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PubMed1984

The role of insertions, deletions, and substitutions in the evolution of R6 related plasmids encoding aminoglycoside transferase ANT-(2").

In 7% of gram-negative bacteria resistance to gentamicin is mainly mediated by plasmid-encoded aminoglycoside transferase ANT-(2"). The genome organization of 15 aadB plasmids (42-110 kb) was analyzed by restriction and hybridization techniques. They appeared to be IncFII-like replicons but were distinct from R6 by virtue of small substitutions in the transfer region. Aminoglycoside resistance genes aadB and aadA were located on Tn21 related elements. Only one of them was able to transpose its resistance genes mer sul aadA and aadB ( Tn4000 ), the other elements were naturally occurring defective transposons. In some of these structures deletions were identified at the termini, at sul, aadA , mer or transposition function--insertions adjacent to aadA or mer. The mode of these rearrangements and their site-specificity were considered with respect to the evolution of the Tn21 transposon family.

Aminoglycosides↗
PubMed1993

Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups.

We have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and 'prototype' plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.

Biological Evolution↗
PubMed1993

Genetic organization and evolution of the cryptic plasmid of Neisseria gonorrhoeae.

Three promoters have been identified on the phenotypically cryptic plasmid of Neisseria gonorrhoeae that can direct transcription in Escherichia coli. These promoters do not seem to function at a detectable level when N. gonorrhoeae is grown in vitro under normal growth conditions. The T7 RNA polymerase expression system has been used to identify the proteins encoded by this plasmid, and a series of subclones were constructed and used to localize the gene encoding each protein. In a search of the sequence databases, we have discovered that the derived amino acid sequence of a 1.2-kb segment of the plasmid shows a high degree of sequence similarity to the Mob proteins encoded by the colicinogenic plasmids ColA, ColE1, and ColK of E. coli. The 1.2-kb segment contains part of a Col mobilization region that has subsequently been inactivated by a small duplication. During the recombination event that formed the cryptic plasmid one of the mobilization genes was fused to a cryptic plasmid open reading frame to form the cppB gene. Despite the manner in which it was formed, we have shown that the cppB gene can be expressed when N. gonorrhoeae is grown under the appropriate environmental conditions.

Amino Acid Sequence↗
PubMed1985

Evolution of an R plasmid from a cryptic plasmid by transposition of two copies of Tn1 in Providencia stuartii.

Examination of a series of isolates of Providencia stuartii collected over an 18 month period from a chronic-care patient at Bristol Royal Infirmary revealed the emergence of resistance to carbenicillin. Resistance was mediated by a 47 kb plasmid which transferred by conjugation to a plasmid-free strain of P. stuartii but not to Escherichia coli. Carbenicillin-sensitive isolates were either plasmid-free or contained a 36 kb cryptic plasmid. Restriction endonuclease mapping of this plasmid showed it to be closely related to 32 kb and 34 kb cryptic plasmids reported previously in P. stuartii from Bristol. Mapping of the R plasmid showed it to be derived from the 34 kb cryptic plasmid by transposition of two copies of Tn1.

Conjugation, Genetic↗
PubMed1998

Structure and evolution of the leucine plasmids carried by the endosymbiont (Buchnera aphidicola) from aphids of the family Aphididae.

In all examined species of the family Aphididae, the bacterial endosymbiont Buchnera aphidicola carries a plasmid encoding the genes leuABCD (involved in leucine biosynthesis) along with repA1, repA2 and ORF1. The gene organisation of the leucine plasmids was conserved, except in Buchnera isolated from Pterocomma populeum, where ORF1 was located in a different position. An inverted repeat (LIR1) located between repA2 and leuA is found in all of the Buchnera leucine plasmids examined. The predicted secondary structure of the LIR1 transcript conforms to a long hairpin loop, suggesting an involvement in transcription termination or messenger stability. Phylogenetic reconstruction based on repA2 sequences suggests that horizontal transfer of Buchnera leucine plasmids has not occurred.

