Genetic Recombination in Bacteria

Bacteria have no sexual reproduction in the sense that eukaryotes do. The have

• no alternation of diploid and haploid generations
• no gametes
• no meiosis

But the essence of sex is genetic recombination, and bacteria do have three mechanisms to accomplish that:

• transformation
• conjugation
• transduction

Transformation

Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surroundings. The ability to deliberately transform the bacterium E. coli has made possible the cloning of many genes — including human genes — and the development of the biotechnology industry.

The first demonstration of bacterial transformation was done with Streptococcus pneumoniae and led to the discovery that DNA is the substance of the genes. The path leading to this epoch-making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith.

The cells of S. pneumoniae (also known as the pneumococcus) are usually surrounded by a gummy capsule made of a polysaccharide. When grown on the surface of a solid culture medium, the capsule causes the colonies to have a glistening, smooth appearance. These cells are called "S" cells.

Streptococcus pneumoniae (pneumococci) growing as colonies on the surface of a culture medium. Left: The presence of a capsule around the bacterial cells gives the colonies a glistening, smooth (S) appearance. Right: Pneumococci lacking capsules have produced these rough (R) colonies. (Courtesy of Robert Austrian, J. Exp. Med. 98:21, 1953.)

However, after prolonged cultivation on artificial medium, some cells lose the ability to form the capsule, and the surface of their colonies is wrinkled and rough ("R"). With the loss of their capsule, the bacteria also lose their virulence. Injection of a single S pneumococcus into a mouse will kill the mouse in 24 hours or so. But an injection of over 100 million (100 x 10⁶) R cells is entirely harmless.

Encapsulated (left) and nonencapsulated (right) pneumococci. The encapsulated forms produce smooth colonies (above). (Courtesy of Robert Austrian, J. Exp. Med. 98:21, 1953.)

The reason? The capsule prevents the pneumococci from being engulfed and destroyed by scavenging cells — neutrophils and macrophages — in the body. The R forms are completely at the mercy of phagocytes.

Pneumococci also occur in over 90 different types: I, II, III and so on. The types differ in the chemistry of their polysaccharide capsule.

Unlike the occasional shift of S -> R, the type of the organism is constant. Mice injected with a few S cells of, say, Type II pneumococci, will soon have their bodies teeming with descendant cells of the same type.

However, Griffith found that when living R cells (which should have been harmless) and dead S cells (which also should have been harmless) were injected together, the mouse became ill and living S cells could be recovered from its body. Furthermore, the type of the cells recovered from the mouse's body was determined by the type of the dead S cells. In the experiment shown, injection of

• living R-I cells and
• dead S-II cells

produced a dying mouse with its body filled with living S-II pneumococci.

The S-II cells remained true to their new type. Something in the dead S-II cells had made a permanent change in the phenotype of the R-I cells. The process was named transformation.

Oswald Avery and his colleagues at The Rockefeller Institute in New York City eventually showed that the "something" was DNA.

In pursuing Griffith's discovery, they found that they could bring about the same kind of transformation in vitro using an extract of the bacterial cells.

Treating this extract with

• enzymes to destroy all polysaccharides (including the polysaccharide of the capsule)
• a lipase to destroy any lipids
• proteases to destroy all proteins
• RNase to destroy RNA

did not destroy the ability of their extracts to transform the bacteria.

But treating the extracts with DNase to destroy the DNA in them did abolish their transforming activity. So DNA was the only material in the dead cells capable of transforming cells from one type to another. DNA was the substance of genes.

Although the chemical composition of the capsule is determined by genes, the relationship is indirect. DNA is transcribed into RNA and RNA is translated into proteins. The phenotype of the pneumococci — the chemical composition of the polysaccharide capsule — is determined by the particular enzymes (proteins) used in polysaccharide synthesis.

Conjugation

Some bacteria, E. coli is an example, can transfer a portion of their chromosome to a recipient with which they are in direct contact. As the donor replicates its chromosome, the copy is injected into the recipient. At any time that the donor and recipient become separated, the transfer of genes stops. Those genes that successfully made the trip replace their equivalents in the recipient's chromosome.

