Bacterial genetics Genetics Genetics is the study of

Bacterial genetics

Genetics • Genetics is the study of biologically inherited traits • It study of what genes are, how they carry information, how they are replicated and passed to subsequent generations of cells • how the expression of genetic information within an organism determines the particular characteristics of that organism

I. Structure And Functions Of The Genetic Material

Nucleic acids structure • There are two major classes: I. Deoxyribonucleic acid (DNA) : The most common macromolecule that encodes genetic information II. Ribonucleic acid (RNA) : plays an essential role in several genetic processes • Nucleotides: Nucleotides are the structural units of nucleic acids. Nucleotides are named according to their nitrogenous base.

Parts of Nucleotide • Each nucleotide has three parts: 1. A nitrogen base: . • Pyrimidines: involving thymine (T), cytosine (C), and uracil (U), • Purines : including adenine (A) and Guanine (G), 2. Deoxyribose (DNA) or ribose (RNA) sugar 3. A phosphate group

I. Deoxyribonucleic Acid (DNA) Structure • Double helix: consists of two long strands wrapped around each other • Base pairing: o o A is always paired with T by two hydrogen bonds, G is always paired with C by three H bonds. • Complementary : each strand act as a template for synthesis the another strand • Antiparallel: One strand is oriented in the 5’ to 3’ direction and its complement is oriented in the 3’ to 5’ direction.

II. Ribonucleic Acid (RNA) • • Single-stranded molecule, shorter than DNA It has ribose sugar rather than deoxyribose. The base uracil (U) replaces thymine (T) in DNA. Three major kinds o messenger RNA (m. RNA), o ribosomal RNA (r. RNA), o transfer RNA (t. RNA). • Small interfering RNA (si. RNA) : o A new class of RNA o Short (20– 25 nts), double-stranded o Act as regulators for gene expression

II. Flow of genetic information

How the genetic information is stored in the DNA ? • Genetic information is stored in DNA as a code. • The unit of code is known as codon. • Consists of a sequence of three bases such as ATG. • Each codon codes for a single amino acid, but more than one codon may exist for a single amino acid.

How the genetic information is stored in the DNA ? • It is first transferred (transcribed ) from one DNA strand to m. RNA strand • Then the m. RNA base sequences serve as a template for amino acid sequences in the polypeptide (during translation process).

The gene • Gene is a segment of DNA carrying a number of codons specifying for a particular polypeptide or RNA. • Composed of two main regions o promoter region (recognised by RNA polymerase to start transcription process o Reading frame (or coding region), contain the codons that will be translated for a particular polypeptide or RNA • A large number of genes constitute a locus and a large number of loci constitute cell genome. • Size of the gene and entire genome is expressed in the number of base pairs (bp) or kilobase pairs (kbp) (1 kbp = 1, 000 base pairs).

Bacterial chromosome • it is a single doublestranded molecule of DNA arranged in a circular form. • containing from 580 kbp to more than 5220 kbp of DNA • It is not separated from the cytoplasm be a membrane as in eukaryotes (no nucleus). • The essential genes that are necessary for cell viability (house keeping genes) are carried on the chromosome • Some bacterial species (pathogenic bacteria) possess specific genes for pathogenic determinants. • These genes are often clustered together in the DNA and are referred to as pathogenicity islands (PIs). • These gene segments encode a collection of virulence factors that are important for pathogenesis, including, antibiotic resistance, adhesins, invasins, and exotoxins.

Non-chromosomal elements of genome • Not all genes in a given cell are confined in the chromosome • Many genes are located on plasmids and transposable elements • These elements can replicate and encode information for production of cellular products • They are not as stable as a chromosome (what does that mean ? )

I. Plasmids • double-stranded circular DNA molecules • separated from the bacterial chromosome. • Replicating independently of the chromosome • Their number per bacterial cell varies from a few to thousands • Their sizes range from a few thousand base pairs to more than 100 kbp • May be incorporated into the chromosome

I. Plasmids • Many plasmids carry genes that code for certain phenotypic characteristics of the host cell. The following plasmid types are medically relevant: § Virulence plasmids. Carry determinants of bacterial virulence, e. g. , enterotoxin genes or hemolysin genes. • Resistance (R) plasmids. Carry genetic information bearing on resistance to anti-infective agents. • R plasmids may carry several R genes at once • Some plasmids have also been described that carry both virulence and resistance gene

II. Transposable elements • Pieces of DNA that move from one genetic elements to another • Do not exist as separate entities within the bacterial cell, must be incorporated to a plasmid or chromosome • 2 types: 1. insertion sequence (IS): contains genes that simply encode for information required for movement among plasmid and chromosome 2. transposons: contains genes for movement as well as genes that encode for other feature such as antibiotic resistance.

