13: The Genetics of Viruses and Prokaryotes

Introduction

· Prokaryotes usually reproduce asexually by cell division.

· They may acquire new genes by simple recombination in a sexual process.

· They may use infective viruses as carriers for prokaryotic genes.

· Viruses are not prokaryotes, but intracellular parasites that can reproduce only within living cells.

Using Prokaryotes and Viruses to Probe the Nature of Genes

· Prokaryotes and viruses have advantages for the study of genetics.

· It is relatively easy to work with the small amounts of DNA exhibited by these groups.

· Bacteria have 1/1000 of the DNA of human cells.

· A virus typically has 1/100 of the DNA of a bacterium.

· Data on large numbers of individuals are easy to obtain.

· Prokaryotes grow rapidly. E. coli doubles every 20 minutes.

· Viruses can replicate far more quickly than bacteria.

· Prokaryotes and viruses are usually haploid, making genetic analyses easier.

· The ease of use of bacteria and viruses in genetic research has propelled the science of genetics and molecular biology during the last 50 years.

· Prokaryotes continue to play a central role as tools for biotechnology in the understanding of genetics, both prokaryotic and eukaryotic.

· Prokaryotes play important ecological roles, including cycling elements in the atmosphere and water.

· Both prokaryotes and viruses present disease challenges to humans.

Viruses: Reproduction and Recombination

· Most viruses are composed of only nucleic acid and a few proteins.

· Some infect cells but postpone reproduction until certain cellular conditions are met.

· Some reproduce shortly after infecting the cell.

· The simplest are viroids, which are made up only of genetic material.

Scientists studied viruses before they could see them

· The tobacco mosaic virus was the first virus to be discovered. (The Instructor’s Resource CD-ROM includes a micrograph of tobacco mosaic virus.)

· Russian botanist Dmitri Ivanovsky, trying to find the cause of tobacco mosaic disease, provided evidence for the existence of this virus in 1892.

· He passed extract from infected plants through a filter fine enough to capture even small bacteria.

· The infectious agent passed through the filter and still caused the disease.

· He chose not to believe that this happened and assumed a faulty filter.

· Martinus Beijerinck repeated the experiment in 1898 by showing the agent could pass through agar gel. He called the infectious agent contagium vivum fluidum, which was later shortened to virus.

· In the late 1930s, the infective agent was crystallized by Wendell Stanley, who won the Nobel Prize for his success.

· The crystallized virus became infectious again when it was dissolved.

· In the 1950s, direct observation by electron microscopes showed how much viruses differed from bacteria. (See Table 13.1.)

Viruses reproduce only with the help of living cells

· Viruses are acellular (noncellular) and do not metabolize energy.

· Viruses do not produce ATP or conduct fermentation, cell respiration, or photosynthesis.

· Whole viruses never directly arise from preexisting viruses.

· Viruses are obligate intracellular parasites that develop and reproduce only within living cells of specific hosts.

· When they reproduce, viruses usually destroy the host cell, releasing infective stages of viruses.

· Many diseases of humans, other animals, and plants are caused by viruses.

· Viruses are unaffected by antibiotics because they lack the structural and biochemical nature of bacteria.

· Outside the cell, the individual viral particles are called virions.

· Virion genetic material is either DNA or RNA and is generally surrounded by a capsid, or protein coat.

· Characteristic shapes are determined by the protein coat. (See Figure 13.1. The Instructor’s Resource CD-ROM includes micrographs and computer models of many different viruses.)

· Many animal viruses have a lipid and protein membrane acquired from the host cell plasma membrane.

There are many kinds of viruses

· Viruses can be classified according to whether they contain DNA or RNA.

· Among DNA-containing viruses, there are single-stranded and double-stranded varieties. One family of DNA virus has circular DNA.

· Some RNA viruses have more than one RNA molecule.

· Viruses can be further classified by overall shape or by the shape of their capsid (protein coat).

· One level of classification is based on whether they have a membranous envelope around the virion.

· Viruses can also be classified according to their hosts.

Bacteriophages reproduce by a lytic cycle or a lysogenic cycle

· Viruses that infect bacteria are called bacteriophages.

· Bacteriophages recognize their host by means of specific binding between proteins in the capsid and receptor proteins on the host’s cell.

· The virions must get their genetic material into the cell, and many do so by attaching with tail assemblies that then inject the DNA into the cell.

· The lytic cycle occurs when the virus infects the cell, takes over the cellular machinery, and then lyses (bursts) the cell, releasing its phage progeny. (See Figure 13.2.)

