Microbial Genetics Lectures John Buchanan Lecture 1 –Mutation ( ) Types of mutations Detection of mutations –Recombination and Plasmids ( ) Plasmids Transposable Elements Transformation Conjugation Recombination

Genetics = branch of biology that deals with heredity, especially the mechanisms of hereditary transmission and the variation of inherited characteristics among similar or related organisms. At the most basic level is the study of genes

Genes are the fundamental unit of heredity –DNA sequence in the chromosome –Transcribed into mRNA –Translated into proteins which make cells work

Genes are copied (DNA replication) almost exactly from parent cell to daughter and from parent to offspring DNA replication is EXTREMELY high fidelity (1 wrong nucleotide every 10 Billion) –The copying of genes from one generation to the next is crucially important –Too many mistakes (MUTATIONS) and gene integrity is lost and the system falls apart

Anyone who has never made a mistake has never tried anything new. - Albert Einstein A mistake may turn out to be the one thing necessary to a worthwhile achievement - Henry Ford Maybe mistakes are not such a bad thing?

–Although multiple redundant systems are in place to prevent alteration of genetic material, mistakes still occur Mutations –The wonder of all of these systems is not just their high fidelity, but also that mistakes are the source of amazing diversity and adaptation to changing environments –Mistakes are the driving forces that lead to the tremendous variation in life we see today –Reoccurring theme of these two lectures – Appreciate the system AND its errors, as both are important

Mutation = A stable, heritable change in the genomic nucleotide sequence

Morphological mutations-result in changes in colony or cell morphology Lethal mutations-result in death of the organism (this is a bit of a dead end) Conditional mutations-are expressed only under certain environmental conditions Biochemical mutations-result in changes in the metabolic capabilities of a cell –1) Auxotrophs-cannot grow on minimal media because they have lost a biosynthetic capability; require supplements –2) Prototrophs-wild type growth characteristics Resistance mutations-result in acquired resistance to some pathogen, chemical, or antibiotic Mutations affect bacterial cell phenotype

How do mutations occur? Spontaneous mutations Spontaneous mutations - Arise occasionally in all cells; are often the result of errors in DNA replication (random changes) Errors in replication which cause point mutations; other errors can lead to frameshifts –Point mutation - mismatch substitution of one nucleotide base pair for another –Frameshift mutation - arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

Types of Mutations Point mutation = affects only 1 bp at a single location –Silent mutation = a point mutation that has no visible effect because of code degeneracy –Missense mutation = a single base substitution in the DNA that changes a codon from one amino acid to another –Nonsense mutation = converts a sense codon to a nonsense or stop codon, results in shortened polypeptide

Base-pair substitution – point mutation

Silent mutation Missense mutation Nonsense mutation

Types of Mutations Point mutation = affects only 1 bp at a single location –Silent mutation = a point mutation that has no visible effect because of code degeneracy –Missense mutation = a single base substitution in the DNA that changes a codon from one amino acid to another –Nonsense mutation = converts a sense codon to a nonsense or stop codon, results in shortened polypeptide Frameshift mutation = arise from accidental insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein

Frame-shift mutation - Insertion

Frameshift mutation - Deletion

How do mutations occur? Induced mutations Induced mutations-caused by mutagens Mutagens – Molecules or chemicals that damage DNA or alter its chemistry –Base analogs are incorporated into DNA during replication and exhibit base- pairing properties different from the bases they replace –Specific mispairing occurs when a mutagen changes a bases structure and thereby alters its pairing characteristics (e.g., alkylating agents) –Intercalating agents insert into and distort the DNA, and thus induce single nucleotide pair insertions or deletions that can lead to frameshifts –Many mutagens (e.g., UV radiation, ionizing radiation, some carcinogens) can severely damage DNA so that it cannot act as a replication template; cell repair mechanisms can restore the DNA, however they are very error prone and lead to mutations

