BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part I. General Biotechnology

CHAPTER 5. FUNDAMENTALS OF GENETIC ENGINEERING

5.1. BIOTECHNOLOGY OF RECOMBINANT DNA CONSTRUCTION

5.1.2. Plasmids and Viruses as Donor Vectors for Genetic Information Transfer

In molecular biology and Introduction/32.html">Genetic Engineering, Plasmids and phage λ are most commonly utilized as vectors (carriers) for genes that have no counterparts in the recipient Cell's DNA and are therefore incapable of participating in Homologous Recombination. Plasmids are small, circular molecules of extrachromosomal DNA that, unlike classical genes, reside in the Cytoplasm of Bacterial Cells and certain Yeasts. Consequently, plasmids are frequently referred to as extrachromosomal genetic elements. The autonomous existence of plasmids stems from the fact that the mechanisms governing their Replication are independent of the regulatory machinery controlling the multiplication of the bacterial chromosome. Plasmids possess The ability to integrate into the host cell genome and persist indefinitely in a state integrated with the bacterial DNA. In this scenario, the genetic material of the plasmid behaves similarly to the chromosomal GENES OF THE bacterium into which it has incorporated. Plasmids known today vary in size, which is a consequence of the differing amounts of Genetic information they contain, as well as in their mechanisms of replication regulation due to variations in the enzyme systems driving these processes. Depending on their molecular weight, plasmids are categorized into small (average Molecular Weight of 5x10⁶) and large (with an established approximate upper molecular weight limit of 150-10⁶ — 170-10⁶).

A plasmid DNA molecule comprises anywhere from 2,250 to 400 thousand nucleotide pairs. In bacterial cells, the preferred DNA configuration takes the form of double-stranded, covalently closed, supercoiled circular molecules, as well as double-stranded open circular DNA molecules. The configuration of the double-stranded, covalently closed circular Structure, which transforms into a supercoil upon twisting, exhibits high stability and remains conformationally altered even under adverse conditions (e.g., alkaline solutions). In bacterial cells, plasmids predominantly exist as covalently closed circular supercoiled structures and open circular DNA molecules. Open circular molecules are generated As a result of phosphodiester bond Cleavage in one of the strands. They do not form a supercoiled structure, reside in a relaxed state, are less stable when exposed to unfavorable factors, and rapidly degrade in alkaline solutions due to the disruption of Hydrogen Bonds.

A bacterial cell may simultaneously harbor plasmids of the same or different types. The copy number of small plasmids typically exceeds ten, whereas large plasmids are, in most cases, represented by one or two copies per bacterial cell. It has been determined that plasmids account for approximately 1-10% of the cellular DNA. For instance, assuming an E. coli cell contains 10 small plasmids with a molecular weight of 5·10⁶ each, and the chromosomal DNA has a molecular weight of 2·10⁹, approximately 2% of the cellular DNA is concentrated within the plasmids. The volume of information housed in small plasmids enables them to encode molecules of two large Proteins, whereas the coding capacity of large plasmids is sufficient for more than 200 large proteins. Sharing certain General characteristics with temperate phages suggests the existence of the same DNA molecule in different phases—plasmid and phage phases. It is known, for example, that phage λ most frequently Functions by integrating its DNA molecule into the chromosomal DNA of a bacterial cell; phage DNA can also replicate autonomously, much like plasmids multiply. Aside from Bacteria, small circular DNA molecules (1.1-2 µm in diameter) are found in the cytoplasm of Eukaryotic cells (yeasts, Neurospora, Euglena, trypanosomes, tobacco, Drosophila, and Xenopus cells, as well as in mouse, monkey, and human cell cultures). Although the PHYSICOCHEMICAL PROPERTIES OF these DNAs have been studied to date, their origin and biological role remain undetermined.

In the 1950s, through the efforts of J. Lederberg, an extrachromosomal genetic structure in bacteria was discovered, initially known as the fertility, conjugation, or genetic transfer factor. The symbol F was proposed to designate this structure. Cells carrying this factor came to be referred to as F+ cells; instances where the aforementioned factor was absent in a cell were designated as F^-. Furthermore, it became apparent that during the conjugation of bacteria from two strains, cells containing the factor

F invariably act as Donors of genetic material, while F^- cells act as recipients. The transfer of genetic material from the donor cell to the recipient cell occurs via conjugation (an analog of the Sexual process in bacteria), with the factor F proving to be the structure possessing sex factor properties. Owing to its extrachromosomal (cytoplasmic) localization, J. Lederberg suggested naming this entity a plasmid. Thus, it was established that the F plasmid determines only a single property of the bacteria carrying it—acting as a donor of genetic material. Extrachromosomal factors similar to F-plasmids, discovered by other researchers (plasmids Δ, T, FP, P), confer donor capabilities regarding genetic material upon the cells harboring them, and also mobilize the transfer of other non-conjugative plasmids into recipient cells.

