BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Chapter 9- An Introduction to Microbial * *Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for the exams.

9.1 and : Unlocking the Secrets of

Genetics is the study of heredity, passing genetic information from parent to offspring. The study of genetics can take place at many levels: organism, cell, , molecular (figure 9.1). The study of microbial genetics can be applied to universal themes, and serves as the basis for in chapter 10.

The Nature of the Genetic Material

Deoxyribonucleic acid, or DNA is the molecule of genetics. It is important to understand its structure, organization and function.

The Levels of Structure and Function of the

As you read the sequence of in a molecule of DNA (e.g. ATGCCCTAA...etc.), there are regions, specific sequences with beginnings and ends, which define genes. These sequences are the regions that encode the information for building . Another way of saying this is that a is a sequence of nucleotides that codes for RNA and in most cases ultimately for the synthesis of a . An organism’s genome is the sum of all its genetic material (DNA). The term chromosome refers to a discrete DNA molecule; whereas tend to have only one, eukaryotes can have many (in humans there are 23 unique, individual ). Structural genes code for proteins; other genes code for tRNAs and rRNAs.

Also found in are additional pieces of extra-chromosomal DNA called . These small (~500- 3000 base pairs), circular pieces of DNA can comprise as much as 10% of the total genetic information in a bacterium.

Resistance plasmids (R plasmids or R factors) are so-called since they give bacteria resistance (i.e. immunity) to a variety of antimicrobial drugs1 (antibiotic resistance). Other functions plasmids encode include: virulence factors (the ability to cause disease), heavy metal resistance, and resistance to attack.

Bacteria can not only transfer plasmids between cells of the same species, but they can also transfer DNA between genera: Shigella, Salmonella, Escherichia, Yersinia, Klebsiella, Serratia, and Proteus can all transfer plasmids. Transfer of antibiotic resistance genes is a growing problem for therapeutics.

Note: Therapeutics Throughout the following sections, take note of the fact that many pharmaceutical agents used to treat infections target DNA replication, transcription, and translation. Treatment of bacterial infections with such drugs is based on the premise that the growth of the will be inhibited by blocking its protein

1 Antibiotic resistance is covered in detail in chapter 12. 1

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus synthesizing machinery selectively, without disrupting the cellular of the patient receiving the therapy.

The genetic makeup of an organism is called its genotype, and when the gene is expressed it produces a phenotype, a characteristic you can see.

The Size and Packaging of

The bacterial chromosome varies in size from fewer than 1,000,000 (106) nucleotide base pairs that code for about 1000 proteins (mycoplasmas) to 4.5 x 106 nucleotide base pairs (E. coli), that code for about 4,500 proteins. (Humans = 3 x 109 nucleotides and approximately 25,000 genes).

The Packaging of DNA

The circular chromosomal DNA of bacteria is packaged by a topoisomerase , specifically called DNA gyrase.

The Structure of DNA: A Double Helix with Its Own Language

Nucleic acids are large organic molecules found in all cells. There are 2 types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are large polymers of subunits called nucleotides. Each nucleotide contains a 5-carbon sugar (either deoxyribose or ribose), a phosphate group, and one of 5 different nitrogen-containing molecules called nitrogenous bases. The sugar and the phosphate form a backbone and the nitrogen bases stick out as side branches. P S-N P S-N fig. 2.24 ; fig. 2.25; fig. 9.B P S = sugar, P = phosphate, N = nitrogenous base S-N P S-N

The nitrogenous bases can be categorized as purines and pyrimidines (fig. 2.25). Purines (2 rings): adenine and guanine (A and G) Pyrimidines (1 ring): thymine, cytosine and uracil (T, C, and U)

They are abbreviated using a capitalized first letter - A, G, T, C, and U. Adenine, guanine, thymine and cytosine are found in DNA (fig 2.24). Adenine, guanine, cytosine and uracil are found in RNA. The sugar in DNA is

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BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus deoxyribose and the sugar in RNA is ribose (fig. 2.24c). Normally2, DNA is a double-stranded helix while RNA is single-stranded.