Features:

• Can only occur between cells of opposite mating types.
• The donor (or "male") carries a fertility factor (F⁺).
• The recipient ("female") does not (F⁻).
• F
• is a set of genes originally acquired from a plasmid and now integrated into the bacterial chromosome;
• establishes the origin of replication for the chromosome.
• A portion of F is the "locomotive" that pulls the chromosome into the recipient cell.
• The rest of it is the "caboose".
• In E. coli, about one gene gets across each second that the cells remain together. (So, it takes about 100 min for the entire genome (4377 genes) to make it. But,
• the process is easily interrupted so
• it is more likely that host genes close behind the leading F genes ("locomotive") will make it than those farther back
• The "caboose" seldom makes it so failing to receive a complete F factor, the recipient cell continues to be "female"
• The DNA that makes it across finds the homologous region on the female chromosome and replaces it (by a double crossover).
• By deliberately separating the cells (in a kitchen blender) at different times, the order and relative spacing of the genes can be determined. In this way, a genetic map — equivalent to the genetic maps of eukaryotes — can be made. But here the map intervals are seconds, not centimorgans (cM).

Demonstration

The figure shows the mechanism of conjugation in E. coli cells where

• The "male" lacks functional genes needed to synthesize the vitamin biotin and the amino acid methionine (Bio⁻, Met⁻) so these must be added to its culture medium.
• The "female" has those genes (Bio⁺, Met⁺) but has nonfunctional (mutant) genes that prevent it from being able to synthesize the amino acids threonine and leucine (Thr⁻, Leu⁻) so these must be added to its culture medium).
• When cultured together, some female cells receive the functional Thr and Leu genes from the male donor.
• A double crossover enables them to replace the nonfunctional alleles.
• Now the cells now can grow on a "minimal" medium containing only glucose and salts.

Transduction

Bacteriophages are viruses that infect bacteria. In the process of assembling new virus particles, some host DNA may be incorporated in them.

The virion head can hold only so much DNA so these viruses

• while still able to infect new host cells
• may be unable to lyze them.

Instead the hitchhiker bacterial gene (or genes) may be inserted into the DNA of the new host, replacing those already there and giving the host an altered phenotype. This phenomenon is called transduction.

Significance of genetic recombination in bacteria.

Transformation, conjugation, and transduction were discovered in the laboratory. How important are these mechanisms of genetic recombination in nature? We don't really know, but

Some thoughts:

• The completion of the sequence of the entire genome of a variety of different bacteria (and archaea) suggest that genes have in the past moved from one species to another. This phenomenon is called lateral gene transfer (LGT).
• The remarkable spread of resistance to multiple antibiotics may have been aided by the transfer of resistance genes within populations and even between species.
• Many bacteria have enzymes that enable them to destroy foreign DNA that gets into their cells. It seem unlikely that these would be needed if that did not occur in nature. In any case, these restriction enzymes have provided the tools upon which the advances of molecular biology and the biotechnology industry depend.

Reductionism

The understanding of complex systems almost always has to await unraveling the details of some simpler system. You may feel that trying to find out how one type of pneumococcus could be converted into another was an exceedingly specialized and esoteric pursuit. But Avery and his coworkers realized the broader significance of what they were observing and, in due course, the rest of the scientific world did as well. By electing to work with a well-defined system: the conversion of R forms of one type into S forms of a different type, these researchers made a discovery that has revolutionized biology and medicine.

Attempting to understand the workings of complex systems by first understanding the workings of their parts is called reductionism. Some scientists (and many nonscientists) question the value of reductionism. They favor a holistic approach emphasizing the workings of the complete system.

But the record speaks for itself. From skyscrapers to moon walks, to computer chips to the advances of modern medicine, progress comes from first understanding the properties of the parts that make up the whole.

