BACTERIAL GENETICS

The lecture prepared by Boena Dera-Tomaszewska, Ph.D. Medical University of Gdask Microbiology Division Department of Molecular Microbiology and Serology, National Salm onella Centre 2009/2010

GENETIC INFORMATION IN BACTERIA

(1)

Deoxyribonucleic acid (DNA)

Cytosine)

Oraganization of genetic information General concepts

stores genetic information as a sequence of nucleotide bases (Adenine, Thymine, Guanine, is generally double stranded, composed of complementary base pairs (A-T or G-C) joined by hydrogen bonds
The length of a DNA molecule is usually expressed in thousands of base pairs, or kilobase pairs (kbp). The single DNA molecule that forms the Escherichia coli chromosome is 4639 kbp.

 Ribonucleic acid (RNA)

GENETIC INFORMATION IN BACTERIA

(2)

transcribes and translates DNA-bound genetic instructions for protein synthesis is generally single stranded substitutes uracil for the thymine base used by DNA; the complementary base pairs for RNA are A-U or G-C is found in three types:
1. messenger RNA (mRNA); is the template that carriers DNA gene sequences to ribosomes, the site of protein synthesis 2. ribosomal RNA (rRNA); is a structural component of ribosomes; acts as a substrate for protein synthesis 3. transfer RNA (tRNA); carriers specific amino acids to the triplet encoded, mRNA-borne message that translates the message into the amino acid structure of proteins

GENETIC INFORMATION IN BACTERIA

Procaryotic genom

(3)

The bacterial genome is the total collection of genes carried by bacterium both on its chromosome and on its extrachromosomal genetic elemnts, if any. Bacteria usually have only one copy of their chromosomes (and therefore a single copy of each gene), they are therefore haploid. The chromosome of a typical bacterium is a single, double-stranded, circular molecule of DNA (covalently closed circular structure), containing from 580 kbp to more than 4600 kbp. Bacteria may also contain extrachromosomal genetic elements: plasmids or bacteriophages. They are independent of bacterial chromosome and can be transmitted from one cell to another. They are nonessential replicons which often determine resistance to antimicrobial agents, production of virulence factors (bacteriocins, toxins, cell surface structures required for adherence or colonization), or other functions.

GENETIC INFORMATION IN BACTERIA

(4)

Each genome contains many operons, which are made up of genes. Operons are groups of one or more structural genes expressed from a particular promoter and ending at a transcriptional terminator. Genes are sequences of nucleotides that have a biologic function; examples are protein-structural genes, ribosomal RNA genes, and recognition and binding sites for other molecules (promoters and operators). Promoters and operators are nucleotide sequences that control the expression of a gene by influencing which sequences will be transcribed into messenger RNA (mRNA).

DNA REPLICATION (1)

The bacterial chromosome is a circular molecule of DNA that functions as a self-replicating genetic element (replicon). Before a cell can divide, it must duplicate all its DNA. Replication of chromosomal DNA in bacteria starts at a specific chromosomal site called the origin. New DNA synthesis occurs at growing replication forks and proceeds bidirectionally until the process is completed. Replication is complete when the two replication forks meet 180 degrees from the origin.

DNA REPLICATION (2)

In general, DNA is replicated by uncoiling of the helix, strand separation by breaking of the hydrogen bonds between the complementary strands, and synthesis of two new strands (leading and lagging) by complementary base pairing.

DNA REPLICATION (3)

DNA replication is semiconservative
When the replication process is complete, two DNA molecules identical to each other and identical to the origin have been produced. This mode of replication is described as semiconservative: one-half of each new molecule of DNA is old, one-half new.

GENE EXPRESSION (1) Expression of genetic determinants in bacteria involves the unidirectional flow of information (DNA RNA protein). Gene expression occurs in two steps:
TRANSCRIPTION of the information encoded in DNA into

molecule of RNA (DNA RNA) TRANSLATION of the information encoded in the nucleotides of mRNA into a defined sequence of amino acids in a protein (RNA protein).

