Recombination, Genetic Technology and Bacterial Diagnosis 8.4, 8.6 and 15 Genetic Recombination During meiosis of human gametes In bacteria, occurs when DNA is transferred between bacteria. Increases diversity in gene pool End result is a new strain different from both the donor and the original recipients Vertical gene transfer Genes/DNA passed from an organism to its offspring Horizontal gene transfer Genes/DNA transferred between organisms Genetic Recombination - Depends on the fact that bacteria have plasmids and are adept at interchanging genes - Provide genes for resistance to drugs and metabolic poisons, new nutritional and metabolic capabilities, and increased virulence and adaptation to the environment Plasmids Self replicating circular pieces of DNA 1 5% the size of bacterial chromosome mini chromosome Bacteria can store up many different plasmids for their use & can transfer these to other bacteria. They can contain any gene that the bacteria don t require but are useful to the survival of the bacteria. For example antibiotic resistance genes, toxin production, etc. Antibiotic Resistance (R) Plasmids Some plasmids can carry many antibiotic resistance genes. When bacteria collect many plasmids and these plasmids have many antibiotic resistance genes, a superbug may originate. Three Types of Genetic Transfer (Recombination) in Bacteria Conjugation Transformation Transduction 1
Conjugation A donor cell contains a F (fertility) plasmid making it F+. A conjugation pilus (genes for which are on the F+ plasmid) forms and the donor cell transfers a copy of the F plasmid to the recipient. Now, both cells have a F plasmid F+ plasmids can have other genes on them too, for example antibody resistance containing genes Hfr Conjugation High frequency recombination (Hfr) donors contain the F factor in the chromosome Donor gives part of its chromosome to the recipient This transfers more genes to the recipient bacteria Very fast evolution for the recipient! Integration of F factor into chromosome Donor Hfr cell Pilus Partial copy of donor chromosome Donated genes Bridge broken Transformation Occurs when naked DNA fragments of one bacteria are close to another living cell. Some bacteria have the ability to pick up naked DNA fragments and recombine the DNA into their own DNA The new recombinant cell now has some new DNA from the disintegrating cell. The now transformed bacteria could have just picked up a new virulence factor or antibody resistance Griffith s Classic Experiment to Test Transformation Principle Mechanism of Transduction Virus mediated gene transfer The virus injects its genetic material into the bacteria The bacterial DNA is fragmented Mechanism of Transduction Viral particles are produced by the bacteria When the cell lyses, the viral particles which have picked up DNA from the original bacterial cell now insert that DNA into a new cell. The new cell may or may not insert the new DNA sequence into its chromosome. Transduction can be a problem when the inserted DNA codes for an antibiotic resistance gene. 2
Transformation and Transduction in Research Electroporation A way to get the genes you want to work with into bacteria. Used in all types of molecular genetics research Transposons Transposons Small segments of DNA that can move (be transposed) from one region of a DNA molecule to another. jumping genes Involved in Changes in traits such as colony morphology, pigmentation, and antigenic characteristics Replacement of damaged DNA Intermicrobial transfer of drug resistance (in bacteria) Genes & Evolution Genes are continually altered due to mutation, recombination, and transposition These changes increase genetic diversity of the gene pool and then through natural selection adventitious genes may be selected for to ensure survival in many different habitats. For pathogens that means they are more virulent! Diagnosing Microbial Diseases How to identify bacteria in patient specimens or in samples from nature? Or the MM project;) - phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry - genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria - immunologic: serological analysis Data from these methods can provide a unique profile for any bacterium Phenotypic Methods: Direct Examination of Specimen Direct observation of fresh or stained specimen Survey of Microbial Diseases: Phenotypic Methods Isolation Media and Morphological Testing - Selective media: encourage the growth of only the suspected pathogen MacConkey Stains most often used - Gram stain - acid fast stain - Differential media: used to identify definitive characteristics and fermentation patterns Mannitol Salts 3
