Plasmid Biology

The presence of Rep-binding iterons is a hallmark, not only of many prokaryotic plasmid orfs but of chromosomal, viral (phage), and eukaryotic ori’s(5, 75, 92) as well. This chapter focuses primarily on the prokaryotic plasmid members of the Rep/iteron family, which is referred to as iteron-containing plasmids (ICPs). Several extensive reviews on a variety of aspects of plasmid biology have been written, and this review serves as an extension of the earlier works. Sequence conservation in the adjacent major and minor grooves has been demonstrated for iterons from prokaryotic and eukaryotic ori’s. It is likely, then, that a diverse set of Rep proteins can utilize the intrinsic instability of specific base pairs within iterons to bind DNA and facilitate its melting. The chapter discusses what appeared to be a rather well-understood system, λdv. It exemplifies the autorepressor model originally developed to explain the control of Escherichia coli chromosomal replication, and discusses some of the additional DNA sequences and host proteins that likely contribute to the regulation of ICPs. The chapter discusses molecular mechanism of ori activation, and focuses on Rep/iteron-mediated replication of minimal ICPs in E. coli. The contributions of plasmids to the development of modern molecular biology were astutely articulated a decade ago. Currently, the need for ongoing and vigorous support of basic and applied research on plasmids continues, with an imperative to solve practical issues of fundamental societal importance. A prime example is the emergence and alarming spread of bacterial resistance to multiple antibiotics.

This chapter reviews antisense-RNA-mediated regulation of plasmid replication. Antisense-RNA control in plasmid replication works through a negative control circuit: Antisense RNAs are constitutively synthesized and metabolically unstable. Antisense-RNA-mediated transcriptional attenuation is another mechanism, which has, so far, only been detected in plasmids of gram-positive bacteria (inc18 family and pT181 family). For antisense-RNA-controlled plasmids that replicate by the theta mechanism, the data on origin characterization and replication mechanism are briefly summarized in this chapter. A three-dimensional model of the N-terminal 63 amino acids (aa) of CopR was constructed, and amino acids involved in DNA binding and dimerization were localized: Arg29 and Arg34 within the HTH-motif are involved in specific recognition of the operator-DNA. Recently, the author showed experimentally that replication control mechanism principally functions in Escherichia coli, albeit with a much lower efficiency than in Bacillus subtilis or Staphylococcus aureus. The interaction between two highly structured antisense and sense RNAs, initiating by defined loop-loop contacts as shown for plasmid R1, is a recurrent one and valid for most cases of plasmid replication control. The degradation pathway of CopA has been studied in detail. A mechanism that involves RNA-RNA interactions in a manner that interferes with translation was also suggested for pC194 and pUBHO, two other RCR-type plasmids. The chapter talks about inhibition of primer formation and pseudoknot formation.

Plasmids that replicate by a rolling-circle (RC) mechanism are ubiquitous in gram-positive bacteria and are also found in gram-negative bacteria as well as in Archaea. This chapter discusses the general anatomy of rolling-circle replicating (RCR) plasmids, architecture of the double-strand origin (dso), the single-strand origin (sso), the initiator proteins and their structure-function relationship, key events during the initiation and termination process, and the role of host proteins in plasmid RC replication. It highlights the gaps in the current understanding of the replication of RCR plasmids and possible future lines of research that may uncover these gaps. The first of the RCR plasmids to be identified were native to the gram-positive bacterium, Staphylococcus aureus. The Rep proteins of the pT181 family act as dimers and utilize Tyr-191 of the two monomers in the initiation and termination events. Biochemical analyses using heterodimers of the pT181 RepC protein have provided insights into the role of individual monomers in plasmid RC replication. Elucidation of the three-dimensional structure of plasmid Rep proteins should considerably increase the understanding of the mechanistic aspects of plasmid RC replication. The availability of crystal structures of the initiators of various plasmid families should provide major insights into the mechanisms of initiation and termination of plasmid RC replication.