The late George Wald, who won the 1967 Nobel Prize in Physiology or Medicine for his discoveries of the molecular basis of detecting light, once worried that his work was overly specialized — studying not vision, not the eye, not the whole retina, not even their rods and cones, but just the chemical reactions of their rhodopsins. But he came to realize "it is as though this were a very narrow window through which at a distance one can see only a crack of light. As one comes closer, the view grows wider and wider, until finally through this same window one is looking at the universe. I think this is the way it always goes in science, because science is all one. It hardly matters where one enters, provided one can come closer....". 

Breakthrough findings on multiple sclerosis

Researchers from the National University of Singapore (NUS) have discovered a new type of immune T helper cells that may help develop treatment for multiple sclerosis.

T helper cells help the activity of other immune cells by helping, suppressing, or regulating immune response.

The researchers, led by NUS medical school professor Fu Xin-Yuan and Doctor Sheng Wanqiang from the Cancer Science Institute of Singapore, found that the immune cells play a crucial role in the immune system and the development of neuronal inflammation.

Together with Dr Zhang Yong-Liang from the department of microbiology at the NUS Yong Loo Lin School of Medicine, the team found that the newly discovered T helper cells is programmed by the STAT5 protein.

The T helper cells can recruit and activate other inflammatory cells to cause neuroinflammation, demyelination and nerve system damage.

The researchers’ work show that if doctors are able to block the STAT5 protein, patients suffering from the autoimmune disease will benefit greatly from it.

MECHANISMS OF GENETIC VARIATIONS

Allele

A DNA coding that occupies a specific place on a chromosome. Alleles hold genetic information such as the color of flower petals.

Genotype

The inheritable information carried in the cells of all living organisms. This stored information is used as a "blueprint" for building and maintaining the organism

Phenotype

The physical attributes of an organism. They are caused by both the organism’s genotype and its environment.

Latent

Present and capable of becoming active, but not yet visible or active.

Patent

Visible and active.

Morphology

What an organism looks like and what it is made of, also known as the form and structure of an organism.

Adaptive Radiation

The rapid diversification of a species to suit many different environments.

Physiology

The functions in living things, or the scientific study of those functions.

Preadaptation

When a creature uses a feature of their anatomy for a purpose seemingly unrelated to its original task.

Built-in Variation in the Gene Pool

For most character traits present in organisms, more than one allele exists. The different genes must have come about by chance alone, because we are dealing with genotype. The genotype of an organism includes both latent and patent genes. Only genes that have been activated are expressed in the phenotype. A new gene must first be expressed before natural selection comes into play.

As far as alleles are concerned, expression is governed by a complex system of dominance versus recessiveness. Furthermore, the frequency of genetic expression can also alter the phenotype. For example, the gene coding for growth hormone can influence the size of the organism. Variation in size does thus not necessarily require new genes, just differential expression of the same genes. An example of built-in variation in the gene pool can be seen in the differences between breeds of dogs. As to how the genes responsible for the variation came in to existence, chance or design are the only options given, since we are dealing with genotype.

By selecting from the built-in natural variation of the gene pool, various breeds of dogs and domestic cattle were produced. Great changes in physiology and morphology are involved, and evolution is here certainly excluded. Differences in dogs are greater than the differences in genera of the Canidae family.

Genetic expression is also influenced, so as to bring about differences in structural expression by the genes in terms of size. Differential hormonal modulation in response to environmental stimuli can alter the time and magnitude of response, effectively producing reproductively isolated communities which would be regarded as different species by evolutionists, but are in effect merely extremes of genetic expression within an existing gene pool. The vast numbers of latent genes would then be accounted for.

Evolutionists recognize that changes in genotype frequencies do occur to produce changes in gene distribution. They, however, explain most changes as resulting from chance mutations, and this is not tenable.

Even evolutionists admit that preadaptation must have played a major role in enabling organisms to survive environmental changes. Preadaptation, however, requires preexisting genes capable of responding to environmental stimuli—precisely what creationists claim. Where did these fully expressional genes come from? 

