Microbial Genetics Dr. Kawther Aabed

Prof. in Microbiology

Study Objectives !  Structure and function of genetic material

DNA and chromosomes

DNA Replication

RNA and protein Synthesis

Transcription

Translation

!  Regulation of bacterial gene expression

!  Mutation

!  Genetic transfer and recombination

!  Molecular analysis methods

Why microbial genetics? ! Microbes are simple, grow fast and easily in

the lab.

! Bacterial haploidy allows the expression of mutations immediately.

! Understanding genetics of microorganisms easy controlling them.

! Cloning of many eukaryotic genes occurs in microbes.

Three Domains of Life

The Svedberg unit (S) offers a measure of particle size based on its rate of travel in a tube

Structure and function of genetic material

DNA Gene

!!Chromosome

Genome

Hierarchical Organisation of the Genome منظمة هرمية من اجلينوم

Genome !  The sum total of genetic material of a cell

!  Mostly in chromosomes

! In cells- DNA (that functions as a self-replicating genetic element) (replicon).

! In Bacterial viruses (bacteriophages or phages) can be either DNA or RNA

!  Can appear in non chromosomal sites (Extrachromosomal genetic elements)

! plasmids and bacteriophages (are nonessential replicons which often determine resistance to antimicrobial agents, production of virulence factors, or other functions)

Chromosome •  cellular structures made up of genes that carry

genetic info •  Eukaryotic chromosomes

– DNA molecule tightly wound around histone proteins

– Located in the nucleus – Can occur in pairs (diploid) or singles (haploid) – Appear linear

•  Bacterial chromosomes – Condensed and secured by means of histone-like

proteins – Single, circular chromosome

Gene !  Gene is the basic physical and functional unit of heredity

!  Genes, which are made up of DNA

! Structural genes: code for proteins or code for RNA

! Regulatory genes: control gene expression

!  Sum of all genes is an organism�s genotype

!  The expression of the genotype creates traits which make up the phenotype. Some genes may not be expressed in the phenotype

!  genes vary in size from a few hundred DNA bases to more than 2 million bases. The Human Genome Project has estimated that humans have between 20,000 and 25,000 genes.

DNA Deoxyribonucleic acid

!  DNA was first identified in the late 1860s by Swiss

chemist Miescher

! Watson and Crick have reached their conclusion of 1953: that the DNA molecule exists in the form of a three-dimensional double helix.

A single nucleotide is made up of three components: a nitrogen-containing base, a five-carbon sugar, and a phosphate group. The nitrogenous base is either a purine or a

pyrimidine. The five-carbon sugar is either a ribose (in RNA) or a deoxyribose (in DNA) molecule.

Basic building blocks

Dr. O. Laneuville

Structures of Sugars: Pentoses

Ribose (D-ribofuranose) Deoxyribose (2'-deoxy-D-ribofuranose) 5432154321

Dr. O. Laneuville

Structures of the Principal Bases: Purines

General Structure 234561789234561789

Adenine (6-aminopurine) Guanine (2-amino-6-oxopurine)

Dr. O. Laneuville

Structures of the Bases: Pyrimidines

General Structure Thymine (5-methyl-2,4-dioxopyrimidine)

Cytosine (4-amino-2-oxopyrimidine) Uracil (2,4-dioxopyrimidine)

123456123456

The nitrogen atoms linked to the C4 of the cytosine and to the C6 of the adenine are taking the amino (NH2) forminstead of the imino (NH) form. Similarly, the oxygen atoms linked to the C6 of the guanine and to the C4 of the thymine are taking keto (C=O) form instead of the enol (C-OH) form.

Cytosine (4-amino-2-oxopyrimidine) Adenine (6-aminopurine)

Guanine (2-amino-6-oxopurine) Thymine (5-methyl-2,4-dioxopyrimidine)

Dr. O. Laneuville

keto -C=O amino -NH2

enol -OH imino =NH

Rule: Enol T: pairs with G and not A Enol G: pairs with T and not C Imino A: pairs with C and not T

All DNA follows Chargaff's Rule, which states that the total number of purines in a DNA molecule is equal to the total

number of pyrimidines.