Other Types of Mutations Point mutation = affects only 1 bp at a single location Frameshift mutation = arise from insertion or deletion within coding region of gene, results in the synthesis of nonfunctional protein Insertion/deletion mutation = Larger stretch of DNA added or deleted from a gene that alters gene expression Forward mutation = a mutation that alters phenotype from wild type Reverse mutation = a second mutation which may make the mutant appear wt (in same gene)

Mutant Detection In order to study microbial mutants, one must be able to detect them and isolate them from the wt organisms Visual observation of changes in colony characteristics Mutant selection-achieved by finding the environmental condition in which the mutant will grow but the wild type will not (useful for isolating rare mutations)

Application of Microbial Genetics Ames Test for carcinogenicity

Auxotroph (tryp- mutant) Looking for reversion mutants Application of Microbial Genetics Selective media to look for mutants

Recombination and Plasmids

Plasmids Plasmids are small ds DNA molecules, usually circular that can exisit independently of the host chromosome. They have their own replication origin so can replicate automonously (episomes) and have relatively few genes (<30) that are not essential to the host. Bacterial cell Plasmid DNA Chromosomal DNA

Types of Plasmids Conjugative plasmids have genes for pili and can transfer copies of themselves to other bacteria during conjugation Fertility factor or F factor - These plasmids can also intergrate into the host chromosome or be maintained as an episome (independent of chromosome) R factor - Also conjugative plasmids which have genes that code for antibiotic resistence for the bacteria harboring them. These do not integrate into the host chromosome. Col Plasmids - harbor Bacteriocins which are proteins that destroy other bacteria (eg cloacins kill Enterobacter species) Virulent plasmids - have genes which make bacteria more pathogenic because the bacteria is better able to resist host defenses or produce toxins/invasins

Bacterial Conjugation The transfer of genetic information via direct cell-cell contact This process is mediated by fertility factors (F factor) on F plasmids Basic Conjugation –F+ / F- mating An F plasmid moves from the donor (F+) to a recipient (F-) The F plasmid is transferred via a sex pilus and then copied; thus the recipient becomes F+ and the donor remains F+ In gram-positive bacteria, the sex pilus is not necessarily required for transmission; generally fewer genes are transferred The F factor codes for pilus formation which joins the donor and recipient and for genes which direct the replication and transfer of a copy of the F factor to the recipient The F factor can remain on a plasmid or it can integrate into the bacterial chromosome via IS sequences. This type of donor is called and Hfr strain (High frequency recombination) F’- When the F factor in an Hfr strain leaves the chromosome, sometimes is makes an error in excision and picks up some bacterial genes

Bacterial chromosome F plasmid F Factor Bacterial chromosome HFR StrainF+ Strain

Hfr X F – Mating Similar to the F + X F – cross Sex pilus F - cell conjugal bridge Hfr F - cell

Transposable Elements Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition) –Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Bacterial chromosome Transposon Bacterial chromosome “Cut and Paste”

Transposable Elements Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition) –Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Insertion sequences - IS elements- short sequence of DNA containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences bp in reversed orientation –Transposase - enzyme that recognizes the IR and promotes transposition Transposase (402 amino acids) Inverted repeat (IR) IR IS bp Bacterial chromosome

TRANSPOSITION MECHANISM OF INSERTION SEQUENCES IR ACAGTTCAG TGTCAAGTC CTGAACTGT GACTTGACA Transposase TCGAT AGCTA Chromosomal DNA Cut Insertion of IS into chromosomal DNA target sequence catalysed by transposase

TRANSPOSITION MECHANISM OF INSERTION SEQUENCES IR ACAGTTCAG TGTCAAGTC CTGAACTGT GACTTGACA Transposase TCGAT AGCTA IR ACAGTTCAG TGTCAAGTC CTGAACTGT GACTTGACA Transposase TCGAT AGCTA TCGAT AGCTA Gap filled by DNA polymerase and DNA ligase