Plasmids, as extrachromosomal elements, bear the burden of ensuring their own faithful segregation at cell division. This chapter reviews partition systems, which are, in general, systems that actively dictate the specific localization of plasmids inside the bacterial cell and coordinate this localization with the bacterial cell cycle. Partition systems also exert incompatibility, which is distinct from the replication-mediated incompatibility that has been used to classify plasmids. Growth of the membrane between attachment sites was proposed to push plasmids apart. It was subsequently shown that membrane growth is dispersive and thus cannot solely account for plasmid movement. An appealing candidate for the plasmid road sign is the bacterial replication apparatus. Experiments in Escherichia coli and Bacillus subtilis indicate that the replication machinery exists as localized factories in the cell. The intracellular localization patterns of the ParA from the E. coli virulence plasmid pB171 provide an intriguing clue as to the mechanism of ParA function. RepA and RepB are not essential for replication but are essential for plasmid stability. They have been shown to influence copy number, but this may be due to effects on the expression of repC. Recent cell biology, biochemical, and structural data show that R1 ParM looks and behaves like actin and suggest a partition model in which ParM acts as a cytoskeletal element to drive the movement of plasmids during the cell cycle.

Observations suggest that the genome is a community of genes that essentially act selfishly and potentially do not have the overall order of the genome as their primary interest. Postsegregational host killing maintains the existence of a genetic element by death. This chapter extracts general rules whereby death is used to govern genomes. It reviews the development of the concept of genetic addiction, and introduces several types of addiction systems. The chapter examines where the addiction modules are located in genomes and how they get there, and discusses their mechanisms of action and their gene expression regulation, which include contributions from structural studies. It sketches various kinds of interactions that take place between addiction systems within a genome and then addresses the central paradox: why a genetic element that is potentially toxic to the genome is ever maintained. The chapter then reviews the evidence that suggests that some forms of genetic addiction have affected genome evolution. Further, the chapter extrapolates these arguments, which are based on bacterial systems, to genome biology in general. Finally, it summarizes the various ways addiction systems can be used in experimental biology, biotechnology, and medicine. The concept of genetic addiction may prove to be one of the most fruitful contributions from plasmid biology to the understanding of life.

Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, an efficient and tightly controlled DNA breakage and joining reaction occurring at the level of determined DNA sequences. Site-specific recombinases, the enzymes that catalyze this type of reaction, fall into two families of proteins: the serine-recombinase and tyrosine-recombinase families. The chapter discusses the mechanisms that generate DNA multimers and their consequence on the segregational stability of bacterial replicons, and also provides an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes and their relationship to other recombination systems. It focuses on site-specific resolution systems of the serine-recombinase family, and plasmid and chromosome resolution systems of the tyrosine recombinase family. The topology of the recombination reaction catalyzed by other resolvases of the serine-recombinase family, such as the ParA protein of RP4/RK2, the resolvase of ISXc5, the Sin recombinase of Staphylococcus aureus, and the β recombinase of pSM19035, was found to be identical to that reported for the cointegrate resolution system of Tn3-family transposons. Studies on plasmid and transposon resolution systems provide fascinating examples of convergent evolution, in which structurally and biochemically unrelated molecular machines have been adapted to bring about functionally similar DNA rearrangements in an exquisitely controlled manner.

Chromosome topology is a fundamental property relevant to a wide range of biological processes including DNA replication, RNA transcription, genetic recombination, transposition, and DNA repair. One aim of this chapter is to summarize the understanding of plasmid topological behavior, and also point out experimental situations in which plasmid topology can be misinterpreted. In enteric bacteria, four distinct topoisomerases are able to change the linking status of plasmid DNA molecules. The known enzymes that alter linking number include Topo I, DNA gyrase, Topo III, and Topo IV. DNA gyrase and Topo IV are related enzymes that break both strands of DNA simultaneously and are classified as type II enzymes. Tests of all four enzymes indicate that Topo III does not normally contribute to the in vivo topology of plasmid DNA. Topo IV can remove negative supercoils from plasmid DNA in vivo, and topological balance inside living cells involves at least DNA gyrase, Topo I, and Topo IV. Intramolecular triplex DNA (H-DNA) may form at sequences containing long stretches of polypurine-polypyrimidine. In the H-form, half of either the purine- or pyrimidine-rich strand becomes unpaired and its complement becomes triple-stranded by forming Hoogsteen base pairs with purines in the major groove of the Watson-Crick base-paired segment. The ability to distinguish between constrained and unconstrained supercoiling is often necessary to fully explain topological changes that can be measured in plasmid DNA.

