Question

Discuss in detail the two different types of operons found in bacterial genomes (inducible operons and repressible operons) and describe how they work. Then describe the three different forms of prokaryotic genetic recombination discussed in the commentary Prokaryotic Genetic Recombination. Hypothesize how recombination might adversely affect the functioning of an operon such as the lac operon or the trp operon. What might be the metabolic implications for an E. coli cell that experiences a disruption of one of these operons?

Answer 1

In bacteria, related genes are often found in a cluster on the chromosome, where they are transcribed from one promoter (RNA polymerase binding site) as a single unit. Such a cluster of genes under control of a single promoter is known as an operon. Operons are common in bacteria, but they are rare in eukaryotes such as humans.

Operons may be inducible or repressible

Some operons are usually "off," but can be turned "on" by a small molecule. The molecule is called an inducer, and the operon is said to be inducible.

• For example, the lac operon is an inducible operon that encodes enzymes for metabolism of the sugar lactose. It turns on only when the sugar lactose is present (and other, preferred sugars are absent). The inducer in this case is allolactose, a modified form of lactose.

Other operons are usually "on," but can be turned "off" by a small molecule. The molecule is called a corepressor, and the operon is said to be repressible.

• For example, the trp operon is a repressible operon that encodes enzymes for synthesis of the amino acid tryptophan. This operon is expressed by default, but can be repressed when high levels of the amino acid tryptophan are present. The corepressor in this case is tryptophan.

These examples illustrate an important point: that gene regulation allows bacteria to respond to changes in their environment by altering gene expression (and thus, changing the set of proteins present in the cell).

Lac Operon

The Lac operon is the classic operon example, and is responsible for the degradation of the milk protein lactose. The Lac operon is an inducible operon; in the absence of lactose the operator is blocked by a repressor protein. The operon is made up of a promoter with operator, and three genes (lacZ, lacY, and lacA) which encode ?-galactosidase, permease, and transacetylase. The three genes are involved in the breakdown of lactose into its metabolites: ?-galactosidase breaks lactose down into glucose and galactose, while the other two proteins aid in the metabolic process. The expression of the Lac operon is controlled by the regulatory gene lacI, located immediately adjacent to the promoter region. LacI encodes an allosteric repressor protein that keep the Lac operon “off”.

In order for the Lac operon to be turned on, an inducer molecule must inactivate the repressor protein. The inducer molecule in this system is allolactose, an isomer of lactose. When lactose and its isomer are present in the cell, allolactose will bind to allosteric sites on the repressor protein, changing its conformation and rendering it inactive. As the repressor protein detaches from the operator, RNA polymerase can bind to the promoter, transcription can occur, and the three lactose degradation genes can be synthesized.

The figure shows the structure of the Lac operon and the adjacent lacR repressor gene.

The Lac operon is also under positive gene regulation. While the removal of the repressor protein in the presence of lactose is required for synthesis of the lacZ, lacY, and lacA genes, the gene expression will remain low. The level of gene expression is controlled by the amount of the preferred energy source, glucose, in the cell. This control is regulated by an allosteric regulatory protein, catabolite activator protein (CAP). When glucose levels in the cell are low, the organic molecule cyclic AMP is in high concentration. Cyclic AMP activates CAP by binding to the allosteric sites, causing CAP to attach to the Lac operon promoter. Unlike the repressor proteins, binding of CAP to the Lac operon stimulates gene expression. When the cell glucose levels increase, the cyclic AMP levels in the cell decrease, and the activator protein will disassociate from the promoter. Transcription will return to low levels, or will turn off if the repressor protein reattaches.

The figure depicts the Lac operon and how its gene expression is under both positive and negative control.

Trp Operon

The Trp operon is responsible for synthesis of the amino acid trytophan when it is not available in the environment. The Trp operon is made up of a promoter with an operator, and five genes that encode enzymes for tryptophan synthesis. The Trp operon is regulated by the regulatory gene trpR, a gene that is located at a distance from the Trp operon.

