Q.12 Give an illustrated account of the process of conjugation in bacteria. (2008)
Related Questions -
Q. Write a short note on bacterial conjugation. (2006)
Q. Write short note on F-factor. (2016)
Ans. Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact. Discovered in 1946 by Joshua Lederberg and Edward Tatum, conjugation is a mechanism of horizontal gene transfer—as are transformation and transduction—although these mechanisms do not involve cell-to-cell contact.
Bacterial conjugation is often incorrectly regarded as the bacterial equivalent of sexual reproduction or mating. It is not actually sexual, as it does not involve the fusing of gametes and the creation of a zygote, nor is there equal exchange of genetic material. It is merely the transfer of genetic information from a donor cell to a recipient. In order to perform conjugation, one of the bacteria, the donor, must play host to a conjugative or mobilizable genetic element, most often a conjugative or mobilizable plasmid or transposon. Most conjugative plasmids have systems ensuring that the recipient cell does not already contain a similar element.
The genetic information transferred is often beneficial to the recipient cell. Benefits may include antibiotic resistance, other xenobiotic tolerance, or the ability to utilize a new metabolite. Such beneficial plasmids may be considered bacterial endosymbionts. Some conjugative elements may also be viewed as genetic parasites on the bacterium, and conjugation as a mechanism that was evolved by the mobile element to spread itself into new hosts.
Mechanism: -
The prototype for conjugative plasmids is the F-plasmid, also called the F-factor. The F-plasmid is an episome (a plasmid that can integrate itself into the bacterial chromosome by genetic recombination) of about 100 kb length. It carries its own origin of replication, the oriV, as well as an origin of transfer, or oriT. here can only be one copy of the F-plasmid in a given bacterium, either free or integrated (two immediately before cell division). The host bacterium is called F-positive or F-plus (denoted F+). Strains that lack F plasmids are called F-negative or F-minus (F-).
Among other genetic information, the F-plasmid carries a tra and a trb locus, which together are about 33 kb long and consist of about 40 genes. The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F bacteria and initiate the conjugation. Though there is some debate on the exact mechanism, the pili themselves do not seem to be the structures through which the actual exchange of DNA takes place; rather, some proteins coded in the tra or trb loci seem to open a channel between the bacteria.
When conjugation is initiated, via a mating signal, a relaxase enzyme creates a nick in one plasmid DNA strand at the origin of transfer, or oriT. The relaxase may work alone or in a complex of over a dozen proteins, known collectively as a relaxosome. In the F-plasmid system, the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY, TraM, and the integrated host factor, IHF. The transferred, or T-strand, is unwound from the duplex plasmid and transferred into the recipient bacterium in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated, either independent of conjugative action (vegetative replication, beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage). Conjugative replication may necessitate a second nick before successful transfer can occur. A recent report claims to have inhibited conjugation with chemicals that mimic an intermediate step of this second nicking event.
If the F-plasmid becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA. The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via recombination.
A culture of cells containing non-integrated F plasmids usually contains a few that have accidentally become integrated, and these are responsible for those low-frequency chromosomal gene transfers which do occur in such cultures. Some strains of bacteria with an integrated F-plasmid can be isolated and grown in pure culture. Because such strains transfer chromosomal genes very efficiently, they are called Hfr (high frequency of recombination). The E. coli genome was originally mapped by interrupted mating experiments, in which various Hfr cells in the process of conjugation were sheared from recipients after less than 100 minutes (initially using a Waring blender) and investigating which genes were transferred.
Inter-Kingdom Transfer: -
The nitrogen fixing Rhizobia are an interesting case, wherein conjugative elements naturally engage in inter-kingdom conjugation[8]. Such elements as the Agrobacterium Ti or Ri plasmids contain elements that can transfer to plant cells. Transferred genes enter the plant cell nucleus and effectively transform the plant cells into factories for the production of opines, which the bacteria use as carbon and energy sources. Infected plant cells form crown gall or root tumors. The Ti and Ri plasmids are thus endosymbionts of the bacteria, which are in turn endosymbionts (or parasites) of the infected plant.
The Ti and Ri plasmids are themselves conjugative. Ti and Ri transfer between bacteria uses an independent system (the tra, or transfer, operon) from that for inter-kingdom transfer (the vir, or virulence, operon). Such transfer creates virulent strains from previously avirulent Agrobacteria.
