Genetic Recombination in Bacteria: 9 Things to Know About

This article throws light upon the nine things to know about genetic recombination in bacteria.

The nine things are: (1) Recombination of Genetic Materials (2) Transformation (3) Transduction (4) Conjugation (5) Mechanism of Genetic Recombination in Bacteria (6) Antibiotic Resistance (7) Phage Genome and Recombination (8) Genetic Maps of Viruses and (9) Fine Structure of rII Locus in T4 Phage.

Thing # 1. Recombination of Genetic Materials:

Like meiotic crossing over in eukaryotes, genetic recombination in bacteria provided the basis for the development of methodology for chromosome mapping. The term genetic recombination, as applied to bacteria and bacteriophages, leads to the replacement of one or more genes present in one strain with those of genetically distinct strain. This is somewhat different from our use of genetic recombination in eukaryotes.

In eukaryotes, the term describes crossing over that results in reciprocal exchange events. The overall effect is the same in both the system; genetic information is transferred from one chromosome to another resulting in an altered genotype. In bacteria there are mainly three phenomenon’s responsible for transfer of genetic information- transformation, transduction and conjugation.

Thing # 2. Transformation:

Transformation was first discovered in Streptococcus pneumonia and led to the discovery that DNA is the substance of the genes. The path leading to this history making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith. Now transformation is also seen in other bacterial genera like Hemophilic, Neisseria, Bacillus, and Staphylococcus.

In transformation, pieces of DNA released from donor bacteria are taken up directly from the extracellular environment by recipient bacteria. This leads to a stable genetic change in the recipient cell. DNA entry is thought to occur at the limited number of sites on the surface of the bacterial cell.

Passage across the cell wall and membrane is an active process requiring energy and specific transport molecule. During the process of entry, one of the two strands of invading DNA molecule is digested by nucleases, leaving only a single strand to participate in transformation. The active single strand aligns with its complementary region of the host bacterial DNA. This process involves several enzymes and the inserted DNA segment replaces its counterpart from the host genome, which is excised and degraded.

The ability of bacteria to take up extra-cellular DNA and to become transformed called transformation. This competence varies with the physiologic state of the bacteria. Many bacteria that are not usually competent can be made to take up DNA by laboratory manipulations, such as calcium shock or exposure to a high-voltage electrical pulse (electroporation). In some bacteria (including Haemophilus and Neisseria) DNA uptake depends on the presence of specific oligonucleotide sequences in the transforming DNA, but in others (including Streptococcus pneumoniae) DNA uptake is not sequence specific.

Competent bacteria may also take up intact bacteriophage DNA (transfection) or plasmid DNA, which can then replicate as extra-chromosomal genetic elements in the recipient bacteria. In contrast, a piece of chromosomal DNA from a donor bacterium usually cannot replicate in the recipient bacterium unless it becomes part of a replicon by recombination. Historically, characterization of “transforming principle” from S. pneumoniae provided the first direct evidence of DNA as a genetic material.

For detecting the recombination, the transforming DNA must be derived from the different strains of bacteria, bearing some genetic variation. Once it is integrated into the chromosome, the recombinant region contains one host strand and one mutant strand. As these strands are from different sources, this helical region is referred to as a hetero-duplex. Following one round of replication, one chromosome is restored to its original configuration, identical to that of the recipient cell and the other contains the mutant gene. Following cell division, one non-mutant (untransformed) cell and one mutant (transformed) cell are produced.

For DNA to be effective in transformation, it must include between 10,000 to 20,000 nucleotides pairs, about 1/200 of the E. coli chromosome. This size is sufficient to encode several genes. Genes that are adjacent or very close to one another on the bacterial chromosomes can be carried on a single segment of DNA of this size. Because of this fact, a single transformation event can result in the co-transformation of several genes simultaneously.

Genes that are close enough to each other to be co-transformed are said to be linked. Here the linkage refers to the proximity of the genes. Linked bacterial genes were first demonstrated in 1954 during studies of Pneumococcus by Rolin Hotchkiss and Julius Marmur. They were examining transformation at the streptomycin and mannitol loci.

Recipient cells were Streptomycin sensitive (str^s) and could not ferment mannitol (mtl^– ). Cells that were streptomycin resistant (str^r) and could ferment mannitol (mtl⁺) were used to derive the transforming DNA. If the str and mtl genes are not linked, simultaneous transformation for both genes will occur with such a minimal probability as to be nearly undetectable.