Amino Acid Sequence↗
PubMed1983

Evolution of antibiotic resistance plasmids in Neisseria gonorrhoeae and Haemophilus species.

The emergence of beta-lactamase producing strains of Haemophilus influenzae and Neisseria gonorrhoeae has required fundamental changes in the antimicrobial therapy of disease caused by these organisms. Ampicillin resistance in both organisms is caused by plasmid mediated production of TEM beta-lactamase. This enzyme is specified by a sequence of mol. wt 3.2 X 10(6) which is capable of inserting itself at multiple sites in DNA replicons without the requirement for significant base sequence homology between donor and recipient replicon. Further, it does so without requirement for conventional recombination enzymes. Analysis of beta-lactamase specifying plasmids of H. influenzae show that they generally have a molecular mass in the order of 30 X 10(6) and contain the complete TnA sequence. They are conjugative but are incapable of mobilizing smaller beta-lactamase plasmids. Previous studies have presented evidence suggesting that these plasmids may have evolved by insertion of the TnA sequence (perhaps introduced from enteric bacteria) into a phenotypically cryptic plasmid of mol. wt 27 X 10(6) resident in rare strains of H. influenzae. In this study, we review data showing a high degree of homology between the small (3--7 X 10(6) mol. wt), nonconjugative beta-lactamase specifying plasmids of N. gonorrhoeae, H. parainfluenzae and H. ducreyl and present new evidence that cryptic plasmids highly homologous to the beta-lactamase plasmids are present in many strains of H. parainfluenzae. This suggests that the small beta-lactamase specifying plasmids of H. parainfluenzae, H. ducreyi and N. gonorrhoeae may have arisen by insertion of TnA into phenotypically cryptic plasmids present in H. parainfluenzae.

Ampicillin↗
PubMed1992

DNA inversion regions Min of plasmid p15B and Cin of bacteriophage P1: evolution of bacteriophage tail fiber genes.

Plasmid p15B and the genome of bacteriophage P1 are closely related, but their site-specific DNA inversion systems, Min and Cin, respectively, do not have strict structural homology. Rather, the complex Min system represents a substitution of a Cin-like system into an ancestral p15B genome. The substituting sequences of both the min recombinase gene and the multiple invertible DNA segments of p15B are, respectively, homologous to the pin recombinase gene and to part of the invertible DNA of the Pin system on the defective viral element e14 of Escherichia coli K-12. To map the sites of this substitution, the DNA sequence of a segment adjacent to the invertible segment in the P1 genome was determined. This, together with already available sequence data, indicated that both P1 and p15B had suffered various sequence acquisitions or deletions and sequence amplifications giving rise to mosaics of partially related repeated elements. Data base searches revealed segments of homology in the DNA inversion regions of p15B, e14, and P1 and in tail fiber genes of phages Mu, T4, P2, and lambda. This result suggest that the evolution of phage tail fiber genes involves horizontal gene transfer and that the Min and Pin regions encode tail fiber genes. A functional test proved that the p15B Min region carries a tail fiber operon and suggests that the alternative expression of six different gene variants by Min inversion offers extensive host range variation.

Amino Acid Sequence↗
PubMed1991

Molecular evolution of tetracycline-resistance plasmids carrying TetM found in Neisseria gonorrhoeae from different countries.

High level tetracycline resistant strains of Neisseria gonorrhoeae (TRNG) have been shown to carry a 40.6 kb (25.2 MDa) conjugative plasmid with a Class M tetracycline resistance determinant. Restriction endonuclease analysis mapping showed that there were at least two different TRNG plasmid types which were found in geographically distinct locations. The physical maps of these two plasmids were compared to a gonococcal conjugative plasmid which did not encode tetracycline resistance. The plasmid type which is endemic in the Netherlands was found to be closely related to the gonococcal conjugative plasmid, which supports the established hypothesis that the 40.6 kb plasmid has evolved by transposition of the TetM determinant into the conjugative plasmid. The plasmid found in the United States has either evolved by substantial divergent evolution or it results from a different transposition event. In the UK there have been isolations of TRNGs carrying either of the two plasmid types reflecting a flow of people both across the Atlantic and in Europe. It is possible that further TetM-containing plasmids will be found in N. gonorrhoeae paralleling the family of TEM beta-lactamase encoding plasmids already described.