Plasmids determine the resistance of bacterial cells to one or multiple drugs simultaneously and have become widespread in recent times; they are carried by nearly all species of bacteria pathogenic to humans and animals.

The synthesis of colicins by E. coli cells is under the control of Col plasmids. Several types of colicins are distinguished: A, B, C, D, E, etc. Some of these named colicins, in turn, feature variants such as colicin E1, E2, E3. Consequently, the designations of the corresponding plasmids include not only the Col symbol, but also the name of the specific type and variant of colicin synthesized by the bacterium—such as ColE1, ColE2, etc. The synthesis of substances biologically similar to colicins (pyocins) is determined by plasmids harbored in pseudomonads; staphylococcal plasmids control the synthesis of staphylococcin, Bacillus subtilis plasmids control megacin, and so forth. A common property of all bacteriocins is their high biological activity. A few molecules of a bacteriocin are sufficient to inhibit the vital activity of a bacterial cell (this high efficacy applies exclusively to bacteria of the same or a closely related species). Colicinogenic (bacteriocinogenic) plasmids and the effects they determine are widespread among bacteria, though not as frequently as R-plasmids.

Certain plasmids (Ent, Hly, K88, K99) are localized exclusively within cells of enteropathogenic E. coli strains. For instance, low-molecular-weight heat-stable and high-molecular-weight heat-stable enterotoxins are synthesized under the control of Ent plasmids. Depending on whether a single plasmid or a complex of two plasmids resides within The Cell, E. coli cells synthesize either one of the two aforementioned enterotoxins or both types simultaneously. The synthesis of α- and β-hemolysins (proteinaceous substances causing erythrocyte hemolysis) by enteropathogenic E. coli cells is determined by the plasmids Hlyα and Hlyβ, respectively. Like Ent plasmids, they may reside within the cell either individually or both simultaneously. Consequently, the cell synthesizes one of the two named enterotoxins, or both types concurrently. The co-presence in the cell of plasmids K88 and K99, which control the synthesis of surface Antigens 88 and 99, along with Ent plasmids, enhances the pathogenicity of such bacterial cells. Plasmids are localized in the cells of many Pseudomonas putida strains, each determining The Biosynthesis of an enzyme designed to metabolize a specific Class of Hydrocarbons. For example, SAL plasmids degrade salicylic acid, XYL degrades xylene and toluene, NAN degrades naphthalene, CAM degrades camphor, and OCT degrades octane, hexane, and decane. All these plasmids can be identified by the traits they control, which manifest phenotypically. Plasmids whose existence in bacterial cells is not phenotypically expressed (requiring Biochemical Methods for identification) are termed cryptic plasmids. These plasmids remain poorly studied.

Before plasmids became indispensable tools in molecular biology and genetic engineering, their general and specialized functions were investigated. Common functions identified across all plasmid types include the ability to replicate (rep), incompatibility (inc), and transfer (tra - derived from transfer).

Plasmids are DNA molecules; therefore, through replication, the number of plasmid copies regularly increases and they are evenly distributed among the progeny of a dividing bacterial cell. The replication mechanism of the small ColE1 plasmid, which is typically present in the cell in a high copy number, has been studied most thoroughly (P. Sengbusch, 1982). The initiation and elongation processes involve products of chromosomal genes, whereas ColE1 replication requires DNA polymerase III and DNA polymerase I. The latter enzyme does not participate in E. coli chromosomal METABOLISM/36.html">DNA replication, serving a repair function there instead. It is suggested that only the initiation of ColE1 plasmid replication is determined by its genetic apparatus, while other functions ensuring plasmid replication are executed with the participation of the bacterial cell's chromosomal apparatus. Certain plasmids present in the cell in low numbers (1-2 or slightly more copies per chromosome) possess their own chromosome-independent replication machinery. Such plasmids primarily include the F, R1, pSC101 factors, and others.

