Bacterial Genetics

Mechanism of mutations

1. Base substitution or a point mutation: Ex. Guanine to cytosine or a guanine to adenine, or DNA polymerase makes a mistake during replication. 2. Deletion or an addition of one or more nucleotides. 3. Disruption of a gene by a transposon. This can cause an insertional mutation. Transposon also called jumping gene.

Result of mutations

1. Missense mutation: change in amino acid sequence of the protein produced. 2. Frame shift mutation: shift the reading frame of the gene. This may change the amino acid sequence. 3. Inversion mutation: segment of DNA is removed and reinserted in the reverse direction. 4. Inactivation of the gene.

Summary

Bacteria are haploid organisms that can sense and quickly adapt to their environment through short-term adaptations. Bacteria can develop inheritable changes in their genome through mutations that they can pass on to daughter cells. Bacteria can acquire genes from other bacteria through transformation, transduction, or conjugation. It is important to understand these and how they affect antibiotic resistance.

Bacterial DNA is..

Bacteria have one, circular, haploid chromosome. →Some do have multiple, and some are linear, but the majority are circular and singular. →Unlike our DNA there is *no untranslated region* or splicing. Most genes are expressed, even the less essential ones. →There is no nucleus, the genetic material is looped and super coiled to fit in and areal called the *nucleoid*. The DNA is attached to the cellular membrane in some locations to promote DNA replication and to aid in segregation of the DNA when it is time for the cell to divide. Wherever the DNA "is hanging out and attaches to the cytoplasmic membrane", it is called the nucleoid. →There are also *no histones*.

Complex transposons

Complex transposons: Carry genes required for mobilization in addition to other genes. Antibiotic resistance, toxins, adhesion, etc. *Pathogenicity Islands* are more complex transposons Larger, contiguous set of genes that encode for virulence properties. These large segments of DNA are clumped together. Code for disease properties in transposon-like elements that can translocate from one bacterium to another. These are only found in pathogenic strains.

Conjugation

Conjugation is bacterial sex at its best. In conjugation, DNA is transferred directly by cell-to-cell contact, resulting in an extremely efficient exchange of genetic information. The exchange can occur between unrelated bacteria and is the major mechanism for transfer of antibiotic resistance. For conjugation to occur, one bacterium must have self-transmissible plasmid, also called F-plasmid (for fertility). F-plasmids encode the enzymes and proteins necessary to carry out the process of conjugation. Bacteria that carry F plasmids are called F(+) cells. In conjugation, an F(+) donor cell will pass its F plasmid to an F (-) recipient cell, thus making the recipient F (+). The self-transmissible plasmid (F plasmid) has a gene that encodes enzymes and proteins that form the sex penis, that is sex pilus. This long protein structure protrudes from the cell surface of the donor F (+) bacterium and binds to and penetrates the cell membrane of the recipient. A nuclease breaks off one strand of the F plasmid DNA and this single strand of DNA passes through the sex pilus to the recipient

Bacteria can quickly modulate gene expression in response to environmental signals.

Ex. Iron presence, oxygen, antibiotics, pH, temperature, and others. A change in phenotype will occur from a change in gene expression.

The transfer of antibiotic resistance to bacteria

Mechanisms (we won't be asked this specifically). Decreased uptake of an antibiotic. Alteration of the antibiotic target. Destroy or modify the antibiotic. This is how methicillin and vancomycin resistant Staph. (MVRSA) has come about. MRSA gained vancomycin resistance from transformation or transduction.

Plasmids

Mobile genetic element: Extrachromosomal element Bacterial DNA usually consists of single circle of double stranded DNA. Smaller adjacent circles of double stranded DNA are called plasmids; they often contain antibiotic resistant genes. →Small, circular DNA that replicate independently of the chromosome. →Usually do not carry essential information, but that will give the bacteria a selective advantage. May carry genes that provide resistance to antibiotics or make proteins that act as toxins or other pathogenic factors. Resistance for more than one antibiotic can be transferred. A single plasmid can carry the genes to more than one antibiotic. →Can replicate independently of the bacterial chromosome, and can be inherited by daughter cells. →Can replicate without the bacterial chromosome being replicated. →Conjugative plasmids (or fertility genes) carry genes that encode for transfer enzymes and sex pili for transfer to other bacterial cells. →Nonconjugative plasmids can be transferred to other cells by transformation or transduction.