A. Structure of DNA transposons. Two inverted tandem repeats (TIR) flank the transposase gene. Two short tandem site duplications (TSD) are present on both sides of the insert. B. Mechanism of transposition: Two transposases recognize and bind to TIR sequences, join together and promote DNA double-strand cleavage. The DNA-transposase complex then inserts its DNA cargo at specific DNA motifs elsewhere in the genome, creating short TSDs upon integration. [

Model of a Hypothetical Conjugative Multiple-Resistance Plasmid • Tn 21, codes for resistance to sulfonamides (sul. I) • In 4 Codes for chloramphenicol acetyltransferase (= cml. A), • Tn 3 Transposon 3; codes for a betalactamase (= bla). • Tn 10 Transposon 10; codes for resistance to tetracyclines (= tet). • rep. A Codes for the replication enzyme of the plasmid. • tra containing 25 tra genes; responsible for the transfer and replication process

The central dogma of molecular biology • The flow of information from DNA to RNA to protein is often referred to as the central dogma of molecular biology • It refers to three major processes include: i. Replication ii. Transcription iii. Translation

i. DNA replication Semiconservative: • each strand serves as a template for synthesizing new DNA strands • Bidirectional: • begins at one point (ori locus) and moves in both directions • Terminated at termination site (ter) in the opposite to ori site.

ii. Transcription • The DNA acts as a template for the transcription of RNA by RNA polymerase • The bases in m. RNA will be complementary to one ( template) strand of DNA Adapted from: http: //www. mun. ca/biology/desmid/brian/BIOL 2060 -21/21_09. jpg Adapted from : https: //courses. lumenlearning. com/microbiology/chapter/rna-transcription/

iii. Translation • The process requires three major components m. RNA, ribosomes, and t. RNAs in addition to various accessory proteins. • The m. RNA carries the coded information for making specific proteins from DNA • Ribosomes: the sites of translation, their structure facilitates the joining of one amino acid to another. • t. RNA: contains (anticodon) at one end amino acid at the other end. • Different amino acid sequence form different protein. Adapted from : http: //hyperphysics. phy-astr. gsu. edu/hbase/Organic/translation. html

Regulation of Gene Expression • Bacteria demonstrate an impressive capacity for adapting to their environment. • A number of regulatory bacterial mechanisms are known, for example posttranslational regulation, transcription termination, and quorum sensing • The most important is regulation of the initiation of transcription by means of activation or repression • Ex. The lac peron

The lac operon • Lactose fermentation requires three enzymes coded by structural genes Lac Z, Lac Y and Lac A of Lac operon respectively • The operon is composed of: 1) The operator, which is a sequence of bases that controls the expression (transcription) of the structural genes 2) The promoter, where the RNA polymerase binds and transcribes the structural genes. • A distant Regulatory gene (not part of the operon) codes for a repressor protein , which is a protein molecule that can combine with either operator region on the chromosome or with the inducer (lactose). The structure of lac operon

• For transcription to occur , RNA polymerase has to attach to DNA at a promoter region and transcribe the DNA. • when lactose (inducer) is not present in the medium, repressor molecule is bound to the operator, preventing the passage of RNA polymerase from promoter to the structural genes. • The repressor molecule has an affinity for lactose, in the presence of which it leaves the operator region free enabling the transcription to take place. • When lactose present is completely metabolized, the repressor again attaches to the operator, switching off transcription.