The actual existence of plasmid replication is evidenced by indirect data, chief among which is the fact that the number of plasmids per chromosome of the host bacterial cell is always a constant value. Conversely, assuming replication were alien to plasmids, any initial quantity of plasmids in a dividing bacterial cell would eventually reach a point where the cell becomes cleared of plasmid structures. In reality, such a phenomenon of plasmid loss from plasmid-bearing cells has not been observed by anyone thus far. Other Examples further support the fact of plasmid multiplication.

The Molecular Mechanism of plasmid multiplication has not yet been fully elucidated. Existing hypotheses allowing for positive and negative control of plasmid replication fail to account for the observed facts. For instance, the hypothesis of positive control posits that the multiplication of F and F' plasmids is governed by their own genetic system, represented by just two genes: one controls the synthesis of a protein product acting as a replication initiator, while the second Gene serves as the replication operator (replicator). The scheme of Genetic control over F or F' plasmid multiplication implies that replication commences when there are no restrictions on the functioning of the initiator (protein substance) and replicator. At the same time, it has been established that during the initiation of ColE1 plasmid DNA Synthesis, replication does not cease despite the presence of a Protein Synthesis inhibitor such as chloramphenicol.

The view that plasmids possess their own replication system is shared by many specialists, yet the functional activity of this system depends on the metabolic state of the bacterial cell. For example, exposing a bacterial culture to conditions unfavorable for its growth is accompanied by an increase in the number of plasmids per bacterial cell. Furthermore, the dependence of plasmid multiplication on bacterial metabolic activity is indicated by the potential involvement of RNA as a primer during plasmid DNA replication.

Understanding The Mechanism of plasmid multiplication is not a narrow issue. Its significance lies in the fact that plasmid DNA replication serves as a model to study fundamental Mechanisms of Genetic material replication in general, which has Practical Applications in developing methods to combat bacterial drug resistance and restricting the spread of bacteria carrying R-plasmids.

The plasmid incompatibility (inc) function is a biological phenomenon manifested during conjugation, implying that two identical plasmids cannot stably coexist within the same cell; upon entering a recipient cell from a donor cell via mating, a plasmid either displaces a similar plasmid (the "resident") or is itself displaced by it. The molecular mechanism of incompatibility remains unclear.

Alongside incompatibility, studies of bacterial interactions revealed a closely related phenomenon termed surface exclusion. Matings between donor cells and recipient cells carrying similar F, R, or Col plasmids are invariably accompanied by the recipient cell's refractoriness to plasmid material from the donor cell. Although convincing data point to plasmid gene control over surface exclusion, the molecular mechanism of this phenomenon has yet to be unequivocally established. Regarding plasmid incompatibility, neither the hypothesis of plasmid competition for a membrane replication site nor the negative replication control hypothesis can fully explain the molecular mechanism of this event. Nevertheless, there is no basis to deny the interrelation between the incompatibility mechanism and the replication mechanism governing plasmid copy number within a single bacterial cell. The discovered phenomenon of plasmid incompatibility forms The basis of their Classification. The hypothesis regarding the phylogenetic relatedness of plasmids belonging to the same group has been substantiated by subsequent research. It was established that plasmids belonging to the same incompatibility group share commonalities in the molecular structure of their DNA; plasmid group membership correlates with bacterial drug resistance. Classifying plasmids based on incompatibility enables tracking their dissemination pathways among bacteria, as well as tracing the spread of plasmid-bearing bacteria where plasmids serve as markers. The latter circumstance facilitates plasmid monitoring. All of this enhances the efficacy of studying the ecology, Epidemiology, and epizootiology of plasmids and plasmid-harboring bacteria.

The transfer (tra) function is a property characteristic of plasmids, yet it is exclusive to large plasmids, with the F plasmid of E. coli being the most thoroughly studied. The molecular weight of this plasmid reaches 65 million, comprising 24 thousand nitrogenous Base Pairs. The transfer factor (transfer Operon, F factor, F plasmid) contains 21 cistrons. The multi-step transfer process begins with the cleavage of One DNA strand at a specific plasmid site known as THE ORIGIN OF transfer (oriT). This strand is subsequently transferred into the acceptor cell. Complementary strands are then replicated on the single-stranded DNA structures remaining in the donor cell as well as those transferred into the acceptor cell.