Mobile genetic elements

Mobile genetic elements have the ability to move from one part of the bacterial genome to other parts of the bacterial genome. MGEs can be chromosomal, extrachromosomal, or both and are not found in all strains of bacteria. *Transposons*: mobile genetic elements that can transfer DNA within a cell, from one position to another in the genome, or between different molecules of DNA.

What are mutations significant?

Mutations are significant because the change in amino acids of bacteria structures can result in resistance to antibiotics. A mutation that causes a bacterium to become resistant to antibiotics will cause that organism to survive and pass on this adventitious quality. Now this bacterium can grow and multiply in the presence of antibiotics. This is usually because an antibiotic binds to a certain protein in the bacteria but this protein is no longer being produced. Genetic mutations cause antigenic and phase variation of major surface components. →Antigenic variation: changes in the composition of structure of a molecule. Example: a surface pili made of cysteines now contains only alanines →Phase variation: the turning on or off of the expression of a molecule. Example: a cell with pili produces daughter cells without pili This can work to confuse the immune system.

Transformation

Naked DNA fragments from one bacterium, released during cell lysis, bind to the cell wall of another bacterium. The recipient bacterium must be competent, which means that it has structure on its cell wall that can bind the DNA and take it up intracellularly. Recipient competent bacteria are usually of the same species as the donor. The DNA that has been brought in can then incorporate itself into the recipient's genome if there is enough homology between strands (can only occur between closely related bacteria). The recipient cell must be in close proximity to the donor cell. The DNA is incorporated into its genome, this can lead to a change in phenotype. Naturally competent bacteria are capable of taking up exogenous DNA without manipulation. Some bacteria only take up exogenous DNA in the laboratory under manipulation.

Summary of bacteria genome important points

Summary →Majority of bacterial chromosomes are haploid, circular, dsDNA. When a mutation occurs it will be transferred to the daughter cells. →Bacteria can sense and respond at the genetic level to changes in their environment. These systems may be future targets for antimicrobials. →Some genes involved in the same process are expressed as operons. Operons can encode for structures or toxins that enhance the bacteria's ability to cause infection.

Evolution of new pathogens

Through transduction a nonpathogenic E. Coli gets foreign DNA to produce strains that cause diarrheal disease or urinary tract infections.

Summary of horizontal gene transfer:

Transformation: transfer of naked DNA. Transduction: mediator is bacteriophage. Conjugation: transfer of a conjugative plasmid from one organism to another.

Transposons

Transposons: mobile genetic elements that can transfer DNA with in a cell, from one position to another in the genome, or between different molecules of DNA. Transposons can move to different regions of the same chromosome and to other areas like a plasmid. Clinically significant in that they can pass antibiotic resistance to another organism and may need more antibiotics to kill. Also known as jumping genes. Transposons can be extrachromosomal and chromosomal elements

Genotype

genetic makeup of the organism.

Mutations

inheritable changes in the base sequences of the DNA

Mutant

organism that exhibits a recognizable change in its characteristics from the normal due to its mutation.

Wildtype

term used to refer to the initial state of the organism before the mutation.

Horizontal Gene Transfer

How bacteria acquire genetic information from other bacteria. As opposed to vertical gene transfer which is from a bacteria directly to its daughter cells.

Life cycle is in two phases of bacteriophages: Lysogenic or temperate

Infection leads to *cell lysis or integration of the phage DNA* into the chromosome now called a *prophage*. Genes that make bacteria more pathogenic can be encoded on a prophage. This can lead to antibiotic resistance. Research is being conducted to use bacteriophages to fight infection instead of antibiotics

what are the smallest kind of transposons?

Insertion Sequences (IS) elements are the simplest kinds of transposons

Life cycle is in two phases for bacteriophages: Lytic or virulent cycle

Lytic or virulent: After binding there is a *rapid replication* that leads to *cell death* and the *production of new phage*.

