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Insertion sequences in prokaryotic genomes.

Insertion sequences (ISs) are small DNA segments that are often capable of moving neighbouring genes. Over 1500 different ISs have been identified to date. They can have large and spectacular effects in shaping and reshuffling the bacterial genome. Recent studies have provided dramatic examples of such IS activity, including massive IS expansion during the emergence of some pathogenic bacterial species and the intimate involvement of ISs in assembling genes into complex plasmid structures. However, a global understanding of their impact on bacterial genomes requires detailed knowledge of their distribution across the eubacterial and archaeal kingdoms, understanding their partition between chromosomes and extra-chromosomal elements (e.g. plasmids and viruses) and the factors which influence this, and appreciation of the different transposition mechanisms in action, the target preferences and the host factors that influence transposition. In addition, defective (non- autonomous) elements, which can be complemented by related active elements in the same cell, are often overlooked in genome annotations but also contribute to the evolution of genome organisation.

Bacteria↗

Five novel plasmid-determined beta-lactamases.

Five novel plasmid-determined beta-lactamases named TLE-1, OXA-4, OXA-5, OXA-6, and OXA-7 were detected in ampicillin-resistant isolates of Escherichia coli and carbenicillin-resistant strains of Pseudomonas aeruginosa. TLE-1 resembled TEM-1 in substrate profile and reactions with inhibitors but differed in isoelectric point (5.55) and enzyme banding pattern on flat-bed electrofocusing.OXA-4, OXA-5, OXA-6, and OXA-7 hydrolyzed oxacillin, methicillin, and cloxacillin readily but differed from OXA-1, OXA-2, and OXA-3 in substrate profiles, inhibitor reactions, and isoelectric points (7.5 to 7.8).OXA-4 and OXA-6 were unusual for members of the OXA group in their sensitivity to inhibition by cloxacillin. OXA-5 and OXA-7 had isoelectric points close to that of SHV-1, emphasizing the need in beta-lactamase classification for studies in addition to isoelectric focusing. These five new enzymes bring the number of plasmid-determined beta-lactamases known in gram-negative organisms to more than 20. The evolution of such enzymatic diversity remains to be explored.

Anti-Bacterial Agents↗

Cyanobacterial plasmids: their widespread occurrence, and the existence of regions of homology between plasmids in the same and different species.

The results of screening of 29 diverse cyanobacterial (blue-green algal) strains for plasmid (CCC DNA) content are reported. Approximately one-half of the strains were shown to contain one or more CCC DNAs. CCC DNAs from four unicellular marine cyanobacteria were characterized in more detail. These strains contained multiple plasmids. Two kinds of Southern hybridization experiments allowed us to show that different plasmids within the same strain, and different plasmids within different strains, can ( but do not always) contain restricted regions of sequence homology. We suggest that these regions of homology may be analogous to the transposable genetic elements of bacterial plasmids. This, together with indirect but compelling evidence for interspecific (or intergeneric) plasmid transfer, indicates that CCC DNAs (although as yet genetically cryptic) may play a role in the ecology and evolution of obligately autotrophic prokaryotes, as they do in the ecology and evolution of the better-known heterotrophic bacteria.

Base Sequence↗

Evolution of virulence factors in Shiga-toxin-producing Escherichia coli.

The major demonstrated or putative virulence factors of Shiga-toxin-producing Escherichia coli (STEC) are the Shiga toxins, products of the locus of enterocyte effacement, and products encoded by the EHEC-hemolysin plasmid. Molecular analysis shows that STEC acquired the majority of these virulence factors by horizontal transfer of genetic material. In the case of Shiga toxins, the phages encoding them are probably responsible for this transfer. For the locus of enterocyte effacement, however, it is not clear how often this transfer took place and which parts of the locus were involved in this transfer. The large EHEC-hemolysin plasmid is clearly a mosaic structure, which arose from multiple recombination events with foreign DNA. Two lineages of this plasmid can be distinguished, one of which is associated with chromosomally encoded virulence factors. Despite the wealth of information available, further comparative studies are needed to decipher definitively the evolution of virulence in STEC.