Bacterial conjugation is one of the fundamental processes for gene dissemination or horizontal gene transfer in nature and involves the transfer of DNA between bacteria in close apposition with one another. This chapter pulls together the information currently available on conjugative systems in gram-negative bacteria; reassess the information available before 1994, the year the sequences of two important gram-negative conjugation systems, F and RP4, appeared; and begin to construct a way of classifying and naming the genes from a plethora of conjugative systems, many of which appear to be chimeras of F and RP4. The junction between mating cells requires that the outer membranes of the two cells come into close apposition. The mechanism of active disaggregation of mating cells is unknown but might be related to the finding that transfer of IncI1 plasmids is terminated by a process requiring de novo protein synthesis in the new transconjugant. The relaxase, which is asymmetric in shape, is situated at the entry portal (base) of the pilus and is translocated as a first step in the conjugative process during mating-pair stabilization. Its inclusion in the conjugative pore could be in response to the triggering of conjugative DNA processing in the donor cell. The chapter discusses DNA processing and transport, the nature of the "signal" that triggers transfer, and mobilizable plasmids. It talks about F-like systems and P-like systems for which detailed analyses of gene regulation exist.

This chapter discusses conjugative systems in various gram-positive genera, and includes some discussion of conjugative transposons insofar as they assume a plasmid-like (circular) intermediate structure during movement. The pheromone-responding plasmids, which frequently encode antibiotic resistance traits as well as the production of cytolysins or bacteriocins, are commonly found in Enterococcus faecalis. The plasmids that have been studied in the greatest detail with respect to regulation of the pheromone response are pADl, pCFlO, pPDl, and pAM373. The hemolysin/bactcriocin (cytolysin)-encoding plasmids exemplified by the well-characterized pADl represent a large and globally disseminated family of pheromone-responding plasmids. Analysis of the sequence suggests an important role for insertion sequence elements in its evolution insofar as the conjugation genes, bacteriocin production genes, and bacteriophage resistance and plasmid maintenance functions are organized in three different regions separated by insertion sequence elements. The mob-oriT region is essential for the pBC16 mobilization by several other Bacillus conjugative plasmids, such as pLS20, pHT73, or pAW63, and represents functions generally required for the transfer of mobilizable plasmids by self-transmissible plasmids in gram-negative and other gram-positive bacteria. The growing problem of multiple antibiotic resistance among human and animal pathogens is a classic example of how quickly horizontal gene transfer can relate to serious health issues.

This chapter emphasizes on the mechanisms that enable promiscuous plasmids of gram-negative bacteria to replicate in diverse hosts. Three different replication strategies have been identified thus far for circular plasmids: theta, strand displacement, and rolling circle. Each replication strategy has specific plasmid-directed initiation events leading to the establishment of the replisome and different host protein requirements. IncP plasmids can replicate in diverse gram-negative bacteria and can be conjugally transferred into an even broader group of organisms. Recent molecular studies have provided evidence that the mechanism utilized for recruitment of the host-encoded replicative helicase to the origin of plasmid RK2 is host specific and is dependent on the form of the TrfA protein present in the cell. Plasmids belonging to the IncQ group, including RSF1010 or R1162, are relatively small in size and have a moderate copy number. The final products of replication initiating at one of the two available ssi sites are a double-stranded replicated molecule and a single-stranded displaced circle. The studies with the IncQ plasmids reveal that providing the proteins essential for the establishment of the replisome allows sufficient flexibility for replication in different bacterial hosts. Recently an Escherichia coli mutant was isolated that allowed efficient replication of wild-type pPS10 at 37°C.

The Leguminosae, with around 18,000 species, is the largest plant family on Earth; its ecological success owes much to the existence of nitrogen-fixing symbioses with prokaryotes. These symbioses occur mainly with members of the Rhizobiaceae family (belonging to the a-proteobacteria). Clearly, research in the molecular biology of rhizobia- legume interactions has illuminated the ways in which bacteria and eukaryotes interact in a symbiotic process. However, this research also showed, almost from the start, the existence of novel forms of genome organization in prokaryotes, such as the finding of multiple large plasmids. Conversely, elimination of the pSym impairs both nodulation and nitrogen fixation of the original bacterial strain. Plasmids p42c, p42e, and p42f influence successful competitiveness between strains for nodulation, while p42f is needed for nitrogen fixation; only the self-conjugative plasmid p42a appears to be dispensable for symbiosis. As everything in biology, the current revolution in genomics has changed the way in which one addresses these problems. On the backbone, genes for nodulation are located in three noncontiguous clusters, which are separate and far apart from three clusters of nitrogen fixation genes. Control by quorum-sensing systems has dominated the study of conjugation in the Rhizobiaceae. However, it must be stressed that conjugative transfer may be modulated by other environmental cues, such as nutritional factors.

