Detoxicol

SDLS 2008
Medicine for the intoxicated
Subject: Microbiology Topic: Microbial Genetics Lecturer: Eleanor Padla, PhD. Lecture Date: June 17, 2005 Transcriber(s): Mon, Caloy No. of pages: 7 BACTERIAL GENETICS Definition o Genetics is the study of heredity and variation o Genes are the units of heredity o Genome is the total complement of genes o Bacterial chromosomes are: naked (w/o nuclear membrane) 10% of cell volume 1500x cell length single, circular, characteristically haploid, tightly wound DNA exist in nucleoid replication by binary fission FLOW OF GENETIC INFORMATION AND REPLICATION Central Dogma: Replication > Transcription > Translation DNA RNA Proteins o DNA Replication is semi conservative, which means the parent strand separates in two strands, each individual replicates, retaining (conserving) one parental strand and forming a newly synthesized strand. REPLICATION: STAGES Inititation A single DNA strand is unwound and separated by the enzyme helicase. Single-stranded binding proteins prevent the reannealing of these strands. Then, with recognition of its origin of replication, there is synthesis of an RNA primer primase. Elongation With DNA Polymerase III, adding nucleotides to the RNA primer in 5 3 manner (leading strand) and 3 5 direstion (lagging strand). The lagging strand has Okazaki fragments.

Termination
Action of DNA Polymerase I which removes the RNA primer. Polymerization refers to the process of adding nucleotides from the 5 3 end. A pre-existing strand is a requisite for this step. ENZYMES Helicase

unwinding of double-stranded DNA

Single-stranded binding proteins prevent reannealing of the single stranded arm by attaching along the chains of impaired DNA strands. Topoisomerase II prevents super coiling Primase

synthesize RNA primer

DNA Polymerase III

for elongation of DNA along the replication fork

 DNA Polymerase I removes the RNA primer after elongation Ligase attaches Okazaki fragments to form a continuous strand Exonuclease Activity 5-3 Removal of primer Yes No Yes Comparison of the functional characteristics of E. coli: DNA polymerase DNA Polymerization 5-3 Exonuclease Activity 3-5 Polymerase Addition of new Removal of nucleotides nucleotides A Yes Yes B Yes Yes C Yes Yes

VARIATION Any change in the genetic make up may change the phenotype and changes the genotype of an organism. There should be constancy (prevention of errors) and a need for variation.

Phenotypic Variations
are variations in the physical make up and are mostly metabolic changes in response to the environment. These changes are reversible but not heritable. Ex. Change in size and shape Genotypic Variations are variations in the genotype (genetic constituent) and may affect only one or two cells (not the population). These changes are not heritable although permanent in: mutation gene transfers by: tranformation transduction conjugation

MUTATION Heritable changes in DNA sequence A mutant is an organism carrying a mutated gene wild-type is a parent organism with normal genes Classification 1. Spontaneous vs. Induced Spontaneous

occurs without an apparent cause These are rare and are said to be errors in replication

Induced

occurs with an identifiable cause (mutagens) which can be a physical or chemical agent

2. Selectable vs. Non-selectable Selectable confers selective advantage to the mutant. (Mutations to drug resistance) Non-selectable no selective advantage over the wild type. (Color loss in pigmented bacteria) Base Substitution / Replacement (Point Mutation) involves substation of one base for another transition o purine purine substitution A to G G to A transversion

3.

purine pyrimidine substitution A to T

o pyrimidine purine substitution 2

 Effects

C to G

Silent Mutations o mutated nucleotide codes the same amino acid, therefore no change in the protein synthesized. o Ex. UAC (tyr) to UAU (tyr) Missense Mutations o there is change in the nucleotide coding for a different amino acid, therefore there is another protein synthesized. o Ex. UAC (tyr) to CAC (his)

Non-sense Mutations o the nucleotide encodes a stop codon, therefore the protein synthesized is incomplete. o Ex. UAC (tyr) to UAG (stop) UAA (stop) UGA (stop) 4. Deletions vs. Insertions Insertions and Deletions are addition or removal of one or more bases. when 1 base is deleted, its called microdeletion when 2 or more, macrodeletion 5. Back Mutations and Suppressor Mutations Reversion is conversion of mutated gene back to the wild type Suppression is conversion of mutant cell into one that is phenotypically identical to the wild type, though the mutation is still present. Transposable Genetic Elements (TGEs) Can transfer from one location to another, or between chromosome or plasmid TGEs are not independent replicons, they cannot replicate when not associated with those that can replicate TGEs have inverted terminal repeats (palindromes) Types of TGEs A. Insertion Sequences another. bacteria. place to another)