Genetic Engineering Applications: -
Conjugation is a convenient means for transferring genetic material into a variety of targets. In the lab, successful transfer has been reported from bacteria into yeast plants, mammalian cells, and isolated mammalian mitochondria. Conjugation has advantages over some other forms of genetic transfer for engineering purposes, namely minimal disruption to the target’s cellular envelope and the ability to transfer relatively large amounts of genetic material. In plant engineering, Agrobacterium-like conjugation is complementary to other standard vehicles, such as tobacco mosaic virus (TMV). While TMV is capable of infecting many plant families, these are primarily herbaceous dicots. Agrobacterium-like conjugation is also primarily used for dicots, but monocot recipients are not uncommon.
Q.13 Write short note on the following -
(a) flagella in bacteria (2005, 2011, 14, 16)
(b) asexual reproduction in bacteria (2011, 14, 15)
(c) citrus canker (2009, 2011)
Ans. (a) Flagella in Bacteria: -
The bacterial flagellum is made up of the protein flagellin. Its shape is a 20 nanometer-thick hollow tube. It is helical and has a sharp bend just outside the outer membrane; this “hook” allows the helix to point directly away from the cell. A shaft runs between the hook and the basal body, passing through protein rings in the cell’s membrane that act as bearings. Gram-positive organisms have 2 of these basal body rings, one in the peptidoglycan layer and one in the plasma membrane. Gram-negative organisms have 4 such rings: the L ring associates with the lipopolysaccharides, the P ring associates with peptidoglycan layer, the M ring is embedded in the plasma membrane, and the S ring is directly attached to the plasma membrane. The filament ends with a capping protein.
The bacterial flagellum is driven by a rotary engine made up of protein (Mot complex), located at the flagellum’s anchor point on the inner cell membrane. The engine is powered by proton motive force, i.e., by the flow of protons (hydrogen ions) across the bacterial cell membrane due to a concentration gradient set up by the cell’s metabolism (in Vibrio species there are two kinds of flagella, lateral and polar, and some are driven by a sodium ion pump rather than a proton pump. The rotor transports protons across the membrane, and is turned in the process. The rotor alone can operate at 6,000 to 17,000 rpm, but with the flagellar filament attached usually only reaches 200 to 1000 rpm.
The components of the bacterial flagellum are capable of self-assembly without the aid of enzymes or other factors. Both the basal body and the filament have a hollow core, through which the component proteins of the flagellum are able to move into their respective positions. During assembly, protein components are added at the flagellar tip rather than at the base.
The basal body has several traits in common with some types of secretory pores, such as the hollow rod-like “plug” in their centers extending out through the plasma membrane. Given the structural similarities between bacterial flagella and bacterial secretory systems, it is thought that bacterial flagella may have evolved from the type three secretory system; however, it is not known for certain whether these pores are derived from the bacterial flagella or the bacterial secretory system.
Through use of their flagella, E. coli are able to move rapidly towards attractants and away from repellents. They do this by means of a biased random walk, with ‘runs’ and ‘tumbles’ brought about by rotating the flagellum counter-clockwise and clockwise respectively.
Flagella Arrangement Schemes: -
Different species of bacteria have different numbers and arrangements of flagella. Monotrichous bacteria have a single flagellum (e.g., Vibrio cholerae). Lophotrichous bacteria have multiple flagella located at the same spot on the bacteria’s surfaces which act in concert to drive the bacteria in a single direction. In many cases, the bases of multiple flagella are surrounded by a specialized region of the cell membrane; the so-called polar membrane.
Amphitrichous bacteria have a single flagellum on each of two opposite ends (only one flagellum operates at a time, allowing the bacteria to reverse course rapidly by switching which flagellum is active). Peritrichous bacteria have flagella projecting in all directions (e.g., Escherichia coli).
In some bacteria, such as the larger forms of Selenomonas, the individual flagella are organized outside the cell body, helically twining about each other to form a thick structure called a “fascicle”. Other bacteria, such as Spirochetes, have a specialized type of flagellum called an “axial filament” that is located in the periplasmic space, the rotation of which causes the entire bacterium to move forward in a corkscrew-like motion.
Counterclockwise rotation of monotrichous polar flagella pushes the cell forward with the flagella trailing behind, much like a corkscrew moving inside cork. Indeed water in the microscopic scale is highly viscous, very different from our daily experience of water. The flagella are left-handed helices, and bundle and rotate together only when rotating counterclockwise. When some of the rotors reverse direction, the flagella unwind and the cell starts “tumbling”. It has also been suggested that even if all flagella would rotate clockwise, they will not form a bundle, due to geometrical as well as hydrodynamical reasons. Such “tumbling” may happen occasionally, leading to the cell seemingly thrashing about in place, resulting in the reorientation of the cell. The clockwise rotation of a flagellum is suppressed by chemical compounds favorable to the cell (e.g. food), but the motor is highly adaptive to this. Therefore, when moving in a favorable direction, the concentration of the chemical attractant increases and “tumbles” are continually suppressed; however, when the cell’s direction of motion is unfavorable (e.g., away from a chemical attractant), tumbles are no longer suppressed and occur much more often, with the chance that the cell will be thus reoriented in the correct direction.