DNA molecules occur as a double-stranded helix in bacteria, protizoans, plants, fungi, and animals. It has a sugar-phosphate backbone with the purines and pyrimidines covalently linked to the deoxyribose sugar. Purines and pyrimidines are complementary to each other in a very specific fashion. As two separate strands of DNA move closer together, the nitrogenous bases can pair up and be held together by hydrogen bonds: A=T and GC. The base pairing of A with T and G with C is always conserved. Hydrogen bonds are easily formed and easily broken (fig. 2.26; fig. 2.27; fig. 9.4). This allows the helix to “unzip” and each strand can be used to produce a new strand through a process called DNA replication:

The physical characteristics of all organisms is determined by the genetic information contained in their cells. Information encoded in sections of the DNA called genes is used by the cell to manufacture the proteins necessary for its survival. This flow of genetic information in cells follows the sequence (figs. 9.8; fig.9.9):

(i) (ii) DNA mRNA Protein

And is made up of two separate events: (i) Transcription is the process whereby DNA → mRNA (ii) Translation is the process whereby mRNA → Protein

DNA Replication: Preserving the Code and Passing it On

Bacteria grow through binary fission. Prior to cell division, the bacterium must replicate its DNA. Replication of the chromosome starts at a site called the origin of replication. An enzyme called DNA gyrase releases chromosome supercoils (“unwinds DNA”). After the DNA is unwound, DNA helicase breaks the hydrogen bonds between nucleotides, allowing for it to “unzip” the DNA double helix, thus separating the two strands. The copying enzyme, DNA polymerase, moves in to synthesize new strands based on the open template. Replication proceeds in two directions around the circumference of the chromosome. Occasionally, incorrect bases are paired which can lead to (see below for more info). (see Table 9.1 for enzyme functions.)

DNA DNA polymerase 2DNA

2 Exceptions occur in where double-stranded DNA, double-stranded RNA, single-stranded RNA, and single-stranded DNA are found. 3

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Replication of DNA A T=A T= T =A T=A T=A A=T A= =T A=T A=T CG C G CG CG T=A  T= =A  T=A T=A GC G C GC GC A=T A=T A=T A=T T=A T=A T=A T=A GC GC GC GC fig. 2.26; GC GC GC GC fig. 9.5; CG CG CG CG fig. 9.6

1. DNA Gyrase relaxes the supercoiled DNA (unwinding). 2. DNA Helicase breaks the hydrogen bonds between bases (unzips the double helix). 3. DNA Polymerase uses complementary base pairs (G to C, C to G, T to A, and A to T) to construct a new strand. 4. Two helices are produced, each containing one old and one new strand.

Therapeutics note: Fluoroquinolones (e.g. Ciprofloxacin) – class of drugs that act by inhibiting bacterial DNA gyrase, thus preventing normal DNA synthesis.

9.2 Applications of the DNA Code: Transcription and Translation

Transcription is the process of producing RNA from a DNA template; and translation is the process of producing a protein from a mRNA.

The Gene-Protein Connection The Triplet Code and the Relationship to Proteins

When DNA is transcribed to mRNA the bases in mRNA are read in triplet, called a codon. Each codon codes for an amino acid (figure 9.9).

The Major Participants in Transcription and Translation

While the role for DNA is information storage, RNA must perform different jobs inside the cell. There are three primary types of RNA, each with a different function (Table 9.2):

1. Messenger RNA (mRNA) is a single stranded copy of the sequence information in a gene. The sequence of nucleotides in an mRNA is used as a template to make a protein during translation (fig. 9.10). 2. Transfer RNA (tRNA) carries the amino acids to the (fig. 9.10). tRNAs are short, folded molecules with two distinct ends: one end of the tRNA attaches to a specific amino acid; the other end has an anticodon, or group of 3 nitrogenous bases that are complementary to a codon on mRNAs.

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3. Ribosomal RNA (rRNA) is a physical part of a functional ribosome. While rRNA is rarely drawn out in diagrams, consider it to be a necessary subunit of a ribosome and that it acts as an enzyme, forming peptide bonds between amino acids.

Summary of differences between DNA & RNA:

DNA RNA Nitrogenous bases A, T, G, C A, U, G, C Pentose sugar Deoxyribose sugar Ribose sugar Structure Double Stranded Helix Single stranded

Transcription: The First Stage of

Transcription refers to the formation of RNA using the DNA sequence as its template. All types of RNA are formed through transcription of a gene, but only mRNA is further translated into protein. Messenger RNA (mRNA) is a transcript (copy) of a structural gene. The enzyme RNA polymerase synthesizes RNA according to the coding sequence of a gene.

Three phases of transcription (fig. 9.11):

1. Initiation – RNA polymerase recognizes the promoter3 region of a gene, binds to the DNA and starts to unwind it. As the DNA is unwound, the hydrogen bonds between nitrogenous bases “unzip” and the RNA polymerase begins synthesis of an RNA chain at the start codon (TAC which transcribes as AUG).

Therapeutics note: Rifamycins – class of drugs used to treat tuberculosis bind to RNA polymerases and prevent the initiation step of transcription. These drugs are selective for bacterial RNA polymerases over human forms of the protein.