However, a low but detectable rate of co-transformation did occur. This suggests that the genes responsible for these characters are linked. In addition to establishing the linkage relationships, relative mapping distances between linked genes can also be determined from the recombination data provided by transformation experiments.

Thing #  3. Transduction:

In transduction, bacteriophages function as vectors to introduce DNA from donor bacteria into recipient bacteria by infection. For some phages, called generalized transducing phages, a small fraction of the virions produced during lytic growth are aberrant and contain a random fragment of the bacterial genome instead of phage DNA. Each individual transducing phage carries a different set of closely linked genes, representing a small segment of the bacterial genome.

Transduction mediated by populations of such phages is called generalized transduction, because each part of the bacterial genome has approximately the same probability of being transferred from donor to recipient bacteria. When a generalized transducing phage infects a recipient cell, expression of the transferred donor genes occurs.

Abortive transduction refers to the transient expression of one or more donor genes without formation of recombinant progeny, whereas complete transduction is characterized by production of stable recombinants that inherit donor genes and retain the ability to express them.

In abortive transduction the donor DNA fragment does not replicate, and among the progeny of the original transductant only one bacterium contains the donor DNA fragment. In all other progeny the donor gene products become progressively diluted after each generation of bacterial growth until the donor phenotype can no longer be expressed.

On selective medium upon which only bacteria with the donor phenotype can grow, abortive transductants produce minute colonies that can be distinguished easily from colonies of stable transductants. The frequency of abortive transduction is typically one to two orders of magnitude greater than the frequency of generalized transduction, indicating that most cells infected by generalized transducing phages do not produce recombinant progeny.

Specialized transduction differs from generalized transduction in several ways. It is mediated only by specific temperate phages, and only a few specific donor genes can be transferred to recipient bacteria. Specialized transducing phages are formed only when lysogenic donor bacteria enter the lytic cycle and release phage progeny.

The specialized transducing phages are rare recombinants which lack part of the normal phage genome and contain part of the bacterial chromosome located adjacent to the pro-phage attachment site. Many specialized transducing phages are defective and cannot complete the lytic cycle of phage growth in infected cells unless helper phages are present to provide missing phage functions.

Specialized transduction results from lysogenization of the recipient bacterium by the specialized transducing phage and expression of the donor genes. Phage conversion and specialized transduction have many similarities, but the origin of the converting genes in temperate converting phages is unknown.

Some of the important transduction phages present in bacteria are as follows:

1. Zinder and Lederberg Experiment:

When certain proviruses turn into virulent viruses, they cause lysis of bacterial cell, and during this they may carry with them segments of bacterial’s DNA. If such virus particles carrying a segment of DNA from one bacteria infects another bacteria then the second bacteria can acquire some of the properties of first one due to transfer of genetic material through the virus. This process is called as transduction and was first discovered in 1952 by Norton Zinder and Joshua Lederberg. Zinder and Lederberg discovered transduction through an experiment popularly called U tube experiment.

In this experiment a U tube was partitioned into two arms by a filter, so that only virus particles and not the bacterial cells could pass from one arm to another. One arm contained lysogenic bacteria carrying latent virus and the other arm contained bacteria susceptible to this virus. Rarely lysogenic bacteria could undergo lysis and release virus particles which crossed the filter and infected the other susceptible strain, which got destroyed releasing virus particles.

These released particles carried genetic material from susceptible strain and through the filter again reached the lysogenic strain and transferred the genetic material to this lysogenic strain. Since two bacterial strains could not come in contact, no conjugation was possible. An enzyme was also used which destroyed free DNA, so that the possibility of the transformation was also eliminated.

Regions as large as one percent of the bacterial chromosome may become enclosed in the viral head. In either case, the ability to infect is unrelated to the type of DNA in the phage head, making transduction possible (Fig. 4.1).

2. Bacteriophage:

Bacteriophages (bacterial viruses, phages) are infectious agents that replicate as obligate intracellular parasites in bacteria. Bacteriophage T4 is one of a group of related bacterial virus. Extracellular phage particles are metabolically inert and consist principally of proteins plus nucleic acid (DNA or RNA, but not both). The proteins of the phage particle form a protective shell (capsid) surrounding the tightly packaged nucleic acid genome.

Phage genomes vary in size from approximately 2 to 200 kilo-bases per strand of nucleic acid and consist of double-stranded DNA, single-stranded DNA, or RNA. The DNA is sufficient in quantity to encode more than 150 average sized genes. Phage genomes, like plasmids, encode functions required for replication in bacteria, but beside that, they also encode capsid proteins and nonstructural proteins required for phage assembly.