This chapter reviews the structure and replication of bacterial linear plasmids. Examples are given of the varying structures of the termini (telomeres) of linear plasmids from representative bacteria, and proposed mechanisms for telomere replication and resolution are described. Although the focus is on linear plasmids, most of what is described in the chapter also applies to linear bacterial chromosomes. The first bacterial linear plasmid was described in 1979 from the antibiotic-producing soil microbe Streptomyces rochei. A few species with linear plasmids or chromosomes have also been found in the alpha, beta, and gamma divisions of the genus Proteobacteria. In bacteria with polyploid stages, such as the mycelium of Streptomyces, linear DNA may segregate with fewer mishaps than circular DNA. Alternatively, one could argue that conversion of a circular replicon to a linear form was an inadvertent but neutral event. Linear plasmids have also been identified in the actinomycetes Rhodococcus fascians, R. erythropolis, Clavibacter michiganensis, Nocardia opaca, Planobispora rosea, and the proteobacteria Thiobacillus versutus and Xanthobacter autotrophics. Linear plasmids generally retain the same features and mechanisms for replication initiation as their circular counterparts. Recent experiments demonstrate that replication initiates from an internal origin and continues around the hairpin telomeres, resulting in a circular dimer. The segregation systems of all linear plasmids characterized to date, regardless of telomere structure, appear to be analogous to those of circular plasmids.

The first part of this chapter is devoted to the 2µm circle partitioning system, a critical component of the plasmid's strategy for stable maintenance in yeast populations. The second part deals with plasmid copy number control, special attention being paid to the Flp recombination system that is believed to trigger a DNA amplification process. The chapter compares the 2µm plasmid with 2µm-like plasmids found in yeast and dwells briefly on the degree and the significance of conservation of structure and function among them. Binary fluorescence tagging of two separate plasmids in the same cell by using cyan fluorescence protein (CFP)-Lac repressor/Lac operators in one case and yellow fluorescent protein (YFP)-Tet repressor/Tet operators in the other is also feasible. Chromatin immunoprecipitation assays revealed that the integral cohesin component Mcd1p associates specifically with the STB DNA in a Rep1p- and Rep2p-dependent manner. Rank and colleagues have characterized 2µm plasmids from several amphiploid industrial strains of Saccharomyces cerevisiae and analyzed their sequence divergence with respect to plasmids from standard haploid laboratory strains. The Flp-FRT site-specific recombination system complements the partitioning system in the dual strategy by which stable high-copy maintenance of the 2μm plasmid is achieved. Plasmids lacking STB tend to dissociate from these sites and wander toward the nuclear periphery. These observations would be in line with the models for plasmid segregation considered in this chapter.

Like bacteria and yeast, mammalian cells can harbor plasmids. These double-stranded circular DNA plasmids or episomes are the genomes of DNA viruses. The genomes of Epstein-Barr virus, the related Kaposi's sarcoma-associated (virus human herpesvirus 8), and papillomavirus can persist indefinitely in latently infected cells due to their ability to replicate and stably segregate during cell division, and this chapter focuses on these viruses. Efficient replication from the dyad symmetry (DS) element requires all four Epstein-Barr nuclear antigen (EBNA)1-binding sites as well as the nonamer repeats that flank the EBNA1 sites. EBNA1 is the only viral protein required to replicate and maintain oriP plasmids and EBV episomes and does so through interactions with the 18-bp palindromic sequences present in the family of repeats (FR) and DS elements of oriP. Replication from oriP requires EBNA1 binding to the DS element, but this interaction alone does not activate the origin, as EBNA1 is bound to the DS throughout most of the cell cycle. The transient replication of bovine papillomavirus (BPV) genomes is dependent on the viral E1 and E2 proteins, and no other viral proteins are required. On the basis of this finding, a mouse cell line was developed that stably expressed E1 and E2 and was used to map the cis-acting requirements for BPV plasmid replication. The latent genomes of several different DNA viruses are stably maintained in mammalian cells as low-copy-number plasmids.