· In a lysogenic cycle, the host cell does not lyse. Instead, it harbors a quiescent virus for many generations.

· Some viruses reproduce using only the lytic cycle; some use both.

· Phages that only have lytic cycles are called virulent viruses. (See Figure 13.3.)

· After infection in a lytic cycle, viral early genes are transcribed. Early gene products often include proteins that shut down host transcription and stimulate viral genome replication.

· Late-stage genes code for the protein coat and an enzyme that causes host cell lysis, resulting in viral release.

· The whole process takes around 30 minutes.

· On rare occasions, two viruses infect the same cell at the same time, providing the opportunity for recombination of genes and the creation of new viral strains.

· Some viruses can infect hosts without killing them.

· The phages that infect in this manner are called temperate viruses, and their bacterial hosts are called lysogenic.

· Lysogenic bacteria have a molecule of noninfective phage DNA called a prophage inserted into their chromosome.

· Under some conditions, the prophage replicates during the bacterium’s normal reproductive cycle without otherwise harming the bacterium.

· Certain conditions will activate the prophage, initiating a lytic cycle that results in the release of a large number of free phages. These phages can then infect other bacteria.

· The lysogenic strategy helps assure long-term survival of a virus by allowing relatively slow, nonlethal replication under some conditions.

· The lytic strategy permits faster but lethal replication under other conditions.

Animal viruses have diverse reproductive cycles

· Almost all vertebrates are susceptible to viral infections.

· Among invertebrates, only arthropods commonly get viral infections.

· Arboviruses infect both insects and mammals.

· The mammal gets infected through transmission via an arthropod (often insect) bite.

· The arthropod is called a vector (carrier) for the disease transmission.

· Animal viruses include those that are just particles of protein surrounding a nucleic acid core as well as those that have a membrane derived from that of the host.

· Some have DNA, and some have RNA, but all have small genomes of limited codes.

· Animal viruses enter cells in three different ways:

· Endocytosis of a naked virion

· Endocytosis of a membrane-encased virus

· Fusion of a membrane-encased virus with the cell's membrane

· Figure 13.4 shows the reproductive cycle of the influenza (RNA type) virus.

· Retroviruses such as HIV (also RNA type) have a more complex reproductive cycle and enter the cell via membrane fusion. (See Figure 13.5.)

Many plant viruses spread with the help of vectors

· Plant viruses spread horizontally or vertically.

· Horizontal transmission is the spread of viruses from one plant to another.

· Vertical transmission is the transfer of viruses from parent plant to offspring.

· A plant virus has to get through the cell wall and plasma membrane of the host.

· Insects are possible vectors. A virion-laden insect feeding on a plant can penetrate the cell wall and insert the virus.

· Another is through contact between damaged tissue of an infected and a noninfected plant.

· Vertical transmission can occur through vegetative or sexual reproduction.

· Once inside a plant cell, viruses can spread by moving through plasmodesmata, the cytoplasmic connections between cells.

· The viruses code for special proteins that change the shape of the plasmodesmata pores.

Viroids are infectious agents consisting entirely of RNA

· Theodore Diener of the U.S. Department of Agriculture discovered viroids and reported their existence in 1971.

· Viroids consist of circular, single-stranded RNA molecules.

· They are just a few hundred nucleotides in length, about 1/1,000 the size of the smallest virus.

· No evidence yet exists that viroid RNA is translated to synthesize proteins.

· They have been found only in plants.

· They cause a variety of plant diseases, but it is not yet known how they cause disease.

· They bear similarities to introns and have some catalytic activities.

Prokaryotes: Reproduction, Mutation, and Recombination

· The Bacteria and Archaea have all the characteristics of living cells.

· Although they reproduce asexually, bacteria do interact and share genomes.

The reproduction of prokaryotes gives rise to clones

· The division of single cells into two identical offspring produces clones, or genetically identical individuals.

· If a number of cells are spread on a semisolid medium containing agar, individual cells give rise to clearly visible colonies. (See Figure 13.6. The Instructor’s Resource CD-ROM includes a photograph of bacterial lawns with and without bacteriophage plaques.)

· If a large number of cells are spread, a confluent lawn (one continuous colony) develops after cell division.

· Bacteria can also be grown in a liquid nutrient medium.

Some bacteria conjugate, recombining their genes

· In 1946, Joshua Lederberg and Edward Tatum demonstrated the exchange of DNA between two living bacteria. (See Figure 13.7.)

· Two auxotrophic strains of E. coli, each requiring different amino acid supplements for growth, were grown together and then plated on minimal medium.