The Genetic Variability of Bacteria Molecular Mechanisms: 1 - mutations 2 - Intercellular transfer of genomic material

1. Mutation • It is random, undirected, heritable variation caused by an alteration in the nucleotide sequences at some points of the DNA of the cell due to addition, deletion or substitution of one or more bases • during DNA replication, some ‘errors’ may occur during copying the progeny strands • For instance, instead of thymine bonding to adenine, it binds to guanine • Different mutations in the a particular gene may produce different effect in the cell

Types of Mutation • Mutations can be divided conveniently into: A. Spontaneous mutation: occur spontaneously in nature in the absence of any mutation-causing agents. B. Induced mutation: enhanced by exposure of cells to several agent (mutagens) include : 1. Physical agents: (i) UV rays; (ii) lonising radiation, e. g. X-rays 2. Chemical agents: Alkylating agents C. Point mutations: • affect just one point (base pair) in a gene. • Leads to change to or substitution of a different base pair. Alternatively, can result in the deletion or addition of a base pair. • It is in general, reversible and is of two classes:

1 - Base pair substitution: • • Depending on the placement of the substituted base silent mutation , cause no change when the m. RNA is translated missense mutation , lead to the insertion of the wrong amino acid nonsense mutation , lead to generate a stop codon and prematurely terminating the polypeptide • 2 - Frameshift mutations (insertion or deletion): • If the number of bases inserted or deleted is not a multiple of three, there will be shift in the reading frame (codons sequence), forming newest of triplet codon. • the new codons will specify the incorrect amino acids, or leading to premature termination for the translation process (truncated protein)

2 - Intercellular transfer of genomic material • DNA can be transferred from one organism to another, and that DNA can be stably incorporated in the recipient, permanently changing its genetic composition. • This Process is called horizontal gene transfer to differentiate it from the inheritance of parental genes, a process called vertical inheritance. • Three broad mechanisms mediate efficient movement of DNA between cells— conjugation, transduction, and transformation.

Conjugation • A process in which one cell, the donor or male cell, makes contact with another, the recipient or female cell • DNA is transferred directly from the donor into the recipient • Certain types of plasmids, known as transfer factors or sex factors, carry the genetic information necessary for conjugation to occur. • These are also called self-transmissible plasmids, and they can mobilize other plasmids or portions of the chromosome for transfer. • Only cells that contain such a plasmid can act as donors; those lacking a sex factor act as recipients. • Such Plasmid codes for specialized fimbria (sex pilus) which projects from the surface of the cell. • The tip of the pilus attaches to the surface of a recipient cell and holds the two cells together so that DNA can then pass into the recipient cell. It A male and a female cell joined by an F pilus (sex pilus).

1. Plasmid transfer • Bacterial Populations can be divided into two types of cells. 1. the donor cell, contains an F or fertility plasmid and is designated F+. 2. the recipient cell, does not contain this plasmid and is called F–. • DNA is transferred only in one direction, from F+ to F– • F Factor (Fertility Factor) • is a transfer factor that contains the genetic information necessary for the synthesis of the sex pilus (Conjugation tube), and for self-transfer. • Cells that contain the F plasmid free in the cytoplasm (F+ cells) have the ability to produce F pili and to transfer the F plasmid to F– cells by conjugation.

Mechanism of DNA transfer during conjugation. A. Connection between two bacterial cells by means of sex pili. . B. Formation of a specific conjugal bridge between donor cell and receptor cell. C. An endonuclease cleaves one strand of the circular DNA double helix at a specific point. The single strand with the “leader region” enters the recipient cell. D. The double-stranded structure of both the transferred single strand the remaining DNA strand is restored by means of complementary DNA synthesis. • The recipient cell, now plasmid-positive, is called a transconjugant

2. Chromosomal transfer • The F factor in some cases can be integrated into the host chromosome in a very small proportion of F+ cells, creating high frequency recombination (Hfr) donor cells • In which, the entire chromosome behaves like an F+ plasmid, and hence, chromosomal genes (fragments) can be transferred (from the site of insertion) in a direction determined by the orientation of insertion in the normal sex factor manner to a recipient cell at a relatively high frequency