Transposable Elements Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition) –Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Insertion sequences - IS elements- short sequence of DNA containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences bp in reversed orientation –Transposase - enzyme that recognizes the IR and promotes transposition Composite transposon (Tn)- contains other genes in addition to transposase like antibiotic resistance genes or toxins

STRUCTURE OF COMPOSITE TRANSPOSONS Bacterial chromosome IS10LIS10R Tn10 9,300 bp Tetracycline resistance gene

Transposable Elements Transposons - DNA segments that carry genes that allow them to move about the chromosome (transposition) –Unlike plasmids or phages, they are unable to reproduce or exist apart from the host chromosome Insertion sequences - IS elements- short sequence of DNA containing only genes required for transposition Flanked by inverted repeats (IR) - identical or similar sequences bp in reversed orientation –Transposase - enzyme that recognizes the IR and promotes transposition Composite transposon (Tn)- contains other genes in addition to transposase like antibiotic resistance genes or toxins Importance Can insert within a gene to cause a mutation or stimulate DNA rearrangement leading to deletions of genetic material Can have termination sequences to block translation or transcription Can have promoters which activate genes near pt of insertion Can move antibiotic resistance genes around Can be on plasmids to aid in insertion of F plasmids into host chromosome Some bear transfer genes (Tn916) and can move between bacteria through conjugation (conjugative transposon)

TRANSPOSABLE GENETIC ELEMENTS CAUSE INSERTION MUTATIONS AATTC TTAAG Chromosomal DNA Tn10 AATTC TTAAG AATTC TTAAG Lactose operon E. coli lac + Mutated lactose operon E. coli lac - Tetracycline resistance gene

DNA Transformation Transformation-a naked DNA molecule from the environment is taken up by the cell and incorporated in some heritable form. This process is random and any portion of the genome may be transferred A competent cell is one that is capable of taking up DNA Competent bacteria must be in a certain stage of growth (usually exponential) and secrete a small protein (competency factor) that stimulates production of new protein required for DNA uptake Gene transfer by this process occurs in soils and marine environments so it is an important route of genetic exchange in nature Artificial transformation - carried out in laboratory to transfer plasmid DNA, a common method for introducing recombinant DNA into bacterial cells. eg CaCl2 or electroporation

Oswald T. Avery

Treatment Competent cell S strain R strainS strain Plasmid Competent cell

Bacterial Recombination-process by which one or more nucleic acid molecules are rearranged or combined to produce a new nucleotide sequence Types of recombination –General recombination involves exchange between homologous DNA sequences –Site-specific recombination is the nonhomologous insertion of DNA into a chromosome; often occurs during viral genome or transposon integration into the host, a process catalyzed by enzymes specific for the host sequence –Replicative recombination accompanies replication and is used by some genetic elements that move about the genome –All can lead to Horizontal Gene Transfer

Bacterial chromosome F plasmid Homologous recombination F Factor Bacterial chromosome HFR StrainF+ Strain General recombination

DNA Recombination: Horizontal Gene Transfer –Horizontal gene transfer-transfer of genes from one independent organism to another (compared to vertical gene transfer-transmission of genes from parents to offspring) –Intracellular fates of transferred DNA Integration into the host chromosome Independent functioning and replication without integration Survival without replication Degradation by host nucleases (host restriction) –Mechanisms of horizontal gene transfer Conjugation is direct transfer from donor bacterium to recipient while the two are temporarily in physical contact Transformation is transfer of a naked DNA molecule Transduction is transfer mediated by a bacteriophages (viruses that infect bacteria)

How do mutations occur? Directed mutations Hypothesis only Organism induces hypermutation in DNA in response to environmental stimuli For example –Bacteria baseline mutation rate = 1 in 10 billion –Bacteria in nutritionally poor medium = 1 in 1 million Some mutations allow survival in poor medium Fundamental question – Can organisms upregulate their mutation rate in response to environmental stress?