Several morphologically distinct types of phage have been described, including polyhedral, filamentous, and complex. Complex phages have polyhedral heads connected to a tail that contains a collar and a contractile sheath surrounding a central core (Fig. 4.2). Tail fibers, which protrude from the tail, contain binding sites in their tips that specifically recognize unique areas of the outer surface of the cell wall of the bacterial host, E. coli.

Infection is initiated by adsorption of phage to specific receptors on the surface of susceptible host bacteria (Fig. 4.3, 4.4). Then contraction of the tail sheath causes the central core to penetrate the cell wall. The DNA in the head is extruded, and then it moves across the cell membrane into the bacterial cytoplasm.

The capsid remains at the bacterial cell surface. Within minutes, all bacterial DNA, RNA and protein synthesis is inhibited and synthesis of viral molecule begins. At the same time, degradation of the host DNA is initiated. After those viral DNA/RNA components of the head, tail and tail fibres are synthesized.

For most phages, assembly of progeny occurs in the cytoplasm, and release of the progeny occurs by bacterial cell lysis by the action of lysozyme (a phage gene product). The latent period is the interval from infection until extracellular progeny appear, and the rise period is the interval from the end of the latent period until all phage is extracellular. The average number of phage particles produced by each infected cell, called the burst size, is characteristic for each virus and often ranges between 50 and several hundreds.

3. Pro-phage:

The relationship between virus and bacterium does not always result in viral reproduction and lysis. In some viruses, the viral DNA, instead of replicating in the bacterial cytoplasm, is integrated into the bacterial chromosome. This feature is called as lysogeny.

Subsequently, each time the bacterial DNA is replicated; the viral DNA is also replicated and passed to daughter bacterial cells following division. No new viruses are produced and no lysis of the bacterial cell occurs. Several terms are used to describe this relationship. The viral DNA integrated into the bacterial chromosomes is called a pro-phage and such viruses are called pro virus.

However, in response to certain stimuli, such as chemical, ultraviolet light treatment, or depletion of nutrients, the viral DNA may lose its integrated status and initiate replication, phage production, and lysis of the bacterium.

Viruses that can either lyse the cell or behave as a pro-phage are called temperate. Those that can only lyse the cell are referred to as virulent. A bacterium harboring a pro-phage that can be lysogenized, is said to be lysogenic; that is, it is capable of being lysed as a result of induced viral reproduction. The viral DNA, which can replicate either in the bacterial cytoplasm or as part of the bacterial chromosome, is sometimes classified as episome (Fig. 4.5).

Thing # 4. Conjugation:

In 1946, Joshua Lederberg and Edward Tatum showed that the bacteria undergo conjugation, a Para sexual process in which the genetic information from one bacterium is transferred to, and recombined with that of another bacterium. In conjugation, direct contact between the donor and recipient bacteria leads to establishment of a cytoplasmic bridge between them and transfer of part or all of the donor genome to the recipient. Donor ability is determined by specific conjugative plasmids called fertility plasmids or sex plasmids.

The F plasmid (also called F factor) of E. coli is the prototype for fertility plasmids in Gram- negative bacteria. Strains of E. coli with an extra-chromosomal F plasmid are called F+ and function as donors, whereas strains that lack the F plasmid are F- and behave as recipients. The conjugative functions of the F plasmid are specified by a cluster of at least 25 transfer (tra) genes which determine expression of F pili (conjugation tube), synthesis and transfer of DNA during mating.

Each F+ bacterium has 1 to 3 F pili that bind to a specific outer membrane protein (the ompA gene product) on recipient bacteria to initiate mating. An intercellular cytoplasmic bridge is formed, and one strand of the F plasmid DNA is transferred from donor to recipient, beginning at a unique origin and progressing in the 5′ to 3′ direction.

The transferred strand is converted to circular double-stranded F plasmid DNA in the recipient bacterium, and a new strand is synthesized in the donor to replace the transferred strand. Both of the ex-conjugant bacteria are F+, and the F plasmid can therefore spread by infection among genetically compatible populations of bacteria. In addition to the role of the F pili in conjugation, they also function as receptors for donor-specific (male-specific) phages.

The F plasmid in E. coli. can exist as an extra-chromosomal genetic element or be integrated into the bacterial chromosome. Because the F plasmid and the bacterial chromosome are both circular DNA molecules, reciprocal recombination between them produces a larger DNA circle consisting of F plasmid DNA inserted linearly into the chromosome. E. coli contains multiple copies of several different genetic elements called insertion sequences (see section on transposons for more detail), at various locations in its chromosome and in the F plasmid.

