Micro - Chapter 9 Genetics
Everything that happens in a cell is determined by the genetic code of that cell. The genetic code determines what metabolic capabilities the cell has, what conditions the cell could live in, what toxins it produces and what mechanisms it has for invading, colonizing and evading the immune system of hosts. genetics is the science of heredity; it includes the study of what jeans are, how they carry information, how they are replicated in past two subsequent generation of cells, or passed between organisms, and how expression of their information within an organism determines the particular characteristics of the organism.
Central dogma of molecular biology: DNA -> RNA -> protein
Bacterial Genomes
The complete set of genetic material in the cell is the genome. The genome includes both chromosomes and extra-chromosomal elements called plasmids.
Not all bacterial cells have plasmids
Plasmids are small circular DNA molecules that carry genes for nonessential cell functions such as antibiotic-resistant genes
Chromosomes are cellular structures that physically carry hereditary information. They contain segments of DNA that code for functional products
Bacterial Chromosomes
Most bacteria have only one circular molecule of DNA. A few species have two or more chromosomes.
The chromosome is inherited from the parent cell, and contains all of the information necessary for the cell to survive and reproduce.
Historically it has been easier to study bacterial DNA because the genome of bacteria is so much smaller than the genome of humans.
Ex: e.coli is roughly 4.7 x 10^6 base pairs long and about 1 mm long, which is 1,000 times longer than the entire cell.
Chromosome contains about 3,500 genes, compared to the human genome, which has around 30,000 genes.
Structure of DNA
Genome is made of DNA
The building blocks of DNA are nucelotides
Each nucleotide is made of a phosphate group, a deoxyribose sugar and one of four nucleic acids (adenine, thymine, guanine, or cytosine)
The phosphates bind to the sugars of the adjacent nucleotide to create the sugar-phosphate backbone of the DNA molecule
Hydrogen bonds between the nucleic acids of the two complementary strands hold the DNA molecule together and allow to molecule to “unzip” for DNA replication and protein synthesis
Adenine is complementary to thymine, which means that adenine always binds to thymine on the opposite (complementary) strand. Cytosine is complementary to guanine.
DNA replication
In order for a cell to reproduce, it must copy its genome so that each of the two daughter cells receives a copy.
DNA replication is semi-conservative, that is, each “new” strand is made up of half new DNA and half old DNA
Directionality of DNA
DNA is directional - the sugar end of a nucleotide is referred to as the 3’ (three prime) end and the phosphate end is the 5’ end.
The 3’ end of a nucleotide binds to the 5’ end of the adjacent nucleotide
Analogy: several people line up holding hands, the left hand of one holds the right hand of the person next to them
DNA is synthesized in the 5’ -> 3’ direction. This means that each new nucleotide is added to the 3’ end of the previous nucleotide.
The leading strand is synthesized continuously; as the DNA molecule unzips to form the replication fork, synthesis continues in the 5’ -> 3’ direction following along behind the replication fork.
On the lagging strand, replication is discontinuous because only the short segments of DNA in the proper direction are available at a time for synthesis
Once replication occurs in these segments, the replication enzymes must wait for the replication fork to unzip more DNA before continuing replication. This “hopscotching” creates several short segments of new DNA called Okazaki fragments.
A DNA molecule consists of two spirally-wound sugar-phosphate chains linked through the hydrogen bonding of four nitrogenous bases. Adenine links with thymine while guanine pairs with cytosine
Molecular events of leading strand synthesis
DNA replication is an anabolic process controlled by enzymes
Specific proteins recognize a region on the chromosome called the origin of replication. The specific site where DNA replication begins will form replication bubbles.
Helicase “unzips” the DNA molecule by breaking the bonds between the complementary base pairs. The separation of strands creates the replication fork
Single strand DNA binding proteins (SSBPs) bind to the DNA strands to stabilize them during replication. Singe-stranded DNA is fragile and tends to break
Primase synthesizes and adds short segments of RNA called primers, which are used to initiate the actual replication of DNA
DNA Polymerase III recognizes the primers and binds to the 3’ end. DNA polymerase synthesizes one of the strands continuously in the 5’ to 3’ direction; this is referred to as the leading strand (steps 5 and 6 occur at the same time)
The other strand that is replicated is the lagging strand; since DNA polymerase cannot add nucleotides to the 5’ end, synthesis must be initiated periodically as the helicase unwinds the DNA. Each re-initiation is preceded by the synthesis of an RNA primer called an Okazaki fragment.
DNA Polymerase I removes the RNA primers and replaces them with DNA
DNA ligase seals the gaps between fragments on the lagging strand.
Because bacterial chromosomes are circular, two replication forks are formed at the origin of replication and DNA replication occurs in a bi-directional fashion around the chromosome. The two forks meet and replication is finished at the terminus of replication.