Degradative plasmids carry genes that confer on the host bacteria the ability to degrade recalcitrant organic compounds not commonly found in nature. Many plasmid-encoded degradative gene clusters are also discrete regulons if they have regulators specialized for the regularion of the genes encoding degradative enzymes. The degradation pathway is composed of two segments when it is delimited by substrates for growth and the integrity as transcriptional units, each of which has a wide range of enzymatic substrate specificity toward alkyl-substituted aromatic compounds. The recent completion of the nucleotide sequencing of the whole pWWO plasmid revealed open reading frames (ORFs) related to plasmid replication, maintenance, and transfer. Other toluene-degradative (TOL) plasmid, such as pWW53 and pDK1 , have been found to have upper and lower pathways at different relative locations on the plasmids, although the organizations of the structural genes for the degradative enzymes in the respective cluster are highly conserved. Most of the 2,4-D plasmids were found in strains isolated by enrichment on 2,4-D as the sole source of carbon and energy, and some of them were found to take part in the degradation of a herbicide with a similar structure, 2-methyI-4-chlorophenoxyacetic acid. The lower pathway includes cleavage of the aromatic ring by dioxygenases with formation of (chloro)maleylacetates. The genes on TOL plasmids, including pWWO, and the related nab and dmp genes have enabled comparative studies, as described. Transposon Tn5271 on plasmid pBRC60 is not flanked by target-site duplications on both sides, which are supposed to be generated during transposition.

Our knowledge of archaeal plasmids is still sketchy compared to that of bacteria and eukarya, and most of it has been accrued quite recently. Moreover, while many archaeal plasmids have been isolated, and several have been sequenced, very few functional studies have been performed, and little is known about their mechanisms of replication, copy-number control, maintenance, partition, or conjugation. Nevertheless, several archaeal plasmids have now been classified with cryptic or conjugative phenotypes, some of which are integrative, and detailed studies on their molecular biology are in progress. This chapter summarizes recent advances in our knowledge of known classes of archaeal plasmids and emphasizes the insights gained into their molecular mechanisms of replication, maintenance, copy-number control, conjugation, and integration, all of which have special archaeal characteristics. The pNRC100 and pNRC200 plasmids have been classified as minichromosomes because they carry essential chromosomal genes including the Cdc6 protein located adjacent to putative multiple replication origins. Few investigations have been reported on the replication mechanisms of archaeal plasmids. The archaeal plasmids that encode integrases and exist in free or integrated states are listed. Aeropyrum pernix and Pyrococcus horikoshii chromosomes each exhibit two int(N)’s, overlapping downstream halves of tRNA genes, and one and two copies of int(C), respectively. Research into archaeal plasmids is entering an exciting phase. The first results reinforce the view emerging from studies of other archaeal systems that they have diverged greatly from corresponding bacterial systems.

This chapter discusses the genome-scale analysis of virulence plasmids: the contribution of plasmid-borne virulence genes to enterobacterial pathogenesis. The spectrum of diseases caused by Escherichia coli and other enteric bacteria is due to the acquisition of a variety of specific virulence genes harbored on plasmids, on bacteriophages, or within distinct chromosomal DNA segments termed pathogenicity islands (PAIs) that are absent from the genomes of commensal E. coli strains. A study showed that StcE is secreted by the plasmid-encoded type II apparatus, and its expression is regulated by chromosomally encoded Ler, which also controls expression of LEE genes that have key roles in the attaching and effacing phenotype of O157:H7. The chapter emphasizes that virulence plasmids of the enteric bacterial pathogens have evolved by acquiring either individual genes or blocks of genes from bacteriophages and other conjugative plasmids by transposition mechanisms. Many of the remnants of such acquisition events are evident in the sequences of the virulence plasmids of Shigella, the pathogenic E. coli, and Yersinia. Genome sequencing will provide many of the E. coli virulence plasmids, with the advantage of economy of effort and expense, since no separate experiments are needed when the plasmids are present in the genomic DNA preparation at the time of library construction. Collection of sequences that will be made available by the worldwide genome sequencing effort should provide a basis for devising new therapeutic solutions to some of the outstanding problems of infectious diseases, including the further transmission of drug resistance.