Medically Important Factors Transferred by Conjugation 1. Colicinogenic (Col) Factor • Several strains of coliform bacteria produce colicins— antibiotic-like substances which are specifically and selectively lethal to other enterobacteria. • Colicin production is determined by a plasmid called the Col factor, which resembles the F factor in promoting conjugation, leading to self-transfer and, at times, transfer of chromosomal segments. 2. Resistance Factors or R Plasmids • Resistance factors (R factors) are plasmids that have significant medical importance as it leads to the spread of multiple drug resistance among bacteria. • This R plasmid consists of two components: RTF+r determinants. • Resistance transfer factor (RTF): The transfer factor that is responsible for conjugal transfer. • Resistance determinant (r): code for resistance against various drugs. • The whole plasmid (RTF+r determinants) is known as the R factor • An R factor can have several r determinants, and resistance to as many as eight or more drugs can be transferred simultaneously.

Transduction • The transfer of a portion of the DNA from one bacterium to another by a bacteriophage • Bacteriophages are viruses that parasitise bacteria and consist of a nucleic acid core and a protein coat. • Bacteriophages exhibit two types of life cycle. • (1) Virulent or lytic cycle; • (2) Temperate or nonlytic cycle: Virulent or lytic cycle: • virus attached to the bacterial surface, inject its DNA inside the bacterial cell , its DNA then controls the bacterial cell activity to synthesis phage particles • large numbers of progeny phages are built up inside the host bacterium, which ruptures to release them.

• During the assembly of bacteriophage progeny inside infected bacteria, ‘packaging errors’ may occur occasionally. • A phage particle may have at its core a segment of the host DNA besides its own nucleic acid. • When this particle infects another bacterium, DNA transfer is effected and the recipient cell acquires new characteristic coded by the donor DNA. • Bacterial genes have been transduced by the phage into the second cell

Temperate or nonlytic cycle • In the temperate or nonlytic cycle, the host bacterium is unharmed. • The phage DNA becomes integrated with the bacterial chromosome as prophage, and is replicated stably as part of the host cell chromosome and is transferred to the daughter cells. • This process is called lysogeny and bacteria harboring prophages are called lysogenic bacteria. • the prophage behaves as an additional segment of the bacterial chromosome, coding for new characteristic

Medical Importance of transduction • 1. Toxigenicity in diphtheria bacilli: Of great medical importance is the lysogenic conversion in diphtheria bacilli, which acquire toxigenicity (and therefore virulence) by lysogenization with the phage beta. Elimination of the phage from a toxigenic strain renders it nontoxigenic. • 2. Production of staphylococci, streptococci and clostridia toxins: It is probable that the production of many toxins by staphylococci, streptococci and clostridia is also dependent upon lysogenic conversion by specific bacteriophages.

Transformation Is the uptake of exogenous DNA by living bacteria from surrounding environment Bacterial cells that have such ability called competent cells Competence can be naturally developed or induced artificially. Natural competence is unusual among bacteria, and some of these strains are require the presence of competence factors, produced only at a specific point in the growth cycle • competence factor (an activating protein) is released by competent cell to induce competence in non- competent cells. • Naturally competent bacteria are found in very few genera include Bacillus subtilis, Haemophilus influenzae, Neisseria gonorrhoeae, Neisseria meningitidis, and Streptococcus pneumoniae. • •

The main steps of bacterial transformation are: Transforming DNA + Competent cells Binding Resistance to exogenous Dnase Fragmentation and uptake Integration Adapted from : http//www. lamission. edu>lifesciences> chapter 8: microbial genetics Expression

Reference • Jawetz, M. & Adelberg’s. 2013. Medical Microbiology , Twenty-Sixth Edition. The Mc. Graw-Hill Companies, Inc. USA • Kumar, S. 2012. Textbook of microbiology. Jaypee Brother Medical Publishers (P) Ltd. New Delhi, India. • Kayser, F. H. , Bienz, K. A. , Eckrt, J. and R. M. Zinkernagel. 2005. Medical Microbiology. Georg Thieme Verlag. Stuttgart, Germany. • Passarge, E. 2001. Color atlas of genetics. Georg Thieme Verlag. Stuttgart, Germany. • http//www. lamission. edu/lifesciences/chapter 8: Microbial genetics