Escherichia coli↗

'Cells' and 'organisms' as a habitat for DNA.

Although the bulk of the hereditary information in bacteria is organized as a single chromosome, it has been known for some years now that bacteria may also carry pieces of self-replicating extrachromosomal DNA. These units are known as plasmids. Sometimes such plasmids carry the information necessary to give rise to mature bacterial viruses under appropriate conditions, but in other cases they specify the production of enzymes and other proteins which alter the bacterial phenotype. Plasmids are often inessential for survival of bacteria, although they may widen the range of environmental conditions under which they flourish. Thus plasmids may be thought of as adventitious additions to the genetic content of bacterial cells. Recently it has become clear that furthur organizational units of DNA are to be found in bacterial cells. These units are called insertion sequences and transposons. Unlike plasmids and the chromosome, however, these DNA units do not carry enough genetic information to specify their own independent replication: they must rely on plasmids or the chromosome for that purpose. Nevertheless they behave in many respects as independent functional units. Although it is possible to think of the chromosome, plasmids and transposons/insertion sequences as three distinct hierarchies of bacterial DNA, genes may move from one hierarchy to another; and such transitions have important implications for the evolution of bacterial populations. Moreover, their study in bacteria may throw much light on the type of DNA interactions occurring in higher cells.

Base Sequence↗

Comparison of multidrug resistance gene regions between two geographically unrelated Salmonella serotypes.

OBJECTIVES: The aim of this study was to identify chromosomally integrated genes conferring multidrug resistance to a Salmonella enterica (S.) serotype Typhimurium isolate, phage type DT193, isolated in Ireland and to compare them with resistance genes conferring plasmid-mediated multidrug resistance to a S. Enteritidis isolate from Italy. METHODS: A complete DNA sequence of the regions containing the resistance genes was obtained from the chromosome of the S. Typhimurium DT193 isolate and from the IncI plasmid of the S. Enteritidis isolate. The plasmid was also characterized by conjugation and incompatibility grouping. RESULTS: Two 10 kb multidrug resistance non-Salmonella Genomic Island 1 type clusters were independently identified in the S. Enteritidis plasmid and in the chromosome of the S. Typhimurium isolate. Detailed characterization identified an IP-type 2 integron containing a dfrA1-aadA1 gene cassette and other common resistance determinants derived from the RSF1010 plasmid. CONCLUSIONS: These multidrug resistance regions originate following chromosomal integration of key resistance markers encountered on plasmids circulating in other Salmonella serotypes. This mechanism of marker acquisition may have future implications for the evolution of similar structures in previously susceptible serotypes, leading to an increased public health risk.

Chromosome Mapping↗

Global lessons from antibiotic resistance: Metformin-hydrolysing genes in transposable elements, a new threat for type II diabetic patients?

OBJECTIVES: To investigate the evolutionary origin, genomic mobility, and potential dissemination of metformin-hydrolysing genes (mfmAB), and to assess whether environmental selection by metformin pollution may drive the emergence of transferable pharmaceutical-degrading traits analogous to antibiotic resistance. METHODS: Large-scale comparative genomics was performed using publicly available bacterial genomes carrying mfmAB homologs. Phylogenomic reconstruction, average nucleotide identity analysis, genomic context comparison, plasmid characterization, and insertion sequence mapping were used to infer evolutionary history and identify mechanisms of horizontal gene transfer. RESULTS: mfmAB homologs were identified in twelve Aminobacter and three Pseudomonas genomes within a conserved ∼8.2 kb gene cluster. Phylogenomic analyses showed that metformin-degrading capacity emerged independently in multiple Aminobacter lineages across distinct continents, consistent with convergent evolution under anthropogenic selective pressure. Genomic comparisons indicated a chromosomal origin of mfmAB, followed by mobilization onto conjugative plasmids through IS1182-mediated transposition. In Pseudomonas, additional IS3/IS6-mediated transposition events integrated mfmAB into diverse plasmid backbones, frequently within composite transposons also encoding guanylurea and biguanide degradation pathways (guuH, bguH). These findings reveal a dynamic modular assembly of metabolic functions facilitating adaptation to pharmaceutical pollutants. CONCLUSIONS: Metformin pollution appears to promote the emergence and mobilization of pharmaceutical-degrading genes through mechanisms paralleling antibiotic resistance evolution. Although no clinical impact has yet been demonstrated, the potential spread of such genes into human-associated microbiomes and their possible co-selection with antibiotic resistance determinants represent an emerging One Health concern. Environmental surveillance of pharmaceutical-degrading genes is warranted to anticipate future threats to drug efficacy.