· Prototrophic bacteria (a strain not requiring supplements) were recovered from these mixtures, demonstrating that genetic recombination had taken place.

· Later experiments showed that the exchange of hereditary information was by direct contact; this form of genetic exchange is called conjugation.

· One bacterial cell—the recipient—had received DNA from the donor.

· Physical contact is initiated by a pilus, which is a fine projection produced by the donor cell. (See Figure 13.8.)

· The DNA transfers through a conjugation tube.

· Only a linear (broken) portion of the genome transfers.

· Once inside, the DNA fragment recombines with a homologous region.

· Enzymes cut the DNA in two places and insert the donor region into the recipient’s circular chromosome. (See Figure 13.9.)

In transformation, cells pick up genes from their environment

· Transformation of bacteria occurs when bacteria take up extracellular DNA and incorporate it. (See Figure 13.10.)

· More than 75 years ago, Griffith obtained the first evidence for transfer of genes between bacteria.

· This was demonstrated by genetically changing nonvirulent pneumococci bacteria to the virulent form with transforming substance from killed virulent bacteria (see Chapter 11).

· The transforming substance was later found to be DNA.

· Incorporation of the virulent DNA inside the host cell is very similar to recombination.

In transduction, viruses carry genes from one cell to another

· During the lytic cycle, some bacteriophages package the host bacteria’s DNA in capsids, or viral protein coats. (See Figure 13.10.)

· Cells infected by such viruses get a segment of another bacteria’s DNA, not the viral DNA.

· This bacterial DNA sometimes recombines with the chromosomal DNA of the host and alters its genetic composition.

Plasmids are extra chromosomes in bacteria

· Plasmids are small, circular chromosomes found in many bacteria. (The Instructor’s Resource CD-ROM includes a micrograph of two bacterial plasmids.)

· Each plasmid has an origin of replication and replicates separately from the primary chromosome.

· Plasmids are not viruses, but they move between bacterial cells during conjugation.

· There are different types of plasmids, called factors, which are classified according to the kinds of genes they carry.

· Metabolic factors carry genes for unusual metabolic functions, such as degrading oils from oil spills.

· F (fertility) factors carry genes for conjugation.

· Around 25 genes, including the ones responsible for the pilus, are on the F factor plasmid.

· Bacteria with this plasmid are called F⁺.

· On occasion, this F plasmid inserts into the main chromosome.

· When this occurs, chromosomal genes can be transferred during conjugation. (See Figure 13.11.)

· R factors are resistance factors.

· R factors carry genes that code for proteins that protect the bacteria.

· Antibiotic resistance genes break down or modify antibiotics, or produce components that interfere with antibiotic activity or prevent their transport.

· These plasmids were discovered in 1957 in Shigella bacteria, which were resistant to several antibiotics.

· Research found that resistance to an entire spectrum of antibiotics could be transferred by conjugation.

· This finding raised the warning that inappropriate use of antibiotics may lead to their becoming ineffective.

Transposable elements move genes among plasmids and chromosomes

· Gene transport can also occur within an individual cell. (See Figure 13.12.)

· Such movement involves a segment of chromosome or plasmid DNA that can insert at new locations.

· The movement of these transposable elements into other genes disrupts normal function.

· Long transposable elements (about 5000 base pairs), which include one or more genes, are called transposons.

· Transposons have contributed to the evolution of plasmids, and there is some evidence that R plasmids developed antibiotic resistance through transposons.

Regulation of Gene Expression in Prokaryotes

· Bacteria can synthesize needed compounds in more than one way.

· For example, the amino groups for amino acids can come from an energy-intensive process that begins by “fixing” N₂ to ammonia (NH₃), or the amino groups can come straight from glutamine. (See Table 3.2.)

· Cells must regulate how they synthesize molecules to suit their condition, environment, and needs.

· Cells can control synthesis or activity of an unneeded protein by regulating or controlling the production of enzymes.

· Cells can block transcription of the gene that codes for a protein.

· This is the more efficient method (using less energy), and the one most extensively used.

· Cells can hydrolyze the mRNA after it is made.

· Cells might prevent translation of mRNA at the ribosome.

· Cells can hydrolyze the protein after it is made.

Regulation of transcription conserves energy

· E. coli prefers glucose, but when glucose availability is low and lactose is available, it can use lactose.

· Lactose is a disaccharide containing galactose b linked to glucose.

· Lactose is transported into the cell by a carrier protein (enzyme) called b-galactoside permease.