Protein synthesis
How cells control their growth and regulate their metabolic activities is found on information within the genome on functional units called genes.
The sequence of nucleotide bases in DNA carries genetic information in units that are referred to as genes.
Think of the genome as a cookbook in which each individual recipe is a gene and whatever is made from the recipe is the protein
Cookbook -> recipe -> food
Structural genes encode the information for specific proteins.
To create a protein, a gene must first be transcribed into a sequence of nucleotide bases in a messenger RNA molecule (mRNA)
The mRNA sequence is then translated into an amino acid sequence of a protein
This sequence of amino acids in a protein molecule determines the shape and chemical characteristics of the protein
Each structural gene specifies a specific protein in the cell that carries out a specific function based on its chemical characteristics and molecular shape.
Central Dogma of DNA: DNA -> mRNA > protein
This function of the specific protein gives the cell the specific trait coded for by the gene
The sequence of bases that represents a specific gene is the genotype
The specific trait (composition, morphology, content and activity of individual enzymes, growth rate, etc.) formed by the genotype of the organism is called the phenotype
Each gene is organized into a promoter, a coding region, and a terminator
This is similar to a sentence. The promotor represents the beginning of the gene, the coding region is the actual gene, or “meaning” of the gene, and the terminator signifies the end of the gene, like a period
Protein synthesis occurs in two phases: transcription and translation
Transcription
During transcription, a single gene is copied from DNA into mRNA.
RNA is similar to DNA in structure, but is made of ribonucleic acid rather than deoxyribonnucleic acid, and RNA uses the nucelic acid uracil instead of thymine..
The base-pairing occurs as it does in DNA except that adenine is complementary to uracil
The entire process is carried out by a single enzyme, RNA polymerase, in 3 steps
Initation: RNA polymerase binds to DNA at the promoter
Elongation: RNA polymerase moves along the template strand of the DNA, synthesizing the complementary single-stranded mRNA. As the RNA polymerase advances along the DNA, RNA nucleotides are added to the growing mRNA
Termination: RNA polymerase reaches the terminator sequence on the DNA and falls off the DNA strand
Translation
The mRNA created in transcription is used as the template to make protein. The mRNA is organized into thee-base long units called codons. Each codon codes for a specific amino acid
Translation begins when mRNA binds to ribosome, and then a transfer RNA (tRNA) -a molecule that carries an amino acid- attaches to the ribosome.
This ribosomal complex positions amino acids in a favorable position so that peptide bonds can be formed between amino acids.
Complementary matching of three nucleotides on the tRNA, called an anticodon, and three nucleotides on the mRNA, called a codon, ensures the correct sequences of amino acids
The mRNA passes along the ribsome in short spurts, one codon at a timE. As this occurs, the first amino acid forms a peptide bond with the second amino acid. At the same time, a new tRNA enters the ribosome. The first tRNA, which no longer carries an amino acid, leaves the ribosome and is recycled inside the cell. Each time the amino acid is transferred to the growing polypeptide, a new tRNA brings an amino acid. As the ribosome proceeds down the mRNA, eventually a stop codon is encountered. At this point the ribosomal complex falls apart, protein synthesis is complete, and the polypeptide is released into the cell where it can be further processed into cell structures
Translation occurs in three steps:
Initiation: this is when the mRNA, first tRNA, and two halves of the ribsome all bind together
Elongation: this is when new tRNA molecules bring in new amino acids to grow the polypeptide chain
Termination: this is when the stop codon is reached and protein synthesis is complete
Regulation of gene expression
Gene regulation is a method that cells use to cope with changing conditions of the environments.
Cells use control mechanisms to synthesize the maximum amount of cell material from a limited supply of nutrients.
Two common examples for studying gene expression are the lac operon and the trp operon.
An operon is a group of genes that are transcribed together as a unit
There are more than one coding region between the promotor and the terminator
Operons may be inducible, which means they are usually “off”, but the genes are expressed when needed, or they made be repressible, meaning that they are usually expressed, but can be repressed.
The (lactose) lac operon: an inducible operon
Many bacteria prefer glucose before they utilize other compounds such as lactose as a growth substrate, when both are present in the medium
The lac operon e.coli has a coding region for three functional proteins or enzymes necessary for the use of lactose as a food source. These enzymes are only needed when lactose is available and the normal food source, glucose, is in short supply. Therefore, the lac operon is not activated until glucose is in short supply, since it would be a waste of energy to produce these enzymes when glucose is plentiful.
Transcription of the lac operon is controlled by two regulatory proteins: activator and repressor.