Convergent evolution↗

Structural and evolutionary relationships of beta-lactamase transposons from Staphylococcus aureus.

A comparison of the beta-lactamase elements detected on three classes of large plasmids together with the chromosomes of penicillin-resistant Staphylococcus aureus revealed substantial physical and genetic relatedness. In most cases, beta-lactamase production could be associated with the presence of a DNA segment of approximately 6.7 kb. Analysis showed that the plasmid-borne determinants constitute nearly identical transposons or transposon-like elements. An element indistinguishable from one of these, Tn4002, which is carried by the pSK1 family of plasmids in clinical isolates from Australian hospitals, was also identified on the staphylococcal chromosome and is implicated in an evolutionary cycle of transposition between chromosomal and extrachromosomal sites in Australian strains of multiresistant S. aureus.

Biological Evolution↗

On-line estimation of the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins by monitoring respiratory energy generation.

Human basic fibroblast growth factor (hFGF-2) was produced in high-cell density cultures of recombinant Escherichia coli using a temperature-inducible expression system. The synthesis rates of proteins were followed by two-dimensional gel electrophoresis of the (35)S-methionine-labeled proteom. After temperature induction of hFGF-2 synthesis, the rate of total protein synthesis per biomass increased by a factor of three, mainly as a result of the additional synthesis of hFGF-2 and heat-shock proteins. The synthesis rates of heat-shock proteins and constitutive plasmid-encoded proteins increased after the temperature upshift also in the control strain without hFGF-2 gene but followed time profiles different from the producing strain. The energy demand for the extra synthesis of plasmid-encoded and heat-shock proteins resulted in an elevated respiratory activity and, consequently, in a reduction of the growth rate and the biomass yield. A procedure was developed to relate the energy demand for the additional synthesis of these proteins to the generation of energy in the respiratory pathway. Specific energy production was estimated based on on-line measurable rates of oxygen consumption, or carbondioxide evolution and growth, respectively. In this way, the metabolic burden resulting from the synthesis of plasmid-encoded and heat-shock proteins was quantified from on-line accessible data.

Electrophoresis, Gel, Two-Dimensional↗

Homogeneous and heterogeneous mini-circle subpopulations in Trypanosoma cruzi kinetoplast DNA.

The small circular components (mini-circles) from Trypanosoma cruzi kinetoplast DNA (kDNA) were cloned in the plasmid vector pBR325. These clones have been used before to demonstrate the rapid evolution of mini-circle subpopulations (S anchez , D.O., Frasch , A.C.C., Carrasco , A.E., Gonzalez Cappa , S.M., Isola , E. and Stoppani , A.O.M. (1984) Mol. Biochem. Parasitol ., in the press). We have now analyzed the cloned molecules and used them to study some structural characteristics of T. cruzi mini-circles and their distribution in total kDNA restriction endonuclease digests. Most molecules partially conserved TaqI, HaeIII and HapII site clusters (constant regions) separated by one-quarter of the total mini-circle length, also detected in total kDNA digests. In addition, in one of the cloned mini-circles, the constant region was present only once, instead of four times as expected. Outside the conserved regions, the mini-circles diverged enough so that no cross-hybridization took place even under relaxed conditions. The recombinant molecules were used to probe total kDNA digests from T. cruzi. Some of them hybridized with most restriction endonuclease kDNA fragments, while one cloned mini-circle ( pTck -14) detected only its homologous subpopulation. The mini-circles detected with the latter probe proved to be nearly homogeneous, and were present in the proportion of 1/20 molecules. These results suggest that some of the generated molecules might have acquired a higher replication rate, giving rise to the homogeneous subpopulation detected. Further mutations, insertions and/or deletions, together with recombination between molecules, would bring this process to an end.