GENE EXPRESSION (2)

TRANSCRIPTION :
DNA is copied into mRNA (or tRNA or rRNA) RNA polymerase binds to the promotor and synthesizes mRNA from the strand of DNA Many copies of mRNA are made (as the first polymerase moves downstream another one binds, etc.) RNA polymerase drops off when it hits the termination region

In complementary base pairing, T, C, G, and A on the template DNA strand specify A, G, C, and U, respectively, on the RNA strand being synthesized.

GENE EXPRESSION
1. Initiation

(3)

The steps of TRANSLATION The small subunit of the ribosome binds to mRNA at a AUG start codon region Met-tRNA (initiator tRNA) binds to start codon Large ribosomal subunit binds and completes the initiation complex In the initiation complex: Met-tRNA is bound to AUG on mRNA in the P site of the ribosom; the next codon is positioned in the A site
P site = peptydyl-tRNA binding site A site = aminoacyl-tRNA binding site

Note: The initiator tRNA is the only member of the tRNA family that can bind directly to the P site. The P site is so-named because, with the exception of initiator tRNA, it binds only to a peptidyl-tRNA molecule; that is, a tRNA with the growing peptide attached. The A site is so-named because it binds only to the incoming aminoacyl-tRNA, that is the tRNA bringing the next amino acid. So, for example, the tRNA that brings Met into the interior of the polypeptide can bind only to the A site.

GENE EXPRESSION
2. Elongation

(4) The steps of TRANSLATION

Another tRNA able to base pair with the next codon on the mRNA arrives at the A site The preceding amino acid (Met) is covalently linked to the incoming amino acid with a peptide bond The initiator tRNA is released from the P site The ribosom moves one codon downstream This shifts the more recently-arrived tRNA (with its attached peptide) to the P site and opens the A site for the arrival of a new tRNA Process repeats, one amino acid being added each time the ribosome moves

GENE EXPRESSION

(5)

The steps of TRANSLATION 3. Termination The end of the message is marked by one or more STOP codons (UAA, UAG, UGA) There are no tRNA molecules with anticodons for STOP codons However, a protein release factor recognizes these codons when they arrive at the A site Binding of this protein releases the polypeptide from the ribosome; the uncharged tRNA is released from the P site The ribosome separates from the mRNA and splits into its subunits, which can later be reassembled for another round of protein synthesis

Regulation of gene expression (1)

Gene regulation can occur at three possible places in the production of an active gene product. 1. The transcription of the gene can be regulated this is known as transcriptional regulation 2. If the gene encodes a protein, it can be regulated at the translational level this is known as translational regulation 3. Gene products can be regulated after they are completely synthesized by either post-transcriptional or posttranslational regulation mechanisms

Regulation of gene expression (2)

Initiation of transcription may be under positive or negative control.

positive control
-is initiation of transcription in response to the binding of an activator protein Genes which expression is under positive control are not transcribed unless an active regulator protein, called an apoinducer, is present. The apoinducer binds to a specific DNA sequence and assists the RNA polymerase in the initiation steps.

negative control
-is inhibition of transcription by binding of a repressor protein
(is exemplified by the lac and trp operons)

Genes under negative control are expressed unless they are switch off by a repressor protein. This repressor protein prevents gene expression by binding to a specific DNA sequence, called the operator, making it impossible for the RNA polymerase to initiate transcription at the promotor.

An operon is a group of genes physically linked on the chromosome and under the control of the same promoter(s)

Regulation of gene expression (3)

OPERONS CAN BE
inducible repressible

Introduction of a substance (inducer) into the growth medium may induce operon to increase the expression of the enzymes necessary for its metabolism [e.g. the lactose (lac) operon]

The end product (co-repressor) of a biosynthetic pathway may signal that a pathway should be shut down or repressed by reducing the synthesis of its enzymes [e.g. the tryptophan (trp) operon]

The lactose (lac ) OPERON

The lactose (lac) operon encodes three enzymes needed for the lactose degradation. That operon is transcribed as a mRNA from the promoter (P) and translated into three proteins: -galactosidase (Z), permease (Y) and transacetylase (A). The lac repressor protein is product of regulatory gene (lac I) in separate regulatory operon.