The F factor, like other plasmids, can integrate into the bacterial chromosome via Illegitimate Recombination at specific sites. Illegitimate recombination is a process based on the exchange of non-homologous DNA segments. The persistence of the F factor in a chromosomally integrated state (the Hfr state) represents an alternative form of plasmid existence, and E. coli Chromosomes harboring an integrated F factor acquire the ability to transfer into cells of a suitable recipient strain—a process occurring at high frequency upon F plasmid integration with the E. coli chromosome. Exposure of the bacterial cell to UV irradiation increases the frequency of F plasmid integration into the cellular chromosome.

Among the known specialized functions encoded by individual plasmids are fertility (the ability of plasmids to transfer genetic material via conjugation), resistance to one or multiple Antibiotics (R₁ factors of R plasmids) and heavy metals (Cd²⁺, Hg²⁺), ultraviolet radiation tolerance, the capacity to produce bacteriocins (substances causing cell death upon action) and antibiotics (methylenomycin, actinorhodin, etc.), toxins and surface antigens (enterotoxins, hemolysin, K88 antigen, etc.), the induction of plant tumors (the Ti plasmid from Agrobacterium tumefaciens induces crown gall formation), the metabolism of unusual carbon sources (many Pseudomonas putida strains harbor plasmids encoding Enzymes for hydrocarbon utilization), and participation in streptomycete sporulation.

Carriers of such specialized functions as resistance to antibiotics and certain other groups of therapeutic agents are R factors or R plasmids (derived from resistance). The mechanism of resistance is driven by plasmid genes encoding the synthesis of specialized enzymes that inactivate antibiotics either through cleavage or via modification via Acetylation, adenylylation, or phosphorylation. Furthermore, certain antibiotics (tetracycline) and sulfonamides fail to exert their typical antibacterial effect due to determined alterations in the bacterial membrane.

The r genes responsible for drug resistance can be combined within plasmid R factors. This leads to The Emergence of multiple drug resistance. As a result of intensive Chemotherapy against bacterial dysentery, one of the strains of the causative agent, Shigella dysenteriae, became simultaneously resistant to all four therapeutic agents used: chloramphenicol, streptomycin, tetracycline, and sulfonamides. Today, The phenomenon of multiple drug resistance is widespread among both pathogenic and non-pathogenic bacteria (E. coli, Salmonella). Japanese researchers found that 65% of Shigella strains and 50% of all other enteric bacteria isolated from patients or convalescent dysentery carriers were resistant to the antibiotics used for Treatment (streptomycin, chloramphenicol, and tetracycline), as well as

to sulfonamides. In plasmids, alongside several r genes determining multiple drug resistance, there is also a resistance transfer factor, known as RTF. The genes of the RTF factor share many similarities with the genes of the general transfer factor F in E. coli and are responsible for conjugation and replication. R plasmids frequently carry the RTF region. In such cases, upon co-cultivation, they can be transmitted to other bacterial species, which indicates the transmissible nature of multiple drug resistance. Most commonly, the RTF region—which ensures bacterial conjugation and DNA replication—and the r determinants are located within the same DNA molecule. However, numerous cases are known (for instance, in Salmonella and Proteus bacteria) where these functional entities reside in separate molecules. Under natural conditions, bacterial populations carrying plasmids in which the RTF factors and r determinants are concentrated in a single plasmid DNA molecule are spatially separated from those bacterial strains where RTF and r are located on different plasmids. Plasmids carrying only a single r gene and lacking the set of genes responsible for the RTF function are characterized by a small size; they confer resistance to only a single drug, and the existing resistance gene cannot be transferred via conjugation. For example, the plasmid RpSC101, which is 8.2 kb long (a small plasmid), confers resistance to only a single drug (the antibiotic tetracycline), yet this resistance trait cannot be transmitted via conjugation due to the absence of the RTF region. In nature, the r gene determining resistance to a single drug, such as an antibiotic, has the ability to interact with plasmids encoding resistance to another antibiotic or a sulfonamide drug. The combination of the drug resistance gene(s) r (typically against chemical compounds) with plasmids carrying the genes of the RTF region ultimately leads to the generation of a plasmid in which the RTF-r combination promotes its rapid dissemination within the bacterial population. Although regulatory mechanisms exist (in particular, the repression of RTF-r functional activity) via mutant forms where this control is weakened or absent, gene transfer of r in natural populations is quite widespread. For instance, in the USA (Atlanta), a penicillin-resistant strain of Neisseria gonorrhoeae, the CAUSATIVE AGENT OF Gonorrhea, was isolated. To treat the infection caused by this penicillin-resistant strain, the antibiotic dosage had to be increased 24-fold (from 200,000 to 4.8 million units). The sharp decrease in penicillin activity is caused by the appearance of the r gene, which determines the synthesis of penicillinase. This enzyme cleaves penicillin and thereby inactivates it.