Simplest; about 1000 bp Short segments of DNA that is transported from 1 site of the chromosome to They could answer for all the immediate recombination that could occur in a carry no genes except those involved in transposition (movement of TGE from one

B. Transposons 10-fold longer than Insertion Sequences transposons are also called jumping genes does not require homology for it to insert into the chromosomal DNA. Inverted repeat genes are markers for transposons to insert themselves into a chromosome flanked by IS elements Insertion Sequences for transposability Central genes code for antibiotic resistance C. Phage-Associated Transposons exist in integrated state fundamentally transposons but have genes required to make infectious phage particles mutator phages Ex. Bacteriophage Mu

Transposition
accompanied by duplication of target site involves transposase, which catalyzes insertion to a new site involves resolvase, in recombination events Two kinds: o conservative transposition transposons are transferred from one place to another without replication of the transposons.

replicative transposition mutations.

transposons are replicated as they transfer. This results in greater number of genes, therefore greater

Gene Transfer Process Fates of DNA Fragments after Transfer 1. Degradation of nucleases if the DNA to be transferred has no homology with the recipient DNA, it will be degraded by endonucleases in the cell wall. 2. Stabilization by circularization

3. Integration by recombination
if the DNA fragments has homology with the recipient DNA recombination will occur. GENETIC RECOMBINATION Genetic elements from two separate sources are brought together in one unit. Recombination (recA) genes express for recA proteins that wrap around the genetic elements so they can recombine. Classifications 1. Generalized (Homologous Recombination) involves Reombination (recA) genes and requires sequence homology. Ex incorporation of homologous DNA via transformation, transduction and conjugation 2. Site-Specific Recombination independent of (recA) genes and requires little, if any, sequence homology. It recombines at highly preferred sites. Ex integration of phage at att site 3. Illegitimate Recombination also independent of (recA) genes and requires little, if any, sequence homology. it occurs at random sites Ex insertion of IS transposons GENE TRANSFERS transfer of DNA from donor to recipient governed by chromosomal, viral and plasmid genes can provide sources of genetic diversity Mechanisms of Gene Transfer 1. Transformation uptake of naked DNA by a recipient cell Process of transferring exogenous DNA to the recipient organisms exogenous DNA a DNA coming from outside the cell, source may be from a previous cell that has lysed coming out of the bacterial chromosomes in the environment. Pieces of DNA in the environment can become a transforming DNA if that DNA can find sequences of homology. Criteria for donor DNA: must be derived from a closely related strain (homologous) size requirement is species specific (depends on the species) double stranded DNA binds more effectively than single stranded DNA

Criteria of Recipient Cell: must be competent (must have the ability to accept foreign DNA) competent in the LOG Phase being competent are sometimes induced, some are naturally occurring a transient state Process for Transformation to Occur: Ther must be uptake of naked DNA a naked DNA is found in the environmet that is in the form of lysed bacterium that has filled its chromosome content. Incorporation of the DNA in the chromosome of the recipient cell. Subsequent expression of the gene coming from the recipient. Formation of hybrid cells from the process of transformation.

Recipient Cell not all cells can take up DNA the cell that undergoes transformation should be competent meaning it has ability to take up foreign DNA.

2. Transduction bacteriophage-mediated transfer of DNA. bacteriophage change the pathogenic properties of bacteria. DNA synapse with homologous areas. Bacteriophage Life Cycle 1. Lytic Cycle possessed by virulent phages lysis of infected cell and release of new phages Stages in lytic cycle a. Phage attaches to the host cell and injects DNA to it b. Phage DNA directs synthesis of viral components of the host cell c. Assembled into virions d. Host cell is lysed releasing new virions 2. Lysogenic possessed by temperate phages integration of phage DNA to bacterial chromosome Stages in lysogenic cycle a. Phage attaches to host cell and injects DNA to it. b. Phage DNA circularizes and enters lytic cycle or lysogenic cycle. If it enters the lysogenic cycle, c. Phage DNA now recombines with bacterial DNA, becoming a prophage. d. Lysogenic bacterium reproduce normally. e. Prophage may again recombine with bacterial DNA and repeats the cycle. The phage that has inserted its chromosome into the bacterial cell is called a Prophage. The process of incorporation is called Lysogeny. This is important in production of transducing particles as well production of toxins. Phage Conversion 1. Generalized Transduction Mediated by virulent phages. Transducing phages carry random DNA fragments The phage as a vector will infect the cell, upon infecting it takes over the machinery of the cell by taking up the bacterial chromosomes so viral eplication can continue by using th genetic materials of the host. The bacterial chromosomes tell the cell to produce more viral particles for its replication. These viral particles will normally get packaged in the phage head but sometimes in the packaging line, there is an error that can occur wherein the bacterial chromosome gets packaged instead of the viral chromosome (defective phage). If the bacterial DNA has sequences of homology, recombination can then occur and this cell will become a transductant (a recipient or hybrid cell coming from generalized transduction, a cell that is different genetically). 2. Specialized Transduction mediated by temperate or lambda phages occurs as a result of lysogeny transducing phages carry selected DNA segments The course would be incorporation of the phage DNA into the genetic constituent of the host. The phage that has inserted its chromosome into the bacterial DNA is called prophage (this is stable and can be replicated). Upon induction there are some inducers coming from the environment like the suppressor genes, for example, sometimes poisoning is reversed, instead of the temperate phage causing virulence, it may choose not be virulent. If it is virulent, it has to get out of the chromosome. In the process of invading, it carries only the phage DNA, and gets replicated causing lysis of the cell. There could be an error in the excision process instead of excising only the phage DNA, it takes along with it portions of the bacterial phagosomes, it could excise either from the right side or the left side, the particle that has the bacterial chromosome on the left will be a possible DNA for transduction.