· Lactose is hydrolyzed to glucose and galactose by the enzyme b-galactosidase.

· Another enzyme is needed to protect the cell from the side effects of the b-galactosidase. It is called thiogalactoside transacetylase.

· When no lactose is present, levels of all three enzymes are low.

· When glucose is low and lactose is high, however, synthesis of all three enzymes occurs rapidly.

· If glucose levels rise again, or lactose levels drop, synthesis stops.

· Lactose is an inducer for the synthesis of an enzyme. (See Figure 13.13.)

· The enzymes produced are inducible enzymes.

· Enzymes made all the time at a constant rate are constitutive enzymes.

A single promoter controls the transcription of adjacent genes

· Structural genes are blueprints that specify the primary structures (amino acid sequence) of a protein molecule. In other words, structural genes are those that can be transcribed into mRNA.

· The three of these that are involved in lactose metabolism are adjacent to each other on the E. coli chromosome.

· All are transcribed together when a single promoter binds RNA polymerase.

· Therefore, their synthesis is coordinated.

· Because there is just a single promoter, all genes are transcribed efficiently onto a single mRNA.

· When these enzymes are not needed, the mRNA synthesis must be shut down.

Operons are units of transcription in prokaryotes

· Transcription shut-down in prokaryotes occurs by placing an obstacle between the promoter and its structural genes.

· Just downstream from the promoter, between the promoter and structural genes, is a DNA site called the operator.

· If a specific protein, the repressor, binds to the operator, it creates an obstacle, and RNA polymerase is blocked from transcribing the structural genes. (See Figure 13.15.)

· When the repressor is not attached to the operator, mRNA synthesis proceeds.

· The whole unit of genes and their DNA controls is called an operon.

Operator–repressor control that induces transcription: The lac operon

· The operon for the three lactose-metabolizing enzymes is called the lac operon.

· The repressor protein has two binding sites: one for the operator and the other for inducers.

· Binding of the repressor by the inducer molecules (e.g., an analog of lactose) changes the shape of the repressor by allosteric modification.

· The change in shape prevents the repressor from binding to the operator. (See Figure 13.17.)

· Thus, RNA polymerase can bind to the promoter and start gene transcription of the lac operon.

· If the concentration of the inducer (lactose) drops, the functioning repressor binds the operator, and the enzymes for lactose metabolism are not synthesized.

· If the concentration of lactose rises, the repressor itself is bound and does not bind the operator. The enzymes for lactose metabolism are synthesized.

· The repressor protein is coded for by the regulatory gene.

· The regulatory gene that codes for the lac repressor is the i (inducibility) gene.

· The i gene just happens to be located near the lac structural genes. However, not all regulatory genes are near the operons they control.

· Regulatory genes like i have their own promoter. The i promoter is called p_i.

· The i gene is expressed constitutively (i.e., expression is constant).

· Figure 13.16 shows the lac operon for E. coli.

· Summary of the lac operon control (see Figure 13.17):

· When no inducer (lactose) is present, lac is off.

· The regulator protein (repressor) turns the operon off.

· The i gene produces the repressor.

· The operator and promoter are DNA sequences that are binding sites for regulatory proteins.

· Adding inducer (lactose) turns the operon on.

Operator–repressor control that represses transcription: The trp operon

· The ability to switch off synthesis of an enzyme can be just as important as the ability to switch it on.

· If synthesis of an enzyme can be turned off, it is said to be repressible.

· The trp operon in E. coli is repressible. (See Figure 13.18.)

· In the absence of tryptophan, RNA polymerase transcribes the trp operon, leading to production of enzymes that synthesize tryptophan.

· When tryptophan is present, it binds to a repressor.

· The repressor is made, whether tryptophan is present or not, by a regulatory gene at another locus.

· The unbound repressor is inactive. When tryptophan binds the repressor, the repressor changes shape and becomes active.

· The repressor in turn binds to the operator of the trp operon, blocking production of the enzymes that synthesize tryptophan.

· As tryptophan concentration rises, production of tryptophan drops off.

· The molecule that binds and activates a repressor is called a corepressor.

· The corepressor may be the end product of the operon (as in the case of tryptophan), or it may be an analog.

· The difference between inducible and repressible systems is small, but significant.

· In inducible systems, an inducer from the cell’s environment prevents a repressor from blocking transcription.

· In repressible systems, a corepressor produced by the cell activates a repressor, enabling it to block transcription.

Protein synthesis can be controlled by increasing promoter efficiency

· Another way to regulate transcription is to make the promoter sequence of the operon work more efficiently.