The lac operon regulation

(a)

The lac operon is an inducible operon under negative and positive regulation (by heaving two mechanisms of controlling the expression of lac Negative regulation: the lac repressor is said to negatively regulate expression of the lac genes because the operon is turned off by the regulatory protein. The presence of lactose* (and thus allolactose=structural isomer of lactose) determines whether or not the lac repressor is bound to the operator.
operon, bacterial cells ensure that the lac gene products are only made when needed).

* lactose itself does not influence regulation of transcription; this function is served by allolactose, which is the inducer of the lac operon

The lac operon regulation

(b)

Positive regulation: glucose is the preferred and most frequently available energy source for bacteria. Whenever glucose is present bacteria metabilize it before using alternative energy sources such as lactose, arabinose, galactose and maltose. So, normally the bacteria use glucose and not lactose. The enzymes to metabolize glucose are made constantly. The cell will use all of the glucose before the lac operon is turned on. To prevent lactose metabolism (when glucose is present), a second level of control of gene expression exists a proteinmediated, positive control mechanism. The catabolite activator protein (CAP) is said to be a transcriptional activator because its presence is required for efficient transcription of the lac operon.

The lac operon regulation

(c)

How does the system work? The presence or absence of glucose affects the lac operon by affecting the concentration in cyclic adenosine monophosphate (cAMP) which is inversely proportional to the concentration of glucose: as the concentration of glucose decreases, the concentration of cAMP increases. A protein called the catabolite activator protein (CAP) forms a complex with cAMP, acquiring the ability to bind to the promoter. The CAP-cAMP complex enhances binding of the RNA polymerase to the promoter, thus allowing an increase in the frequency of tanscription initiation. The complex CAP-cAMP exerts a positive control over the expression of the lac operon by enhancing RNA polimerase activity.

The tryptophan (trp ) OPERON

Different mechanisms of control are found in many genes for enzymes involved in the biosynthesis of amino acids, such as tryptophan.

The tryptophan (trp) operon contains five structural genes that code for enzymes involved in the synthesis of tryptophan. These genes are transcribed from a common promoter into a mRNA which is translated to the five proteins needed for the three tryptophan biosynthetic enzymes. The trp regulatory gene encoding the Trp repressor is not located near the trp operon.

The trp operon regulation

Although tryptophan is essential for protein synthesis, too much tryptophan in the cell can be toxic, therefore its synthesis must be regulated. The tryptophan (trp) operon is a repressible operon under dual transcriptional control mechanisms: at the DNA level the trp repressor protein is said to negatively regulate expression of the trp genes, because the operon is turned off by the regulatory protein (negative control) at the mRNA level in a process termed attenuation, in which mRNA synthesis is prematurely terminated (attenuation mechanism control).

Negative control

The effect of tryptophan on expression from the trp operon

in the absence of co-repressor

(tryptophan) the repressor is inactive and does not bind to the operator, resulting in transcription of the structural genes.

in the presence of the co-repressor

(tryptophan) the conformation of the inactive repressor is changed; the repressor becomes active and binds to the operator, resulting in repression of transcription of the trp mRNA by the RNA polymerase

Attenuation mechanism control (a) The trp operon is also under control of attenuation mechanism. Upstream of the structural genes are the promoter (P), the operator (O) and a leader (L), which can be transcribed into a short (14 amino acids) peptide containing two tryptophans near its distal end.

Attenuation mechanism control (b)

The leader mRNA possesses four domains (1,2,3 and 4), which can pair (forming a double-stranded RNA loops) differently according to the tryptophan availability, leading to an early termination of transcription of the trp operon or its full transcription. The formation of one stem-loop structure (hairpin) can preclude the formation of others. If domain 2 forms base pairs with domain 1 it is not available to base pair with domain 3. Similarly if domain 3 forms base pairs with domain 2 it is not available to base pair with domain 4. Thus three possible stem/hairpin structures can be formed in the RNA domain 1:domain 2 (a pause hairpin), domain 2:domain 3 (an antiterminator hairpin) and domain 3:domain 4 (a terminator hairpin).