Homologous recombination between insertion sequences in the chromosome and the F plasmid leads to preferential integration of the F plasmid at chromosomal sites where insertion sequences are located. The chromosomal sites where insertion sequences are found vary among strains of E. coli. The Figure 4.6 shows that the F plasmid is representative of specific conjugative plasmids that control donor ability in E. coli.

F- strains lack the F plasmid and are genetic recipients. F+ strains harbor the F plasmid as a cytoplasmic element, express F pili, and are genetic donors. The F plasmid can become integrated into bacterial chromosome at various locations to produce Hfr (high-frequency recombination) donor strains.

Abnormal excision of F plasmid can result in formation of F’ plasmids that contain segments of bacterial chromosome and the corresponding bacterial genes. The arrowhead in F plasmid defines origin for transfer of DNA during conjugation. F plasmid and chromosomal DNA are indicated by heavy and fine lines, respectively.

An E. coli strain with an integrated F plasmid retains its ability to function as a donor in conjugal matings. Because donor strains with integrated F factors can transfer chromosomal genes to recipients with high efficiency, they are called Hfr (high-frequency recombination) strains. Transfer of single-stranded DNA from an Hfr donor to a recipient begins from the origin within the F plasmid and proceeds as described above, except that the transferred DNA is the hybrid replicon consisting of F plasmid integrated into the bacterial chromosome.

Transfer of this entire replicon, including the bacterial chromosome, requires approximately 100 minutes. The identity of the first chromosomal gene to be transferred and the polarity of chromosomal transfer are determined by the site of integration of the F plasmid and its orientation with respect to the bacterial chromosome. Because the mating bacteria usually separate spontaneously before the entire chromosome is transferred, conjugation typically transfers only a fragment of the donor chromosome into the recipient.

The probability that a donor gene will enter the recipient bacterium during conjugation decreases, as its distance from the F origin (and therefore the time of its transfer) increases. Mating cells can also be broken apart experimentally by subjecting them to strong shearing forces in a mechanical blender; this is called interrupted mating.

Formation of recombinant progeny requires recombination between the transferred donor DNA and the genome of the recipient bacterium. Analysis of progeny from matings that are interrupted after different intervals demonstrates which chromosomal genes are transferred first by particular donor strains, the sequential times of entry for genes that are transferred subsequently, and the progressively lower probability that genes transferred later will appear in recombinant progeny.

The circularity of the genetic map of E. coli was originally deduced from the overlapping, circularly permuted groups of linked genes that were transferred early by individual donor strains in which the F factor was integrated at different chromosomal locations.

In matings between F+ and F- bacteria, only the F plasmid is transferred with high efficiency to recipients. Chromosomal genes are transferred with very low efficiency, and it is the spontaneous Hfr mutants in F+ populations that mediate transfer of donor chromosomal genes. In matings between Hfr and F- strains, the segment of the F plasmid containing the tra region is transferred last, after the entire bacterial chromosome has been transferred.

Most recombinants from matings between Hfr and F- cells fail to inherit the entire set of F plasmid genes and are phenotypically F. In matings between F+ and F- strains, the F plasmid spreads rapidly throughout the bacterial population, and most recombinants are F+. Integrated F plasmids in Hfr strains can sometimes be excised from the bacterial chromosome. If excision precisely reverses the integration process, F+ cells are produced. On rare occasions, however, excision occurs by re-combinations involving insertion sequences or other genes on the bacterial chromosome that are located at some distance from the original integration site.

In such cases segments of the bacterial chromosome can become incorporated into hybrid F plasmids that are called F’ plasmids. By similar processes, segments of the bacterial chromosome can sometimes become incorporated into R plasmids to produce hybrid R’ plasmids. Conjugative R’ plasmids can function as fertility plasmids because they can integrate into the bacterial chromosome by homologous recombination and mediate transfer of chromosomal genes during matings with recipient bacteria.

F’ plasmids, R’ plasmids, specialized transducing phages, and recombinant plasmids or phages constructed by gene cloning are hybrid replicons that can include segments of the bacterial chromosome. Therefore, any of these genetic elements can be used to construct the partially diploid bacterial strains that are required for complementation tests and other purposes.

Conjugation also occurs in Gram-positive bacteria. Gram-positive donor bacteria produce adhesins that cause them to aggregate with recipient cells, but sex pili are not involved. In some Streptococcus species, recipient bacteria produce extracellular sex pheromone that attracts another bacterium.