Animals↗

Pathogenic diversity of Escherichia coli and the emergence of 'exotic' islands in the gene stream.

Escherichia coli is a highly adaptive bacterial species that is both a member of the commensal intestinal flora and a versatile pathogen associated with numerous types of intestinal and systemic infections in humans and other animals. The spectrum of diseases caused by E. coli is due to the acquisition of specific virulence genes harbored on plasmids, bacteriophages, or within distinct DNA segments termed pathogenicity islands (PAIs) that are absent from the genomes of commensal E. coli strains. PAIs are likely to have been transferred horizontally and may have integrated into the E. coli chromosome through bacteriophage or plasmid integration or transposition. The contribution of intergenic inheritance to the adaptation and evolution of E. coli, types of PAIs associated with different groups of pathogenic E. coli and approaches to identify unique sequence islands (USIs), some of which might confer pathogenicity, in E. coli and other bacteria are presented.

Animals↗

The conformation of T4 bacteriophage dihydrofolate reductase from circular dichroism.

The secondary and tertiary structure of T4 bacteriophage dihydrofolate reductase is investigated by vacuum ultraviolet circular dichroism (CD) spectroscopy and probability analysis of the primary amino acid sequence. The far ultraviolet CD spectrum of the enzyme in the range of 260-178 nm is analyzed by the generalized inverse and variable selection methods developed by our laboratory. Variable selection yields an average content of 26% alpha-helix, 21% antiparallel beta-sheet, 10% parallel beta-sheet, 20% beta-turns, and 32% "other" structures within the T4 protein. The characteristic peaks of the CD spectrum indicate that the enzyme has a lot of antiparallel beta-sheet, which is typical of the alpha + beta tertiary class of globular proteins. The secondary structure of the protein is also analyzed by using four statistical methods on the amino acid sequence. Although the secondary structures predicted by each individual statistical method vary to a considerable extent, the fractions of each structure jointly predicted by a majority of the methods are in excellent agreement with our CD analysis. The alternating arrangement for some segments of alpha-helix and beta-sheet predicted from primary structure to be within the enzyme is characteristic of proteins containing parallel beta-sheet. This supports our conclusion that the protein contains both parallel and antiparallel beta-sheet structures, but finding both types of beta-sheet also means that the protein may have the variation on alpha/beta tertiary structure recently found in EcoRI endonuclease and thymidylate synthase. These observations, in conjunction with other physical properties of the T4 reductase, suggest that the enzyme perhaps shares an evolution in common with the dihydrofolate reductases derived from type I R-plasmids rather than with the host-cell protein.

Circular Dichroism↗

Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) that have characteristics of mtDNA introns and retroid elements. The plasmids contain a single long open reading frame (710 amino acids), whose amino-terminal half has structural similarity to reverse transcriptases. Using antibodies against synthetic peptides and trpE fusion proteins, we detected an 81-kDa protein encoded by this open reading frame in mitochondrial preparations from the plasmid-containing strains. This 81-kDa protein cosegregates with reverse transcriptase activity in sexual crosses and comigrates with reverse transcriptase activity in sodium dodecyl sulfate-polyacrylamide gels, where it can be assayed after renaturation of the protein. In glycerol gradients under nondenaturing conditions, the reverse transcriptase activity sediments at approximately 145 kDa, close to the value expected for a dimer of the 81-kDa protein. The 81-kDa protein represents most of the 710-amino acid open reading frame, but may be missing some amino acids at the amino terminus. The regions upstream and downstream of the putative reverse transcriptase domain lack sequences characteristic of gag, protease, RNase H, or integrase domains found in other retroid elements. The plasmid-encoded 81-kDa protein seems to be a novel type of reverse transcriptase that may provide insight into the evolution of these enzymes.

Cloning, Molecular↗

Horizontal transfer of Shiga toxin and antibiotic resistance genes among Escherichia coli strains in house fly (Diptera: Muscidae) gut.

