What part of the chromosome is thought to be the location where cells lose their ability to replicate?

Plasmid Vectors.

Plasmids are small, circular molecules of double-stranded DNA derived from larger plasmids that occur naturally in bacteria.68 Most plasmid-cloning vectors are designed to replicate inE. coli.69 All of the enzymes required for replication of the plasmid DNA are produced by a host bacterium. The classic example of plasmid vector is pBR322, which was one of the first such vectors to be recognized. The three important features of plasmid vectors are as follows:

Origin of replication. This origin permits the efficient replication of plasmid to a large number of copies of cells, by the plasmid's replicon, a region of approximately 1000 bp encoding the site at which DNA replication is initiated.

Presence of selectable marker. Most plasmid vectors encode a gene that confers bacterial resistance to antibiotic. This allows selection of clones carrying the plasmid in the medium containing antibiotic.

Cloning, or restriction enzyme, cleavage site. All cloning vectors must have at least one cloning site (a specific DNA sequence that is recognized and cut by a restriction endonuclease), where the foreign DNA is inserted.

Three classes of restriction enzymes bind to DNA at the recognition sequence and hydrolyze the phosphodiester bond on both strands of DNA. Such restriction sites usually have twofold symmetry; that is, the restriction sites are palindromic. Class II restriction endonucleases, which recognize a DNA sequence of four to eight nucleotides, are preferred for DNA technology. The restriction enzyme EcoRI, isolated fromE. coli, cleaves DNA at the sequence 5′-GAATTC.69 The EcoRI scans the plasmid until it finds the GAATTC sequence, where it hydrolyzes the phosphodiester bond between deoxyguanosine and deoxyadenosine on both strands of the DNA, creating a 4-bp (AATT) single-stranded overhang. Because EcoRI is palindromic, the overhanging single-stranded ends (sticky ends) are complementary to each other and can hybridize or anneal to each other by base pairing. Now the DNA to be cloned (cleaved from its source by EcoRI) is inserted into a plasmid vector whose DNA sequence has been cut by restriction endonuclease. The DNA fragment anneals to the vector through DNA ligase, which catalyzes the covalent joining of the vector DNA to the new piece of DNA (chimeric DNA). The gene (DNA fragment to be cloned) now becomes a passenger on the vector molecule, ready to be introduced into bacteria (DNA transformation).

Electroporation is the most efficient of the several techniques used to achieve DNA transformation.70 The chimeric DNA is mixed with bacteria in a cuvette, and an electric potential is created across the wall of the container, allowing the DNA to enter the bacteria (transfection). The bacteria are then grown in the presence of antibiotic (e.g., ampicillin, neomycin) for which the resistant gene is present in the chimeric DNA.71 This will allow the bacteria with recombinant plasmid to proliferate, whereas any bacteria that were not transformed with the recombinant plasmid will die. The clones or colonies of bacteria containing cloning vector can be isolated for further characterization. There are several ways to characterize clones, but the most common technique is to culture individual bacterial clones, isolate their DNA, and analyze the clones. Once the plasmid DNA has been purified, its structure can be analyzed by digesting the DNA with restriction endonuclease (e.g. EcoRI) and then subjecting it to agarose gel electrophoresis. DNA is visualized by staining the gel with ethidium bromide.

2 General Properties of Plasmids

Plasmids are usually circular molecules of DNA, although occasionally, plasmids that are linear or made of RNA exist. They may be found as single or multiple copies and may carry from half a dozen to several hundred genes. Plasmids can only multiply inside a host cell. Most plasmids inhabit bacteria, and indeed around 50% of bacteria found in the wild contain one or more plasmids. Plasmids are also found in higher organisms such as yeast and fungi. The 2 micron circle of yeast (discussed later) is a well-known example that has been modified for use as a cloning vector.

Most plasmids are circular, made of DNA, and much smaller than chromosomes.

The copy number is the number of copies of the plasmid in each bacterial cell. For most plasmids, it is 1 or 2 copies per chromosome, but it may be as many as 50 or more for certain small plasmids such as the ColE plasmids. The number of copies influences the strength of plasmid-borne characteristics, especially antibiotic resistance. The more copies of the plasmid per cell, the more copies there will be of the antibiotic resistance genes, and therefore, the higher the resulting level of antibiotic resistance.