This causes the donor phenotype to attach to another bacterium, that harbor an appropriate conjugative plasmid. The conjugative plasmid prevents the donor cells from producing the corresponding pheromone. All the three types of bacterial re-combinations are presented in the Figure 4.7.

Thing # 5. Mechanism of Genetic Recombination in Bacteria:

Generalized recombination involves donor and recipient DNA molecules that have homologous nucleotide sequences. Reciprocal exchanges can occur between any homologous donor and recipient sites. In E. coli, the product of the recA gene is essential for generalized recombination, but other gene products also participate. Site-specific recombination involves reciprocal exchanges only between specific sites in donor and recipient DNA molecules. The recA gene product is not required for site-specific recombination.

Integration of the temperate bacteriophage λ into the chromosome of E. coli is a well-studied example of site-specific recombination (Fig. 4.8). The specific attachment (att) sites on the E. coli chromosome and λ phage DNA have a common core sequence of 15 nucleotides, within which reciprocal recombination occurs, flanked by adjacent sequences that are not homologous in the phage and bacterial genomes.

In phage λ the product of the int gene (integrase) is required for the site-specific integration event in lysogenization; the products of the int and xis (excisionase) genes are both needed for the complementary site-specific excision event. This event occurs during induction of lytic phage development in lysogenic cells.

Illegitimate recombination is the term used to describe non-homologous, aberrant recombination events such as those involved in formation of specialized transducing phages. The mechanisms of illegitimate recombination are unknown. In the Figure 4.8 λ DNA is shown by thin lines and chromosomal DNA by thick lines. Attachment (att) sites are closed boxes for the bacterial chromosome and open boxes for the λ chromosome.

The gal and bio operons, which determine utilization of galactose and biosynthesis of biotin, are located adjacent to the bacterial attachment site. In an infected E coli the λ DNA becomes circular by joining ends m and m’, and site-specific recombination between phage and bacterial att sites results in insertion of the X genome into the bacterial chromosome. The arrangement of the prophage DNA (m and m’ located internally) is, therefore, a circular permutation of λ virion DNA (m and m’ located terminally).

Thing # 6. Antibiotic Resistance:

Bacteria are single-celled microorganisms, and most bacterial species are either spherical (called cocci) or rod-shaped (called bacilli) (Fig. 4.9). The extraordinary ability of certain bacteria to develop resistance to antibiotics has been a hot topic on the minds of doctors, hospital staff, reporters, and the general public for several years. It is also used as textbook example of evolution in action. These bacteria are being studied by evolutionary scientists with the hope that they will reveal secrets as to how molecules-to-man evolution could have happened.

Antibiotic resistance is the ability of a microorganism to withstand the effects of antibiotics. It is a specific type of drug resistance. Antibiotic resistance evolves via natural selection acting upon random mutation, but it can also be engineered by applying an evolutionary stress on a population. Once such a gene is generated, bacteria can then transfer the genetic information in a horizontal fashion (between individuals) by plasmid exchange.

If a bacterium carries several resistance genes, it is called multi-resistant or, informally, a superbug. Antibiotic resistance can also be introduced artificially into a microorganism through transformation protocols. This can aid in implanting artificial genes into the microorganism. If the resistance gene is linked with the gene to be implanted, the antibiotic can be used to kill off organisms that lack the new gene.

Antibiotics are natural substances secreted by bacteria and fungi to kill other bacteria that are competing for limited nutrients (The antibiotics used to treat people today are typically derivatives of these natural products). Scientists are dismayed to discover that some bacteria have become resistant to antibiotics through various alterations, or mutations, in their DNA. Hospitals have become a breeding ground for antibiotic resistant bacteria.

These bacteria proliferate in an environment filled with sick people who have poor immune systems and where antibiotics have eliminated competing bacteria that are not resistant. Bacteria that are resistant to modern antibiotics have even been found in the frozen bodies of people who died long before those antibiotics were discovered or synthesized.

Antibiotics were first discovered through a fortunate experiment by Alexander Fleming in 1928. His work eventually led to the large-scale production of penicillin from the mold Penicillium notatum in the 1940s. As early as the late 1940s resistant strains of bacteria began to appear. After four years, when drug companies started to mass produce penicillin, in 1943, the first signs of penicillin-resistant bacteria started to show up.

The first bacteria that fought penicillin were called Staphylococcus aureus. This bug is usually harmless but can cause an illness such as pneumonia. In 1967, another penicillin-resistant bacterium found. It was called pneumococcus and it broke out in a small village in Papua New Guinea. Other penicillin resistant bacteria found are Enterococcus faecium and a new strain of gonorrhea.

