage of bla(SHV-12) and bla(SHV-2a). We also studied, using PCR, the upstream non-coding region of several SHV beta-lactamase genes for the presence of IS26 sequence. The flanking IS26 sequence in the immediate upstream region of the bla(SHV) gene was not detected in five standard strains producing SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. However, IS26 was detected in all 69 clinical strains producing SHV-2a or SHV-12 isolated from three university hospitals in Korea during 1993-1999. The above findings suggest a direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5, and it is considered to be one of the reasons for the absolute predominance of SHV-2a and SHV-12 in Korea.

Base Sequence↗

Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis.

The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR-amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram-positive (H. chlorum) --> high G+C Gram-positive --> Deinococcus-Thermus --> green non-sulphur bacteria (Cf. aurantiacus ) --> cyanobacteria --> spirochaetes --> Chlamydia-Cytophaga-Aquifex-flavobacteria-green sulphur bacteria (Cb. tepidum) --> proteobacteria (alpha, delta and epsilon) and --> proteobacteria (beta and gamma). The members of the Heliobacteriaceae family that contain a Fe-S type of reaction centre (RC-1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative bacteria or diderm prokaryotes, green non-sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin-quinone type of reaction centre (RC-2), are indicated to have evolved very early. Thus, the organisms containing either RC-1 or RC-2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.

Amino Acid Sequence↗

Integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution.

Integrons are horizontal gene transfer (HGT) systems containing elements necessary for site-specific recombination and expression of foreign DNA. The overall phylogenetic distribution of integrons and range of genes that can be transferred by integrons are unknown. This report contains an exploration of integrons in an environmental microbial community and an investigation of integron evolution. First, using culture-independent techniques, we explored the diversity of integrons and integron-transferred genes in heavy-metal-contaminated mine tailings. Using degenerate primers, we amplified integron integrase genes from the tailings. We discovered 14 previously undescribed integrase genes, including six novel gene lineages. In addition, we found 11 novel gene cassettes in this sample. One of the gene cassettes that we sequenced is similar to a gene that codes for a step in a pathway for nitroaromatic catabolism, a group of compounds associated with mining activity. This suggests that integrons may be important for gene transfer in response to selective pressures other than the presence of antibiotics. We also investigated the evolution of integrons by statistically comparing the phylogenies of 16S rRNA and integrase genes from the same organisms, using sequences from GenBank and various sequencing projects. We found significant differences between the organismal (16S rRNA) and integrase trees, and we suggest that these differences may be due to HGT.

Bacteria↗

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects.

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.

Animals↗

Characterization of circulating immune complexes in leprosy patients and their correlation with specific antibodies against Mycobacterium leprae.

Circulating immune complex (CIC) levels and their antibody and antigenic composition were evaluated in patients with leprosy as well as in any individuals living with them; they were precipitated with 3.5% polyethylene glycol (PEG) and, after affinity chromatography isolation and purification, analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with monoclonal antibodies (Mabs). The presence of CICs was demonstrated throughout the clinical and immunopathological leprosy spectrum at levels related to bacterial load, and in leprosy patients they showed a positive correlation with specific anti-PGL and anti-65 kDa antibodies. The isolation and analysis, however, failed to identify any Mycobacterium leprae antigenic components; although two specific antibodies anti-PGL-1 and anti-65 kDa were identified as possible CIC constituents and may be potentially useful in the follow-up of leprosy patients, especially to check bacterial load evolution, PGL-1 being an authentic antigen of this mycobacterium. Also, the involvement of 65 kDa in CICs, being homologous with the human heat shock protein (HSP) 60 kDa family, suggests an autoimmune mechanism in leprosy pathogenesis. Furthermore, those results support the inclusion of CIC antibody reactivity studies to enhance the sensitivity of serology.

Adolescent↗

Protein H--a surface protein of Streptococcus pyogenes with separate binding sites for IgG and albumin.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

Amino Acid Sequence↗