Chapter

Microbial Genetics

Basis of heredity

DNA replication

Protein synthesis

Transmsission of genetic material

Mutation

Genetic information The blueprint for synthesis of all components of a cell or virus

• Encoded in DNA (or RNA of RNA viruses) as sequences of nucleotides containing the bases A, T(U), G, C

Ex. ---A-T-G-C-T-T-G-C---
         . .   .   .   .   .   .   .   .
      ---T-A-C-G-A-A-C-G---

• Different sequences contain different information

Genetics The study of genetic information, including:

1. how it is organized

2. how it is expressed

3. how it changes

4. how it is reproduced and inherited

5. how it is transferred from one cell to another

• Two strands, each consisting of nucleotides linked together by phosphodiester bonds

-Strands are held together by hydrogen bonds between bases in adjacent strands

A bonds to T

G bonds to C

-Strands run in opposite directions (5' to 3' and 3' to 5')

-The nucleotide sequences of the strands are complimentary to each other

Ex.
5' -------A-T-G-C-T-T-G-C--------3'
             .  .   .   .   .   .  .   .
3'-------T-A-C-G-A-A-C-G--------5'

Cellular organisms have DNA as the genetic material (some viruses have RNA)

Present in cell as large molecules called chromosomes

Most bacteria have one circular chromosome

Genetic information is arranged on the chromosome in units called genes

Gene --the basic unit of heredity

Contains information for synthesis of one protein

Nucleotide sequence of a gene specifies the sequence of amino acids of a protein

Ex. A-T-G-G-T-T-G-T-T-T-C-G-C-C-C---

Methionine-Valine-Valine-Serine-Proline---

Genes also contain additional nucleotide sequences that control expression in the cell

-i.e. When and how often the gene is used to make a protein

Proteins are sometimes called gene products

(Note. A few genes specify sequences of ribosomal RNA and transfer RNA, rather than proteins)

How much information is needed by a cell?

Escherichia coli K-12 has 1 chromosome

~ 4.6 million nucleotides in length

~ 4,400 genes (1 gene ~ 1000 nucleotides long)

 

Human cell has 23 pairs (46) chromosomes

~ 3.2 billion nucleotides

~ 30,000 to 40,000 genes

We have ~1,000 times more DNA, but only ~10 times more genes!?

Many microorganisms reproduce by binary fision of the parent cell, producing two daughter cells (Fig. 2.26)

Before a cell divides it must precisely duplicate (replicate) its chromosome so that each daughter cell will receive a copy having the same nucleotide sequence

Replica: an exact copy of an object

Replication = DNA synthesis

Replication of a Bacterial Chromosome

• Each strand serves as a template for synthesis of a new strand

The base sequences specify the sequences of the new strands

The specificity of base pairing allows the chromosome to be precisely copied

• Replication is semiconservative

Each copy contains 1 original and one new strand

• DNA polymerase Enzyme that catalyzes synthesis of new DNA
• Replication begins at the ori (origin of replication) and proceeds in opposite directions around the chromosome
• Replication is semiconservative

Both chromosomes have one original and one new strand

Expression of genetic information

• Most genes contain information that specifies the sequence of amino acids of a protein
• Expression of genetic information = protein synthesis
• During protein synthesis, genetic information flows from DNA to RNA to protein
• Protein synthesis involves two steps

1. Transcription: Nucleotide sequence of DNA is copied as a sequence of messenger RNA (mRNA)

2. Translation: Protein is synthesized using the mRNA nucleotide sequence to specify the amino acid sequence

1. Transcription: DNA sequence is copied as an RNA sequence (Fig. 9.14)

• 1 DNA strand of the gene is used as a template for synthesis of mRNA (the other strand of DNA is called the sense or coding strand)
• Base pairing rules result in a strand of mRNA (called the transcript) that has the same sequence of bases as that of the sense strand of the gene

Ex.

DNA --ATG CAT GCG--     Sense strand
           --TAC GTA  CGC--     Template strand

mRNA --AUG CAU GCG-- Transcript (remember that U substitutes for T in RNA)

• RNA polymerase is the enzyme that catalyzes synthesis of mRNA
• Transcription of a gene starts when RNA polymerase binds to DNA at a sequence called the promoter which is located at the beginning of the gene
  

2. Translation: Message is decoded and protein is synthesized (Fig. 9.15)

mRNA nucleotide sequence ------> protein amino acid sequence

Ex. AUG CAU GCG ... ---------> Met-His-Ala ...