The regulatory region of the lac operon has two switches
The activator protein can bind to the activator binding site of DNA and facilitate transcription
The repressor protein can bind to the operator site of DNA and block transcription
When glucose is present, the activator protein cannot bind to the activator binding site of DNA. Thus, RNA polymerase cannot bind to the promoter, and the transcription of the lac operon does not occur.
If there is no lactose in the medium, the lactose repressor binds to the operator site of DNA and blocks transcription
When glucose is absent and lactose is present, the activator protein binds to the activator binding site of DNA and facilitates transcription
Lactose inhibits the repressor from binding to the operator site. RNA polymerase can now bind to the promote and carry out transcription
The (tryptophan) trp operon: a repressible operon
The trp operonis responsible for making enzymes involved in the synthesis of tryptophan, an amino acid used to make proteins
The trp operon is usually on, and the repressor protein remains inactive and does not bind to the DNA. however, if the concentration of tryptophan in the cell becomes too great, tryptophan will bind to the repressor protein and activate it. This will allow the repressor to bind to the RNA and revent RNA polymerase from initiating transcription. Therefore the trp operon is down down until the concetrnation of tryptophan is depleted by the organism
Phenotypic variation
Phenotypic variation occurs when environmental factors regulate gene expression and influence the phenotype of the microorganism
Influenced by several variable including temperature, pH, nutrient media, osmotic pressure, and expose to UV light
Dramatic changes, affecting the great majority of the cells of a pure bacterial culture, frequencetly occur when the cells are transferred from one environment to another
Phenotypic variations vary according to the environmental condition and are reversible. A particular gene which already exists is turned on or turned off according to the environmental needs. A mutation, on the other hand, is an alteration in the genetic make-up of the bacterium
Phenotypic variation can observed in Serratia macescens, one of the few bacteria that produce bright pigments, most commonly red, white, or pink.
Commonly found in intestine and in soil
Causes about 2% of nosocomial infections of the bloodsteam, lower respiratory tract, urinary tract, surgical wounds, and skin and soft tissues of adult patients
Outbreaks have occurred in pediatric wards
Has a fondness for bread and communion wafers, where the pigmented, aged colonies have been mistaken for drops of blood
Grow well in damp basements, on food stored in damp places, and in bathrooms
Pigment produced: prodigiosin, offers protection against excessive UV in sunlight, serves as an antibiotic, enhances flagella production, and has cytotoxic qualities
Very helpful in establishing the bacterias’s domain in soil as it competes with fungi
Biosynthesis of prodigiosin is a comprehensive pathway that has at least 10 different enzymes involved in its synthesis
In Serratia marcescens, pigment production is a temperature-regulated gene
If incubated at normal body temp (37 C), colines are creamish whitish
If incubated at room temp (25 C), colines are dark red
Serratia marcescens is negative for lactose fermentation, it appears positive due to production of this pigment
Genetic diversity and evolution
In order to evolve bacteria rely on mutation and gene transfer
Everything that a cell can do is because of the directions in the cell’s DNA. Sometimes, during DNA replication errors occurs which change the sequence of bases on the DNA molecule
A change in the sequence of DNA leads to a change in the sequence of mRNA, which may lead to a chance in the amino acid sequence of the polypeptide
Most mutations are either harmful or deadly to the bacterial cell
Most common type of mutation is the point mutation, where only one base pair is affected.
Point mutations can be:
Substitutions: substituting one base for another
Insertions: adding an extra base layer
Deletions: leaving out a base
Mutation in the DNA can cause:
Silent mutations: one base is substituted for another, but the resulting amino acid remains the same
Missense mutations: a new amino acid is substituted in place of the old one
Nonsense mutations: a STOP codon is generated in place of an amino acid
Frameshift mutations: by inserting or deleting a base pair, the entire three-base reading frame shifts either to the left or right, causing all of the amino acis downstream of the shift to be changed
Causes of mutation
Some mutations are spontaneous, just accidents that happen during DNA replication. Others are induces, or caused by outside agents known at mutagens
Mutagens can be chemicals that interfere with DNA replication, or they can be radiation, such as UV light that causes damage to DNA
Mutations are accidents that occur during DNA replication
Most mutations are harmful or deadly and therefore said to be deleterious
DNA repair
Cells have a variety of repair mechanisms to fix damaged DNA
Two mechanisms for repairing DNA in which the incorrect base has been added to the DNA strand are base-excision repair, in wich the single, incorrect base is removed and replace, and mismatch repair, in which an entire section of DNA surrounding the incorrect DNA is removed and replaced
Two mechanisms exist for repairing thymine-dimers that are the result of UV exposure
Light repair is when the enzyme photolyase is activated by visible light and cleaves the bond between adjacent thymine molecules, allowing the DNA to return to its original shape
Dark repair is when the cell does not have photolyase, and a repair enzyme removes the entire section of DNA containing the thymine-dimer and replaces it with a new DNA
Gene transfer
The other mechanisms by which bacterial cells generate genetic diversity are through gene transfer. These mechanisms are important because much of the spread is antibiotic resistance among bacteria is due to these mechanisms
Horizontal gene transfer occurs when genes are transferred from one microbe to another microbe of the same generation
Vertical gene transfer is the transfer of genes from part cell to progeny
Three types of gene transfer: transformation, transduction, and conjugation
Transformation is the uptake of naked DNA by a competent cell, a cell that is capable of taking in naked DNA
During natural transformation of a DNA fragment, double-stranded DNA pairs with a homologous region on the recipient DNA and is integrated into the recipient genome by the breakage and reunion mechanism called homologous recombination.