Spore-forming bacteria are the causative agents of some of the most dramatic life-threatening human and animal infections and toxemias, including diseases such as tetanus, botulism, gas gangrene, pseudomembranous colitis, and anthrax. This chapter reviews the current state of knowledge of virulence plasmids of the Clostridia and the bacilli. Although other pathogens are mentioned, the focus is on the major human pathogens, Clostridium perfringens and Bacillus anthracis, primarily because most is known about virulence plasmids in these species. The major toxins involved in C. perfringens type A-mediated gas gangrene, the α-toxin and perfringolysin O, together with other extracellular toxins such as collagenase and hyaluronidase, are chromosomally encoded. Along with tetanus, botulism, and gas gangrene, anthrax is one of the four classical diseases that are caused by spore-forming bacteria. Expression of the plasmid-determined toxin genes in Bacillus thuringiensis is coordinately regulated with the sporulation process and therefore clearly involves chromosomal regulatory genes. Toxin-producing strains often carry multiple cry genes and large amounts of the δ-endotoxins are produced in stationary phase, to the extent that the crystalline inclusion may account for more than 20% of the cell's dry weight.

This chapter focuses on virulence plasmids of Yersinia species with particular emphasis on their architecture, the virulence factors encoded by them, and comparisons between species and strains where possible. The involvement of plasmids in the virulence of Yersinia has long been known and is well established. The best-characterized of these is the low calcium response (LCR) plasmid that is common to the three pathogenic Yersinia species. The synthesis and regulation of effector Yop secretion by the type III system are extremely complex. The largest difference between the pLcr molecules that have been sequenced to date is in the position and orientation of some of the more important genes. The common mechanism of partitioning between the sequenced pLcr plasmids is interesting because the common partitioning system has been shown to be responsible for plasmid incompatibility between F and pYVe. The chapter addresses the putative virulence factors encoded by pFra as well as make postgenomic sequence comparisons where possible. The most prominent phage element that can be identified easily as a remnant on pFra is a portion of a lambda-like phage element. Recently, two independent self-transmissible antibiotic resistance plasmids (RTFs) have been identified in natural isolates of Y. pestis in Madagascar.

Gram-positive bacteria are leading causes of many types of human infection, including pneumonia; skin and nasopharyngeal infections; and, among hospitalized patients, bloodstream, urinary tract, and surgical wound infections. As variable traits of the species, many of these virulence properties are encoded by mobile genetic elements, such as virulence plasmids and pathogenicity islands. This chapter reviews virulence plasmids in nonsporulating gram-positive bacteria and examines their contribution to the pathogenesis of disease. More recent studies have determined the nature of the bacteriocin activity linked with exfoliative toxin B (ETB) virulence plasmids. Some Enterococcus faecalis strains produce a cytolysin with both bactericidal and toxin activity against eukaryotic cells. The cytolysin operon occurs along with aggregation substance on pheromone-responsive plasmids and within the recently described 150-kb pathogenicity island on which enterococcal surface protein (Esp) and aggregation substance are found. Aggregation substance expression has also been shown to correlate with an enhanced uptake of enterococci by intestinal epithelial cells. However, this increase in uptake did not result in an increase in translocation across intestinal epithelium in vitro. Virulence plasmids may then represent "selfish DNA" of limited benefit to the bacterium that takes advantage of an otherwise stable, intimate association to ensure its perpetuation, with selection limiting its presence to a small proportion of the population so as not to jeopardize the commensal existence of the vast majority.

This chapter summarizes the latest information about Ti-encoded functions, with emphasis on the mechanistic studies of proteins associated with pathogenesis and intercellular signaling. The ability to transfer T-DNA across kingdom boundaries is a hallmark feature of the Agrobacterium tumefaciens infection process. Moreover, the presence of overdrive is correlated with increased production of single-stranded T-DNA, the T-strand, which corresponds to the translocation-competent form of the T-DNA. A. tumefaciens strains in the vicinity of the transformed plant cells can then import the opines by mechanisms described for use as sources of carbon and energy and, in some cases, nitrogen. The converse model, that VirA interacts directly with phenolic signals, is supported by two genetic lines of study. First, virA genes from different strains of A. tumefaciens, when introduced into an isogenic background, encode VirA proteins that sense different types of inducers. Second, a recent study reported the reconstitution of the VirA/VirG two-component system in Escherichia coli. VirE1 interacts with an N-terminal domain and at least one internal domain of VirE2. Finally, VirD4 recently was shown to assemble at the poles of A. tumefaciens. One of the most striking features of the Ti plasmids is the evolution of an extensive regulatory network that serves to link the activities of the five conserved gene sets. Studies on the regulatory circuitries governing Ti plasmid gene expression will lead to a refined, mechanistic understanding of the paradigmatic regulatory factors VirA and VirG, OccR and AccR, and TraR.