• Ribosome is the site of translation (i.e protein synthesis) in the cell (Fig. 4.18)

Two subunits (1 large and 1 small) composed of proteins and ribosomal RNA (rRNA)

• Transfer RNA (tRNA) is bound to an amino acid and brings it to the ribosome

mRNA + Ribosome + tRNA-aa --------> Protein

The amino acids are covalently linked by peptide bonds during protein synthesis

How does mRNA determine which of the 20 amino acids to incorporate into a protein?

The Genetic Code (Fig. 9.13)

• Codon A sequence of 3 nucleotide bases in mRNA that specifies a particular amino acid of a protein (or that acts a stop signal that ends protein synthesis --see stop codons below)
  
• 4 bases are possible in a codon: A, U, G, C

Ex. AUG = Methionine, AUC = Isoleucine, AAG = Lysine

• 64 different codons are possible
• 61 of the codons specify the 20 different amino acids present in proteins
• So, the genetic code is redundant: most amino acids have more than 1 codon

Ex.
-Phenylalanine UUU UUC

-Isoleucine AUU AUC AUA

-Alanine GCU GCC GCA GCG

Special codons

Start codon

AUG = Methionine (Met)

Specifies 1st amino acid of all proteins (with occasional exceptions)

Met may be removed after synthesis of protein is completed, so all mature proteins do not necessarily have it as their first amino acid

Stop codons

UAA, UAG, UGA

Do not specify any amino acid

In an mRNA transcript, a stop codon occurs immediately after the codon that specifieds the last amino acid of a protein

Signals the end of translation (protein synthesis)

Anticodon

Present in tRNA (transfer RNA)

Also a sequence of three bases

Complementary to and base pairs with a corresponding codon in mRNA

Each tRNA is attached to the amino acid specified by its complimentary mRNA codon

mRNA         ---AUG CAU GCG---
tRNA               UAC

A tRNA with the UAC antocodon is attached to the amino acid methionine

For every codon of mRNA that specifies an amino acid, there is a matching tRNA with a compilmentary anticodon

tRNAs bring amino acids to the ribosome during protein synthesis

Ribosomes (Fig. 9.13)

Site of protein synthesis

Composed of a large and a small subunit

Subunits contain proteins and ribosomal RNA (rRNA) that form sites where mRNA and tRNA bind during protein synthesis

• Some antibiotics kill bacteria by inhibiting protein synthesis

Ex. Erythromycinm streptomycin, chloramphenicol, neomycin

Genotype Potential characteristics of an organism represented by its genes

Ex.
fla is a gene that codes for synthesis of the protein that makes up flagella

Phenotype Observable characteristics of an organism due to expression of its genes

Ex.
Obseved presence of flagella indicates that the cell has the fla gene and that it was expressed

• If we detect or isolate a gene encoding the information for production of flagella proteins, we can list the gene as a characteristic of an organism's genotype
• If we see flagella under the microscope, we are observing one of the organisms phenotypic traits and can deduce that the organism contains a functional fla gene

Exchange and Uptake of DNA by Bacteria

• Bacteria can exchange or transfer DNA between cells or take up free DNA from their environment to acquire new genes and the phenotypic traits they confer

Ex. bla encodes beta lactamase, an enzyme that destroys penicillin

A cell becomes resistant to penicillin when it acquires a bla gene and expresses it

Pathogens that acquire antibiotic resistance genes are a big problem in medicine

They can make an antibiotic useless for treatment of infections caused by the resistant strain

Three different mechanisms are used by bacteria to acquire new genes

1. Conjugation: Transmission of plasmid DNA (see below) from 1 cell to another (Fig. 9.22)

2. Transduction: Transfer of DNA to a cell by a bacteriophage virus (Fig. 9.24)

When new viral particles are produced in an infected cell, some of the bacterium's DNA may be incorporated into the virus and transferred to another cell when that virus infects it

3. Transformation: Uptake of naked DNA in the environment by a cell

When a bacterial cell dies and lyses, some of the released DNA may be taken into nearby bacterial cells

In biotechnology, transformation is used to introduce entire plasmids into bacterial cells

Plasmids

• DNA molecules that are separate from the chromosome (most are circular)
• Smaller than chromosomes, with fewer genes
• Contain and ori and are replicated in the cell
• May be 1, a few or many copies in one cell
• The genes carried on plasmids are an advantage to the cell under certain circumstances

Ex.