· When glucose is high, even when lactose is available, the lac operon fails to transcribe frequently.

· When glucose is low, and lactose is available, lac structural genes are transcribed.

· Low glucose levels cause elevated levels of cyclic AMP (cAMP).

· The molecule AMP is the monophosphate form of the familiar ATP. Cyclic AMP has a phosphodiester linkage between its own 5¢ and 3¢ carbons.

· When glucose is low and cAMP is high, cAMP binds to a protein called CRP. CRP is short for cAMP receptor protein. (See Figure 13.19.)

· The CRP–cAMP complex binds the DNA just upstream of the promoter.

· Binding of this site makes it easier for RNA polymerase to bind the promoter and thus increases rates of transcription.

· When glucose is abundant, cAMP levels drop.

· The CRP-cAMP complex does not form.

· Without the CRP-cAMP complex, RNA polymerase cannot bind to the promoter efficiently.

· The lac structural genes are not transcribed.

· This is called catabolite repression.

· Table 13.2 summarizes positive and negative control in the lac operon.

· cAMP is used widely by both eukaryotes and prokaryotes as a signaling molecule.

Control of Transcription in Viruses

· Viruses also have gene regulation mechanisms.

· Even such “simple” biological agents must activate genes in the right order.

· Early genes must be transcribed before later ones.

· Bacteriophage lambda is a temperate phage, which can undergo either a lytic or a lysogenic cycle.

· Temperate viruses need to regulate when to undertake a lytic cycle.

· When host bacteria are growing in rich medium, lambda enters a lytic cycle.

· When the host is less healthy, lambda “lays low” as a lysogenic prophage.

· A “genetic switch” determines lambda’s behavior. (See Figure 13.20.)

· Two regulatory proteins compete for two operator/promoter sites on the phage DNA.

· One operator controls the lytic gene activities; the other, lysogenic cycles.

· The two regulatory proteins have opposite effects on the two operators.

· cI represses the lytic operator/promoter and activates the lysogenic operator/promoter.

· Cro activates the lytic operator/promoter and represses the lysogenic operator/promoter.

· The relative concentrations of cI and Cro determine the outcome.

· When the host is healthy, a lot of Cro is made, lysogeny is blocked, and lysis ensues.

· When Cro synthesis is low in an unhealthy host, the phage enters a lysogenic cycle.

Prokaryotic Genomes

· Viral genomes were the first to be sequenced.

· In 1995, Haemophilus influenzae (a bacterium) was the first free-living organism to be completely sequenced. (See Figure 13.21.)

· This has revealed details of how bacteria allocate and organize their genes.

· Three types of information can be obtained from a genomic sequence.

· Open reading frames (coding regions of genes) can be recognized by promoter regions and start and stop codons.

· Amino acid sequences can be deduced from the DNA sequence.

· Gene control sequences of promoters and terminators can be identified.

Functional genomics relates gene sequences to functions

· Haemophilus influenzae, which infects humans, has a circular chromosome of 1,830,137 base pairs.

· It has 1,743 protein-coding regions.

· When the sequencing of H. influenzae was finished, 42% of the genes coded for proteins with unknown functions.

· Roles for most of the unknown proteins have now been identified.

· This learning process is called annotation.

· Functional genomics is the assignment of roles to genes and the description of how they work in the organism.

· In addition to H. influenzae, the sequences for Mycoplasma genitalium (580,070 base pairs) and E. coli (4,639,211 base pairs) have been completed.

· With several genome sequences identified, it is now possible to compare genomes to see what genes one organism has or is missing and relate the results to physiology.

· This process is known as comparative genomics.

The sequencing of prokaryotic genomes has medical applications

· Scientists are discovering genes for proteins in prokaryotes that cause infectious diseases. These are potential targets for new drugs.

· New vaccines might be possible as cell surface antigen coding genes are discovered.

What genes are required for cellular life?

· There are some universal genes needed by all organisms. (See Figure 13.22.)

· There are some universal gene segments, like those coding for an ATP binding site.

· M. genitalium has just 470 genes, the smallest known genome.

· Some genes are dispensable under certain conditions. For example, those for lactose utilization are not needed by E. coli when it is grown on glucose.

· Using mutagens to knock out genes, it has been determined that M. genitalium can survive in the laboratory with just 337 genes!

· This number is termed the “minimal essential genome.”

The Instructor’s Resource CD-ROM includes micrographs and computer models of many different viruses, a micrograph of two bacterial plasmids, and a photograph of bacterial lawns with and without viral plaques.