Homologous

Similarity in structure between parts of different organisms, such as the similarity between a human hand and a bat wing.

Genotype

The inheritable information carried in the cells of all living organisms. This stored information is used as a "blueprint" for building and maintaining the organism

Phenotype

The physical attributes of an organism. They are caused by both the organism’s genotype and its environment.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

Ligase

An enzyme that joins two larger molecules together with a chemical bond.

DNA

Deoxyribonucleic acid (DNA) is a molecule that stores genetic make up, or code. All living things have DNA.

Chiasma

The point where two chromatids are interwoven in a cell.

Chromatid

One of the two identical halves of a replicated chromosome.

Nucleotide

Molecules that join together to make up the structure of DNA and RNA.

Reproductive Exchange

Through sexual reproduction, genetic material is exchanged. This induces genetic recombination. The significance of this is obvious: the exchange of material increases the variation. This holds particular advantages to populations and is considered by evolutionists to be an innovation that greatly enhances the evolutionary process.

We know what sexual reproduction achieves. It increases the variation. However, increased variation in the genotype is of no value until it is expressed in the phenotype. The new varieties must be expressed in the offspring before natural selection can feast on this increased variation. The process that brings about the variation (sexual reproduction) is not subject to selection, only the result thereof (the increased variation in the offspring) is subject to selection.

The exchange of gametes requires a modified form of cell division which is the process of meiosis. During meiosis, the number of chromosomes is halved, resulting in the gametes having half the chromosomes. Sexual fusion of two gametes then restores the number of chromosomes. Variation in the genome is greatly increased by two processes occurring during meiosis: independent assortment and crossing over. Both these processes are extremely complex, but in themselves are not subject to selection. They rearrange the genetic material, resulting in new combinations of the material. As this reshuffling occurs at the level of the genotype, it is not subject to natural selection until the new combinations have been expressed in the phenotype. 

i) Independent Assortment

Independent assortment is achieved when chromosomes line up in homologous pairs and move independently to the one pole or the other. The process is governed by complex enzyme systems which in turn must also have come about by chance. The possible variation that can be achieved by independent assortment depends on the number of chromosomes present. In humans, there are 46 chromosomes, which would arrange themselves in 23 homologous pairs. There are thus 80 trillion possible variations.

ii) Crossing Over

Crossing Over is an awe-inspiring process. When homologous chromosomes are lined up during meiosis, they can, in a very precise way, exchange genetic material. There are five steps in achieving this:

a) Enzymes open the double helix of DNA in the aligned chromosomes to permit intermolecular base pairing.

b) One strand of each helix is cut at equivalent positions.

c) The enzyme ligase joins them to form a half-chromatid chiasma (because only one strand of each chromatid cross over), resulting in a cross-shaped molecule.

d) The cross-shaped molecule is cut in half by an enzyme, leaving a break in one strand of each recombinant.

e) The break is sealed by ligase.

The process has to be extremely precise. If even one nucleotide is transferred incorrectly, the genetic message becomes useless.

Somehow the genetic material from one parental chromosome and the genetic material from the other parental chromosome are cut up and pasted together during each meiosis, and this is done with complete reciprocity. In other words, neither chromosome gains or loses any genes in the process. In fact, it is probably correct to say that neither chromosome gains or loses even one nucleotide in the exchange.

Plasmid

A DNA molecule that is separate from the chromosomal DNA, and can replicate without the chromosomal DNA.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

DNA

Deoxyribonucleic acid (DNA) is a molecule that stores genetic makeup, or code. All living things have DNA.

Nucleotide

Molecules that join together to make up the structure of DNA and RNA.

Transposons

Sequences of DNA that can move around to different positions within the genome of a single cell. In the process, they can cause mutations and change the amount of DNA in the genome.

Transposons

Chunks of DNA that can move around within a genome.

