Microbial Genetics Dr. Bhavesh Patel Principal V.P. and R.P.T.P. Science College Vallabh Vidyanagar –

Structure and Function of Genetic Material DNA & RNA DNA & RNA DNA DNA –deoxyribonucleic acid RNA RNA –ribonucleic acid Nucleotides Nucleotides –Phosphate group –Pentose sugar –Nitrogenous base

Structure of DNA Double stranded (double helix) Double stranded (double helix) polymers of nucleotides polymers of nucleotides 5’ to 3’ (strands shows polarity and are anti-parallel) 5’ to 3’ (strands shows polarity and are anti-parallel) Complimentary Base Pairing Complimentary Base Pairing –A – T (double bond) –G – C (tripal bond) Thus in a DNA A=T, G=C or A+G=T+C Erwin Chargaff

3 Types of RNA 1. mRNA 1. mRNA –Contains the codons 2. rRNA 2. rRNA –Ribosomes (70S) 3. tRNA 3. tRNA –Transfer amino acids to the ribosomes for protein synthesis –Anti-codon

DNA act as Genetic material Hershey & Chase,1952

DNA Replication Bacteria have only Bacteria have only 1 chromosome (ccDNA) E. coli E. coli –about 4 million base pairs –1 mm long (1000 times longer than the cell) longer than the cell) –DNA takes up only about 10 % of cells volume

DNA Replication-Hypotheses

Meselson-Stahlexperiment,1957 Meselson-Stahlexperiment,1957

DNA Replication occurs at the Replication Fork (5’ to 3’) DNA Helicase DNA Helicase 1. Leading Strand ( 5’ to 3’ ) 1. Leading Strand ( 5’ to 3’ ) –DNA Polymerase 2. Lagging Strand ( 5’ to 3’) 2. Lagging Strand ( 5’ to 3’) –RNA Polymerase (RNA Primer) –DNA Polymerase (extends primer and digests RNA) –DNA Ligase Okazaki Fragments Okazaki Fragments

Enzymes involved in replication

Bidirectional replication Origin of replication Origin of replication Termination site Termination site

Genetic Code DNA DNA mRNA mRNA tRNA tRNA Triplet code Triplet code codons codons anticodons anticodons

Genetic Code Codons code for a specific amino acid Codons code for a specific amino acid 20 amino acids 20 amino acids 3 base code - 4 bases ( A,U,G,C ) 3 base code - 4 bases ( A,U,G,C ) 64 possible combinations ( 4 3 ) 64 possible combinations ( 4 3 ) Amino acids are coded for by more than one codon Amino acids are coded for by more than one codon Genetic Code is Degenerative Genetic Code is Degenerative Genetic Code is Universal Genetic Code is Universal

Protein Synthesis DNA Transcription mRNA Translation Protein DNA Transcription mRNA Translation Protein Central Dogma Central Dogma of Molecular of MolecularGenetics

Transcription One strand of DNA is used as a template to form a complimentary strand of mRNA One strand of DNA is used as a template to form a complimentary strand of mRNA mRNA ----> messenger RNA mRNA ----> messenger RNA

Translation rRNA rRNA –ribosomal RNA (forms the ribosomes) (70S) tRNA tRNA –transfer RNA (transfers amino acids to the ribosomes for protein synthesis) –anticodon 3 base sequence that is complimentary to the codon on mRNA 3 base sequence that is complimentary to the codon on mRNA

Regulation of Gene Expression All Genes are not always being expressed All Genes are not always being expressed Genes turned on all the time - Constitutive Genes turned on all the time - Constitutive Other genes can be regulated: Other genes can be regulated: –Turned On –Turned Off

lac operon Lac - Lactose (dissacharide) Lac - Lactose (dissacharide) –Glucose and Galactose operon - series of structural genes all under the control of a Regulatory Gene operon - series of structural genes all under the control of a Regulatory Gene lac operon is normally turned off lac operon is normally turned off lac operon is an Inducible operon lac operon is an Inducible operon

Tryptophan operon Tryptophan - amino acid Tryptophan - amino acid operon - series of structural genes all under the control of a Regulatory Gene operon - series of structural genes all under the control of a Regulatory Gene Tryptophan operon is normally turned on Tryptophan operon is normally turned on Tryptophan operon is a repressible operon Tryptophan operon is a repressible operon

Mutations - a change in the base sequence of DNA Mutations can be: Mutations can be: 1. Harmful 1. Harmful 2. Lethal 2. Lethal 3. Beneficial 3. Beneficial 4. Silent (neutral) 4. Silent (neutral) –UUU - Phe –UUC - Phe

Types of Mutations Normal DNA

Base Substitution Missense Mutation (C to T)

Nonsense Mutation (T to A) Base Substitution

Frame Shift Mutation ATG CAT GCA TGC ATT TCC TGC TTA AAA ATG CAT GCA TGC ATT TCC TGC TTA AAA 1. Addition Mutation 1. Addition Mutation AAT GCA TGC ATG CAT TTT CCT GCT TAA AAT GCA TGC ATG CAT TTT CCT GCT TAA Reading Frame is Shifted Reading Frame is Shifted 2. Deletion Mutation 2. Deletion Mutation TGC ATG CAT GCA TTT CCT GCT TAA TGC ATG CAT GCA TTT CCT GCT TAA Reading Frame is Shifted Reading Frame is Shifted

Direct selection of Mutant

Replica plating technique

Auxotroph isolation

Isolation of conditionally lethal mutants

Ames Test

Genetic Transfer in Bacteria Genetic Transfer - results in Genetic Variation Genetic Transfer - results in Genetic Variation Genetic Variation - is needed for Evolution Genetic Variation - is needed for Evolution 3 Ways Genetic Transfer can occur in Bacteria 3 Ways Genetic Transfer can occur in Bacteria –1. Transformation –2. Conjugation –3. Transduction