Bacterial Proteins↗
PubMed1999

Proteic toxin-antitoxin, bacterial plasmid addiction systems and their evolution with special reference to the pas system of pTF-FC2.

Genes encoding toxin-antitoxin proteins are frequently found on plasmids where they serve to stabilize the plasmid within a bacterial population. The toxin-antitoxin proteins do not increase the likelihood of a progeny cell receiving a plasmid but rather function as post-segregational killing mechanisms which decrease the proportion of cells that survive after losing the plasmid. These toxin-antitoxin couples therefore act as plasmid addiction systems. Several new proteic toxin-antitoxin systems have been identified and these systems appear to be ubiquitous on the chromosomes of bacteria and archaea. When placed on plasmids, these chromosomal systems also have the ability to stabilize plasmids and in at least one case, chromosomal- and plasmid-based toxin-antitoxin systems have been shown to interact. Recent findings regarding toxin-antitoxin systems and questions that have arisen as a result of these findings are reviewed.

Bacterial Proteins↗
PubMed1996

Identification of a native Dichelobacter nodosus plasmid and implications for the evolution of the vap regions.

Studies on the role of various virulence factors of the ovine pathogen, Dichelobacter nodosus, have suffered from the absence of a mechanism for the introduction of DNA into this organism. As an initial step in the development of genetic methods, we have identified and cloned a native 10-kb plasmid, pJIR896, from a clinical isolate. This plasmid was found to be a circular form of vap region 1/3 that is found in the reference strain, A198. However, pJIR896 lacked the duplicated region present in the A198 sequence and instead contained a 1.7-kb putative insertion sequence, IS1253, which shared similarity to a number of unusual IS elements. A model is proposed for the evolution of vap region 1/3 which involves the integration of a plasmid, such as pJIR896, and subsequent rearrangements resulting from the deletion or transposition of IS1253.

Base Sequence↗
PubMed1979

Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences.

Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.

Anti-Bacterial Agents↗
PubMed1989

[Genetic systems of biodegradation: organization and regulation of expression].

The review discusses the current state of genetic analysis of degradative processes. Attention is mainly given to the structural and functional organization of the plasmid systems of degradation of organic compounds in gram-negative bacteria. The data available on the regulation mechanisms of D plasmids' metabolic operons, based on the most studied models of xyl and nah operons, are critically analyzed. The problems of evolution of plasmid D systems are considered conceptually as well as the principles of the experimental strategy of developing new metabolic pathways under laboratory conditions. The prospects of constructing the strains capable of efficient degradation of xenobiotics are considered in brief.

Biodegradation, Environmental↗
PubMed2004

Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli.

Plasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera. They frequently mediate resistance to multiple antimicrobials and can result in the acquisition by a pathogen of resistance to all or most clinically relevant antimicrobials. Unfortunately, there are still large gaps in our understanding of how new multi-resistance plasmids evolve. Five Australian clinical institutions collaborated in this study of multi-resistance plasmids in clinical isolates of Escherichia coli. We characterized 72 resistance plasmids in terms of the antimicrobial resistance profile they conferred, their size and their incompatibility group. Restriction fragment length polymorphisms were used to determine the genetic relationships between the plasmids. Relationships between the host cells were determined using multi-locus enzyme electrophoresis. A lack of correlation between the evolutionary history of the host cells and their plasmids suggests that the horizontal transfer of resistance plasmids between strains of E. coli is common. The resistance plasmids were very diverse, with a wide range of resistance profiles and a lack of discrete evolutionary lineages. Multi-resistance plasmids did not evolve via the co-integrative capture of smaller resistance plasmids; rather, the roles of recombination and the horizontal movement of mobile genetic elements appeared to be most important.

