# Mechanisms of Microbial Genetics

## Protein Synthesis (Translation)

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the genetic code and explain why it is considered almost universal
2. Explain the process of translation and the functions of the molecular machinery of translation
3. Compare translation in eukaryotes and prokaryotes
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.

### The Genetic Code

Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring amino acids. Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon. The relationship between an mRNA codon and its corresponding amino acid is called the genetic code.
The three-nucleotide code means that there is a total of 64 possible combinations (43, with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon (). This redundancy in the genetic code is called degeneracy. Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobble position, is less critical. In some cases, if the nucleotide in the third position is changed, the same amino acid is still incorporated.
Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codons or nonsense codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the start codon to initiate translation. The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.
The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon.

### The Protein Synthesis Machinery

In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.

### Ribosomes

A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).
Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a polyribosome (or polysome). In both bacteria and archaea, before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes (). The reason why transcription and translation can occur simultaneously is because both of these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.

### Transfer RNAs

Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.
Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.
An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.

### The Mechanism of Protein Synthesis

Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

### Initiation

The initiation of protein synthesis begins with the formation of an initiation complex. In , this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying  (fMet-tRNAfMet) (). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.
In eukaryotes, initiation complex formation is similar, with the following differences:
1. The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
2. Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

### Elongation

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In , the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

### Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex.
In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in  and listed in .

### Protein Targeting, Folding, and Modification

During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational modifications include:
1. removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular compartment
2. proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins, into a distinct three-dimensional structure
3. proteolytic processing of an inactive polypeptide to release an active protein component, and
4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.

### Key Concepts and Summary

1. In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
2. The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost universal among living organisms.
3. Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
4. Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a cognate amino acid. All tRNAs with a specific anticodon will carry the same amino acid.
5. Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
6. In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the mRNA.
7. During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the ribosomal complex.
8. Termination of translation occurs when the ribosome encounters a stop codon, which does not code for a tRNA. Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
9. In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the cytoplasm or in association with the rough endoplasmic reticulum.
10. Polypeptides often require one or more post-translational modifications to become biologically active.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking


# Mechanisms of Microbial Genetics

## Mutations

### Learning Objectives

By the end of this section, you will be able to:
1. Compare point mutations and frameshift mutations
2. Describe the differences between missense, nonsense, and silent mutations
3. Describe the differences between light and dark repair
4. Explain how different mutagens act
5. Explain why the Ames test can be used to detect carcinogens
6. Analyze sequences of DNA and identify examples of types of mutations
A mutation is a heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature. A change in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on translation. Because proteins carry out the vast majority of cellular functions, a change in amino acid sequence in a protein may lead to an altered phenotype for the cell and organism.

### Effects of Mutations on DNA Sequence

There are several types of mutations that are classified according to how the DNA molecule is altered. One type, called a point mutation, affects a single base and most commonly occurs when one base is substituted or replaced by another. Mutations also result from the addition of one or more bases, known as an insertion, or the removal of one or more bases, known as a deletion.

### Effects of Mutations on Protein Structure and Function

Point mutations may have a wide range of effects on protein function (). As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would have no effect on the protein’s structure, and is thus called a silent mutation. A missense mutation results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme’s active site, then the effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to some degree. Sometimes the effects of missense mutations may be only apparent under certain environmental conditions; such missense mutations are called conditional mutations. Rarely, a missense mutation may be beneficial. Under the right environmental conditions, this type of mutation may give the organism that harbors it a selective advantage. Yet another type of point mutation, called a nonsense mutation, converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional.
Deletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of three nucleotides may lead to the insertion or deletion of one or more amino acids and may not cause significant effects on the resulting protein’s functionality. However, frameshift mutations, caused by insertions or deletions of a number of nucleotides that are not a multiple of three are extremely problematic because a shift in the reading frame results (). Because ribosomes read the mRNA in triplet codons, frameshift mutations can change every amino acid after the point of the mutation. The new reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly always nonfunctional.

### Causes of Mutations

Mistakes in the process of DNA replication can cause spontaneous mutations to occur. The error rate of DNA polymerase is one incorrect base per billion base pairs replicated. Exposure to mutagens can cause induced mutations, which are various types of chemical agents or radiation (). Exposure to a mutagen can increase the rate of mutation more than 1000-fold. Mutagens are often also carcinogens, agents that cause cancer. However, whereas nearly all carcinogens are mutagenic, not all mutagens are necessarily carcinogens.

### Chemical Mutagens

Various types of chemical mutagens interact directly with DNA either by acting as nucleoside analogs or by modifying nucleotide bases. Chemicals called nucleoside analogs are structurally similar to normal nucleotide bases and can be incorporated into DNA during replication (). These base analogs induce mutations because they often have different base-pairing rules than the bases they replace. Other chemical mutagens can modify normal DNA bases, resulting in different base-pairing rules. For example, nitrous acid deaminates cytosine, converting it to uracil. Uracil then pairs with adenine in a subsequent round of replication, resulting in the conversion of a GC base pair to an AT base pair. Nitrous acid also deaminates adenine to hypoxanthine, which base pairs with cytosine instead of thymine, resulting in the conversion of a TA base pair to a CG base pair.
Chemical mutagens known as intercalating agents work differently. These molecules slide between the stacked nitrogenous bases of the DNA double helix, distorting the molecule and creating atypical spacing between nucleotide base pairs (). As a result, during DNA replication, DNA polymerase may either skip replicating several nucleotides (creating a deletion) or insert extra nucleotides (creating an insertion). Either outcome may lead to a frameshift mutation. Combustion products like polycyclic aromatic hydrocarbons are particularly dangerous intercalating agents that can lead to mutation-caused cancers. The intercalating agents ethidium bromide and acridine orange are commonly used in the laboratory to stain DNA for visualization and are potential mutagens.