This chapter provides an overview of plasmid classification systems and then describes the various mechanisms of plasmid-mediated resistance to antibacterial agents. The major pathways for the evolution of bacterial resistance plasmids are discussed. In general, plasmid-mediated resistance to antibacterials is a result of three major mechanisms: (i) destruction or modification of the antibacterial agents, (ii) prevention of the antibacterial agent from reaching its target in the bacterial cell, and (iii) production of an altered bacterial target. Resistance to tetracyclines, macrolides, glycopeptides, and quinolones are examples of this type of resistance. The majority of the approximately 60 known gene cassettes code for antibiotic resistance determinants. Genes in integrons have been found to encode resistance to a wide variety of antibiotics, including aminoglycosides, chloramphenicol, erythromycin, and the β-lactams. The aadA1 gene cassette (encoding spectinomycin resistance), carried by Tn21, is one of the most widespread resistance genes. Although the epidemiology of integrons has only recently been investigated, several reports have emphasized the importance of integrons in the dissemination of antibiotic resistance. IncHI1 plasmids are representative of antibiotic resistance plasmids that play a central role in the emergence and reemergence of bacterial pathogens. The chapter highlights the dynamic processes involved in plasmid evolution to acquire resistance markers. Transposons, integrons, conjugation, and other mechanisms of resistance spread are all employed by pathogens to respond to continued antibiotic usage in the clinic and in the environment.

This chapter describes the analysis of pJM1 plasmid and pCoIV-K30 plasmid-mediated iron uptake systems. The iron transport-biosynthesis operon (ITBO) and other anguibactin biosynthetic genes located downstream are bracketed by the highly related ISV-A1 and ISV-A2 insertion sequences. The peptide siderophore anguibactin is synthesized via a nonribosomal peptide synthetase mechanism, an RNA-independent template chain growth process with an assembly line organization of different catalytic and carrier protein domains whose placement and function determine the number and sequence of the amino and carboxylic acids incorporated into the peptide product. Anguibactin can be thought as derived from the precursors dihydroxy benzoic acid (DHBA), histidine, and cysteine. The pJM1 plasmid-mediated proteins AngR, AngM, and AngN play a role in subsequent biosynthetic steps although AngR, in addition to its biosynthetic function, is also essential for regulation of iron transport gene expression. By deletion analysis the TAFr region has been narrowed down to two small subregions that could harbor this TAFr factor. At high iron concentrations iron represses the ITBO mRNA levels and concomitantly induces the synthesis of another antisense RNA (RNAα) that might constitute another novel component of the bacterial iron regulatory circuit. The genetic determinants for the aerobactin iron uptake system of plasmid pColV-K30 were cloned as recombinant plasmid pABN1.

The distinction between plasmids and chromosomes has been blurred by the discovery of megaplasmids and small chromosomes through the use of pulsed-field gel electrophoresis. This chapter considers why plasmids have survived at all if they are not essential to their host and how they have evolved. Population genetics seeks to explain the evolution of species by considering the competition between individuals in a population and the effect that genetic differences have on this competition. An insight into the process can be gained from comparison of so-called trb operons of many self-transmissible bacterial plasmids of gram-negative bacteria. The par function may be inserted diametrically opposite or it may occur close to the replicon. It may be, for example, that a location close to the rep region allows the par genes to function better because partitioning of early replicated DNA is easier than for late replicated DNA. The selective pressure that promotes plasmid evolution does not work just at the level of competition between bacteria. It is widely accepted that to allow drift to occur requires gene duplication. However, for any other than an absolutely unit copy number plasmid, effective gene duplication is a way of life. The circumstances that place a premium on such evolution involve constantly changing physical/chemical and biological environments inherent to most microbial communities.