2. Elongation – the mRNA assembly continues by addition of nucleotides that are complementary to the DNA template. (Note: uracil is substituted for thymine in RNA). As elongation of the RNA continues, the part of the DNA that has already been read “re-zips” and rewinds into a double helix and the growing RNA molecule “peels away” from the reformed double helix.

3. Termination – upon reaching specific DNA sequences in the gene, the RNA polymerase releases the DNA. The newly formed mRNA transcript is released and the DNA rewinds.

3 Promoter – a short sequence of nucleotides “upstream” of the actual start of the gene. These sequences provide a recognition and binding site for RNA polymerase. 5

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T=A T=A A A=T A=U T CG unzips CG G transcription via RNA polymerase T=A  T= A A GC G C A=T A=T T=A T=A GC GC GC GC CG CG T=A T=A CG CG T=A T=A

A U G A C U A C C G A G C G U U G A messenger RNA

Translation: The Second Stage of Gene Expression

After mRNA is transcribed, it then needs to be translated into whichever protein the molecule codes for. This is the essence of the flow of genetic information. For our purposes, one mRNA codes for one protein. Translation of mRNA into protein takes place on : protein complexes that serve as (i) docking stations for the mRNA transcript and tRNAs, and (ii) enzyme catalysis centers for the formation of peptide bonds between amino acids. Note: the ribosome is a frequent target of antibiotics (see below). A complete ribosome is composed of two principle protein subunits (one large; one smaller) and rRNA (fig. 9.12).

If you begin with the first three nucleotides of any mRNA – AUG – the message is then read in groups of three nucleotides, each referred to as a codon. Codons, or triplets, in the mRNA sequence each code for a specific amino acid, i.e. it takes at least 3 nucleotides to code for a single amino acid in the future protein; these groups of three nucleotides are called a codon.

For example, the mRNA sequence: AUGCCCGUAGGG is comprised of four codons: AUG, CCC, GUA, GGG these codons code for the amino acids (in order): Methionine, Proline, Valine, Glycine

The lists which codons code for each amino acid (fig. 9.13, pg. 267). Note: the code is redundant, i.e. for any given amino acid, more than one three letter code can serve for it. AUG, the start codon, is always the first codon of any mRNA. Also, there are three codons that do not code for any amino acid (UAA, UAG, and UGA); these are the stop codons (also known as nonsense codons) used to terminate the translation of proteins.

Transfer RNA (tRNA) molecules are folded strands of RNA that serve as carriers for amino acids. They bring the amino acids into close proximity on the ribosome “dock” so that a peptide bond can be formed between 6

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus them. On one end of the tRNA there is a sequence of three nitrogenous bases called the anticodon which is complementary to the sequence found in the mRNA. Thus, the sequence in the mRNA is translated to the amino acid carried by that particular tRNA. The tRNA with the anticodon UAC will always only carry a methionine. The tRNA with the anticodon CAU (the complement to the mRNA codon GUA) will always only carry the amino acid valine, etc. (fig. 9.14)

Three phases of translation: See fig. 9.15

1. Initiation begins when the small ribosome subunit binds to AUG of an mRNA and tRNA (AUG/UAC-met) enters the P site (on the ribosome) and binds to the start codon. A second tRNA binds to the A site and then the large subunit of the ribosome binds completing the initiation complex.

Therapeutics note: 4 The antibiotics erythromycin and spectinomycin prevent translation by interfering with mRNA attachment to ribosomes.

2. Elongation – after the initiation complex is complete, the two amino acids are covalently linked together in a dehydration synthesis reaction by the enzymatic activity of rRNA (built into the ribosome). The covalent bond between two amino acids is referred to as a peptide bond. Once the peptide bond forms between the first two amino acids, the tRNA in the P site is released and the ribosome shifts down. The tRNA which now has a dipeptide attached to it is in the P site and the A site is empty. A new tRNA (with an anticodon corresponding to the next mRNA codon exposed in the A site) binds to the ribosome, thus positioning the amino acid it is carrying close to the dipeptide. As a new peptide bond is formed, the tripeptide remains attached to the tRNA in the A site, the empty tRNA in the P site is released, and the ribosome once again shifts down so that the A site is vacant for the next tRNA. This process continues for the length of the mRNA molecule (which may be many thousands of nucleotides long).

Therapeutics note: Chloramphenicol, lincomycin, and tetracycline bind to the ribosome in a way that blocks the elongation of the polypeptide. Another whole class of antibiotics that work this way are the aminoglycosides (e.g. streptomycin, kanamycin, gentamycin).