How are genes that are not always needed regulated?

1. There is an enhancer or a repressor in the promoter region. 2. Expression of certain genes in operons.

Examples of how these mutations occur at the genetic level.

Figure A: Phenotypic modulation due to a deletion. When the pathogenic island is deleted the bacteria cannot cause infection. Figure B: Phase variation due to a DNA inversion. This is reversible and the bacteria can go from infective to non-infective. Figure C: Phase variation due to point mutations. This is also reversible, the example has the capsule and no capsule when it is mutated. Figure D: Phase variation due to insertion of IS256. This codes for a slime layer, is reversible. Figure E: Antigenic variation due to rearrangements. There are four types of genes in a silent locus, so the only way they can be expressed is if they are recombined into an expression locus. Each is different. It is important to know that while many mutations will kill bacteria, some mutations can be beneficial for the bacteria making them harder to treat and/or resistant to therapy

Bacterial gene expression

Genes that are necessary for survival are constitutively expressed. The genes that are not always needed are regulated.

Bacteriophage

Resemble most viruses in having a protein coat called a capsid that surrounds the molecule of DNA or RNA. The phage will then bind its tail fibers to specific receptors on the bacterial cell surface this is called absorption. The phage then undergoes penetration and injects contents. Two types of phages virulent and temperate phages. *Structure*: protein capsid that houses genome may have base and tail, and no membrane envelope. *Genome*: DNA or RNA, single or double stranded, linear or circular.

Expression of certain genes in operons

Sequence of DNA that contains multiple genes to produce multiple proteins for a single purpose. Under control of the same promoter and terminator. Not all genes are in operons, and eukaryotic cells do not have this. *Polycistronic mRNA* is the mRNA that is expressed from the groups of genes in an operon. It has information for more than one gene or protein. *Lac operon* is a common example, but will not be specifically tested on the exam. Bacteria use glucose for nutrition when it is available. When lactose is present and glucose is absent there will be expression so that the bacteria can use lactose for nutrition. When lactose is present it causes an *allosteric change to the repressor* so that it will *no longer bind on the operon*. *Lac operon* will then be *expressed* and lactose will be able to get into the cell and be metabolized. There is both a positive and a negative control - the lac operon is only active when lactose is present and glucose is absent. Some virulence genes are also expressed through an operon.

Quorum sensing

Signal molecule binds to the regulator and is harmless at low numbers of bacteria, because there is no gene expression. If the bacteria get to a higher density then more signal is made. This will turn on the aggressive phenotype and more will be made, therefore the bacteria become more pathogenic. The signal molecules up-regulate the expression of virulence genes that cause the pathological effects of the bacteria such as biofilm generation and toxin production. Example of biofilm formation: Once the bacteria get to a certain density that is high enough they make more slime. This slime makes the bacteria more resilient to immune cells and antibiotics. Does not allow them to get in.

Transduction

transduction occurs when a virus that infects bacteria called a bacteriophage, carries a piece of bacterial DNA from one bacterium to another. After bacteriophage penetration into a host bacterium, the phage DNA is transcribed, replicated, and translated into capsids and enzymes. At this time the bacterial DNA is repressed and eventually destroyed. Sometimes pieces of the bacterial DNA are left intact. If these pieces are the same size as the phage DNA, they can accidentally be packed into the phage capsid head (in addition to the viral DNA/RNA). Following lysis of the cell and release of the phages, the one phage with bacterial DNA in its head can then infect another bacterium. it will inject the piece of bacterial DNA that it is "accidentally" carrying. If there is some homology between the newly injected strand and the recipient bacterial genome, the piece may become incorporated. The gene on that piece could encode a protein that the recipient did not have such as a protein that inactivates as antibiotic. In generalized transduction the bacteriophage will only be carrying bacterial DNA, so the recipient cell will survive (since no viral genes that encode for replication and lysis are present). This type of genetic transfer is more effective than transformation because the transferred DNA piece is protected from destruction during transfer by the phage capsid that holds it.

Phenotype

visible properties of an organism.