Antibiotic resistance genes on R plasmids

Cell survives exposure to the antibiotic

Virulence genes that allow a pathogen to infect a host

Some encode toxins the kill host cells or enzymes that digest host tissue

• Plasmids can be transferred from 1 cell (donor) to another (recipient) by conjugation

The plasmid is transferred through a sex pilus (conjugation bridge)

The donor retains a copy of the plasmid

One way in which antibiotic resistant strains develop and spread in hospitals

• Naked plasmids can also be taken up by cells via transformation

Transposons (Fig. 9.26)

• Segments of DNA that carry mobile genes ("jumping genes")
• Can move from 1 chromosomal location to another or between a plasmid and a chromosome
• Antibiotic resistance genes may be located on transposons making them easier to spread to other bacteria
  

Recombintion

• Joining together of two pieces of DNA
• Process that allows DNA to integrate into chromosomes and plasmids
• Transposons and virus DNA integrate into a chromosome or plasmid via recombination

• Recombinate DNA can be created in a test tube by mixing DNA fragments, enzymes and ATP
• Gene cloning involves creating recombinant plasmids and introducing them into a host bacterial cell where they can be replicated and the gene expressed
• Used in biotechnology to produce valuable proteins on an industrial scale

Ex. Human insulin gene has been cloned and transferred to E. coli to manufacture insulin for use by diabetics

Mutation

• A permanent change in the nucleotide sequence of DNA
• Spontaneous mutations occur when mistakes are made during replication that are not corrected

1. Nucleotide with wrong base may be incorporated

---T-A-C-G-A-G-G-C---
---A-T-G-A-T-C-C-G---

2. Extra nucleotides may be added

3. Nucleotides may be deleted

• Mutation rate is low because replication mistakes are usually corrected
• Most mutations that aren't corrected lower fitness of cell and are eventually eliminated from population by competition with other organisms
• Rarely, a mutation is beneficial, providing the cell with a selective advantage over its wild type competitors --the mutated gene is then passed on to subsequent generations
• Mutation rate can be increased by exposure of cell to mutagens (Table 9.3)

Physical mutagens

Ex. X rays, ultraviolet light

Chemical mutagens

Ex. nitrous acid, mustard gas

Types of mutations

1. Point mutation

• 1 base change in the sequence

Ex.

Met   Thr    Cys   Leu
ATG-ACG-TGC-CTG---- Wild type (normal) sequence

ATG-CCG-TGC-CTG---- Alters the amino acid sequence
          Lys

May or may not affect structure and function of a protein

Change at acitve site of an enzyme may eliminate catalytic function

Change at the beginning or end may not affect tertiary structure or function

2. Frameshift mutation

• Results from insertion or deletion of nucleotide(s)

Shifts the reading frame of codons during translation or results in shortened proteins that aren't functional

Drastically alters amino acid sequence and may form a stop codon before the end of the gene

Ex.

Insertion of A between the 2nd and 3rd codons

ATG-ACG-TGC-CTG---
Met     Thr   Cys  Leu

ATG-ACG-ATG-CCT-G---
Met    Thr    Met    Pro

Some effects of mutation on microbial cells

• No effect (silent mutation or one that doesn't disrupt function of crucial cellular components)
• Death: most mutations are deleterious to cell

-Billions of years of evolution have selected for the most efficient phenotypic traits (and the genes that determine them) that allow a microorganism to survive in its environment and to compete with other organisms

• Change in morphology

Ex. loss of ability to produce a capsule or flagella

• Loss or gain of virulence (ability to cause disease)

Ex. ability to evade immune system of host

• Increased resistance to antibiotics

Ex. antibiotic's target in cell may be changed so that the antibiotic can't affect it

• Requirement for a specific nutrient for growth

Ex. loss of ability to synthesize an amino acid

An auxotroph is a mutant that requires a specific substance for growth that is not normally required by the wild type cell

Ex. histidine auxotroph --histidine must be available in environment or medium for growth of the mutant

Prototroph = wild type, doesn't have the mutation, can make the nutrient and can grow in a medium lacking the nutrient

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Last updated: Feb. 27, 2007/jdh