 This DNA gets packaged into the head in the process of packaging. If this DNA gets packaged and sliced thin, then the defective pouch again in contact with another recipient and it can reject the DNA. 3. Conjugation transfer of DNA fragments between two mating pairs. requires cell to cell contact. involves F plasmid-encoded sex pillus common in gram (-) cells Two Mating Types male (donor): fertility (F) plasmid-carrying cell and does not give entire chromosome female (recipient): lacks the F plasmid (so F) but has the receptor site (OmpA) for adherence An F plasmid is a genetic element frequently used in DNA transfer. Small, circular, double-stranded DNA. Conjugative, containing the tra gene for sex pillus production. Replicates independently of the chromosome. Occurs in autonomous or integrated states. High Frequency Recombination (Hfr) plasmids Donors with an integrated F plasmid. F plasmid has replicated with the chromosome. Through this, one can transfer large portions of donor genes at high frequencies. F(+) plasmids Plasmids Donors with an autonomous F plasmid. Found in the cytoplasm. Have segments of chromosomal DNA (extrachromosomal markers). From imprecise excisions of Hfr. Can transfer restricted portions of donor genes. Small, circular, double-stranded DNA, may also integrate Size: 2-300 kb. Capable of independent replication. Have extra-chromosomal existence. Not essential to survival of cell. May contribute to the phenotype of the cell. Confer selective advantage. Properties determined by Plasmids Replication - maintenance properties Host Range - narrow (in related species) or broad (in different species) Copy Number - low (1-2 copies) or high (more than 2) Incompatibility Group compatible or not compatible incompatible groups DNA homology, pillus type, size Conjugal Properties Conjugative - often large plasmids (>35 kb) mediate their own transfer from cell to cell form sex pillus have tra genes, coding for transfer machinery Non-conjugative-smaller plasmids (6-18 kb) can be transmitted by transduction or transformation. no tra genes

In non-conjugative plasmids, conjugal transfer can be mobilized by co-existing conjugative plasmid, with mob genes. Transfer occurs if conjugative plasmid provides a tube and transfer the non-conjugative plasmid into the cell. Other Properties: 1. Resistance Properties Drug Resistance (e.g. antibiotics) Resistance to heavy metal cations/anions 2. Metabolic Properties

 3.

antibiotic and bacteriocin production

Pathogenic Properties toxin production

Evidence of Plasmid-Mediated Phenotypes Plasmid Instability and Curing Plasmid Loss of Protoplasting Plasmid Transfer Plasmid Isolation Genetic Determinants of Resistance Chromosomal Genes Plasmid Genes Kinds of Drug Resistance Non-Genetic o phenomenon of bacterial resistance organism that are metabolically inactive at the time of exposure to the drug. It could be phenotypically resistant but the offspring is very sensitive. Occurrence of small population of cells which are phenotypicaly resistant to drugs but if they resume active multiplication they are again sensitive to the drug. o loss of target structure

Genetic o achieved by chromosomal factors accomplished by spontaneous mutations, resistance occurs at a very low frequency. o extra-chromosomal taken cared of by the resistance plasmid (R plasmids) R Plasmid Determined Resistance in Enteric Bacteria Components of R Plasmids o Resistance Transfer Factor plasmid replication transfer by conjugation o Resistance genes

Resistance Determinants o permanence of resistance o where all genes are clustered o acquired mobile genetic elements

Mechanisms of R Plasmid-Mediated Resistance 1. altering target site of antibiotics 2. modifications of antibiotics 3. prevention of cell entry of antibiotics 4. specifying enzymes which provide resistance for specific hosts

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