### Radiation

Exposure to either ionizing or nonionizing radiation can each induce mutations in DNA, although by different mechanisms. Strong ionizing radiation like X-rays and gamma rays can cause single- and double-stranded breaks in the DNA backbone through the formation of hydroxyl radicals on radiation exposure (). Ionizing radiation can also modify bases; for example, the deamination of cytosine to uracil, analogous to the action of nitrous acid.K.R. Tindall et al. “Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage.”  Ionizing radiation exposure is used to kill microbes to sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA, proteins, and other cellular components (see Using Physical Methods to Control Microorganisms).
Nonionizing radiation, like ultraviolet light, is not energetic enough to initiate these types of chemical changes. However, nonionizing radiation can induce dimer formation between two adjacent pyrimidine bases, commonly two thymines, within a nucleotide strand. During thymine dimer formation, the two adjacent thymines become covalently linked and, if left unrepaired, both DNA replication and transcription are stalled at this point. DNA polymerase may proceed and replicate the dimer incorrectly, potentially leading to frameshift or point mutations.

### DNA Repair

The process of DNA replication is highly accurate, but mistakes can occur spontaneously or be induced by mutagens. Uncorrected mistakes can lead to serious consequences for the phenotype. Cells have developed several repair mechanisms to minimize the number of mutations that persist.

### Proofreading

Most of the mistakes introduced during DNA replication are promptly corrected by most DNA polymerases through a function called proofreading. In proofreading, the DNA polymerase reads the newly added base, ensuring that it is complementary to the corresponding base in the template strand before adding the next one. If an incorrect base has been added, the enzyme makes a cut to release the wrong nucleotide and a new base is added.

### Mismatch Repair

Some errors introduced during replication are corrected shortly after the replication machinery has moved. This mechanism is called mismatch repair. The enzymes involved in this mechanism recognize the incorrectly added nucleotide, excise it, and replace it with the correct base. One example is the methyl-directed mismatch repair in . The DNA is hemimethylated. This means that the parental strand is methylated while the newly synthesized daughter strand is not. It takes several minutes before the new strand is methylated. Proteins MutS, MutL, and MutH bind to the hemimethylated site where the incorrect nucleotide is found. MutH cuts the nonmethylated strand (the new strand). An exonuclease removes a portion of the strand (including the incorrect nucleotide). The gap formed is then filled in by DNA pol III and ligase.

### Repair of Thymine Dimers

Because the production of thymine dimers is common (many organisms cannot avoid ultraviolet light), mechanisms have evolved to repair these lesions. In nucleotide excision repair (also called dark repair), enzymes remove the pyrimidine dimer and replace it with the correct nucleotides (). In E. coli, the DNA is scanned by an enzyme complex. If a distortion in the double helix is found that was introduced by the pyrimidine dimer, the enzyme complex cuts the sugar-phosphate backbone several bases upstream and downstream of the dimer, and the segment of DNA between these two cuts is then enzymatically removed. DNA pol I replaces the missing nucleotides with the correct ones and DNA ligase seals the gap in the sugar-phosphate backbone.
The direct repair (also called light repair) of thymine dimers occurs through the process of photoreactivation in the presence of visible light. An enzyme called photolyase recognizes the distortion in the DNA helix caused by the thymine dimer and binds to the dimer. Then, in the presence of visible light, the photolyase enzyme changes conformation and breaks apart the thymine dimer, allowing the thymines to again correctly base pair with the adenines on the complementary strand. Photoreactivation appears to be present in all organisms, with the exception of placental mammals, including humans. Photoreactivation is particularly important for organisms chronically exposed to ultraviolet radiation, like plants, photosynthetic bacteria, algae, and corals, to prevent the accumulation of mutations caused by thymine dimer formation.

### Identifying Bacterial Mutants

One common technique used to identify bacterial mutants is called replica plating. This technique is used to detect nutritional mutants, called auxotrophs, which have a mutation in a gene encoding an enzyme in the biosynthesis pathway of a specific nutrient, such as an amino acid. As a result, whereas wild-type cells retain the ability to grow normally on a medium lacking the specific nutrient, auxotrophs are unable to grow on such a medium. During replica plating (), a population of bacterial cells is mutagenized and then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies. Cells from these colonies are removed from this master plate, often using sterile velvet. This velvet, containing cells, is then pressed in the same orientation onto plates of various media. At least one plate should also be nutritionally complete to ensure that cells are being properly transferred between the plates. The other plates lack specific nutrients, allowing the researcher to discover various auxotrophic mutants unable to produce specific nutrients. Cells from the corresponding colony on the nutritionally complete plate can be used to recover the mutant for further study.

### The Ames Test

The Ames test, developed by Bruce Ames (1928–) in the 1970s, is a method that uses bacteria for rapid, inexpensive screening of the carcinogenic potential of new chemical compounds. The test measures the mutation rate associated with exposure to the compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames test uses as the test organism a strain of  that is a histidine auxotroph, unable to synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are plated onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that arise in the absence of the potential mutagen (). Chemicals that are more mutagenic will bring about more mutants with restored histidine synthesis in the Ames test. Because many chemicals are not directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested for their potential carcinogenic properties by using other models, including animal models like mice and rats.