MUTATIONS (1)
Mutation is any change in the base sequence of the DNA.
Many mutations occur spontaneously in nature. Spontaneous mutation occurs naturally about one in every million to one in every billion divisions and is probably due to low level natural mutagens normally present in the environment. Induced mutations are caused by mutagens, substances that cause a much higher rate of mutation. Mutations can be induced by physical or chemical agents.
Among the physical agents used to induce mutations in bacteria are: heat,

ultraviolet light, X-rays and gamma rays.

Chemicals that are mutagens can be grouped into three classes:

1. nucleotide-base analogues (they are incorporated into the DNA during replication and lead to mispairing and frequent mistakes during DNA replication), 2. polycyclic flat molecules (e.g., ethidium bromide or acridine derivatives; they are examples of frameshift mutagens), 3. DNA-reactive chemicals (they act directly on the DNA, resulting in a modification of the normal base into a chemically different structure).

MUTATIONS (2)
Spontaneous mutation
1. Mechanisms of mutation a). Substitution of a nucleotide (point mutations): substitution of one nucleotide for another during DNA replication. This is the most common mechanism of mutation (a, b, c). b). Deletion or addition of a nucleotide (frameshift mutations): deletion or addition of a nucleotide during DNA replication (d). 2. Results of mutation One of four things can happen as a result of these mechanisms of mutation and the resulting change in the deoxyribonucleotide base sequence mentioned above:
a. a missense mutation occurs b. a nonsense mutation occurs c. a silent (sense) mutation occurs d. a frameshift mutation occurs

MUTATIONS (3)
A missense mutation This is usually seen with a single substitution mutation and results in one wrong codon and one wrong amino acid. A nonsense mutation If the change in the nucleotide base sequence results in transcription of a stop or nonsense codon, the protein would be terminated at that point in the message. A silent mutation This is sometimes seen with a single substitution mutation when the change in the DNA base sequence results in a new codon still coding for the same amino acid. A frameshift mutation A small deletion or insertion that is not in multiples of three produces that kind of mutation. This results in a change in the reading frame, usually leading to a nonsense peptide and premature truncation of the protein.

MUTATIONS (4)
NULL MUTATION Null mutations, which completely destroy gene function, arise when there is an extensive insertion, deletion, or gross rearrangement of the chromosome structure. Insertion of long sequence of DNA (many thousands of base pairs) by recombination, by transposition, or during genetic engineering can produce null mutations by separating the parts of a gene and inactivating the gene. For example, a null mutation in a gene that usually encodes a specific enzyme leads to the production of a nonfunctional enzyme or no enzyme at all.

Repair mechanisms of DNA

They can be divided into five groups:
Direct DNA repair is the enzymatic removal of damage, such as
pyrimidine dimers and alkylated bases. followed by synthesis of a new DNA strand (two types of excision-repair mechanisms exist: generalized and specialized)

Excision repair is the excision of a DNA segment containing the damage, Recombinational or postreplication repair is the retrieval of missing The SOS response is the induction of many genes (approximately 15)
after DNA damage or interruption of DNA replication.

information by genetic recombination when both DNA strands are damaged.

Error-prone repair is the last resort of a bacterial cell before it dies. It is

used to fill in gaps when a DNA template is not available for directing an accurate repair.

Exchange of genetic information

Genetic exchange is one mechanism by which new genotypes of species are formed (mutation is the other one). Part of the genetic material of a donor cell can be transferred to a recipient cell. After the transfer, recombination between the donor and recipient DNA may occur followed by succeeding cell division.