3. Termination – When an mRNA sequence such as UAA, UAG, or UGA (stop codons) is encountered by the ribosome, there are no tRNAs to bind and the message is at its end. The ribosome separates into subunits and detaches from mRNA; the completed protein is released.

Note: Transcription and translation in prokaryotes can often take place simultaneously (fig. 9.16). As the newly formed mRNA is synthesized and peels away from the DNA, ribosomes complex with it and the translation process can start. Also, mRNAs can be long enough to be fed through many ribosomes at once, forming a polyribosomal complex which serves to speed up production of proteins (many proteins are thus produced from one mRNA transcript). In eukaryotes, transcription takes place within the nucleus, while translation of the mRNA takes place in the cytoplasm on ribosomes associated with the rough endoplasmic reticulum.

4 Used almost exclusively for Neisseria gonorrhoeae infections. 7

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Eukaryotic Transcription and Translation: Similar yet Different

Eukaryotic mRNAs are made in the nucleus where they are processed before being translated in the cytoplasm by the ribosomes. Eukaryotic mRNAs have their introns removed, and the exons are spliced together by spliceosomes. The final processed mRNA is exported out of the nucleus for translation (figure 9.17).

A reminder on the levels of protein structure:

The simplest way to think of a protein is as a chain of amino acids connected by peptide bonds (fig. 2.22). This would be the protein’s primary structure. However, proteins are not just “beads on a string”. Depending on the R-groups of each amino acid, the molecule will be able to form hydrogen and sulfur bonds resulting in complex secondary and tertiary structures. The three- dimensional structure of a protein is a function of its primary sequence in that it is that sequence which determines which R groups will be present. (See fig. 2.23)

 primary structure – is the sequence of amino acids  secondary structure - formation of hydrogen bonds result in β-pleated sheets and alpha helices (singular = α-helix).  tertiary structure - is a three-dimensional shape caused by the formation of sulfur and hydrogen bonds between amino acids more widely separated in the primary sequence. (The amino acids cysteine and methionine contain sulfur.)  quaternary structure - results from separate proteins associating to form large multi-polypeptide complexes

*Note: We will not cover section 9.3 Genetic Regulation of Protein Synthesis and Metabolism.

9.4 Mutations: Changes in the Genetic Code

A is a permanent change in the organism's genome. On the molecular level, a mutation is any change in the DNA sequence of a gene. (Table 9.4). Often, mutations are deleterious, i.e. they have a negative impact on the organism (e.g. cancer in humans). However, mutation is also one of the only ways new genes are created; thus are sometimes beneficial to the organism.

An organism that has a natural, non-mutated characteristic is called the wild type, and the organism that has the mutation is called the mutant strain.

Mutation in bacteria has led to the development of antibiotic resistant strains of nearly ALL known species.

Causes of Mutations

Spontaneous mutations can happen as a result of base pair mismatches during DNA replication or during a recombination event error.

Induced mutations are caused by physical and chemical agents called mutagens (ultraviolet light, nitrous acid, ethidium bromide, ionizing radiation, etc. See table 9.3).

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Categories of Mutations

Point mutations are small (one to few) base pair changes in DNA. A change in DNA sequence can change the amino acid that is coded. Types of point mutations are:  missense mutation: a mutation that codes for a different amino acid from the wild type sequence, can have various effects on the outcome.  nonsense mutation: a mutation that codes for a stop codon, produces a shorter, truncated protein.  silent mutation: the RNA sequence changes, but it still codes for the same amino acid because of the redundancy of the genetic code (figure 9.13).  back-mutation: when a gene has mutated back to its original composition.

Frameshift mutations occur when one or more bases are inserted or deleted from a DNA sequence (table 9.4).

Repair of Mutations

DNA has a light repair mechanism that can correct mutations as a result of exposure to ultraviolet (UV) light. Another repair mechanism system is excision repair, which removed mutated bases, and the correct complementary nucleotide is added.

Positive and Negative Effects of Mutations

Some mutations can cause the organism to die, or not be able to survive in its environment as well as the non- mutated organism. But, some mutations are beneficial to the organism giving them a ‘leg-up’, in the environment so they have a better chance of survival.

9.5 DNA Recombination Events

For any organism to adapt to new environments it is important to maintain . Genetic diversity means having different forms of genes (or combinations of genes). Most eukaryotic organisms have two means for increasing genetic diversity: mutation and exchange of DNA by sexual recombination of parental gametes. In order to increase their genetic diversity, many prokaryotes have evolved the ability to pick up foreign DNA through several unique ways; this creates more genetic variability than spontaneous mutations alone. Three methods of genetic recombination in bacteria are: conjugation, , and transformation. (Table 9.5)

While these forms of genetic recombination occur in nature, it is important to note that all three are also used in the laboratory. In this manner, biologists can shape a bacteria’s genome in order to study its physiology (and virulence as a pathogen). Manipulating bacterial genomes is also the basis for all recombinant genetic engineering and as such is the basis for all .