Currently, it is estimated that more than 70% of the bacteria that cause hospital acquired infections are resistant to at least one of the antibiotics used to treat them. Antibiotic resistance continues to expand for a multitude of reasons, including over-prescription of antibiotics by physicians, non-completion of prescribed antibiotic treatments by patients, use of antibiotics in animals as growth enhancers (primarily by the food industry), increased international travel, and poor hospital hygiene.

Several studies have demonstrated that patterns of antibiotic usage greatly affect the number of resistant organisms which develop. Overuse of broad-spectrum antibiotics, such as second- and third-generation cephalosporin’s, greatly hastens the development of methicillin resistance. Other factors contributing towards resistance include incorrect diagnosis, unnecessary prescriptions, improper use of antibiotics by patients, the impregnation of household items and children’s toys with low levels of antibiotics, and the administration of antibiotics by mouth in livestock for growth promotion.

Mutation:

Antibiotic resistance can be a result of unlinked point mutations in the pathogen genome and a rate of about 1 in 108 per chromosomal replication. The antibiotic action against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which will result in a fully resistant colony.

The antibiotic works by binding to a protein so that the protein cannot function properly. The normal protein is usually involved in copying the DNA, making proteins, or making the bacterial cell wall. Proteins are involved in all important functions for the bacteria to grow and reproduce.

If the bacteria have a mutation in the DNA which codes for one of those proteins, the antibiotic cannot bind to the altered protein; and the mutant bacteria survive. In the presence of antibiotics, the process of natural selection will occur, favoring the survival and reproduction of the mutant bacteria.

During the anthrax scare shortly after the September 11, 2001, attacks in the U.S., Ciprofloxacin (Cipro) was given to potential victims. Cipro belongs to a family of antibiotics known as quinolones, which bind to a bacterial protein called gyrase, decreasing the ability of the bacteria to reproduce. This allows the body’s natural immune defenses to overtake the infectious bacteria as they are reproducing at a slower rate. Quinolone-resistant bacteria have mutations in the genes encoding the gyrase protein.

The mutant bacteria survive because the Cipro cannot bind to the altered gyrase. This comes at a cost as quinolone-resistant bacteria reproduce more slowly. Resistance to this family of antibiotics is becoming a major problem with one type of bacteria which causes food poisoning. This bacteria increased its resistance to quinolones 10-fold in just five years.

Recent research has shown that the SOS pathway may be essential in the acquisition of bacterial mutations which lead to resistance to some antibiotic drugs. The increased rate of mutation during the SOS response is caused by three low-fidelity DNA polymerases: Pol II, Pol IV and Pol V. Researchers are now targeting these proteins with the aim of creating drugs that prevent SOS repair. By doing so, the time needed for pathogenic bacterial to evolve antibiotic resistance could be extended, and thus improve the long term viability of some antibiotic drugs.

By Horizontal Gene Transfer Share Resistance Genes:

Bacteria can also become antibiotic resistant by gaining mutated DNA from other bacteria. Bacteria can exchange DNA. No new DNA is generated it is just exchanged. This mechanism of exchanging DNA is necessary for bacteria to survive in extreme or rapidly changing environments like a hospital (or like those found shortly after the Flood). The mechanisms of mutation and natural selection aid bacteria populations in becoming resistant to antibiotics.

The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:

1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases.

2. Alteration of target site: e.g. alteration of PBP—the binding target site of penicillin’s—in MRSA (Methicillin resistant Staphylococcus aureus) and other penicillin-resistant bacteria.

3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-amino benzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria is inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid.

4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.

Prevention of Antibiotic Resistance and Development of New Drugs:

Since antibiotics became so prosperous, all other strategies to fight bacterial diseases were put aside. Now since the effects of antibiotics are decreasing and antibiotic resistance is increasing, new research on how to battle bacteria has started. Improving infection control, discovering new antibiotics, and taking drugs more appropriately are ways to prevent resistant bacteria from spreading.

Rational use of antibiotics may also reduce the chances of development of opportunistic infection by antibiotic-resistant bacteria due to dysbacteriosis. In developing nations, approaches are being made to control infections such as hand washing by health care people, and identifying drug resistant infections quickly to keep them away from others. The World Health Organization has begun a global computer program that reports any outbreaks of drug-resistant bacterial infections. Phage therapy, an approach that has been extensively researched and utilized as a therapeutic agent for over 60 years, especially in the Soviet Union, is an alternative that might help with the problem of resistance.