Genetic exchanges among bacteria occur by several mechanisms: TRANSFORMATION - the transfer of naked DNA TRANSDUCTION - the transfer of genetic information through bacterial
viruses (bacteriophages):
a. generalized transduction - mediated by lytic phages (each part of the bacterial genome has approximately the same probability of being transferred from donor to recipient bacteria) b. specialized transduction - mediated by specific temperate phages (only a few specific donor genes can be transferred to recipient bacteria)

CONJUGATION
Animations available at:
Point G (2,3,4)

- the passage of plasmids through direct physical contact

between two bacteria (F+conjugation; Hfr conjugation; R plasmid
conjugation) http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/index.html

TRANSFER OF DNA BETWEEN BACTERIAL CELLS

Comparison of conjugation, transduction, and transformation
Transfer procedure Conjugation Transduction Process
DNA transferred from one bacterium to another DNA transferred by a virus from one cell to another

Type of cells involved

procaryotic

Nature of DNA transferred

chromosomal or plasmid

procaryotic

any gene in generalized transduction; only certain genes in specialized transduction any DNA

Transformation purified DNA taken up

must be at least 500 nucleotides in length)

by a cell (DNA molecules

procaryotic or eukaryotic
(e.g., human)

GENETIC ENGINEERING
The basic tools of genetic engineering are: cloning vectors (which can be used to deliver the DNA sequences into receptive
bacteria and amplify the desired sequence)

restriction enzymes (which are used to cut DNA reproducibly at defined

sequences, e.g.: BamHI, EcoRI, HindIII, AccI, HincII, PstI, SmaI, Sau3AI, XmaI)

DNA ligase (the enzyme that links the fragment to the cloning vector).

Genetic engineering has been used to isolate and express the genes (in bacteria, yeast or insects cells) for useful proteins such as insulin, interferon, growth hormones and interleukin. Large amount of pure immunogen for vaccine can be prepared without the need to work with the intact disease organisms. Gene therapy is one of the many applications of genetic engineering it can be used to treat or prevent diseases. Recombinant DNA technology has also become essential to laboratory diagnosis, forensic science, agriculture and many other disciplines.

References
1. 2. 3. 4.

Patric R. Murray et al., Bacterial Genetics. In: Medical Microbiology 4th ed., Chapter 5, Mosby, Inc., U.S., 2002, pp. 35 46 Gary E. Kaiser, Microbial genetics and microbial matabolism, Unit 4: BIOL 230 Lectures, http://student.ccbcmd.edu/courses/bio141/lecguide/unit6/index.html Gene Mayer, Genetic Regulatory Mechanisms. MBIM Microbiology and Immunology on-line, Bacteriology, Chapter Nine, University of South Carolina.
http://www.med..sc.adu.85/mayer/geneticreg.htm

PHSchool Home: DNA Structure and Replication. From Gene to Protein: Transcription. From Gene to Protein: Translation. The lac Operon in E.coli . Pearson Edducation, Inc. Publishing as Pearson Prentice Hall.
http://www.phschool.com/science/biology_place/biocoach/index.html

5. 6. 7. 8. 9.

http://www.blackwellpublishing.com/trun/pdfs/Chapter12.pdf

Randall K. Holmes, Michael G. Jobling, Genetics. In: Medical Microbiology, Chapter 5, Section I, Bacteriology. http://www.gsbs.utmb.edu/microbook/ch005.htm Nuncy Trun, Janine Trempy, Gene Expression and Regulation. In: Fundamental Bacterial Genetics, Chapter 12, Bleckwell Publishing, pp. 191-212 M. Tevfik Dorak, Viral and Bacterial Genetics. Genetic Engineering.

http://dorakmt.tripod.com/genetics/notes08.html Microbial Genetics, http://www.2.hawaii.edu%7Ejohnb/micro/m130/m13lect9.html

Geo.F.Brooks, Janet S.Butel, Stephen A.Morse, Microbial Genetics. In: Jawtz, Melnick, & Adelbergs Medical Microbiology, twenty-third edition, Chapter 7, McGraw-Hill Companies, Inc., U.S., 2004, pp. 96-118

Images accompanying that lecture have been acquired from numerous sources. My thanks to everyone who has shared images with me for teaching purposes.

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