This chapter reviews some aspects of the many major secondary DNA replicons that have been characterized from organisms that possess multireplicon genomes. Nonprimary replicons are often referred to as secondary chromosomes if they are essential for cell viability or as megaplasmids. A modern source of ambiguity in genomic biology is whether certain replicons represent megaplasmids or second chromosomes. Multircplicon genomes in bacteria could conceivably arise by a number of mechanisms, but two general mechanisms seem most plausible. A major secondary replicon may derive from an ancestral chromosome via an excision event where the excised DNA possesses an origin of replication that is either a duplicated copy of the oriC region or a second, redundant origin that was previously resident on that part of the ancestral chromosome. Chromosome I has an origin of replication typical of other bacterial chromosomes, and the region encodes the dnaA, dnaN, recF, and gyrA genes. A greater proportion of chromosome II is also devoted to genes encoding transporters and solute binding proteins and to genes encoding enzymes required in central intermediary metabolism. The linear chromosome encodes exoC and other genes required for synthesis of several cell surface polysaccharides and also the cellulose synthesis genes that are required for host attachment. Copies of genes required for the synthesis of some amino acids and for certain enzyme cofactors are carried uniquely on the megaplasmid as are the flagellar genes.

While plasmid vectors were initially designed for gene cloning and DNA analysis in Escherichia coli, shuttle vectors for gene transfer between E. coli and other model organisms for gene function analysis and protein production were quickly developed. In an attempt to shed some light on the future development and utilization of plasmid vectors, this chapter summarizes some of the historical events in plasmid vector development and provides some critical considerations of the various types of vectors for gene cloning and gene expression. Gene expression is a complex process that involves dynamic steps that can be regulated at multiple levels, which include transcriptional (transcription initiation, elongation, and termination), posttranscriptional (RNA splicing, RNA translocation, RNA stability), translational (translation initiation, elongation, and termination), and posttranslational (protein splicing, translocation, stability, and modifications). A few general considerations may be used to achieve the desired level and duration of gene expression for a particular task. They are: (i) gene copy number and plasmid stability; (ii) transcription rate, inducibility, and RNA stability; (iii) translation efficiency and protein stability; (iv) protein solubility, functionality, and downstream utility; (v) gene delivery efficiency and protein targeting; (vi) host cell engineering; and (vii) cell growth condition and optimization. A number of approaches have been routinely used to generate a diversified pool of a target gene or a gene family. These approaches include random mutagenesis, exon swap, gene shuffling, and molecular evolution.

This chapter provides a broad overview of many applications of plasmids for genetic analysis, primarily in bacteria. Ever since DNA sequencing became accessible to most research laboratories, reverse genetic analysis has become a standard experimental approach to study bacterial gene function. Similar suicide vectors have also been used for nontargeted insertional mutagenesis by cloning random chromosomal DNA fragments into the plasmid. The use of suicide vectors also allows for easy identification of the insertion mutations. Plasmids that utilize different combinations of double-counter selective markers have been used for diverse applications, including the search for extremely rare suppressor mutations of essential Escherichia coli genes, and to improve the efficiency of allelic exchange on bacterial artificial chromosomes (BACs). Although temperature-sensitive vectors represent the majority of conditionally replicating plasmids, other plasmids that exhibit conditional replication have been described. Cloning by recombination was also achieved using the highly efficient DNA uptake and recombination systems in Acinetobacter calcoaceticus. Site-specific recombination machinery has also been incorporated into several expression vector systems to achieve very tight regulation of gene expression. Although antibiotic resistance is typically used to maintain selection for plasmids grown in culture, there are disadvantages to the use of antimicrobial agents for certain industrial, medical, and biotechnological applications.

This chapter reviews and discusses different aspects of containment, highlighting those systems devoted to contain organisms that remove toxic pollutants. There are two main strategies to diminish the potential risks associated with the deliberate or unintentional release of genetically modified organisms (GMOs) into the open environment. The lethal functions most extensively used for developing active containment circuits are those that disrupt the membrane potential, specially the two-component toxin-antidote systems involved in post-segregational killing of plasmid-free cells and their chromosomal counterparts. Biological containment systems have been engineered on plasmids using the Plac promoter and the Lacl repressor from Escherichia coli, and they are triggered by addition of isopropyl-β-D-thiogalactopyranoside (IPTG). The most advanced containment systems are those developed for bacteria that degrade pollutants. Although the biological containment systems increase the predictability of GMOs, one of the main concerns about the release of such GMOs to the environment is how recombinant DNA can spread among indigenous bacterial populations. Lethal donation circuits, such as those described for gene containment, constitute interesting tools to explore the ecological and evolutionary consequences of shifting the natural equilibrium between genetic change and genetic constancy toward the latter. Many lethal functions and regulatory circuits have been used and combined to design efficient containment systems. Active containment systems are a major tool to reduce the uncertainty associated with the introduction of monocultures, genetically engineered or not, into target habitats.