The size of plasmids varies enormously. The F-plasmid of Escherichia coli is fairly average in this respect and is about 1% the size of the E. coli chromosome. Most multicopy plasmids are much smaller (ColE plasmids are about 10% the size of the F-plasmid). Very large plasmids, up to 10% of the size of a chromosome, are sometimes found, but they are difficult to work with and few have been properly characterized (see Box 23.02).

Box 23.02

Plasmid or Chromosome?

When the genome of the Gram-negative bacterium Vibrio cholerae, the causative agent of cholera, was sequenced, it was found to consist of two circular chromosomes of 2,961,146 and 1,072,314 bp. Together, this totals approximately 4 million base pairs and encodes about 3900 proteins—about the same amount of genetic information as E. coli. Many genes that appear to have origins outside the enteric bacteria, as deduced from their different base composition, were found on the smaller chromosome. Many of these genes lack homology to characterized genes and are of unknown function. The smaller chromosome also carries an integron gene capture system (see Chapter 25: Mobile DNA) and hosts “addiction” genes that are typically found on plasmids (discussed later). Furthermore, the smaller chromosome replicates by a different mechanism from the large chromosome. In fact, the smaller chromosome shares a replication system with a family of widely distributed plasmids. It seems likely that the smaller chromosome originated as a plasmid that has grown to its present size by accumulating genes from assorted external sources. Perhaps it is better to regard the smaller chromosome as a “megaplasmid.” Genome-sequence data suggest that some 10% of bacteria carry such megaplasmids, although the size varies considerably. In most of these cases, the larger chromosome carries almost all of the genes needed for vital cell functions such as protein, RNA, and DNA synthesis. In a few cases such megaplasmids can transfer themselves to related bacteria by conjugation.

Some plasmids are present in one or two copies per cell, whereas others occur in multiple copies.

Plasmids carry genes for managing their own lifecycles and some plasmids carry genes that affect the properties of the host cell. These properties vary greatly from plasmid to plasmid, the best known being resistance to various antibiotics. Cryptic plasmids are those that confer no identifiable phenotype on the host cell. Cryptic plasmids presumably carry genes whose characteristics are still unknown. Plasmids that are modified for different purposes are used in molecular biology research and are often used to carry genes during genetic engineering.

The host range of plasmids varies widely. Some plasmids are restricted to a few closely related bacteria; for example, the F-plasmid only inhabits E. coli and related enteric bacteria like Shigella and Salmonella. Others have a wide host range; for example, plasmids of the P-family can live in hundreds of different species of bacteria. Although “P” is now usually regarded as standing for “promiscuous,” due to their unusually wide host range, these plasmids were originally named after Pseudomonas, the bacterium in which they were discovered. They are often responsible for resistance to multiple antibiotics, including penicillins.

Certain plasmids can move themselves from one bacterial cell to another, a property known as transferability. Many medium-sized plasmids, such as the F-type and P-type plasmids, can do this and are referred to as Tra+ (transfer-positive). Since plasmid transfer requires over 30 genes, only medium or large plasmids possess this ability. Very small plasmids, such as the ColE plasmids, simply do not have enough DNA to accommodate the genes needed. Nonetheless, many small plasmids, including the ColE plasmids have mobilizability, meaning they can be mobilized by self-transferable plasmids [i.e., they are Mob+ (mobilization-positive)]. However, not all transfer-negative plasmids can be mobilized. Some transferable plasmids (e.g., the F-plasmid) can also mobilize chromosomal genes. It was this observation that allowed the original development of bacterial genetics using E. coli. The mechanism of plasmid transfer and the conditions necessary for transfer of chromosomal genes are therefore discussed in Chapter 28, Bacterial Genetics.

Some plasmids can transfer themselves between bacterial cells and a few can also transfer chromosomal genes.

2.1 Plasmid Families and Incompatibility

Two different plasmids that belong to the same family cannot co-exist in the same cell. This is known as incompatibility. Plasmids were originally classified by incompatibility and so plasmid families are often known as incompatibility groups and are designated by letters of the alphabet (F, P, I, X, etc.). Plasmids of the same incompatibility group have very similar DNA sequences in their replication and partition genes, although the other genes they carry may be very different. It is quite possible to have two or more plasmids in the same cell as long as they belong to different families. So a P-type plasmid will happily share the same cell with a plasmid of the F-family (Fig. 23.03).

