Why do bacterial plasmids carry some genes and not others?

Previous explanations of why bacterial genes for certain "optional" traits tend to occur on plasmids rather than chromosomes are based on an outdated misunderstanding of natural selection. They also fail to explain why certain characters that are ubiquitous in some bacterial species tend to occur on plasmids. This paper shows that all major classes of traits usually associated with plasmids rather than chromosomes confer adaptations to locally restricted conditions. A new "local adaptation" model of plasmid evolution, based on simultaneous application of modern selection theory at the levels of gene, plasmid, cell, and clone reproduction, shows that genes coding local adaptations will reproduce more successfully when on plasmids than when on chromosomes, due to plasmids' greater horizontal mobility.     

Lineage‐specific plasmid acquisition and the evolution of specialized pathogens in Bacillus thuringiensis and the Bacillus cereus group

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co‐evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large‐scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co‐evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.     

'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.     

A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution

Bacterial circular chromosomes have sporadically become linearised during prokaryote evolution. Unrelated bacteria, including the spirochete Borrelia burgdorferi and the actinomycete Streptomyces, have linear chromosomes. Linear chromosomes may have been formed through integration of linear plasmids. Linear chromosomes use linear plasmid strategies to resolve the 'end-of-replication problem', but they have generally retained from their circular ancestors a central origin of replication. Streptomyces linear chromosomes are very unstable and at high frequency undergo amplifications and large deletions, often removing the telomeres. At least in Streptomyces, chromosome linearity is reversible: circular chromosomes arise spontaneously as products of genetic instability or can be generated artificially by targeted recombination. Streptomyces circularised chromosomes are very unstable as well, indicating that genetic instability is not confined to the linearised chromosomes. Bacterial linear chromosomes may contain telomere-linked regions of enhanced genomic plasticity, which undergo more frequent genetic exchanges and rearrangements and allow differential evolution of genes, depending on their chromosomal location.     

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.     

Gene and cell survival: lessons from prokaryotic plasmid R1

Plasmids are units of extrachromosomal genetic inheritance found in all kingdoms of life. They replicate autonomously and undergo stable propagation in their hosts. Despite their small size, plasmid replication and gene expression constitute a metabolic burden that compromises their stable maintenance in host cells. This pressure has driven the evolution of strategies to increase plasmid stability--a process accelerated by the ability of plasmids to transfer horizontally between cells and to exchange genetic material with their host and other resident episomal DNAs. These abilities drive the adaptability and diversity of plasmids and their host cells. Indeed, survival functions found in plasmids have chromosomal homologues that have an essential role in cellular responses to stress. An analysis of these functions in the prokaryotic plasmid R1, and of their intricate interrelationships, reveals remarkable overall similarities with other gene- and cell-survival strategies found within and beyond the prokaryotic world.     

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.     

Das bakterielle Mobilom

Mobile elements Meanwhile, we know that mobile elements are present in all forms of life. The genomes of mammals consist of up to 50% of mobile elements, those of plants up to 90%. In bacteria mobile elements establish themselves as autonomously replicating replicons (plasmids and plasmid‐prophages) or are constituents of replicons such as IS elements, transposons, conjugative transposons, integrative and conjugative elements (ICEs), genomic islands and integrons. Why distinct bacterial species still exist? Most probably, still unknown mechanisms ensure that some genes never become mobile.     

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.     

Occurrence of sep insecticidal toxin complex genes in Serratia spp. and Yersinia frederiksenii

Some strains of Serratia entomophila and S. proteamaculans cause amber disease of the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae). Three genes required for virulence, sepABC, are located on a large plasmid, pADAP. Sequence analysis suggests that the sepABC gene cluster may be part of a horizontally mobile region. This study presents evidence for the putative mobility of the sep genes of pADAP. Southern blot analysis showed that orthologues of the sep genes reside on plasmids within S. entomophila, S. liquefaciens, S. proteamaculans, and a plasmid from Yersinia frederiksenii. Three plasmids hybridized to the pADAP sep virulence-associated region but not the pADAP replication and conjugation regions. Subsequent DNA sequence analysis of the Y. frederiksenii sep-like genes, designated tcYF1 and tcYF2, showed that they had 88% and 87% DNA identity to sepA and sepB, respectively. These results indicate that the sep genes are part of a discrete horizontally mobile region.     

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.     

Diversity of bacterial type II toxin-antitoxin systems: a comprehensive search and functional analysis of novel families

Type II toxin-antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as 'addiction', and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the 'guilt by association' principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.     

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.     

Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea

Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic-producing strains, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.