Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution

Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosome, termed‘pathogenicity islands’(Pais). Pathogenicity islands are found in Gram-negative as well as in Gram-positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non-pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G+C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e.g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication or IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.     

Evolution in bacterial plasmids and levels of selection

Gene flow between different reproductive units such as bacterial plasmids and chromosomes presents unusual problems for evolutionary analysis. Far more than in eukaryotes, reproductive advantages at several levels of selection--genes, transposons, plasmids, cells, and clones--must be considered simultaneously to understand plasmid evolution. No level consistently prevails in conflict situations, and some reproductive units carry genes that restrain their own reproduction or survival, apparently to enhance the reproduction or survival of the higher-level reproductive units that carry them. Despite gene flow between plasmids and chromosomes, genes for certain functions show strong tendencies to occur on plasmids while others consistently occur on chromosomes. Functions generally associated with plasmids are diverse, but all are useful only in locally restricted contexts; it is argued that the selective consequences of the greater horizontal (within generation) transmission of plasmids are responsible for this pattern. The tendency for prokaryote transposons, which are also horizontally mobile, to carry genes similar to those commonly on plasmids supports this argument. The apparent trends in eukaryote plasmids and transposons to lack these same characters also accords with predictions of the local adaptation hypothesis, because genes on these genetic units are generally no more horizontally mobile than chromosomal genes. There are theoretical reasons to expect that plasmid genes tend to evolve more rapidly than chromosomal genes. "The selfish interests of genes have manifestly produced 'vehicles' in the forms of organelles, cells, individuals and yet higher units. If evolution is to predict as well as describe, then selfish interests must be understood in the framework of the constraints and opportunities generated by these 'vehicles'" (Buss, 1987, p. 182).     

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non‐essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.     

Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution

Restriction–modification (RM) systems are composed of genes that encode a restriction enzyme and a modification methylase. RM systems sometimes behave as discrete units of life, like viruses and transposons. RM complexes attack invading DNA that has not been properly modified and thus may serve as a tool of defense for bacterial cells. However, any threat to their maintenance, such as a challenge by a competing genetic element (an incompatible plasmid or an allelic homologous stretch of DNA, for example) can lead to cell death through restriction breakage in the genome. This post-segregational or post-disturbance cell killing may provide the RM complexes (and any DNA linked with them) with a competitive advantage. There is evidence that they have undergone extensive horizontal transfer between genomes, as inferred from their sequence homology, codon usage bias and GC content difference. They are often linked with mobile genetic elements such as plasmids, viruses, transposons and integrons. The comparison of closely related bacterial genomes also suggests that, at times, RM genes themselves behave as mobile elements and cause genome rearrangements. Indeed some bacterial genomes that survived post-disturbance attack by an RM gene complex in the laboratory have experienced genome rearrangements. The avoidance of some restriction sites by bacterial genomes may result from selection by past restriction attacks. Both bacteriophages and bacteria also appear to use homologous recombination to cope with the selfish behavior of RM systems. RM systems compete with each other in several ways. One is competition for recognition sequences in post-segregational killing. Another is super-infection exclusion, that is, the killing of the cell carrying an RM system when it is infected with another RM system of the same regulatory specificity but of a different sequence specificity. The capacity of RM systems to act as selfish, mobile genetic elements may underlie the structure and function of RM enzymes.     

Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats

Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

Background  

In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery and production of recombinant proteins. Usually, bacteria are manipulated using plasmid expression vectors. The major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. We have developed a genetic system based on suicide vectors on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable Gram-positive bacteria.     

Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.     

Periodic extinctions of transposable elements in bacterial lineages: evidence from intragenomic variation in multiple genomes

Most previous work on the evolution of mobile DNA was limited by incomplete sequence information. Whole genome sequences allow us to overcome this limitation. I study the nucleotide diversity of prominent members of five insertion sequence families whose transposition activity is encoded by a single transposase gene. Eighteen among 376 completely sequenced bacterial genomes and plasmids carry between 3 and 20 copies of a given insertion sequence. I show that these copies generally show very low DNA divergence. Specifically, more than 68% of the transposase genes are identical within a genome. The average number of amino acid replacement substitutions at amino acid replacement sites is Ka = 0.013, that at silent sites is Ks = 0.1. This low intragenomic diversity stands in stark contrast to a much higher divergence of the same insertion sequences among distantly related genomes. Gene conversion among protein-coding genes is unlikely to account for this lack of diversity. The relation between transposition frequencies and silent substitution rates suggests that most insertion sequences in a typical genome are evolutionarily young and have been recently acquired. They may undergo periodic extinction in bacterial lineages. By implication, they are detrimental to their host in the long run. This is also suggested by the highly skewed and patchy distribution of insertion sequences among genomes. In sum, one can think of insertion sequences as slow-acting infectious diseases of cell lineages.     

Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element.

