Bacterial Chromosomes and Extra-chromosomal Elements

• Module: Foundations of Infections & Infestations BMS171
• Lecture title: Bacterial Chromosomal and Extra-chromosomal elements
• Instructor contact: Professor Doctor Mohammed Mahmoud El-Naggar, at [email protected], or mobile 01126625177, or Sundays, 10:00-12:00 AM

Learning Outcomes:

• Students should get familiar with the bacterial genome and differences between prokaryotic and eukaryotic DNA/RNA
• Compare and contrast between prokaryote and eukaryote DNA replication, transcription, and translation
• Extra-chromosomal elements should be identifiable
• Plasmids classified
• Transposon structures must be definable/describable

Key Topics:

• Prokaryotic/eukaryotic DNA and mRNA structure, DNA replication, transcription, and translation differences
• Bacterial extra-chromosomal elements (plasmids and transposons)

Case Scenario

• A 9-year-old girl exhibits a sore throat and cough, voice hoarseness, fever with sweats and throat pain.
• She is not responding to ampicillin, though she has responded in the past.
• Question: Why is the girl not responding to ampicillin?

Genome

• The genome is the total genetic information in an organism.

Prokaryotic genome (Bacterial)

• Consists of a single copy (Haploid) circular DNA molecule
• Ranges from 580-4600 Kbp
• Many bacteria have extra chromosomal DNA materials called plasmids and transposons

DNA: Prokaryotes versus Eukaryotes

• Number of copies inside the cell: Prokaryotes have a single copy (haploid), Eukaryotes have 2 copies (diploid)
• Shape: Prokaryotes have a circular shape, Eukaryotes have a linear shape
• Location: Prokaryotes are freely in the cytoplasm (within the nucleoid), Eukaryotes are within a nucleus
• Presence of histones (DNA binding proteins): No histones in Prokaryotes, histones bound in Eukaryotes
• Presence of repetitive DNA and introns (non coding DNA): Prokaryotes have little repetitive DNA and no introns, Eukaryotes contain large amounts of repetitive DNA and introns
• Extrachromosomal elements (plasmids): Present in Prokaryotes, Absent in Eukaryotes

RNA

• Three main classes of RNA molecules exist in all prokaryotic and eukaryotic organisms:
• Messenger RNA (mRNA)
• Transfer RNA (tRNA)
• Ribosomal RNA (rRNA)

mRNA: Prokaryotes versus Eukaryotes

• Number of encoded proteins: Prokaryotes are polygenic/poly-cistronic (single mRNA code for different proteins), Eukaryotes are monogenic/mono-cistronic (single mRNA code for one protein)
• Beginning of mRNA translation: during transcription in Prokaryotes, mRNA is first transported to the cytoplasm and starts translation in Eukaryotes
• Life span: very short (constant breakdown by nucleases) in Prokaryotes, much longer (stable) in Eukaryotes
• Post transcriptional modifications: Minor in Prokaryotes, Major: Polyadenylation, capping and splicing in Eukaryotes

DNA Replication: Prokaryotes versus Eukaryotes

• Time: Continuous in Prokaryotes, During S phase of cell cycle in Eukaryotes
• Location: Cytoplasm in Prokaryotes, Nucleus in Eukaryotes
• Origin: Prokaryotes have one point (Ori C), Eukaryotes have multiple points
• Direction: Bidirectional (proceed in two opposing directions at the same time) in Prokaryotes, Unidirectional in Eukaryotes
• DNA polymerases: 2 in Prokaryotes, 4 or more in Eukaryotes

DNA Replication: Prokaryotes versus Eukaryotes- Enzymes

• Topoisomerases regulate the winding and unwinding of DNA during the movement of the replication fork.
• Type II topoisomerase called DNA gyrase, cuts both DNA strands in prokaryotes.
• Type I topoisomerases cut a single strand of DNA in eukaryotes.
• Okazaki fragments are longer in prokaryotes and shorter in eukaryotes.
• Termination: single termination site midway in the circular chromosome for prokaryotes, several termination sites for eukaryotes