Whether the house fly, Musca domestica L., gut is a permissive environment for horizontal transfer of antibiotic resistance and virulence genes between strains of Escherichia coli is not known. House flies were immobilized and force fed suspensions of defined, donor strains of E. coli containing chloramphenicol resistance genes on a plasmid, or lysogenic, bacteriophage-born Shiga toxin gene stx1 (bacteriophage H-19B::Ap1). Recipient strains were E. coli lacking these mobile elements and genes but having rifampicin as a selectable marker. Plasmid transfer occurred at rates of 10(-2) per donor cell in the fly midgut and 10(-3) in the fly crop after 1 h of incubation postfeeding. Bacteriophage transfer rate was approximately 10(-6) per donor cell without induction, but induction with mitomycin C increased rates of transfer to 10(-2) per donor cell. These findings show that genes encoding antibiotic resistance or toxins will transfer horizontally among bacteria in the house fly gut via plasmid transfer or phage transduction. The house fly gut may provide a favorable environment for the evolution and emergence of pathogenic bacterial strains through acquisition of antibiotic resistance genes or virulence factors.

Animals↗

A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.

Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors.

Conjugation, Genetic↗

Gene arrangement in sea star mitochondrial DNA demonstrates a major inversion event during echinoderm evolution.

The mitochondrial (mt) DNA from the sea star Pisaster ochraceus has been isolated, restriction-mapped, and cloned into plasmid vectors. Both ribosomal RNA genes, the genes for 12 of the 13 mitochondrial proteins, and 11 of the tRNA genes have been localized by DNA sequence analyses. The sequence arrangement of the genes is markedly different from that seen in sea urchin mitochondrial DNA. A segment of the DNA molecule extending from tRNA(pro), including the tRNA cluster, ND1, ND2, and 16S genes, is inverted in relation to the sea urchin genome. The resulting gene order in the sea star is 12S, 16S, ND2, tRNA cluster, COI. As a result of the inversion, the transcriptional polarity of ND1, ND2, and 16S genes are opposite to that of the 12S and COI genes. The arrangement and transcriptional polarity of the other genes mapped here is the same as seen in urchin.

Animals↗

The Yersinia high-pathogenicity island (HPI): evolutionary and functional aspects.

The high-pathogenicity island (HPI) is a genomic island essential for the mouse-virulence phenotype in Yersinia and indispensable for pathogenicity of Yersinia and certain pathotypes of Escherichia coli. In contrast to most genomic islands, the HPI is a functional island widely disseminated among members of the family of Enterobacteriaceae. The HPI-encoded phage P4-like integrase together with excisionase and recombination sites make up the genetic mobility module of the island, while the siderophore yersiniabactin biosynthesis and uptake system comprises its functional part with respect to fitness and pathogenicity. The HPI-integrase promotes integration of the island into attB sites represented by three to four asn tDNAs in Yersinia pestis and E. coli. An additional enzyme, excisionase, is essential for efficient excision of the HPI from the initial site of integration. Furthermore a unique type of HPI has been characterized in the E. coli strain ECOR31 carrying a functional conjugative mating pair formation (Mpf) and a DNA-processing system, both of which are characteristic of integrative and conjugative elements (ICE). A model of conjugative transfer for the dissemination of HPIs is proposed in which the excised HPI is mobilized to a new recipient either trapped by a transmissive asn tDNA-carrying plasmid or autonomously as an ICE named ICEEcl.

Animals↗

Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation.

The efficiency of homologous recombination in Campylobacter coli following the introduction of DNA by natural transformation was determined by using a series of nonreplicating integrative vectors containing DNA fragments derived from the C. coli catalase gene. Homologous recombination occurred with as little as 286 homologous bp present and was not detected when 270 bases of homology was provided. Instead, when plasmids with little or no homology to the chromosome were introduced by natural transformation, the vector DNA became chromosomally integrated at random sites scattered throughout the C. coli genome. Southern analysis and nucleotide sequencing revealed that recombination had occurred between nonhomologous sequences and can therefore be described as illegitimate. There were at least five different recombination sites on plasmid pSP105. The ability of C. coli to acquire heterologous plasmids by natural transformation, and maintain them by chromosomal integration following illegitimate recombination, has fascinating implications for the genomic diversity and evolution of this species.

Base Sequence↗