The phenomenon of microbial resistance to penicillin was also established in Haemophilus influenzae, the causative agent of meningitis. Other examples of drug resistance gene migration are likewise known. Specifically, it was established that the gene responsible for bacterial resistance to tetracycline can transfer from an R plasmid into a phage multiplying within Salmonella cells, then into the Salmonella chromosome, from the chromosome into the $\lambda$ phage, subsequently into the trp operon of E. coli, and finally back into the $\lambda$ phage. The migration pathway of the chloramphenicol resistance gene has also been traced.

The phenomenon of chromosomal migration of resistance genes to certain chemical substances, discovered by Japanese researchers under the leadership of S. Mitsuhashi, has been confirmed in laboratories worldwide. Prokaryotic genetic elements capable of migration and encoding resistance to specific chemical compounds were named Transposons, and the symbol Tn came to be used to designate them. Among the currently known transposons, those isolated from various plasmids have been studied the most: Tn1, Tn2, and Tn3 confer resistance to ampicillin; Tn4 confers simultaneous resistance to several chemical compounds—streptomycin, ampicillin, and sulfonamides; Tn5 and Tn6 confer resistance to the antibiotic kanamycin; Tn7 to trimethoprim and streptomycin; Tn9 to chloramphenicol; and Tn10 to tetracycline. Transposons consist of approximately 2,600–5,200 nucleotide pairs.

When investigating the causes of the high migration capacity of transposons, repeated nucleotide sequences were discovered at their ends. Furthermore, it had previously been established that certain R plasmids contain repeatedly recurring nucleotide sequences (inverted repeats) comprising 800–1,400 nucleotide pairs. A characteristic feature of these repetitive sequences is their genetic inertness, as they do not encode any traits. To date, the five distinct sequences (IS1, IS2, IS3, IS4, and IS5) known as insertion sequences, or introns (from the English "intervening sequence"), have been the most thoroughly studied. These elements have been found in E. coli, Salmonella typhimurium, Citrobacter freundii, certain plasmids (F, R), and temperate phages ($\lambda$). For instance, the E. coli chromosome contains eight copies of IS1 and five copies of IS2—elements that differ from Viruses and plasmids in their inability to replicate autonomously. They are the smallest Mobile Genetic Elements, with a length of about 1 kb. Moreover, as mentioned earlier, IS sequences do not carry any genes. However, they cannot be dismissed as inert genetic material because, representing a novel type of sequence, they influence the expression of neighboring genes, can act as novel promoters, block the Transcription of distal genes within a transcriptional unit, induce deletions and inversions leading to chromosomal rearrangements, and possess the ability to integrate into and excise from the bacterial genome at various sites independently of the recA gene.

It is worth noting that IS sequences lack a fixed Location: they are found in various regions of DNA molecules. Transposons incorporated into these polynucleotide sequences move along with them, and the underlying mechanism of transposon insertion relies on illegitimate recombination, since integration Specificity is determined primarily by DNA-Protein Interactions rather than base pairing. As previously mentioned, IS elements have the capability of inserting into various sites within The Genome, although incorporation occurs more frequently in some sites than in others.

It is suggested that the high precision with which nucleotide fragments are inserted and excised indicates the involvement of proteins that specifically interact with the terminal regions of IS elements.

Determination of The nucleotide sequence at the terminal regions of transposons revealed their repetitive nature. For instance, the ends of the transposon conferring ampicillin resistance (Tn3) are inverted repeats comprising 38 nucleotide pairs. However, no Homology was found between the terminal NUCLEOTIDES of the Tn3 transposon and the adjacent Regions of the recipient DNA. Thus, a transposon can be defined as a DNA segment consisting of one or more resistance genes flanked on both sides by IS elements. The Study of transposons, their migration mechanisms and pathways, as well as the identification of eukaryotic transposons, is essential for resolving both theoretical and practical issues.