### Key Concepts and Summary

1. A mutation is a heritable change in DNA. A mutation may lead to a change in the amino-acid sequence of a protein, possibly affecting its function.
2. A point mutation affects a single base pair. A point mutation may cause a silent mutation if the mRNA codon codes for the same amino acid, a missense mutation if the mRNA codon codes for a different amino acid, or a nonsense mutation if the mRNA codon becomes a stop codon.
3. Missense mutations may retain function, depending on the chemistry of the new amino acid and its location in the protein. Nonsense mutations produce truncated and frequently nonfunctional proteins.
4. A frameshift mutation results from an insertion or deletion of a number of nucleotides that is not a multiple of three. The change in reading frame alters every amino acid after the point of the mutation and results in a nonfunctional protein.
5. Spontaneous mutations occur through DNA replication errors, whereas induced mutations occur through exposure to a mutagen.
6. Mutagenic agents are frequently carcinogenic but not always. However, nearly all carcinogens are mutagenic.
7. Chemical mutagens include base analogs and chemicals that modify existing bases. In both cases, mutations are introduced after several rounds of DNA replication.
8. Ionizing radiation, such as X-rays and γ-rays, leads to breakage of the phosphodiester backbone of DNA and can also chemically modify bases to alter their base-pairing rules.
9. Nonionizing radiation like ultraviolet light may introduce pyrimidine (thymine) dimers, which, during DNA replication and transcription, may introduce frameshift or point mutations.
10. Cells have mechanisms to repair naturally occurring mutations. DNA polymerase has proofreading activity. Mismatch repair is a process to repair incorrectly incorporated bases after DNA replication has been completed.
11. Pyrimidine dimers can also be repaired. In nucleotide excision repair (dark repair), enzymes recognize the distortion introduced by the pyrimidine dimer and replace the damaged strand with the correct bases, using the undamaged DNA strand as a template. Bacteria and other organisms may also use direct repair, in which the photolyase enzyme, in the presence of visible light, breaks apart the pyrimidines.
12. Through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients, specific loss-of-function mutants called auxotrophs can be identified.
13. The Ames test is an inexpensive method that uses auxotrophic bacteria to measure mutagenicity of a chemical compound. Mutagenicity is an indicator of carcinogenic potential.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer

### Critical Thinking


# Mechanisms of Microbial Genetics

## How Asexual Prokaryotes Achieve Genetic Diversity

### Learning Objectives

By the end of this section, you will be able to:
1. Compare the processes of transformation, transduction, and conjugation
2. Explain how asexual gene transfer results in prokaryotic genetic diversity
3. Explain the structure and consequences for bacterial genetic diversity of transposons
Typically, when we consider genetic transfer, we think of vertical gene transfer, the transmission of genetic information from generation to generation. Vertical gene transfer is by far the main mode of transmission of genetic information in all cells. In sexually reproducing organisms, crossing-over events and independent assortment of individual chromosomes during meiosis contribute to genetic diversity in the population. Genetic diversity is also introduced during sexual reproduction, when the genetic information from two parents, each with different complements of genetic information, are combined, producing new combinations of parental genotypes in the diploid offspring. The occurrence of mutations also contributes to genetic diversity in a population. Genetic diversity of offspring is useful in changing or inconsistent environments and may be one reason for the evolutionary success of sexual reproduction.
When prokaryotes and eukaryotes reproduce asexually, they transfer a nearly identical copy of their genetic material to their offspring through vertical gene transfer. Although asexual reproduction produces more offspring more quickly, any benefits of diversity among those offspring are lost. How then do organisms whose dominant reproductive mode is asexual create genetic diversity? In prokaryotes, horizontal gene transfer (HGT), the introduction of genetic material from one organism to another organism within the same generation, is an important way to introduce genetic diversity. HGT allows even distantly related species to share genes, influencing their phenotypes. It is thought that HGT is more prevalent in prokaryotes but that only a small fraction of the prokaryotic genome may be transferred by this type of transfer at any one time. As the phenomenon is investigated more thoroughly, it may be revealed to be even more common. Many scientists believe that HGT and mutation are significant sources of genetic variation, the raw material for the process of natural selection, in prokaryotes. Although HGT is more common among evolutionarily related organisms, it may occur between any two species that live together in a natural community.
HGT in prokaryotes is known to occur by the three primary mechanisms that are illustrated in :
1. Transformation: naked DNA is taken up from the environment
2. Transduction: genes are transferred between cells in a virus (see The Viral Life Cycle)
3. Conjugation: use of a hollow tube called a conjugation pilus to transfer genes between cells

### Transformation

Frederick Griffith was the first to demonstrate the process of transformation. In 1928, he showed that live, nonpathogenic  bacteria could be transformed into pathogenic bacteria through exposure to a heat-killed pathogenic strain. He concluded that some sort of agent, which he called the “transforming principle,” had been passed from the dead pathogenic bacteria to the live, nonpathogenic bacteria. In 1944, Oswald Avery (1877–1955), Colin MacLeod (1909–1972), and Maclyn McCarty (1911–2005) demonstrated that the transforming principle was DNA (see Using Microorganisms to Discover the Secrets of Life).
In transformation, the prokaryote takes up naked DNA found in its environment and that is derived from other cells that have lysed on death and released their contents, including their genome, into the environment. Many bacteria are naturally competent, meaning that they actively bind to environmental DNA, transport it across their cell envelopes into their cytoplasm, and make it single stranded. Typically, double-stranded foreign DNA within cells is destroyed by nucleases as a defense against viral infection. However, these nucleases are usually ineffective against single-stranded DNA, so this single-stranded DNA within the cell has the opportunity to recombine into the bacterial genome. A molecule of DNA that contains fragments of DNA from different organisms is called recombinant DNA. (Recombinant DNA will be discussed in more detail in Microbes and the Tools of Genetic Engineering.) If the bacterium incorporates the new DNA into its own genome through recombination, the bacterial cell may gain new phenotypic properties. For example, if a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and then incorporates it into its chromosome, it, too, may become pathogenic. Plasmid DNA may also be taken up by competent bacteria and confer new properties to the cell. Overall, transformation in nature is a relatively inefficient process because environmental DNA levels are low because of the activity of nucleases that are also released during cellular lysis. Additionally, genetic recombination is inefficient at incorporating new DNA sequences into the genome.
In nature, bacterial transformation is an important mechanism for the acquisition of genetic elements encoding virulence factors and antibiotic resistance. Genes encoding resistance to antimicrobial compounds have been shown to be widespread in nature, even in environments not influenced by humans. These genes, which allow microbes living in mixed communities to compete for limited resources, can be transferred within a population by transformation, as well as by the other processes of HGT. In the laboratory, we can exploit the natural process of bacterial transformation for genetic engineering to make a wide variety of medicinal products, as discussed in Microbes and the Tools of Genetic Engineering.