Fragments of DNA from donor cell + competent recipient cell → uptake of donor DNA fragment by recipient cell → recombination of donor DNA fragment with recipient cell chromosome
If there are any differences between the nucleotide sequences of the donor and recipients DNAs, the mismatch repair system comes into play
The repair system removes either the donor or the recipient strand, and replace it with the complementary sequence
Since either strand may be repaired, some cells contain the new, donor DNA and others maintain the original DNA sequences
For genera sich as as Streptococcus, Acinetobacter, Bacillus, Haemophilus, transformation appears to be a major means by which genetic transfer occurs
Also known to occur in genera such as Neisseria, Escherichia, and Pseudomonas
Some genera of bacteria are naturally competent, which means they are capable of undergoing transformation in the normal environment. They can take in free DNA from their environment and incorporate it into their chromosome
Other genera can be made artificially competent, altered in a lab to make them able to undergo transformation
Transformation discovered by Frederil Griffith in 1928 while down experiments on mice with two strains of Streptococcus pneumonia
The S strain of the cells was smooth, because they had a capsule. The capsule allowed the cells to evade the immune system and cause pneumonia and death in the test mice
The R strain was rough, because it had no capsule, and therefore could not hide from the immune system and cause disease
Griffith did several experiments
Injected test mice with S strain of s. Pneumonia. Mice developed pneumonia and died. Found S strain bacteria in lungs of dead mice
Injected test mice with R strain of s. Pneumonia. Mice did not develop and symtpoms. Mice were sacrificed and lungs examined for signs of bacteria. There were no bacteria in the lungs of the R strained-infected mic
Heat-killed the S strain bacteria and injected into mice. Mice did not develop symptoms. No signs of bacteria found in lungs
Mixed heat-treated cells of strain S and live cells of strain R and mice developed pneumonia and died. Upon examination, found S strain bacteria in the lungs of dead mice
S. pneumonia is naturally competent, thereby transforming itself into a different cell type
In Griffith’s experiment, the R cells were able to take in the gene for capsule production, transforming themselves into the S strain and acquiring the ability to evade the immune system to cause disease
Transduction occurs when bacterial DNA is transferred between cells by way of bacteriophage
The goal of the phage is to take over the bacterial cell and use the cell to make more phage particles, but in some instances, the phage accidentally incorporate segments of bacterial DNA into the phage head. When the phage carrying the bacterial DNA infections another bacterial cell, that cell receives bacterial DNA rather than phage DNA
Lysogenic conversion occurs when bacteria receive genes from bacteriophage that increase the pathogenicity of the bacterium
Ex: Corynebacterium biphtheriae causes illness by producing a toxin that disrups protein synthesis in the cells of infected individuals. However, it only produces the toxin after it has itself been infected by phage beta. The toxic gene is actually produced by the phage, not the bacterium
Conjugation
Conjugation occurs when genes on plasmids are transferred from a live donor cell to a live recipient cell through a sex pilus (conjugation pilus)
Plasmids are extrachromosomal segments of DNA
Vary in size
Can carry genes for toxic production, antibiotic resistance, and metabolic enzymes
The gene that mediates the formation of the sex pilus is called the fertility gene and is found on the F (fertility) plasmid.
Cells that have F plasmids (donors) are said to be F+ and cells lacking the plasmid (recipients) are F-
Special type of conjugation, Hfr (high-frequency recombination) occurs when the fertility plasmid splices itself into the chromosome of the donor cell. Then, when the plasmid replicated and is transferred to the recipient cell, part of the donor’s chromosomal DNA is transferred as well
Current examples of antibiotic resistance due to plasmid transfer
VRE: vancomycin-resistant enterococci
Penicillin- resistance in S. pneumoniae
Resistance to ampicillin, tetracycline, and kanamycin by Pseudomonas and Enterobacteriae
1941: penicillin given to first patient
1956: vancomycin introduces
1960: methicillin introduced
When and why does DNA replication take place?
Binary fission