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Transmission of Genetic Material in Bacteria

1. Conjugation (fig. 9.22): This method requires cell-to-cell contact between 2 living cells of the same species: a donor cell and a recipient cell. It enables the passage of many genes; sometimes entire chromosomes can be transferred. Generally, it is not the entire chromosome but plasmids that are transferred.

Conjugative plasmids have genes that give bacteria the ability to transfer genes from one cell to another. One group of such plasmids are fertility factors or F factors (F plasmids). They give the bacteria the ability to produce a protein called pilin that assembles to form sex pili. A sex pilus is a hollow, tubular structure produced by a donor through which genes are transferred to the recipient cell.

Donor cells containing F factors are termed F+; recipient cells lacking the are F-. After the transfer of an F plasmid, the recipient cell becomes F+. While the process of transferring the ability to make pili seems unconstructive, the fact that the F plasmid occasionally becomes “entangled” with chromosome and thus transfers some of those genes as well, ensures a greater degree of genetic diversity. Also, while the conjugation tube is in place, smaller R plasmids can be transferred between cells independent of the F factor or pieces of chromosome.

2. Transformation (fig. 9.23 for Griffith’s experiment) Bacteria can pick up DNA from their environment (figure 9.24).

Griffith’s experiment Working in the late 1920s, English biochemist Frederick Griffith made a seminal discovery on how bacteria exchange DNA that was to provide the basis for determining DNA was the genetic material. He worked with two strains of Streptococcus pneumoniae, a bacterium which can cause pneumonia in humans and in mice. One strain, termed the S strain since it produced smooth colonies on agar plates, formed capsules and was deadly. The other, the R strain (its colonies were rough in appearance), lacked the capsule and thus was avirulent, i.e. was not capable of causing pneumonia. Using these two strains his experiment went as follows:

Mouse is exposed to: Result: live S strain (encapsulated Streptococcus pneumoniae) mouse dies live R strain (unencapsulated Streptococcus pneumoniae) mouse lives dead S strain (heat killed) mouse lives dead S strain + live R strain mouse dies

The question was: If both dead S strain and live R strain have no effect on the mice when used alone, why are they deadly when used in combination?

The answer: Small pieces of naked DNA from the killed bacteria can be taken up and incorporated into the chromosome of the living cells. Although the exact mechanism remains unknown, bacteria that acquire free DNA from their environment are said to be transformed; the process is transformation. Cells are competent (able to transform) during short periods of time during exponential

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BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus growth. DNA from dead bacteria (usually the small, stable R plasmids) can remain intact for some time in solution. In this way, genetic information from dead cells can be incorporated into living cells and increase genetic variability.

This talent of prokaryotes is unique. No other organisms on the planet can acquire the DNA of another species and express the proteins it codes for. It would be like you drinking the DNA of a zebra and waking up with black and white stripes.

The fact that bacteria can express proteins from other bacteria and even organisms from other kingdoms is used as the basis of all modern biotechnology. For instance, today all used by diabetics is made by bacteria. You simply clone the human insulin gene into a plasmid, transform E.coli with that plasmid, then grow the cells in large volumes. The bacteria don’t know they have a human gene inside them and transcribe and translate the gene and secrete human insulin into the media, from which it is easily purified.

3. Transduction Transduction is the transfer of bacterial genes from a donor cell to a recipient cell via a bacteriophage (a.k.a. “phage”) – a specifically adapted to target bacteria (fig 9.25 for generalized transduction). The phage invades a bacteria cell (donor cell). During viral replication, the bacterial chromosome fragments. As the new viral particles assemble themselves, pieces of bacterial chromosome are incorporated into some viruses. The donor cell ruptures (lyses) and releases the viruses. The bacteriophage with the bacterial chromosome from the donor cell attacks a second bacterium (the recipient cell) and injects the DNA it acquired from the donor. The transferred DNA can now insert itself into the chromosome of the recipient cell. This form of sexual recombination explains how bacteria incapable of conjugation acquire new R plasmids.

4. Transposons are also called “jumping genes” for their ability to hop, or transpose, from one piece of genetic material to another, or within the same genetic material. Often they carry antibiotic resistance genes. Transposons can harm the host DNA if it “jumps” into a critical gene (figure 9.27).

*Note: We will not cover section 9.6 The Genetic Code of Animal Viruses. We will cover this in chapter 6.

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