### Transduction

Viruses that infect bacteria (bacteriophages) may also move short pieces of chromosomal DNA from one bacterium to another in a process called transduction (see ). Recall that in generalized transduction, any piece of chromosomal DNA may be transferred to a new host cell by accidental packaging of chromosomal DNA into a phage head during phage assembly. By contrast, specialized transduction results from the imprecise excision of a lysogenic prophage from the bacterial chromosome such that it carries with it a piece of the bacterial chromosome from either side of the phage’s integration site to a new host cell. As a result, the host may acquire new properties. This process is called lysogenic conversion. Of medical significance, a lysogenic phage may carry with it a virulence gene to its new host. Once inserted into the new host’s chromosome, the new host may gain pathogenicity. Several pathogenic bacteria, including  (the causative agent of diphtheria) and  (the causative agent of botulism), are virulent because of the introduction of toxin-encoding genes by lysogenic bacteriophages, affirming the clinical relevance of transduction in the exchange of genes involved in infectious disease. Archaea have their own viruses that translocate genetic material from one individual to another.

### Conjugation

In conjugation, DNA is directly transferred from one prokaryote to another by means of a conjugation pilus, which brings the organisms into contact with one another. In , the genes encoding the ability to conjugate are located on a bacterial plasmid called the F plasmid, also known as the fertility factor, and the conjugation pilus is called the F pilus. The F-plasmid genes encode both the proteins composing the F pilus and those involved in rolling circle replication of the plasmid. Cells containing the F plasmid, capable of forming an F pilus, are called Fs or donor cells, and those lacking an F plasmid are called Fs or recipient cells.

### Conjugation of the F Plasmid

During typical conjugation in E. coli, the F pilus of an F+ cell comes into contact with an F– cell and retracts, bringing the two cell envelopes into contact (). Then a cytoplasmic bridge forms between the two cells at the site of the conjugation pilus. As rolling circle replication of the F plasmid occurs in the F+ cell, a single-stranded copy of the F plasmid is transferred through the cytoplasmic bridge to the F− cell, which then synthesizes the complementary strand, making it double stranded. The F− cell now becomes an F+ cell capable of making its own conjugation pilus. Eventually, in a mixed bacterial population containing both F+ and F− cells, all cells will become F+ cells. Genes on the E. coli F plasmid also encode proteins preventing conjugation between F+ cells.

### Conjugation of F’ and Hfr Cells

Although typical conjugation in E. coli results in the transfer of the F-plasmid DNA only, conjugation may also transfer chromosomal DNA. This is because the F plasmid occasionally integrates into the bacterial chromosome through recombination between the plasmid and the chromosome, forming an Hfr cell (). “Hfr” refers to the high frequency of recombination seen when recipient F− cells receive genetic information from Hfr cells through conjugation. Similar to the imprecise excision of a prophage during specialized transduction, the integrated F plasmid may also be imprecisely excised from the chromosome, producing an F’ plasmid that carries with it some chromosomal DNA adjacent to the integration site. On conjugation, this DNA is introduced to the recipient cell and may be either maintained as part of the F’ plasmid or be recombined into the recipient cell’s bacterial chromosome.
Hfr cells may also treat the bacterial chromosome like an enormous F plasmid and attempt to transfer a copy of it to a recipient F− cell. Because the bacterial chromosome is so large, transfer of the entire chromosome takes a long time (). However, contact between bacterial cells during conjugation is transient, so it is unusual for the entire chromosome to be transferred. Host chromosomal DNA near the integration site of the F plasmid, displaced by the unidirectional process of rolling circle replication, is more likely to be transferred and recombined into a recipient cell’s chromosome than host genes farther away. Thus, the relative location of bacterial genes on the Hfr cell’s genome can be mapped based on when they are transferred through conjugation. As a result, prior to the age of widespread bacterial genome sequencing, distances on prokaryotic genome maps were often measured in minutes.

### Consequences and Applications of Conjugation

Plasmids are an important type of extrachromosomal DNA element in bacteria and, in those cells that harbor them, are considered to be part of the bacterial genome. From a clinical perspective, plasmids often code for genes involved in virulence. For example, genes encoding proteins that make a bacterial cell resistant to a particular antibiotic are encoded on R plasmids. R plasmids, in addition to their genes for antimicrobial resistance, contain genes that control conjugation and transfer of the plasmid. R plasmids are able to transfer between cells of the same species and between cells of different species. Single R plasmids commonly contain multiple genes conferring resistance to multiple antibiotics.
Genes required for the production of various toxins and molecules important for colonization during infection may also be found encoded on plasmids. For example, verotoxin-producing strains of  (VTEC) appear to have acquired the genes encoding the Shiga toxin from its gram-negative relative  through the acquisition of a large plasmid encoding this toxin. VTEC causes severe diarrheal disease that may result in hemolytic uremic syndrome (HUS), which may be lead to kidney failure and death.
In nonclinical settings, bacterial genes that encode metabolic enzymes needed to degrade specialized atypical compounds like polycyclic aromatic hydrocarbons (PAHs) are also frequently encoded on plasmids. Additionally, certain plasmids have the ability to move from bacterial cells to other cell types, like those of plants and animals, through mechanisms distinct from conjugation. Such mechanisms and their use in genetic engineering are covered in Modern Applications of Microbial Genetics.