The Trp operon is an example of a repressible operon; it is on unless turned off by a repressor protein. The repressor protein is synthesized by trpR. While the repressor protein is always present in the cell, it is synthesized in an inactive form. When a corepressor is present, in this case tryptophan, it binds to the repressor protein in an allosteric site. This changes the conformation of the protein such that it can bind to the operator and block transcription by preventing the binding of RNA polymerase to the promoter. In this way the cell saves energy by not producing tryptophan when it is already present.

The figure depicts the Trp operon with the repressor gene, the promoter, operator, and five tryptophan-synthesizing genes.

3 Ways in which the Genetic Recombination in Bacteria Takes Place

Genetic recombination in bacteria is a process where genetic materials, contained in two separate genomes, are brought together within one unit. In bacteria the recombination takes place by (1) transformation, (2) transduction and (3) conjugation.

1. Transformation:

The genetic transfer in bacteria also occurs by transformation in which the DNA molecule of the donor cell, when liberated by its disintegration, is taken up by another recipient cell and its offspring inherit some characters of the donor cell. When different strains of bacteria are found in a mixed stage either in culture or in nature, some of the resultant offspring possess a combination of characters of the parent strains. This phenomenon is known as recombination.

The phenomenon of transformation was first recorded by Griffith (1928). Avery, Macleod and McCarty (1944) demonstrated that the transforming principle being DNA in the sequence of events in bacterial transformation.

2. Transduction:

The genetic transfer in bacteria is achieved by a process known as transduction. Laderberg and Zinder’s (1952) experiment in U-tube Salmonella typhimurium indicated that bacterial viruses or phages are responsible for the transfer of genetic material from one to the other lysogenic and lytic phages. Thus the host acquires a new genotype. Transduction has been demonstrated in many bacteria.

In this process, the DNA molecule that carries the hereditary characters of the donor bacterium is being transferred to the recipient cell through the agency of the phage particle.

When a bacterial cell is being infected with a temperate virus either lytic-cycle or lysogeny starts. Thereafter, host DNA breaks down into small fragments along with the multiplication of virus. Some of these DNA fragments are incorporated with the virus particles becoming transducing one.

When bacteria lyse these particles along with normal virus particles are released when this mixture of transducing and normal virus particles is allowed to infect the population of recipient cells, most of the bacteria are infected with normal virus particles and with the result lysogeny or lytic-cycle occurs again. A few bacteria are infected with transducing particles, transduction takes place and the DNA of virus particles undergo genetic Recombinations with the bacterial DNA.

3. Bacterial conjugation:

Wollman and Jacob (1956) have described conjugation in which two bacteria lie side by side for as much as half an hour. During this period of time a portion of genetic material is slowly passed from one bacterium which is designated as a male to a recipient designated as a female. This was established that the male material entered the female in a liner series.

The genetic recombination between donor and recipient cells takes place as follows: The Hfr DNA after leaving apart in fragment to recipient cell again reforms in circular manner. In F-strain genetic recombination takes place between donor fragment and recipient DNA. Gene transfer is a sequential process and a given Hfr strain always donates genes in a specific order. A single stranded donor DNA (F factor) is integrated into the host chromosome with the help of nuclease enzyme.

Hypothesis

The various forms of recombination would interfere with the lac and trp repressors causing an inability of e. coli to know when to produce proteins to metabolize lactose or to produce tryptohan when needed.

Metabolic implications

In case of disruption of an operon for example, lac operon, the E. coli. will be anable to use lactose as a nutrient source. The reason for this is the absence of enzymes in bacteria which help in breakdown of lactose. And, in case of disrupted trp operon, E. coli will be unable to synthesis tryptophan amino acid and will be dependent on diet for the supply of this amino acid. In absence of any amino acid, protein synthesis may halt in the bacterial system and thus can cause death of the bacteria.