Anti-Bacterial Agents↗
PubMed2005

Whole plasmid mutagenic PCR for directed protein evolution.

Protein function can be engineered through iterated cycles of random mutagenesis and screening (directed evolution). Optimization of protein expression is essential for the development of sensitive and precise high throughput assays. Here we optimize the performance of a plasmid-borne Escherichia coli lacZ gene in two rounds of directed evolution. First, its promoter was "randomized" by whole plasmid polymerase chain reaction (PCR) and intra-molecular self-ligation. A genetically stable constitutive expression vector was isolated in an in vivo genetic selection. Second, the entire plasmid was randomly mutated in a slightly mutagenic long polymerase chain reaction. The PCR products were digested with a restriction enzyme, self-ligated by T4 DNA ligase and transformed into E. coli. The resulting library of beta-galactosidase (beta-gal) mutants consisted mostly ( approximately 80%) of hypomorphs, suggesting that the mutation rate was appropriate for directed evolution applications. We isolated and characterized 14 variants with increased activity in reactions with 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). The purified protein derived from one clone exhibited a 100-fold improvement in k(cat) over its parent in reactions with para-nitrophenyl-beta-d-galactopyranoside (pNP-gal). This latter result clearly demonstrates the utility of whole plasmid mutagenic PCR for directed protein evolution.

Cloning, Molecular↗
PubMed2003

Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase.

BACKGROUND: The ars gene system provides arsenic resistance for a variety of microorganisms and can be chromosomal or plasmid-borne. The arsC gene, which codes for an arsenate reductase is essential for arsenate resistance and transforms arsenate into arsenite, which is extruded from the cell. A survey of GenBank shows that arsC appears to be phylogenetically widespread both in organisms with known arsenic resistance and those organisms that have been sequenced as part of whole genome projects. RESULTS: Phylogenetic analysis of aligned arsC sequences shows broad similarities to the established 16S rRNA phylogeny, with separation of bacterial, archaeal, and subsequently eukaryotic arsC genes. However, inconsistencies between arsC and 16S rRNA are apparent for some taxa. Cyanobacteria and some of the gamma-Proteobacteria appear to possess arsC genes that are similar to those of Low GC Gram-positive Bacteria, and other isolated taxa possess arsC genes that would not be expected based on known evolutionary relationships. There is no clear separation of plasmid-borne and chromosomal arsC genes, although a number of the Enterobacteriales (gamma-Proteobacteria) possess similar plasmid-encoded arsC sequences. CONCLUSION: The overall phylogeny of the arsenate reductases suggests a single, early origin of the arsC gene and subsequent sequence divergence to give the distinct arsC classes that exist today. Discrepancies between 16S rRNA and arsC phylogenies support the role of horizontal gene transfer (HGT) in the evolution of arsenate reductases, with a number of instances of HGT early in bacterial arsC evolution. Plasmid-borne arsC genes are not monophyletic suggesting multiple cases of chromosomal-plasmid exchange and subsequent HGT. Overall, arsC phylogeny is complex and is likely the result of a number of evolutionary mechanisms.

Adenosine Triphosphatases↗
PubMed1983

Investigation of nosocomial infections by plasmid analysis.

Molecular biological techniques, including agarose gel electrophoresis, restriction endonuclease analysis and DNA-DNA hybridization (Southern blotting and heteroduplex analysis) are being applied very successfully to the investigation of nosocomial infections. 'Plasmid fingerprinting' by electrophoresis, with or without restriction endonuclease analysis, can be used to identify epidemic strains of bacteria and 'epidemic plasmids' which have spread through several different bacterial species. This technique is rapid and inexpensive, and can be applied to drug-sensitive as well as resistant strains. This approach is especially useful for investigation of organisms for which no standard typing system is available. DNA-DNA hybridization techniques can be used to study the evolution of plasmids in the hospital environment, and to demonstrate the presence and spread of translocatable DNA sequences (transposons) carrying drug resistance determinants from plasmid to plasmid within a bacterial cell.

Cross Infection↗