### Transposition

Genetic elements called transposons (transposable elements), or “jumping genes,” are molecules of DNA that include special inverted repeat sequences at their ends and a gene encoding the enzyme transposase (). Transposons allow the entire sequence to independently excise from one location in a DNA molecule and integrate into the DNA elsewhere through a process called transposition. Transposons were originally discovered in maize (corn) by American geneticist Barbara McClintock (1902–1992) in the 1940s. Transposons have since been found in all types of organisms, both prokaryotes and eukaryotes. Thus, unlike the three previous mechanisms discussed, transposition is not prokaryote-specific. Most transposons are nonreplicative, meaning they move in a “cut-and-paste” fashion. Some may be replicative, however, retaining their location in the DNA while making a copy to be inserted elsewhere (“copy and paste”). Because transposons can move within a DNA molecule, from one DNA molecule to another, or even from one cell to another, they have the ability to introduce genetic diversity. Movement within the same DNA molecule can alter phenotype by inactivating or activating a gene.
Transposons may carry with them additional genes, moving these genes from one location to another with them. For example, bacterial transposons can relocate antibiotic resistance genes, moving them from chromosomes to plasmids. This mechanism has been shown to be responsible for the colocalization of multiple antibiotic resistance genes on a single R plasmid in  strains causing bacterial dysentery. Such an R plasmid can then be easily transferred among a bacterial population through the process of conjugation.
 summarizes the processes discussed in this section.

### Key Concepts and Summary

1. Horizontal gene transfer is an important way for asexually reproducing organisms like prokaryotes to acquire new traits.
2. There are three mechanisms of horizontal gene transfer typically used by bacteria: transformation, transduction, and conjugation.
3. Transformation allows for competent cells to take up naked DNA, released from other cells on their death, into their cytoplasm, where it may recombine with the host genome.
4. In generalized transduction, any piece of chromosomal DNA may be transferred by accidental packaging of the degraded host chromosome into a phage head. In specialized transduction, only chromosomal DNA adjacent to the integration site of a lysogenic phage may be transferred as a result of imprecise excision of the prophage.
5. Conjugation is mediated by the F plasmid, which encodes a conjugation pilus that brings an F plasmid-containing F into contact with an F- cell.
6. The rare integration of the F plasmid into the bacterial chromosome, generating an Hfr cell, allows for transfer of chromosomal DNA from the donor to the recipient. Additionally, imprecise excision of the F plasmid from the chromosome may generate an F’ plasmid that may be transferred to a recipient by conjugation.
7. Conjugation transfer of R plasmids is an important mechanism for the spread of antibiotic resistance in bacterial communities.
8. Transposons are molecules of DNA with inverted repeats at their ends that also encode the enzyme transposase, allowing for their movement from one location in DNA to another. Although found in both prokaryotes and eukaryotes, transposons are clinically relevant in bacterial pathogens for the movement of virulence factors, including antibiotic resistance genes.

### Multiple Choice

### Fill in the Blank

### True/False

### Short Answer


# Mechanisms of Microbial Genetics

## Gene Regulation: Operon Theory

### Learning Objectives

By the end of this section, you will be able to:
1. Compare inducible operons and repressible operons
2. Describe why regulation of operons is important
Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two pure bacterial cultures inoculated from the same starting colony contain the same DNA, with the exception of changes that arise from spontaneous mutations. If each cell in a multicellular organism has the same DNA, then how is it that cells in different parts of the organism’s body exhibit different characteristics? Similarly, how is it that the same bacterial cells within two pure cultures exposed to different environmental conditions can exhibit different phenotypes? In both cases, each genetically identical cell does not turn on, or express, the same set of genes. Only a subset of proteins in a cell at a given time is expressed.
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms allows for cellular differentiation, in single-celled organisms like prokaryotes, it primarily ensures that a cell’s resources are not wasted making proteins that the cell does not need at that time.
Elucidating the mechanisms controlling gene expression is important to the understanding of human health. Malfunctions in this process in humans lead to the development of cancer and other diseases. Understanding the interaction between the gene expression of a pathogen and that of its human host is important for the understanding of a particular infectious disease. Gene regulation involves a complex web of interactions within a given cell among signals from the cell’s environment, signaling molecules within the cell, and the cell’s DNA. These interactions lead to the expression of some genes and the suppression of others, depending on circumstances.
Prokaryotes and eukaryotes share some similarities in their mechanisms to regulate gene expression; however, gene expression in eukaryotes is more complicated because of the temporal and spatial separation between the processes of transcription and translation. Thus, although most regulation of gene expression occurs through transcriptional control in prokaryotes, regulation of gene expression in eukaryotes occurs at the transcriptional level and post-transcriptionally (after the primary transcript has been made).

### Prokaryotic Gene Regulation

In bacteria and archaea, structural proteins with related functions are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript (). In this way, regulation of the transcription of all of the structural genes encoding the enzymes that catalyze the many steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time, or none will be needed. For example, in , all of the structural genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the  of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine in 1965. Although eukaryotic genes are not organized into operons, prokaryotic operons are excellent models for learning about gene regulation generally. There are some gene clusters in eukaryotes that function similar to operons. Many of the principles can be applied to eukaryotic systems and contribute to our understanding of changes in gene expression in eukaryotes that can result pathological changes such as cancer.
Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.
In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers.

### Regulation by Repression

Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby preventing the transcription of the structural genes. Such operons are classified as either repressible operons or inducible operons. Repressible operons, like the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However, when the product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue to make more, the expression of the operon is repressed. Conversely, inducible operons, like the  of , often contain genes encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose. These enzymes are only required when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate.