Figure 23.03. Plasmid Incompatibility

Plasmids with different origins of replication and different replication genes are able to inhabit the same bacterial cell and are considered compatible (left). During cell division, both types of plasmid replicate; therefore, each daughter cell will inherit both plasmids, just like the mother cell. On the other hand, if two plasmids have identical origins and replication genes they are incompatible and will not be replicated during cell division (right). Instead, the two plasmids are partitioned into different daughter cells.

Plasmids are classified into families whose members share very similar replication genes.

2.2 Occasional Plasmids Are Linear or Made of RNA

Although most plasmids are circular molecules of DNA there are occasional exceptions. Linear plasmids of double-stranded DNA have been found in a variety of bacteria and in fungi and higher plants. The best-characterized linear plasmids are found in those few bacteria such as Borrelia and Streptomyces that also contain linear chromosomes. Linear DNA replicons in bacteria are not protected by telomeres like the linear chromosomes of eukaryotes. Instead, a variety of individual adaptations protect the ends from endonucleases.

Linear plasmids have special structures to protect the ends of the DNA.

In Borrelia, there are not actually any free DNA ends. Instead, hairpin sequences of single-stranded DNA form loops at the ends of both linear plasmids and chromosomes (Fig. 23.04A). Some animal viruses, such as the iridovirus that causes African swine fever, have similar structures. Different species of Borrelia cause Lyme’s disease and relapsing fever. Their linear plasmids appear to encode both hemolysins that damage blood cells and surface proteins that protect the bacteria from the host immune system. Thus, as is true of many other infectious bacteria, the virulence factors of Borrelia are also largely plasmid-borne.

Figure 23.04. End Structures of Linear Plasmids

(A) Linear plasmids of Borrelia form hairpin loops at the ends. (B) Linear plasmids of Streptomyces are coated with proteins that protect the DNA ends. If linear plasmids had exposed double-stranded ends, this would trigger recombination, repair, or degradation systems.

The linear plasmids of Streptomyces are indeed genuine linear DNA molecules with free ends. They have inverted repeats at the ends of the DNA that are held together by proteins. In addition, special protective proteins are covalently attached to the 5′ ends of the DNA. The net result is a tennis racket structure (Fig. 23.04B). The DNA of adenovirus, most linear eukaryotic plasmids, and some bacterial viruses show similar structures.

Linear plasmids are also found among eukaryotes. The fungus Flammulina velutipes, commonly known as the enoki mushroom, has two very small linear plasmids within its mitochondria. Several higher plants are also known that have linear plasmids in their mitochondria. The dairy yeast, Kluyveromyces lactis, has a linear plasmid that normally replicates in the cytoplasm. However, on occasion the plasmid relocates to the nucleus where it replicates as a circle. Circularization is due to site specific recombination involving the inverted repeats at the ends of the linear form of the plasmid. The physiological role of these plasmids is obscure.

RNA plasmids are rare and most are poorly characterized. Examples are known from plants, fungi, and even animals. Some strains of the yeast Saccharomyces cerevisiae contain linear RNA plasmids. Similar RNA plasmids are found in the mitochondria of some varieties of maize plants. RNA plasmids are found as both single-stranded and double-stranded forms and replicate in a manner similar to certain RNA viruses. The RNA plasmid encodes RNA-dependent RNA polymerase that directs its own synthesis. Unlike RNA viruses, RNA plasmids do not contain genes for coat proteins. Sequence comparisons suggest that most RNA plasmids are merely defective versions of RNA viruses that have taken up permanent residence after losing the ability to move from cell to cell as virus particles.

Plasmids

Extrachromosomal elements were present in bacteria before the advent of antibiotics.11,14 The introduction of antibiotics into clinical medicine in the 20th century created selection pressures, however, that favor the dissemination of resistance genes via mobile genetic elements.2,3,13 Plasmids are particularly well adapted to serve as agents of genetic exchange and resistance-gene dissemination.1,3 Plasmids are autonomously replicating genetic elements that generally consist of covalently closed, circular, double-stranded DNA molecules ranging from less than 10 kilobase pairs (kbp) to more than 400 kb. They are extremely common in bacteria.14 Although multiple copies of a specific plasmid, or multiple different plasmids, or both may be found in a single bacterial cell, closely related plasmids often cannot coexist in the same cell. This observation led to a classification scheme of plasmids based on incompatibility (Inc) groups.1,4