Two hydrogen bonds connect T to A; three hydrogen bonds connect G to C. The sugar-phosphate backbones (grey) run anti-parallel to each other, so that the 3’ and 5’ ends of

the two strands are aligned.

DNA Structure

Double stranded (double helix) Chains of nucleotides

Three different conformations of the DNA double helix. (A) A-DNA is a short, wide, right-handed helix. (B) B-DNA, the structure proposed by Watson and Crick, is the most common conformation in most living cells. (C) Z-DNA,

unlike A- and B-DNA, is a left-handed helix.

Structure of the B Form of the DNA Double Helix

!  2 chains of complementary polynucleotides and turning around the same axis in the right orientation.

!  The H bonds are additive and contribute to the stability of the double helix.

!  The helix makes one complete turn every 10 nucleotides. The distance between 2 adjacent bases on the same chain is equal to 0.34 nm. A complete turn of the helix measures 3.4 nm.

!  The distance between the phosphorus atom located at the exterior of the helix and the axis of the helix is 1 nm. The width of the double helix is equal to 2 nm.

!  Both purines and pyrimidines are hydrophobic (reduce their interactions with water)

!  At the same time, each nucleotide has two very hydrophilic groups: a negatively charged phosphate and a sugar (carbohydrate) group. Both form will interact strongly with water

!  The most obvious way is to stack the hydrophobic surfaces of the bases in the center of the molecule and place the sugars and phosphates at the edges, in contact with water.

!  DNA acquires its negative charge by dissociating into phosphate esters.

!  Supercoiling: The helix itself is wound up into coils

Between 40 and 50 supercoiled loops radiating from the central protein core.

Bacterial Genomes !  Prokaryotes are haploid

!  Genes are carried on a single double stranded circular chromosome

!  The chromosome is attached to plasma membrane and not enclosed in a nucleus.

!  Bacteria may contain extra-chromosomal genetic elements such as plasmids and viral DNA.

!  Some plasmids can integrate into the bacterial chromosome.

!  DNA replicates directionally (5` to 3`) starting from a single replication origin.

!  Introns are very rare.

!  The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm

Borrelia burgdorfei (Lyme disease) ~1.0 Mb linear plus 14-21 small linear & circular plasmids (ranging in size from ~

5 – 60 kb, NCBI site)

Not all bacteria have single, circular chromosomes…

Girons Microbiol 140:1803, 1994

Deinococcus radiodurans 2 circular chromosomes (2.6 & 0.4 Mbp),

White Science 286:1571, 1999

etc.

Small segment of H. influenza genome:

Energy metabolism Replication

Amino acid biosynthesis

1.8 Mbp

First bacterial genome sequenced July 1995: Haemophilus influenza

Table 8.4

CATEGORIES OF BACTERIAL GENES

!  Eukaryotes, each cell carries more than one linear chromosomes located within a membrane-bound nucleus

!  Eukaryotic genomes contain introns

!  Aerobic fungal cells carry mitochondria which contain circular chromosomes

!  Fungal cells may contain plasmids

!  Fungi show alternation of generations: switching between haploid and diploid stages.Most fungi are haploid during the principal stage of their lifecycle.

Fungal Genomes

Viral Genomes !  Viruses have huge variety of genomic structures.

!  They have small and simple genomes of single or double stranded DNA or RNA.

!  Viral genomes can be linear or circular and some are segmented.

!  Segmented genomes are formed of two or more nucleic acid molecules.

!  All viral genomes are haploid, except retroviruses which show an alternation of generations between a diploid free living phase and a haploid integrated phase.

Active Learning Time

60 : 60 : 30 : 30