Transcription: Prokaryotes versus Eukaryotes

• RNA Polymerase: one type in Prokaryotes, 3 types in Eukaryotes
• Location: Cytoplasm in Prokaryotes, Nucleus in Eukaryotes
• Post transcription modification: Minor in Prokaryotes, Major in Eukaryotes
• Relation to translation: Occurs simultaneously in Prokaryotes. Transcription occurs before translation, RNA is transcribed in the nucleus and then translated in tEukaryotes
• Promoter: 3 different elements in Prokaryotes, Many different elements in Eukaryotes
• Nascent RNA introns: Absent in Prokaryotes, Present in Eukaryotes

Extrachromosomal elements definition

• All the DNA material present in a cell other than chromosomal DNA; The most famous elements include Plasmids, Transposons, and Bacteriophages (virus infecting bacteria)

Plasmids

• Pieces of DNA that exist separately from the chromosome
• Contain an origin of replication
• Replicate independently from the chromosome

Plasmids Classification

Based on size:

• From few hundred base pairs up to 3000 Kb Based on copy number per cell:
• Stringent plasmids: 1-2 copies / cell
• Low copy number plasmids: 10-15/copies/cell
• High copy number plasmid: up to 50 copies/cell
• Extremely high copy number plasmid: up to 100-200 copies/cell Based on compatibility of plasmids:
• Compatible plasmids: cell can maintain more than one plasmid in the same cell if carrying different origins of replication
• Incompatible plasmids: Inability of two plasmids to be maintained in the same cell if carrying the same origin of replication. Based on shape of plasmids:
• Covalently closed circular (CCC) form: the most common form, ds completely closed circular forms
• Semi circular form: transient form, one strand is completely closed, the other strand is opened
• Linear: Double stranded linear DNA, unstable, attacked by exonucleases Based on host range:
• Broad host range plasmids: replicate in a wide range of bacteria
• Narrow host range plasmids: only replicate in one or a few closely related bacteria Moving plasmids from cell to cell:
• Conjugative plasmids: mobilize from one cell to another by conjugation (have the tra genes)
• Non–conjugative plasmids: cannot be mobilized
• Shuttle vector: plasmids that propagate in two different host species (yeast and bacteria) Artificial and natural plasmids:
• Natural plasmids: Present naturally in bacterial and some yeast cells like F- plasmid (F-pili during conjugation)
• Artificial plasmids: Designed artificially, present naturally but designed artificially, by adding antibiotic resistant markers or DNA sequence to be target of restriction endouncleases. Used as vectors in gene cloning

Plasmids Importance

• Plasmids encode gene products responsible for resistance to antibiotics, heavy metals (metal reductase) and ultraviolet (DNA repair enzymes).
• Plasmids lead to conjugation, production of toxins, enzymes and bacteriocin, and biochemical reactions i.e. sugar fermentation
• Plasmids act as vectors in molecular biology for gene therapy and DNA vaccines

Transposons

• Extra chromosomal small pieces of DNA capable of moving itself from one location in DNA to another, (movable elements or jumping genes)
• Structure: there are 3 forms of transposable elements:
• Insertion sequence (IS)
• Composite transposons (Tn)
• Non composite transposons

Insertion sequence (IS)

• The simplest form.
• Encode only proteins needed for its own transposition
• Have the same repeats at their ends (direct repeats or inverted repeats)
• Examples: IS 1, 3, and 10

Composite transposons

• Contain 2 IS at both ends and a central piece of DNA which encodes for antibiotic resistance or virulence factors
• Examples:
• Tn 5 encodes for kanamycin resistance
• Tn 10 encodes for tetracycline resistance

Non – composite transposons

• Have no IS at their ends
• Encode for transposition proteins, and carry genes for antibiotic resistance, virulence factors and catabolic enzymes
• Examples:
• Tn 3 carries Ampicillin resistance gene
• Tn 7 carries streptomycin and trimethoprim resistance

Questions & Answers

• Question: Which of the following plasmids are stringent plasmids? Answer: Plasmids that contain one or two copies inside the cell.
• Question: Prokaryotic DNA is characterized by what? Answer: Absence of introns.
• Question: What is an extrachromosomal small piece of DNA capable of moving from one location in DNA to another? Answer: Transposon.