Plasmids may determine a wide range of functions besides antibiotic resistance, including virulence and metabolic capacities. All plasmids possess an origin of replication for DNA polymerase to bind and replicate plasmid DNA. Plasmids must also retain a set of genes that facilitate their stable maintenance in host bacteria. The transfer of plasmid DNA between bacterial species is a complex process, and the genes needed for transfer (tra genes) make conjugative plasmids larger than nonconjugative ones. Some small plasmids may be able to transfer to other bacteria via the use of the conjugation apparatus provided by coresident conjugative plasmids or even conjugative transposons. Many plasmid-encoded functions enable bacterial strains to persist in the environment by resisting noxious agents, such as heavy metals. Mercury released from dental fillings may increase the number of antibiotic-resistant bacteria in the mouth.19 Compounds such as hexachlorophene and quaternary ammonium compounds are used as topical bacteriostatic agents, and plasmid-mediated resistance to these agents has increased significantly.2 Recently, mathematical modeling and long-term gene transfer experiments within complex, multispecies environments such as the gut microbiome have confirmed that R plasmids serve another critical role in retention of resistance genes once antibiotic therapy is discontinued.20 R plasmid horizontal transfer by conjugation occurs at a surprisingly high rate, allowing R plasmids to transfer and continuously “infect” a sufficient proportion of antibiotic-susceptible host bacteria to maintain the resistance genes within bacterial genomes even after the antibiotic has been discontinued. This explains the problem of lingering persistence of resistance genes in patients, even after good antibiotic stewardship programs are working to curtail unnecessary antibiotic use.20 This chapter primarily focuses on the molecular aspects of antibiotic resistance. Readers interested in practical recommendations to limit the spread of antibiotic-resistance genes in clinical settings are referred toChapters 14, 51, and 298Chapter 14Chapter 51Chapter 298.

Incompatibility

Incompatibility among plasmids is usually manifested as the inability of a plasmid to be established in a cell that already contains another plasmid or as destabilization of a resident plasmid by a second, incoming plasmid. Experimentally, it has been possible to classify plasmids according to incompatibility groups. Incompatible plasmids, i.e., members of the same incompatibility group, share one or more elements of the plasmid replication or partitioning systems. Incompatibility is usually symmetric: in the absence of external selective pressure, two incompatible plasmids are lost from cell progeny at the same frequency. This symmetry is explained in the following way. In any given cell, copies of one plasmid or the other are selected at random for replication or partition. Occasional increases in the number of copies of one plasmid at the expense of the other cannot be corrected because the copy number control mechanism cannot distinguish between the two plasmids. Thus each host colony recovered will contain only one plasmid type. Since each plasmid predominates over the other with the same probability, the number of progeny cells, and therefore the number of colonies, carrying one plasmid or the other will be equal.

Cases have also been found in which incompatibility is unidirectional. For example, cloned DNA fragments encoding essential plasmid replication or partitioning functions tend to exclude plasmids requiring those functions. Unidirectional incompatibility is also created by mutations that cause replication defects (the mutant plasmid cannot compete with a coresident, incompatible plasmid) or that alter interactions between a copy control regulator and its target (the mutant plasmid is less sensitive to the inhibitor encoded by a coresident incompatible plasmid).

Plasmids

Many gonococci possess a 24.5-mDa conjugative plasmid and can thereby conjugally transfer other non–self-transferable plasmids with high efficiency; chromosomal genes are not mobilized. Many gonococci carry a plasmid (Pcr) that specifies production of a TEM-1 type of β-lactamase (penicillinase). The two most common Pcr plasmids have molecular weights of 3.2 and 4.4 mDa and are closely related to each other and to similar plasmids found in certainHaemophilus spp., includingHaemophilus ducreyi. In fact, it is suspected that gonococci first acquired Pcr plasmids fromH. ducreyi.27 Pcr plasmids are commonly mobilized to other gonococci by the conjugative plasmid.

Gonococci with plasmid-mediated high-level resistance to tetracycline, with minimum inhibitory concentrations (MICs) of 16 mg/L or greater, carry the 24.5-mDa conjugative plasmid into which thetetM transposon has been inserted.28 ThetetM determinant also confers tetracycline resistance to a variety of other bacteria, including someStreptococcus andMycoplasma spp. and various genital organisms such asGardnerella vaginalis andUreaplasma urealyticum. Because of its location on the conjugative plasmid, high-level tetracycline resistance is readily transferred among gonococci. ThetetM determinant functions by encoding a protein that protects ribosomes from the effect of tetracycline. Finally, all gonococci contain a small (2.6 mDa) cryptic plasmid of unknown function.