The Bacteroides species harbor a family of conjugative transposons called tetracycline resistance elements (Tcr elements) that transfer themselves from the chromosome of a donor to the chromosome of a recipient, mobilize coresident plasmids, and also mediate the excision and circularization of members of a family of 10- to 12-kbp insertion elements which share a small region of DNA homology and are called NBUs (for nonreplicating Bacteroides units). The NBUs are sometimes cotransferred with Tcr elements, and it was postulated previously that the excised circular forms of the NBUs were plasmidlike forms and were transferred like plasmids and then integrated into the recipient chromosome. We used chimeric plasmids containing one of the NBUs, NBU1, and a Bacteroides-Escherichia coli shuttle vector to show that this hypothesis is probably correct. NBU1 contained a region that allowed mobilization by both the Tcr elements and IncP plasmids, and we used these conjugal elements to allow us to estimate the frequencies of excision, mobilization, and integration of NBU1 in Bacteroides hosts to be approximately 10(-2), 10(-5) to 10(-4), and 10(-2), respectively. Although functions on the Tcr elements were required for the excision-circularization and mobilization of NBU1, no Tcr element functions were required for integration into the recipient chromosome. Analysis of the DNA sequences at the integration region of the circular form of NBU1, the primary insertion site in the Bacteroides thetaiotaomicron 5482 chromosome, and the resultant NBU1-chromosome junctions showed that NBU1 appeared to integrate into the primary insertion site by recombining within an identical 14-bp sequence present on both NBU1 and the target, thus leaving a copy of the 14-bp sequence at both junctions. The apparent integration mechanism and the target selection of NBU1 were different from those of both XBU4422, the only member of the conjugal Tcr elements for which these sequences are known, and Tn4399, a mobilizable Bacteroides transposon. The NBUs appear to be a distinct type of mobilizable insertion element.     

Construction of transposons carrying the transfer functions of RP4

The transfer genes and origin of transfer of the wide host range plasmid RP4 have been cloned into the transposons Tn1 and Tn5. The newly constructed transposons can be used to mutagenize bacterial plasmids or the chromosome in species such as Escherichia coli or Rhizobium. It is then possible to mobilize the plasmid or chromosome using the transfer functions provided in cis by the transposon. These constructs may aid chromosome mapping in many gram-negative species by allowing the wider use of the RP4 conjugal transfer system combined with the potential ability to select the site of insertion and thus the site of the origin of transfer.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.     

Genes without frontiers?

For bacteria, the primary genetic barrier against the genetic exchange of DNA that is not self-transmissible is dissimilarity in the bacterial DNA sequences concerned. Genetic exchange by homologous recombination is frequent among close bacterial relatives and recent experiments have shown that it can enable the uptake of closely linked nonhomologous foreign DNA. Artificial vectors are mosaics of mobile DNA elements from free-living bacterial isolates and so bear a residual similarity to their ubiquitous natural progenitors. This homology is tightly linked to the multitude of different DNA sequences that are inserted into synthetic vectors. Can homology between vector and bacterial DNA enable the uptake of these foreign DNA inserts? In this review we investigate pUC18 as an example of an artificial vector and consider whether its homology to broad host-range antibiotic resistance transposons and plasmid origins of replication could enable the uptake of insert DNA in the light of studies of homology-facilitated foreign DNA uptake. We also discuss the disposal of recombinant DNA, its persistence in the environment and whether homologies to pUC18 may exist in naturally competent bacteria. Most DNA that is inserted into the cloning site of artificial vectors would be of little use to a bacterium, but perhaps not all.     

Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance

Antibiotic-resistant Gram-positive bacteria are responsible for morbidity and mortality in healthcare environments. Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pneumoniae can all exhibit clinically relevant multidrug resistance phenotypes due to acquired resistance genes on mobile genetic elements. It is possible that clinically relevant multidrug-resistant Clostridium difficile strains will appear in the future, as the organism is adept at acquiring mobile genetic elements (plasmids and transposons). Conjugative transposons of the Tn916/Tn1545 family, which carry major antibiotic resistance determinants, are transmissible between these different bacteria by a conjugative mechanism during which the elements are excised by a staggered cut from donor cells, converted to a circular form, transferred by cell-cell contact and inserted into recipient cells by a site-specific recombinase. The ability of these conjugative transposons to acquire additional, clinically relevant antibiotic resistance genes importantly contributes to the emergence of multidrug resistance.     

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn3-Derived Inverted-Repeat Miniature Elements (TIMEs)

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.     

Transfer of "artificial transposons" constructed on the basis of insertion element IS1

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.     

Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells

Defective herpes simplex virus type 1 genomes are composed of head-to-tail tandem repeats of small regions of the nondefective genome. Monomeric repeat units of class I defective herpes simplex virus genomes were cloned into bacterial plasmids. The repeat units functioned as replicons since both viral and convalently linked bacterial plasmid DNA replicated (with the help of DNA from nondefective virus) when transfected into rabbit skin cells. Recombinant plasmids were packaged into virions and were propagated from culture to culture by infection with progeny virus. Replication was evidently by a rolling circle mechanism since plasmid DNA was present in a high-molecular-weight form in transfected cells. Circular recombinant plasmid DNA replicated with a high degree of fidelity. In contrast, linear plasmid DNA underwent extensive deletions of both viral and bacterial sequences when transfected into rabbit skin cells. Derivative plasmids, a fraction of the size of the parental plasmid, were rescued by transforming Escherichia coli with DNA from the transfected rabbit skin cells. These plasmids functioned as shuttle vectors since they replicated faithfully in both eucaryotic and procaryotic cells.     

Infectious plasmid resistance and efflux pump mediated resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.