General Features of Gene Transfer in Bacteria Unidirectional Unidirectional –Donor to recipient Donor does not give an entire chromosome Donor does not give an entire chromosome –Merozygotes Gene transfer can occur between species Gene transfer can occur between species

Transformation Genes are transferred from one bacterium to another as “naked” DNA Genes are transferred from one bacterium to another as “naked” DNA Frederick Griffith (1928) Frederick Griffith (1928)

Transformation Definition: Gene transfer resulting from the uptake of nacked DNA from a donor. Definition: Gene transfer resulting from the uptake of nacked DNA from a donor. Factors affecting transformation Factors affecting transformation –DNA size and state Sensitive to nucleases Sensitive to nucleases –Competence of the recipient (Bacillus, Haemophilus, Neisseria, Streptococcus) Competence factors Competence factors Induced competence Induced competence

Griffith’s Experiment 2 Strains of Streptococcus pneumoniae 2 Strains of Streptococcus pneumoniae –1. Virulent strain with a capsule - Pneumonia –2. Avirulent strain without a capsule - no disease

Electroporation

Conjugation One bacterium passes some DNA (or a plasmid) to another bacterium

Conjugation Definition: Gene transfer from a donor to a recipient by direct physical contact between cells Definition: Gene transfer from a donor to a recipient by direct physical contact between cells Mating types in bacteria Mating types in bacteria –Donor F factor (Fertility factor) F factor (Fertility factor) –F (sex) pilus Donor Recipient –Recipient Lacks an F factor Lacks an F factor

Physiological States of F Factor Autonomous (F + ) Autonomous (F + ) –Characteristics of F + x F - crosses F - becomes F + while F + remains F + F - becomes F + while F + remains F + Low transfer of donor chromosomal genes Low transfer of donor chromosomal genes F+

Physiological States of F Factor Integrated (Hfr) (High Frequency of Recombination) –Characteristics of Hfr x F - crosses F - rarely becomes Hfr while Hfr remains Hfr F - rarely becomes Hfr while Hfr remains Hfr High transfer of certain donor chromosomal genes High transfer of certain donor chromosomal genes F+F+ Hfr

Physiological States of F Factor - Autonomous with donor genes (F’) - Autonomous with donor genes (F’) –Characteristics of F’ x F - crosses F - becomes F’ while F’ remains F’ F - becomes F’ while F’ remains F’ High transfer of donor genes on F’ and low transfer of other donor chromosomal genes High transfer of donor genes on F’ and low transfer of other donor chromosomal genes Hfr F’

Mechanism of F + x F - Crosses DNA transfer DNA transfer –Origin of transfer –Rolling circle replication Pair formation – Conjugation bridge F+F+ F-F- F+F+ F-F- F+F+ F+F+ F+F+ F+F+

Mechanism of Hfr x F - Crosses DNA transfer DNA transfer –Origin of transfer –Rolling circle replication Homologous recombination Homologous recombination Pair formation – Conjugation bridge HfrF-F- F-F- F-F- F-F-

Microbe Library, American Society for Microbiology

Mechanism of F’ x F - Crosses DNA transfer DNA transfer –Origin of transfer –Rolling circle replication Pair formation – Conjugation bridge F’ F-F- F-F-

Conjugation Significance Significance –Gram - bacteria Antibiotic resistance Antibiotic resistance Exponential increase under selective pressure Exponential increase under selective pressure –Gram + bacteria Production of adhesive material by donor cells Production of adhesive material by donor cells

Transduction DNA is transferred from one bacterium to another by a virus DNA is transferred from one bacterium to another by a virus Bacteriophage Bacteriophage –virus that only infects bacteria

Generalized Transduction Release of phage Release of phage  Phage replication and degradation of host DNA Assembly of phages particles Assembly of phages particles Infection of recipient Infection of recipient Homologous recombination Homologous recombination  Infection of Donor Potentially any donor gene can be transferred

Specialized Transduction Excision of the prophage Excision of the prophage gal bio gal bio gal bio gal bio gal Replication and release of phage Replication and release of phage Infection of the recipient Infection of the recipient Lysogenization of the recipient Lysogenization of the recipient –Homologous recombination also possible

Transposable Genetic Elements Definition: Segments of DNA that are able to move from one location to another Definition: Segments of DNA that are able to move from one location to another Properties Properties –“Random” movement –Not capable of self replication (not a replicon) –Transposition mediated by site-specific recombination Transposase Transposase –Transposition may be accompanied by duplication

Types of Transposable Genetic Elements Insertion sequences (IS) Insertion sequences (IS) –Definition: Elements that carry no other genes except those involved in transposition –Nomenclature - IS1 –Structure (flanking inverted repeats) – Importance Insertional Mutation Plasmid insertion Phase variation Transposase ABCDEFG GFEDCBA

Types of Transposable Genetic Elements Transposons (Tn) Transposons (Tn) –Definition: Elements that carry other genes in addition to those involved in transposition –Nomenclature - Tn10 –Structure Composite Tns Composite Tns – Importance Antibiotic resistance IS Resistance Gene(s) IS Resistance Gene(s)

Plasmids Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) Definition: Extrachromosomal genetic elements that are capable of autonomous replication (replicon) Episome - a plasmid that can integrate into the chromosome Episome - a plasmid that can integrate into the chromosome

Classification of Plasmids Transfer properties Transfer properties –Conjugative –Nonconjugative Phenotypic effects Phenotypic effects –Fertility –Bacteriocinogenic plasmid –Resistance plasmid (R factors)

Structure of R Factors RTF RTF –Conjugative plasmid –Transfer genes RTF R determinant R determinant R determinant –Resistance genes –Transposons

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