Incompatibility

Different plasmids that share common inheritance elements are unable to coexist in the same cell. If the replication system of two plasmids is similar, both plasmids will be considered as identical by the regulatory system and individual molecules will be chosen at random as templates. In consequence, after several rounds of replication the total copy number of both plasmids will be that of each one of them, but there will be an uneven representation of each plasmid, which makes it more probable that at the time of division daughter cells receive more copies of one of the plasmids. This process will repeat exacerbating the differences and eventually one of the daughter cells will be left with only one of the plasmids. After a number of generations, most cells will possess one or the other plasmid. Likewise, two different plasmids with identical partition systems will be segregated as if they were identical, leading to the imbalances that result in the loss of one of the plasmids. In both cases described here, half of the cells will end up with one of the plasmids and half with the other. However, there are other situations in which one of the plasmids may have an advantage and prevail in a larger percentage of the cells.

A Bacterial elements

Plasmids encode two features that are important for their propagation in bacteria. One is the bacterial origin of replication, usually derived from a high-copy plasmid, such as pUC plasmid (Vieira and Messing, 1982). The second required element is a selectable marker, usually a gene that confers resistance to an antibiotic, such as kanamycin or neomycin. These “prokaryotic” plasmid segments permit the production of large quantities of a given plasmid in bacteria. The prokaryotic origin of replication is a specific DNA sequence that binds to factors that regulate replication of plasmid and, in turn, control the number of copies of plasmid per bacterium.

Plasmid Incompatibility

Two plasmids that cannot be stably maintained in the absence of selection for both are said to be incompatible and to belong to the same incompatibility group. Incompatibility is often used to determine relatedness among plasmids, particularly in replication functions. If identical inhibitors control two plasmids, they cannot be maintained in the same cell without selection for both. Since the inhibitors regulate the total plasmid copy number, the ratio of the two plasmid kinds varies from cell to cell because the plasmids are chosen at random for replication. Eventually, this disproportion leads to the loss of one or the other plasmid kind. Since all plasmids make replication inhibitors, they are all capable of exhibiting incompatibility.

Plasmids

Plasmids are bacterial DNA molecules that are smaller than the chromosome(s). Generally, they are dispensable for bacterial growth at least under some conditions. Instead, they typically encode properties that allow growth or otherwise give the bacteria selective advantages under niche-specific conditions. In some rhizobia (e.g., Rhizobium and Sinorhizobium species) nod, nif, and fix genes are found on a plasmid termed the symbiotic plasmid. This plasmid is distinct in each strain, having a different size and different additional genes that have no apparent role in symbiosis. These other genes generally outnumber the genes devoted to the symbiosis. In Bradyrhizobium and Mesorhizobium species, the nod, nif, and fix genes are found on the chromosome.

In some of the rhizobia, a significant portion of the genome is contained on plasmids. Plasmids larger than 1 Mb arbitrarily are termed megaplasmids. S. meliloti has two of them, which account for almost half of its 6.7 Mb genome. Plasmid A is 1.35 Mb and plasmid B is1.68 Mb. These plasmids are larger than the entire genomes of many obligately symbiotic bacteria and even some free-living bacteria. Plasmid A is the typical symbiotic plasmid with nod, nif, and fix genes, whereas plasmid B has genes for exopolysaccharides required in the symbioses of this species.

In the two Rhizobium strains whose entire genomic nucleotide sequences have been determined, the plasmids are smaller, but there are more of them. Strain R. leguminosarum 3841 has 12 plasmids that together account for about 40% of the 7.8 Mb genome. Strain R. etli CFN42 has six plasmids that account for about one third of the 6.5 Mb total genome. In both of these strains, one of the plasmids is a typical symbiotic plasmid carrying the nod, nif, and fix genes, as well as genes not required in the symbiosis.

Plasmid

Plasmid pTOM31c is a 114 Kb plasmid containing the TOM pathway. Plasmid pTOM31c constitutively encodes for toluene ortho-monoxygenase (tom A) and catechol 2,3 dioxygenase (C230) genes, as well as for all of the other genes needed for the aerobic, cometabolic mineralization of TCE [17]. In addition, pTOM31c contains a Tn5 transposon carrying the kanamycin resistance marker. A detailed map of plasmid pTOM31c can be found in [14]. This plasmid was used in Pc 17616 strain to determine its activity and expression in suspended and biofilm cultures.