Transposable Elements

Transposable elements are sometimes called "jumping genes." They consist of segments of DNA that can move from one position on a chromosome to another. In 1951, Nobel prize-winning Dr. Barbara McClintock proposed that genes are not fixed on chromosomes, but that they can move around on the chromosome. At first her findings were discarded because they contradicted the genetic concept of the day. Today, her discovery of what she calls transposable elements has an established place in science.

Transposable elements allow antibiotic resistance and increased variation. The genes move because they are part of a small circular auxiliary genome called a plasmid, which enters and leaves the main genome at a specific place where there is a nucleotide sequence that is also present on the plasmid. Other genes move within small fragments of the genome called transposons. Together, transposons and plasmids produce genetic recombinations.

Integration at a new position also transfers the gene to that new position. The repositioning may be random, but occurs at sequence-specific insertion points which means that the process is orderly. The splicing and repositioning is carried out by enzyme systems and involves the transfer of complete information. 

Speciation

The evolutionary process by which new species arise.

Genome

A full set of chromosomes. A regular cell contains two full genomes.

Recombination of Chromosomes

Changes in chromosomal structure have been cited as important contributing factors in providing variation, and as a mechanism for speciation.

Changes in chromosomes can include changes in chromosome number or arm number, deletions, duplications, inversions, or even radical reorganizations of the genome.

CIRCULAR BACTERIAL CHROMOSOME

A circular bacterial chromosome, showing DNA replication proceeding bidirectionally, with two replication forks generated at the "origin". Each half of the chromosome replicated by one replication fork is called a "replichore".

Circular bacterial chromosomes are the bacterial chromosomes contained in a circular DNA molecule. Unlike the linear DNA of vertebrates, typical bacterial chromosomes contain circular DNA.

Most bacterial chromosomes contain a circular DNA molecule - there are no free ends to the DNA. Free ends would otherwise create significant challenges to cells with respect to DNA replication and stability. Cells that do contain chromosomes with DNA ends, or telomeres (most eukaryotes), have acquired elaborate mechanisms to overcome these challenges. However, a circular chromosome can provide other challenges for cells. After replication, the two progeny circular chromosomes can sometimes remain interlinked or tangled, and they must be resolved so that each cell inherits one complete copy of the chromosome during cell division.

Replication of a circular bacterial chromosome

Bacterial chromosome replication is best understood in the well-studied bacteria Escherichia coli and Bacillus subtilis. Chromosome replication proceeds in three major stages: initiation, elongation and termination. The initiation stage starts with the ordered assembly of "initiator" proteins at the origin region of the chromosome, called oriC. These assembly stages are regulated to ensure that chromosome replication occurs only once in each cell cycle. During the elongation phase of replication, the enzymes that were assembled at oriC during initiation proceed along each arm ("replichore") of the chromosome, in opposite directions away from the oriC, replicating the DNA to create two identical copies. This process is known as bidirectional replication. The entire assembly of molecules involved in DNA replication on each arm is called a "replisome." At the forefront of the replisome is a DNA helicase that unwinds the two strands of DNA, creating a moving "replication fork". The two unwound single strands of DNA serve as templates for DNA polymerase, which moves with the helicase (together with other proteins) to synthesize a complementary copy of each strand. In this way, two identical copies of the original DNA are created. Eventually, the two replication forks moving around the circular chromosome meet in a specific zone of the chromosome, approximately opposite oriC, called the terminus region. The elongation enzymes then disassemble, and the two "daughter" chromosomes are resolved before cell division is completed.

Initiation

The E. coli bacterial replication origin, called oriC consists of DNA sequences that are recognised by the DnaA protein, which is highly conserved amongst different bacterial species. DnaA binding to the origin initiates the regulated recruitment of other enzymes and proteins that will eventually lead to the establishment of two complete replisomes for bidirectional replication.

DNA sequence elements within oriC that are important for its function include DnaA boxes, a 9-mer repeat with a highly conserved consensus sequence 5' - TTATCCACA - 3', that are recognized by the DnaA protein. DnaA protein plays a crucial role in the initiation of chromosomal DNA replication. Bound to ATP, and with the assistance of bacterial histone-like proteins [HU] DnaA then unwinds an AT-rich region near the left boundary of oriC, which carries three 13-mer motifs, and opens up the double-stranded DNA for entrance of other replication proteins.