Phage Therapy was widely used in the United States until the discovery of antibiotics, in the early 1940s. Bacteriophages or “phages” are viruses that invade bacterial cells and, in the case of lytic phages, disrupt bacterial metabolism and cause the bacterium to lyse. Phage Therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections.

Bacteriophage therapy is an important alternative to antibiotics in the current era of multidrug resistant pathogens. British studies also demonstrated significant efficacy of phages against Escherichia coli, Acinetobacter spp., Pseudomonas spp and Staphylococcus aureus. Phage therapy may prove as an important alternative to antibiotics for treating multidrug resistant pathogens.

The ability of microorganisms to exchange their genetic material has provided them an armament to fight new drugs and pose serious problem to human health. By understanding the mechanism of genetic recombination we can use modern tools of biology to control bacterial diseases and use them for production of useful drugs.

Thing # 7. Phage Genome and Recombination:

The genome of phage A is a double-stranded DNA molecule about 47,000 base pairs in length. In the phage particle, A DNA has single-stranded, complementary ends 12 bases in length, termed mature or cohesive ends m and m’.

Within an infected cell, DNA forms a circle through pairing of the single-stranded DNA, and is replicated and transcribed as a circular molecule during the replication-oriented early phase of a development.

After this early stage, a development may proceed along the productive (or lytic) pathway or along the alternative lysogenic pathway. The encapsulation-oriented late stage of the productive pathway involves a transcription switch to synthesis of head, tail, and lysis proteins and a replication switch to a rolling-circle mode that generates multimeric genomes (concatemers) that are the obligatory precursors for the cleaved, linear molecules packaged into a phage head.

The lysogenic pathway involves a repression of transcription and a site-specific recombination event that inserts A DNA into the host E. coli genome. This integrative recombination between the phage and host attachment sites (att) generates a genetic structure that is permuted from the linear order found in the phage particle because the phage attachment site (a a’ or P P) is approximately in the center of the mature DNA molecule.

The prophage has structurally distinct attachment sites: a left attL site (b a’ or B P’) and a right attR site (a b’ or P B’); these in turn can recombine to detach the prophage DNA when the virus is induced to lytic development, regenerating the phage att P site (a a’ or P P’) and the original host att B site (b b’ or B B’).

Viruses can undergo genetic recombination. It was first described by A.D. Hershey and R. Rotman in 1949. It was discovered during mixed infection experiments, in which two distinct mutant strains were allowed to simultaneously infect the same bacterial culture.

In it two loci are involved therefore recombination is referred to as intergenic. For example, if two strains of a virus having genotypes a⁺b” and a^–b⁺ are allowed to infect the same host cell, the resulting progeny of virus will also have recombinants a⁺b⁺ and air , in addition to parental combinations a⁺b^– and ar b⁺. This phenomenon of genetic recombination has been used in preparing linkage maps of viruses. Linkage maps in T2 and T4 phages have been found to be circular, as in the case in E. coli chromosome.

Thing # 8. Genetic Maps of Viruses:

The viruses, being very small particles, are often considered not suitable for inheritance studies. However plaque (clear area produced on opaque lawn of bacteria on the surface of a petri plate of solid medium) morphology like large vs. small, fuzzy vs. sharp, host range and virulence are characteristics features of bacteriophages, which have been used for inheritance and recombination in viruses can be illustrated by a cross in T2 phage, attempted by Hershey. This cross (h^–r⁺ x h⁺r^–) involved host range and plaque morphology.

Here h⁺ denotes that these viruses can infect only strain 1 and h^– can infect both strains 1 and 2. r⁺ lyses slowly producing small plaques and r lyses rapidly producing large plaques. For producing genetic map bacterial strain 1 was infected by both phage genotypes (mixed infection or double infection) and the lysate was analyzed by spreading it onto a bacterial lawn composed of a mixture of strain 1 and 2 (h^– will produce clear and h⁺ will produce cloudy plaques). Four plaque types were distinguishable (Fig. 4.10);

1. Clear and small: h^–r⁺

2. Cloudy and large: h⁺r^–

3. Cloudy and small: h⁺r⁺

4. Clear and large: h^– r^–

The first two of these four are parental phenotypes and last two are recombinants, so that the recombination frequency (RF) can be calculated as follows: RF= (h⁺r⁺) + (h^–r^–) / total plaques. In order to prepare the linkage map, several rapid lysis genotypes (r1, r2, r3, in order of discovery) were available and the corresponding strains were called r_a r_b and r_c Utilizing these three strains with h⁺ and h^– genotypes, following crosses were made: r_a^– h⁺ x r_a⁺ h^–, r_b^– h⁺ x r_b⁺ h^–, r_c^– h⁺ x r_c⁺ h^–, r_c^– r_b⁺x r_c⁺r_b ^–. The recombination frequencies were calculated and following two probable linear orders were available: r_a^– r_c^– h^–r_b and r_c^– h^– r_b^–r_a. This can be explained only if the circular map shown in circular genetic map for a T-even phage (Fig. 4.11). S. Benzer resolving the fine structure of rll locus has also studied recombination in T4.