### The trp Operon: A Repressible Operon

 can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each other in the  (). When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur and tryptophan is not synthesized.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon. Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.

### The lac Operon: An Inducible Operon

The  is an example of an inducible operon that is also subject to activation in the absence of glucose (). The lac operon encodes three structural genes necessary to acquire and process the disaccharide lactose from the environment, breaking it down into the simple sugars glucose and galactose. For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available.
In the absence of lactose, the lac repressor is bound to the operator region of the lac operon, physically preventing RNA polymerase from transcribing the structural genes. However, when lactose is present, the lactose inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to bind to the operator DNA. Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator region and begin transcription of the lac structural genes.

### The lac Operon: Activation by Catabolite Activator Protein

Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted. Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed only when the alternative substrate is available. In the 1940s, Jacques Monod was the first to demonstrate the preference for certain substrates over others through his studies of E. coli’s growth when cultured in the presence of two different substrates simultaneously. Such studies generated diauxic growth curves, like the one shown in . Although the preferred substrate glucose is used first,  grows quickly and the enzymes for lactose metabolism are absent. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose. Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.
The ability to switch from glucose use to another substrate like lactose is a consequence of the activity of an enzyme called Enzyme IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism (see Catabolism of Carbohydrates), and EIIA becomes phosphorylated. Phosphorylated EIIA activates adenylyl cyclase, an enzyme that converts some of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of AMP and important signaling molecule involved in glucose and energy metabolism in E. coli. As a result, cAMP levels begin to rise in the cell ().
The lac operon also plays a role in this switch from using glucose to using lactose. When glucose is scarce, the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon (). In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. Binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present (removing the lac repressor protein) and glucose levels must be depleted (allowing binding of an activating protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. Because of low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate transcription of these operons. See  for a summary of the regulation of the lac operon.

### Global Responses of Prokaryotes

In prokaryotes, there are also several higher levels of gene regulation that have the ability to control the transcription of many related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a regulon.

### Alarmones

When sensing impending stress, prokaryotes alter the expression of a wide variety of operons to respond in coordination. They do this through the production of alarmones, which are small intracellular nucleotide derivatives. Alarmones change which genes are expressed and stimulate the expression of specific stress-response genes. The use of alarmones to alter gene expression in response to stress appears to be important in pathogenic bacteria. On encountering host defense mechanisms and other harsh conditions during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling. Knowledge of these responses is key to being able to fully understand the infection process of many pathogens and to the development of therapies to counter this process.

### Alternate σ Factors

Since the σ subunit of bacterial RNA polymerase confers specificity as to which promoters should be transcribed, altering the σ factor used is another way for bacteria to quickly and globally change what regulons are transcribed at a given time. The σ factor recognizes sequences within a bacterial promoter, so different σ factors will each recognize slightly different promoter sequences. In this way, when the cell senses specific environmental conditions, it may respond by changing which σ factor it expresses, degrading the old one and producing a new one to transcribe the operons encoding genes whose products will be useful under the new environmental condition. For example, in sporulating bacteria of the genera  and  (which include many pathogens), a group of σ factors controls the expression of the many genes needed for sporulation in response to sporulation-stimulating signals.

### Additional Methods of Regulation in Bacteria: Attenuation and Riboswitches

Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also mechanisms to control both the completion of transcription as well as translation concurrently. Since their discovery, these mechanisms have been shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes.
One such regulatory system is attenuation, whereby secondary stem-loop structures formed within the 5’ end of an mRNA being transcribed determine if transcription to complete the synthesis of this mRNA will occur and if this mRNA will be used for translation. Beyond the transcriptional repression mechanism already discussed, attenuation also controls expression of the  in  (). The trp operon regulatory region contains a leader sequence called trpL between the operator and the first structural gene, which has four stretches of RNA that can base pair with each other in different combinations. When a terminator stem-loop forms, transcription terminates, releasing RNA polymerase from the mRNA. However, when an antiterminator stem-loop forms, this prevents the formation of the terminator stem-loop, so RNA polymerase can transcribe the structural genes.
A related mechanism of concurrent regulation of transcription and translation in prokaryotes is the use of a riboswitch, a small region of noncoding RNA found within the 5’ end of some prokaryotic mRNA molecules (. A riboswitch may bind to a small intracellular molecule to stabilize certain secondary structures of the mRNA molecule. The binding of the small molecule determines which stem-loop structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.

### Other Factors Affecting Gene Expression in Prokaryotes and Eukaryotes

Although the focus on our discussion of transcriptional control used prokaryotic operons as examples, eukaryotic transcriptional control is similar in many ways. As in prokaryotes, eukaryotic transcription can be controlled through the binding of transcription factors including repressors and activators. Interestingly, eukaryotic transcription can be influenced by the binding of proteins to regions of DNA, called enhancers, rather far away from the gene, through DNA looping facilitated between the enhancer and the promoter (). Overall, regulating transcription is a highly effective way to control gene expression in both prokaryotes and eukaryotes. However, the control of gene expression in eukaryotes in response to environmental and cellular stresses can be accomplished in additional ways without the binding of transcription factors to regulatory regions.

### DNA-Level Control

In eukaryotes, the DNA molecules or associated histones can be chemically modified in such a way as to influence transcription; this is called epigenetic regulation. Methylation of certain cytosine nucleotides in DNA in response to environmental factors has been shown to influence use of such DNA for transcription, with DNA methylation commonly correlating to lowered levels of gene expression. Additionally, in response to environmental factors, histone proteins for packaging DNA can also be chemically modified in multiple ways, including acetylation and deacetylation, influencing the packaging state of DNA and thus affecting the availability of loosely wound DNA for transcription. These chemical modifications can sometimes be maintained through multiple rounds of cell division, making at least some of these epigenetic changes heritable.