A specialized plasmid function, namely the biosynthesis of bacteriocins, has been identified in numerous bacterial species. Chemically, bacteriocins are proteinaceous substances with molecular weights ranging between 40,000 and 100,000. Currently, the bacteriocinogenic properties of plasmids are best understood in E. coli cells. Bacteriocins encoded by the genes of these plasmids are called colicins, and the plasmids encoding them are designated as ColE1, ColE2, and so forth. The action of colicins involves distinct mechanisms. For instance, the biological activity of ColE1 is mediated through the inhibition of Oxidative Phosphorylation. Additionally, this plasmid is utilized as a vector in genetic engineering. The bacteriocinogenic effect of ColE2 is achieved via DNA cleavage, whereas ColE3 exerts a degradative effect on the 3' end of 16S rRNA, thereby disrupting protein synthesis because the rRNA loses its ability to specifically interact with mRNA; the MECHANISM OF ACTION of ColK is based on the disruption of bacterial Membrane Functions. Colicins effectively target bacteria of the same species or closely related species, such as Proteus and Shigella.

The toxin-producing function of bacteria, which determines their pathogenicity, is realized through plasmid-encoded biosynthesis of enterotoxins, hemolysins, and antigens. E. coli plasmids encoding enterotoxin synthesis represent a transfer factor (RTF) integrated with various genes carrying genetic information for the synthesis of two protein factors: one (ST) is a heat-stable substance, while the other (LT) is a heat-labile substance. Exposure to heat-labile enterotoxins results in more severe clinical consequences than exposure to ST-enterotoxins. E. coli toxins that elicit a hemolytic effect, much like enterotoxins, are encoded by plasmid genes and reside on transmissible plasmids. Gamma ($\gamma$), beta ($\beta$), and alpha ($\alpha$) hemolysins have been identified. To produce a hemolysin exhibiting pathogenic activity, the participation of multiple cistrons is required: two ensure the biosynthesis of the functionally active hemolysin, while a third facilitates the translocation of the synthesized hemolysin across the bacterial membrane into the external environment. Evidence suggests that the manifestation of pathogenic properties by the synthesized hemolysin requires the participation of two other plasmids, which in wild-type E. coli strains coexist within the same bacterial cell alongside the hemolysin determinant. This and other examples indicate that plasmid-encoded toxins alone are often insufficient for a bacterial cell to achieve full pathogenicity. Nevertheless, the precise contribution of both plasmids and chromosomes to The conversion of non-pathogenic bacteria into pathogenic ones remains to be fully elucidated. Practically nothing is known about the mechanism of action of surface antigens (K88 and K99) and their molecular properties, although the plasmids encoding these antigens have been studied to a certain extent.

Plasmids can directly or indirectly influence the biosynthesis of antibiotics, which are primarily synthesized by streptomycetes. It has recently been established that plasmids are involved in the biosynthesis of methylenomycin, chloramphenicol, tetracycline, and macrolides. The antibiotic-producing function is frequently inferred from its impairment following plasmid elimination from cells using ethidium bromide, acridine orange, or other agents, although the exact mechanism of this reaction remains incompletely understood.

A circumstance of great practical importance is the ability of certain bacteria (many strains of Pseudomonas putida) to metabolize various classes of hydrocarbons, as well as the fact that this trait is determined by transmissible plasmids. Currently, breeding experiments have yielded Pseudomonas putida strains harboring a plasmid that encodes the enzymes for xylene and toluene degradation (the XYL plasmid), as well as the NAH plasmid, which encodes the enzymes for naphthalene degradation. A significant achievement from both theoretical and practical standpoints is the introduction into a bacterial cell—harboring both the XYL and NAH plasmids—of a newly engineered hybrid plasmid that incorporates fragments of the incompatible plasmids OCT and CAM. Because of the incompatibility principle, the OCT and CAM plasmids cannot simultaneously coexist within a single bacterial cell; thus, these findings serve as an example of overcoming plasmid incompatibility.

A bacterium harboring both functionally active XYL and NAH plasmids, alongside a hybrid plasmid incorporating fragments of the OCT and CAM plasmids, is capable of metabolizing a broader spectrum of hydrocarbon classes than strains containing only a single plasmid. Consequently, it is characterized by a high growth rate when utilizing crude oil as a growth medium. This "superbug" is proposed for use in cleaning up oil spills and degreasing tanker holds.