In some Vibrio (particularly Vibrio parahemolyticus[) and related proteobacteria such as Aeromonas, two flagellar systems co-exist, using different sets of genes and different ion gradients for energy. The polar flagella are constitutively expressed and provide motility in bulk fluid, while the lateral flagella are expressed when the polar flagella meets too much resistance to turn. These provide swarming motility on surfaces or in viscous fluids.
(b) Asexual Reproduction in Bacteria: -
Bacteria can reproduce at tremendous speeds. Some bacteria can reproduce as often as once every 20 minutes! However, bacteria have to have certain conditions in which to reporduce. These conditions are not often met, and that is one thing that keeps bacteria from growing out of control.
Bacteria reproduce using two basic methods: asexual reproduction and sexual reproduction.
Asexual reproduction involves only one individual or parent. The offspring generated by asexual reproduction are exact duplicates of the parent. Binary fission is the process by which a bacteria splits into two cells. Each cell gets an exact copy of the parent cell’s genetic material.
Binary fission theoretically results in two identical cells. However, the DNA of bacteria has a relatively high mutation rate. This rapid rate of genetic change is what makes bacteria capable of developing resistance to antibiotics and helps them exploit invasion into a wide range of environments.
Binary fission begins with DNA replication. DNA replication starts from an origin of replication, which opens up into a replication bubble (note: prokaryotic DNA replication usually has only 1 origin of replication, whereas eukaryotes have multiple origins of replication). The replication bubble separates the DNA double strand, each strand acts as template for synthesis of a daughter strand by semiconservative replication, until the entire prokaryotic DNA is duplicated.After this replicational process, cell growth occurs.
Each circular DNA strand then attaches to the cell membrane. The cell elongates, causing the two chromosomes to separate.
Cell division in bacteria is controlled by the FtsZ, a collection of about a dozen proteins that collect around the site of division. There, they direct assembly of the division septum. The cell wall and plasma membrane starts growing transversely from near the middle of the dividing cell. This separates the parent cell into two nearly equal daughter cells, each having a nuclear body.
The cell membrane then invaginates (grows inwards) and splits the cell into two daughter cells, separated by a newly grown cell plate.
(n) Citrus Canker: -
Citrus canker is a disease affecting citrus species that is caused by the bacterium Xanthomonas axonopodis. Infection causes lesions on the leaves, stems, and fruit of citrus trees, including lime, oranges, and grapefruit. While not harmful to humans, canker significantly affects the vitality of citrus trees, causing leaves and fruit to drop prematurely; a fruit infected with canker is safe to eat but too unsightly to be sold.
Plants infected with citrus canker have characteristic lesions on leaves, stems, and fruit with raised, brown, water-soaked margins, usually with a yellow halo or ring effect around the lesion. Older lesions have a corky appearance, still in many cases retaining the halo effect. The bacterium propagates in lesions in leaves, stems, and fruit. The lesions ooze bacterial cells that, when dispersed by windblown rain, can spread to other plants in the area. Infection may spread further by hurricanes. The disease can also be spread by from contaminated equipment, and by transport of infected or apparently healthy plants. Due to latency of the disease, a plant may appear to be healthy, but actually be infected.
Citrus canker bacteria can enter through a plant’s stomata or through wounds on leaves or other green parts. In most cases, younger leaves are considered to be the most susceptible. Also, damage caused by Citrus Leaf Miner larvae (Phyllocnistis citrella) can be sites for infection to occur. Within a controlled laboratory setting, symptoms can appear in 14 days following inoculation into a susceptible host. In the field environment, the time for symptoms to appear and be clearly discernible from other foliar diseases varies; it may be on the order of several months after infection. Lower temperature increases the latency of the disease. Citrus canker bacteria can stay viable in old lesions and other plant surfaces for several months.
Symptoms: -
Citrus canker symptoms appear on the fruit, leaves, and twigs of infected plants, and typically consist of small, round, blister-like formations called lesions. Lesions usually become apparent about 7 to 14 days after infection. As the lesions mature, the epidermis or outer layer of tissue ruptures, producing a craterlike spot lined with tan-colored tissue that is usually surrounded by an oily, water-soaked margin and a yellow ring that looks like a halo. On fruit, the lesions appear scab-like or corky. On leaves, old lesions sometimes fall out, leaving behind a scattering of round holes.