### Key Concepts and Summary

1. Gene expression is a tightly regulated process.
2. Gene expression in prokaryotes is largely regulated at the point of transcription. Gene expression in eukaryotes is additionally regulated post-transcriptionally.
3. Prokaryotic structural genes of related function are often organized into operons, all controlled by transcription from a single promoter. The regulatory region of an operon includes the promoter itself and the region surrounding the promoter to which transcription factors can bind to influence transcription.
4. Although some operons are constitutively expressed, most are subject to regulation through the use of transcription factors (repressors and activators). A repressor binds to an operator, a DNA sequence within the regulatory region between the RNA polymerase binding site in the promoter and first structural gene, thereby physically blocking transcription of these operons. An activator binds within the regulatory region of an operon, helping RNA polymerase bind to the promoter, thereby enhancing the transcription of this operon. An inducer influences transcription through interacting with a repressor or activator.
5. The trp operon is a classic example of a repressible operon. When tryptophan accumulates, tryptophan binds to a repressor, which then binds to the operator, preventing further transcription.
6. The lac operon is a classic example an inducible operon. When lactose is present in the cell, it is converted to allolactose. Allolactose acts as an inducer, binding to the repressor and preventing the repressor from binding to the operator. This allows transcription of the structural genes.
7. The lac operon is also subject to activation. When glucose levels are depleted, some cellular ATP is converted into cAMP, which binds to the catabolite activator protein (CAP). The cAMP-CAP complex activates transcription of the lac operon. When glucose levels are high, its presence prevents transcription of the lac operon and other operons by catabolite repression.
8. Small intracellular molecules called alarmones are made in response to various environmental stresses, allowing bacteria to control the transcription of a group of operons, called a regulon.
9. Bacteria have the ability to change which σ factor of RNA polymerase they use in response to environmental conditions to quickly and globally change which regulons are transcribed.
10. Prokaryotes have regulatory mechanisms, including attenuation and the use of riboswitches, to simultaneously control the completion of transcription and translation from that transcript. These mechanisms work through the formation of stem loops in the 5’ end of an mRNA molecule currently being synthesized.
11. There are additional points of regulation of gene expression in prokaryotes and eukaryotes. In eukaryotes, epigenetic regulation by chemical modification of DNA or histones, and regulation of RNA processing are two methods.

### Multiple Choice

### Fill in the Blank

### Short Answer

### Critical Thinking


# Modern Applications of Microbial Genetics

## Introduction

Watson and Crick’s identification of the structure of DNA in 1953 was the seminal event in the field of genetic engineering. Since the 1970s, there has been a veritable explosion in scientists’ ability to manipulate DNA in ways that have revolutionized the fields of biology, medicine, diagnostics, forensics, and industrial manufacturing. Many of the molecular tools discovered in recent decades have been produced using prokaryotic microbes. In this chapter, we will explore some of those tools, especially as they relate to applications in medicine and health care.
As an example, the thermal cycler in  is used to perform a diagnostic technique called the polymerase chain reaction (PCR), which relies on DNA polymerase enzymes from thermophilic bacteria. Other molecular tools, such as restriction enzymes and plasmids obtained from microorganisms, allow scientists to insert genes from humans or other organisms into microorganisms. The microorganisms are then grown on an industrial scale to synthesize products such as insulin, vaccines, and biodegradable polymers. These are just a few of the numerous applications of microbial genetics that we will explore in this chapter.


# Modern Applications of Microbial Genetics

## Microbes and the Tools of Genetic Engineering

### Learning Objectives

By the end of this section, you will be able to:
1. Identify tools of molecular genetics that are derived from microorganisms
2. Describe the methods used to create recombinant DNA molecules
3. Describe methods used to introduce DNA into prokaryotic cells
4. List the types of genomic libraries and describe their uses
5. Describe the methods used to introduce DNA into eukaryotic cells
The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic.
One example of a transgenic microorganism is the bacterial strain that produces human insulin (). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.

### Molecular Cloning

Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in 1973 when they successfully cloned genes from the African clawed frog () into a bacterial plasmid that was then introduced into the bacterial host . Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism; it makes use of a number of molecular tools that are derived from microorganisms.

### Restriction Enzymes and Ligases

In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends ().
Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

### Plasmids

After restriction digestion, genes of interest are commonly inserted into plasmids, small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome (see Unique Characteristics of Prokaryotic Cells). In recombinant DNA technology, plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 (). Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow.
Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes.
This polylinker site is often found within a reporter gene, another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial  gene encoding beta-galactosidase, an enzyme that naturally degrades lactose but can also degrade a colorless synthetic analog X-gal, thereby producing blue colonies on X-gal–containing media. The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be isolated. This blue-white screening method is described later and shown in . In addition to these features, some plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign DNA fragments.

### Molecular Cloning using Transformation

The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation, a process in which bacteria take up free DNA from their surroundings. In nature, free DNA typically comes from other lysed bacterial cells; in the laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.
Some bacteria, such as  spp., are naturally competent, meaning they are able to take up foreign DNA. However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane. This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. These methods yield chemically competent or electrocompetent bacteria, respectively.
Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening is then used for lacZ-encoding plasmid vectors such as pUC19. Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site. These colonies typically result from the digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. To be sure the correct DNA was incorporated into the plasmid, the DNA insert can then be sequenced.

### Molecular Cloning Using Conjugation or Transduction

The bacterial process of conjugation (see How Asexual Prokaryotes Achieve Genetic Diversity) can also be manipulated for molecular cloning. F plasmids, or fertility plasmids, are transferred between bacterial cells through the process of conjugation. Recombinant DNA can be transferred by conjugation when bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid. F plasmids encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F plasmid. On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid, transferring a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell. The use of conjugation to transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells.
Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the transduction process (see How Asexual Prokaryotes Achieve Genetic Diversity). In the laboratory, DNA fragments of interest can be engineered into phagemids, which are plasmids that have phage sequences that allow them to be packaged into bacteriophages. Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Depending on the type of phage, the recombinant DNA may be integrated into the host bacterial genome (lysogeny), or it may exist as a plasmid in the host’s cytoplasm.

