Pharmaceuticals Microbiology-I PHT 226 Dr. Rasheeda Hamid Abdalla Assistant Professor E-mail rasheedahamed12@hotmail.com

OBJECTIVES Microbial Genetics Nucleic Acids Structure Transcription Translation Genetic Transfer and Recombination PLASMID

Bacterial Genetics

Microbial Genetics Genetics : is the study of what genes are, how they carry information, how their information is expressed, and how they are replicated and passed to subsequent generations or other organisms. DNA is the genetic material for all living organisms. In bacteria, there is a single circular (closed) chromosome; which is a polymer of DNA (nucleic acid). Gene: is a segment of DNA, a sequence of nucleotides, that codes for a functional product, usually a protein.

When gene is expressed, DNA is transcribed to produce an mRNA; mRNA is then translated into proteins. The DNA in a cell is replicated before the cell divides, so each daughter cell receives the same genetic information. Clone : is a population of cells that are genetically identical. Genome: the complete set of genes or genetic material present in a cell or organism..

Genotype: the specific set of genes an organism possesses. Phenotype: The collection of characteristics of an organism that an investigator can observe. The DNA in a chromosome exists as one long double helix associated with various proteins that regulate it is activity.

Nucleic Acids Structure DNA made from subunits called nucleotides. Each nucleotide contains: 1. Purine (Adenine or A, Guanine or G) or Pyrimidine (Cytosine or C , Thymine or T) bases. 2. Deoxyribose sugar. 3. 1, 2, or 3 phosphate groups. RNA nucleotides containing ribose sugar

Nucleotides are named according to # of phosphates: e. g Nucleotides are named according to # of phosphates: e.g., dATP = deoxy adenosine triphosphate, whereas dAMP = deoxy adenosine monophosphate. Nucleotides in RNA don't have deoxyribose, don't have prefix "d"; names like ATP, ADP, refer to RNA nucleotides.

Nucleotides Linked by Phosphodiester Bond

Purine and pyrimidine bases are attached to deoxyribose sugar ,free to rotate . In DNA ,from specific base pairs :A with T (2H-bonds), G with C (3H-bonds) Two chains of DNA face in opposite directions , called antiparallel (define by which way 3’ and 5’ sides of sugar molecule are facing) In a linear DNA molecule ,one strand has 3’-end , other (complementary) strand has 5’-end

DNA Replication During DNA replication, the two strands of the double helix separate at the replication fork , each strand used as a template by DNA polymerases to synthesize two new strands of DNA according to the rules of nitrogenous base pairing The result of DNA replication is two new strands of DNA ,each having abase sequence complementary to one of the original strands.

To form a chemically stable structure, the two DNA strands are antiparallel (have opposite polarity). That is, one strand running from the 5'-phosphate to 3'-OH is paired with the other strand arranged with its 3'-OH opposite the 5'-phosphate of the first strand, and its 5'-phosphate opposite the 3'-OH of the first strand.

Characteristics of Double Helix

Because each double-stranded DNA molecule contains one original and one new strand, the replication process is called semiconservative. DNA polymerase proofreads new molecules of DNA and removes mismatched bases before continuing DNA synthesis. Each daughter bacterium receives a chromosome identical to the parent's. DNA is synthesized in one chemical direction called 5' to 3' (5' is phosphate end; 3' is hydroxyl end of deoxyribose).

Transcription Is the first step of gene expression, in which a particular segment of DNA is copied into RNA (mRNA) by the enzyme RNA polymerase. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language

During transcription, the enzyme RNA polymerase synthesizes a strand of RNA from one strand of double-stranded DNA, which serves as a template. RNA is synthesized from nucleotides containing the bases A, C, G, and U, which pair with the bases of the DNA sense strand. The starting point for transcription, where RNA polymerase binds to DNA, is the promoter site; the region of DNA that is the endpoint of transcription is the terminator site; RNA is synthesized in the 5' —> 3' direction.