Thing # 9. Fine Structure of rII Locus in T4 Phage:

T4 phage rll locus for the rough plaque phenotype of the phage colonies was most extensively studies for the fine structure of gene. The most refined analysis of a single gene ever conducted is the one undertaken during 1950’s by Seymour Benzer for a locus in T4 bacteriophage infecting E. coli. This locus is known as rll locus and a mutant at this locus is responsible for the formation of rough plaques or colonies.

This locus had largest number of rapid lysing (r) mutants, and is called rll locus. It can be distinguished from other r loci, by the inability of rll mutants to produce plaques on lysogenic ‘K’ strain of E. coli, which carries lamda prophage. The rll mutant make large plaques with fuzzy edges on E. coli, strain B. The wild type phage T4 rll⁺ will make small plaques with sharp edges, both on B and / k strains.

Further, when ‘K’ was infected simultaneously by rll⁺ and rll. Large plaques were formed, since rll* helps in lysis. So that rll may express. These distinguishing features enabled Benzer to distinguish mutants and wild type phages with high efficiency.

Complementation Test:

To determine complementation relations between rll mutant alleles, Benzer used two different rll mutants, arbitrarily designated as rll_x and rll_y. He allowed mixed infection of K strain by these two mutants. In most cases, this did not result into lysis and plaque formation; in some cases it did lead to plaque formation. If two mutants did not formed plaques on mixed infection, they were placed in the same group, but if plaques are produced the two mutants involved in mixed infection were placed in two different groups.

In this manner, two groups A and B can be established in rll region. All mutants with the help of complementation test could be classified in these two groups, in such a manner that two mutants from group A or two mutants from group B could not cause plaque formation but mixed infection by one mutant from each group could cause plaque formation.

Since group A and B are separated on the basis of cis-trans test, these were called as cistron A and cistron B. From the complementation test, it is clear that in rll region two cistron A and B are independent functionally and must be responsible for sequential synthesis for two separate products (different proteins).

Therefore, all mutants belonging to one cistron share a common deficiency, which is different from deficiency in the second cistron. When two mutants of same group are mixed, deficiency persists in them. When two mutants of different groups are mixed, they complement each other and may express wild phenotype i.e. lysis and plaque formation.

Recombination Test:

Once rll mutants were classified into cistron A and cistron B, Benzer analysed the mutants belonging to same cistron. Mutants were subjected to recombination test to find out whether they are located on same site or different sites separable by a distance. This distance can be resolved by re-combinations.

Benzer classified all the mutants into two categories:

(1) Revertant type-point mutations

(2) Non revertant type-deletion mutant (multi-site mutations).

Deletion mutations were determined by several tests including their non-revertant nature. For recombination analysis, Benzer used non revertant mutants. These deletions were arranged in set of overlapping deletions representing segments of different sizes in rll region as shown in the Figure 4.12.

The principle involved in this technique was that if a point mutation lies within the region of a deletion in another mutant, then mixed infection with these two will not be able to give rise to a wild type (both defective at same site). If the point mutation falls outside the deletion region of second mutant, it will be able to give wild type recombinant (both defective at different sites).

Using non revertant mutants having successive overlapping deletions of smaller lengths, one could locate a mutant to a fairly small region. All point mutations located in this particular small segment of rll region, could then be subject to recombination test. For this purpose, two mutants at a time were used for mixed infection on E. coli B strain.

The lysate produced on plaques was used for infection on K strain to find out the frequency of wild type phage particles produced, because this will represent the recombination frequency. Benzer estimated recombination frequencies as low as 0.0001%. Thus, Benzer was able to divide rll region into cistrons A and B and same cistron into hundreds of mutational sites separable due to recombination.

By this method -2400 mutations were mapped in rll region and were found to occupy 300 different sites called mutational sites. There were regions having high mutational frequencies called as hot spots whereas other sites having no mutations were called zero sites.