### Creating a Genomic Library

Molecular cloning may also be used to generate a genomic library. The library is a complete (or nearly complete) copy of an organism’s genome contained as recombinant DNA plasmids engineered into unique clones of bacteria. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.
One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme (). After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study. This makes it possible for researchers to screen these different clones to discover the one containing a gene of interest from the original organism’s genome.
To construct a genomic library using larger fragments of genomic DNA, an E. coli bacteriophage, such as lambda, can be used as a host (). Genomic DNA can be sheared or enzymatically digested and ligated into a pre-digested bacteriophage lambda DNA vector. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. coli host cells on a plate. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. coli lawn, forming a plaque. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.
To focus on the expressed genes in an organism or even a tissue, researchers construct libraries using the organism’s messenger RNA (mRNA) rather than its genomic DNA. Whereas all cells in a single organism will have the same genomic DNA, different tissues express different genes, producing different complements of mRNA. For example, all human cells’ genomic DNA contains the gene for insulin, but only cells in the pancreas express mRNA directing the production of insulin. Because mRNA cannot be cloned directly, in the laboratory mRNA must be used as a template by the retroviral enzyme reverse transcriptase to make complementary DNA (cDNA). A cell’s full complement of mRNA can be reverse-transcribed into cDNA molecules, which can be used as a template for DNA polymerase to make double-stranded DNA copies; these fragments can subsequently be ligated into either plasmid vectors or bacteriophage to produce a cDNA library. The benefit of a cDNA library is that it contains DNA from only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to study the sequence and structure of the genome in its entirety. The construction of a cDNA genomic library is shown in .

### Introducing Recombinant Molecules into Eukaryotic Hosts

The use of bacterial hosts for genetic engineering laid the foundation for recombinant DNA technology; however, researchers have also had great interest in genetically engineering eukaryotic cells, particularly those of plants and animals. The introduction of recombinant DNA molecules into eukaryotic hosts is called transfection. Genetically engineered plants, called transgenic plants, are of significant interest for agricultural and pharmaceutical purposes. The first transgenic plant sold commercially was the Flavr Savr delayed-ripening tomato, which came to market in 1994. Genetically engineered livestock have also been successfully produced, resulting, for example, in pigs with increased nutritional valueLiangxue Lai, Jing X. Kang, Rongfeng Li, Jingdong Wang, William T. Witt, Hwan Yul Yong, Yanhong Hao et al. “Generation of Cloned Transgenic Pigs Rich in Omega-3 Fatty Acids.”  and goats that secrete pharmaceutical products in their milk.Raylene Ramos Moura, Luciana Magalhães Melo, and Vicente José de Figueirêdo Freitas. “Production of Recombinant Proteins in Milk of Transgenic and Non-Transgenic Goats.”

### Electroporation

Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts is far more challenging and requires more intrusive techniques for success. One method used for transfecting cells in cell culture is called electroporation. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a short-lived positive charge on one side of the cell’s interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the cell ().

### Microinjection

An alternative method of transfection is called microinjection. Because eukaryotic cells are typically larger than those of prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette, as shown in .

### Gene Guns

Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls. One approach involves treating plant cells with enzymes to remove their cell walls, producing protoplasts. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts at high speeds. Recipient protoplast cells can then recover and be used to generate new transgenic plants ().

### Shuttle Vectors

Another method of transfecting plants involves shuttle vectors, plasmids that can move between bacterial and eukaryotic cells. The tumor-inducing (T originating from the bacterium  are commonly used as shuttle vectors for incorporating genes into plants (). In nature, the Ti plasmids of A. tumefaciens cause plants to develop tumors when they are transferred from bacterial cells to plant cells. Researchers have been able to manipulate these naturally occurring plasmids to remove their tumor-causing genes and insert desirable DNA fragments. The resulting recombinant Ti plasmids can be transferred into the plant genome through the natural transfer of Ti plasmids from the bacterium to the plant host. Once inside the plant host cell, the gene of interest recombines into the plant cell’s genome.

### Viral Vectors

Viral vectors can also be used to transfect eukaryotic cells. In fact, this method is often used in gene therapy (see Gene Therapy) to introduce healthy genes into human patients suffering from diseases that result from genetic mutations. Viral genes can be deleted and replaced with the gene to be delivered to the patient;William S.M. Wold and Karoly Toth. “Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy.”  the virus then infects the host cell and delivers the foreign DNA into the genome of the targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing and dividing host cells. However, use of viral vectors for gene therapy can pose some risks for patients, as discussed in Gene Therapy.

### Key Concepts and Summary

1. Biotechology is the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an organism’s genome through genetic engineering has been made possible due to advances in recombinant DNA technology, which allows researchers to create recombinant DNA molecules with new combinations of genetic material.
2. Molecular cloning involves methods used to construct recombinant DNA and facilitate their replication in host organisms. These methods include the use of restriction enzymes (to cut both foreign DNA and plasmid vectors), ligation (to paste fragments of DNA together), and the introduction of recombinant DNA into a host organism (often bacteria).
3. Blue-white screening allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a reporter gene that is disabled by insertion of the DNA fragment.
4. Genomic libraries can be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
5. cDNA libraries can be generated to represent the mRNA molecules expressed in a cell at a given point.
6. Transfection of eukaryotic hosts can be achieved through various methods using electroporation, gene guns, microinjection, shuttle vectors, and viral vectors.

### Multiple Choice

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