Translation: Translation is the process in which the information in the nucleotide base sequence of mRNA is used to dictate the amino acid sequence of a protein.

- The mRNA associates with ribosomes, which consist of rRNA and protein. - Three-base segments of mRNA that specify amino acids are called codons. - The genetic code refers to the relationship among the nucleotide base sequence of DNA, the corresponding codons of mRNA, and the amino acids for which the codons code. - The genetic code is degenerate; that is, most amino acids are coded for by more than one codon.

Genetic Transfer and Recombination: Genetic recombination: is the transfer of DNA from one organism to another. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms. Natural mechanisms of genetic recombination in bacteria include: transformation, transduction & conjugation.  Plasmids: A plasmid is an "extra-chromosomal" piece of bacterial DNA. Plasmids are stably maintained within bacterial cells, replicating fast enough that they are passed on to bacterial progeny as the bacteria divide. Like bacterial chromosomes, plasmids are circular, double-stranded DNA.

PLASMID (Cont’d) There are several types of plasmids, including conjugative plasmids, dissimilation plasmids, plasmids carrying genes for toxins or bacteriocins, and resistance factors. The major difference between chromosomes and plasmids is that they are much smaller than chromosomes, and they tend to carry genes that are not essential except in certain environments. R PLASMID: The genes on R plasmids confer resistance to antibiotics or other bacterial growth inhibitors. A bacterium with an R plasmid for penicillin resistance is able to survive treatment by that antibiotic

Plasmids (Cont’d) A cell possessing the F plasmid (F+, male) can form a conjugation bridge (F pilus) to a cell lacking the F plasmid (F−, female), through which genetic material may pass from one cell to another

Transformation In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. For transformation to happen, bacteria must be in a state of competence, which might occur as a time-limited response to environmental conditions such as starvation and cell density. Bacteria that are not naturally competent (e.g., E. coli) often can be manipulated in the laboratory in such a way that they become able to pick up environmental DNA.

Transformation

Conjugation Conjugation: is the process by which one bacterium transfers genetic material to another through direct contact. One type of genetic donor cell is an F+; recipient cells are F-. F+ cells contain plasmids called F factors; these plasmids are transferred to the F- cells during conjugation. Donating bacteria is described as being male, and the recipient then becomes an F+ male and can make a sex pilus. Conjugation serves to convert the recipient bacteria also to a male. - When the plasmid becomes incorporated into the chromosome, the cell is called an Hfr (high-frequency recombinant).

Conjugation

Bacteriophages are viruses that infect bacteria. Transduction Transduction is the transfer of fragments of DNA from one bacterium to another bacterium by a bacteriophage. Bacteriophages are viruses that infect bacteria. Generalized Transduction by Lytic Bacteriophage is done following these steps: 1. A lytic bacteriophage adsorbs to a susceptible bacterium. 2. The bacteriophage genome enters the bacterium. The genome directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes. 3. Occasionally during maturation, a bacteriophage head or capsid assembles around a fragment of donor bacterium's nucleoid or around a plasmid instead of a phage genome by mistake.

Transduction(Cont’d) 4. The bacteriophages are released. 5. The bacteriophage carrying the donor bacterium's DNA adsorbs to a recipient bacterium. 6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium. DNA is exchanged for some of the recipient's DNA

Basic Structure What is a Bacteriophage Basic Structure What is a Bacteriophage? It A small virus that only infects bacteria”

E. Coli erupting

Types of Bacteriophages Different Types of Bacteriophages: Lytic- infect the cell and force the replication of the viruses until the cell lyses (or splits the cell open). Lysogenic- infect the cell and integrates its genetic material into the bacterial DNA, remaining dormant until the cell shows signs of stress, when the phage becomes active and begins making copies of itself.

Infection Cycle Adsorption- tail fibers attaching to the bacterium, reversible attachment Irreversible Attachment- base plate irreversibly attaches to the bacterium Sheath Contraction- hollow tail fiber is pushed through the bacterial envelope Nucleic Acid Injection- goes from the head through the tail, into the bacterium

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