This region also contains four “GATC” sequences that are recognized by DNA adenine methylase (Dam), an enzyme that modifies the adenine base when this sequence is unmethylated or hemimethylated. The methylation of adenines is important as it alters the conformation of DNA to promote strand separation, and it appears that this region of oriC has a natural tendency to unwind.

Elongation

When the replication fork moves around the circle, a structure shaped like the Greek letter theta Ө is formed. John Cairns demonstrated the theta structure of E. coli chromosomal replication in 1963, using an innovative method to visualize DNA replication. In his experiment, he radioactively labeled the chromosome by growing his cultures in a medium containing 3H-thymidine. The nucleoside base was incorporated uniformly into the bacterial chromosome. He then isolated the chromosomes by lysing the cells gently and placed them on an electron micrograph (EM) grid which he exposed to X-ray film for two months. This Experiment clearly demonstrates the theta replication model of circular bacterial chromosomes.

As described above, bacterial chromosomal replication occurs in a bidirectional manner. This was first demonstrated by specifically labelling replicating bacterial chromosomes with radioactive isotopes. The regions of DNA undergoing replication during the experiment were then visualized by using autoradiography and examining the developed film microscopically. This allowed the researchers to see where replication was taking place. The first conclusive observations of bidirectional replication were from studies of B. subtilis. Shortly after, the E. coli chromosome was also shown to replicate bidirectionally.

The E. coli DNA polymerase III holoenzyme is a 900 kD complex, possessing an essentially a dimeric structure. Each monomeric unit has a catalytic core, a dimerization subunit, and a processivity component. DNA Pol III uses one set of its core subunits to synthesize the leading strand continuously, while the other set of core subunits cycles from one Okazaki fragment to the next on the looped lagging strand. Leading strand synthesis begins with the synthesis of a short RNA primer at the replication origin by the enzyme Primase (DnaG protein).

Deoxynucleotides are then added to this primer by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. Leading strand synthesis then proceeds continuously, while the DNA is concurrently unwound at the replication fork. In contrast, lagging strand synthesis is accomplished in short Okazaki fragments. First, an RNA primer is synthesized by primase, and, like that in leading strand synthesis, DNA Pol III binds to the RNA primer and adds deoxyribonucleotides.

When the synthesis of an Okazaki fragment has been completed, replication halts and the core subunits of DNA Pol III dissociates from the β sliding clamp [B sliding clap is the processivity subunit of DNA Pol III]. The RNA primer is remove and replaced with DNA by DNA polymerase I [which also possesses proofreading exonuclease activity] and the remaining nick is sealed by DNA ligase, which then ligates these fragments to form the lagging strand.

Termination

Termination is the process of fusion of replication forks and disassembly of the resplisomes to yield two separate and complete DNA molecules. It occurs in the terminus region, approximately opposite oriC on the chromosome. The terminus region contains several DNA replication terminator sites, or "Ter" sites. A special "replicaiton terminator" protein must be bound at the Ter site for it to pause replication. Each Ter site has polarity of action, that is, it will arrest a replication fork approaching the Ter site from one direction, but will allow unimpeded fork movement through the Ter site from the other direction. The arrangement of the Ter sites forms two opposed groups that forces the two forks to meet each other within the region they span. This arrangement is called the "replication fork trap."

Replication of the DNA separating the opposing replication forks, leaves the completed chromosomes joined as ‘catenanes’ or topologically interlinked circles. The circles are not covalently linked, but cannot be separated because they are interwound and each is covalently closed. The catenated circles require the action of topoisomerases to separate the circles [decatanation]. In E.coli, DNA topoisomerase IV plays the major role in the separation of the catenated chromosomes, transiently breaking both DNA strands of one chromosome and allowing the other chromosome to pass through the break.