Survey of Microbial Diseases: Phenotypic Methods Physiological/Biochemical Characteristics Traditional mainstay of bacterial identification Enzyme production and other biochemical properties are reliable ways to ID microbes Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities: - fermentation of sugars - capacity to digest complex polymers - production of gas - sensitivity to antibiotics - nutrient sources Phenotypic Methods: Biochemical Testing Unknown microbe + different substrates Results (+/ ) DNPG ADH LDC ODC CIT H2S URE TDA IND VP GEL GLU MAN INO SOR RHA SAC MEL AMY ARA + + + + Enzyme mediated metabolic reactions often visualized by a color change - microbe is cultured in a medium with a special substrate, then tested for a particular end product - microbial expression of the enzyme is made visible by a colored dye Flowchart: We will use one to ID our MM! Cocci Gram (+) Gram ( ) Catalase (+), Catalase ( ), irregular clusters, pairs, chain tetrads arrangement Aerobic, Anaerobic, oxidase (+), oxidase ( ), catalase (+) catalase ( ) Streptococcus Strictly Facultative aerobic anaerobic Neisseria Veillonella Branhamella Micrococcus Staphylococcus Moraxella Planococcus Phenotypic Methods: Phage Typing - Testing for sensitivity to various phage groups - a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block - cleared areas corresponding to lysed cells indicate sensitivity to that phage - Ex. S. aureus Phage Group I vs. Group II Bacterial Tools in the Lab and Genotypic Diagnosis Tools of the Lab: Electrophoresis DNA is separated according to size by running it trough a gel. The gel is made of agarose The gel is placed in an electrical field that pushes the negatively charged DNA towards the positive electrode. 4
Tools of the Lab: Restriction Endonucleases Enzymes capable of recognizing foreign DNA and breaking the bonds between adjacent nucleotides on both strands of DNA Protects bacteria against incompatible DNA of bacteriophages Allows biotechnologists to cleave DNA at desired sites Necessary for recombinant DNA technology Recognize and clip at palindromes cut four to five bases on the 3 strand and on the 5 strand, leaving overhangs on each end adhesive tails will base-pair with complementary tails on other DNA fragments or plasmids Tools of the Lab: Restriction Fragment Length Polymorphisms Restriction fragment length polymorphisms (RFLPs): differences in the cutting patterns of specific restriction endonucleases RFLP allows the comparison of different cutting sites at specific areas in the genome Tools of the Lab: Polymerase Chain Reaction Specific DNA Replication of a particular portion of the DNA Rapidly increases the amount of DNA in a sample without the need for making cultures or carrying out complex purification techniques Sensitive enough to detect cancer from a single cell or diagnose an infection from a single bacteria Rapid enough to replicate target DNA from a few copies to billions of copies in a few hours Tools of the Lab: Polymerase Chain Reaction First the DNA is heated so that it will open up it s A s, T s, G s, and C s. This is called denaturing the DNA https://www.youtube. com/watch?v=iqsu3 Kz9NYo Tools of the Lab: Polymerase Chain Reaction Second the DNA is cooled a small amount so that primers which match a portion of the target DNA can bind to the template DNA. Primers: DNA strands 15 30 bases long that serve as landmarks where DNA amplification should begin Tools of the Lab: Polymerase Chain Reaction Third the DNA Polymerase uses free nucleotides in the solution to build the desired DNA sequence. This is called DNA polymerization This is called annealing 5
Tools of the Lab: Polymerase Chain Reaction One cycle of denaturation, annealing, and polymerization will double the DNA fragments between the primers Since the reaction goes from 50 C to 95 C and there are about 35 cycles total there is a need for a DNA polymerase that can be heated to 95 C and not denature. We get this DNA polymerase from thermophilic bacteria Thermus aquaticus! Genotypic Methods of Identifying Bacteria: PCR Bacterial DNA from a sample can be PCRed at a particular area in the genome Using restriction endonucleases the PCR producted are cut and an RFLP emerges This is a DNA fragment of that bacteria Morning Glory hot springs, habitat for Thermus aquaticus Hybridization: Genotypic Methods of Identifying Bacteria: Genetic Probes Used to identify bacterial species by analyzing the sequences of nitrogenous bases in DNA Probes: small fragments of singlestranded DNA or RNA complementary to the specific DNA sequence of a particular microbe Probes with florescent or radioactive tags are added to the RFLP to visible changes in the DNA sequence in that area After PCR all of the banding patterns look the same, but are all of the bands the same sequence? Tools of the Lab: Recombinant DNA Technology Remove genetic material from one organism and combine it with that of a different organism Bacteria can be genetically engineered to mass produce substances such as hormones, enzymes, and vaccines difficult to synthesize by usual industrial methods Tools of the Lab: Recombinant DNA Technology Genetic clones and cloning: involves removal of a selected gene from an animal, plant, or microorganism and grow it in a host microorganism gene must be inserted into a vector (usually a plasmid or a virus) vector inserts the gene into the cloning host cloning host is usually a bacterium or yeast which can translate the gene into the desired protein Tools of the Lab: Cloning Vectors Small, well characterized, easy to manipulate Readily accepted DNA by the cloning host Contain an origin of replication Contain a selective antibiotic resistant gene Ex. Plasmids, phages 6
Tools of the Lab: Producing Recombinant DNA Start with a cloning vector (special plasmid) and DNA with your gene of choice. Cut the cloning vector and your desired gene out of the parent chromosome with specific enzymes. Tools of the Lab: Producing Recombinant DNA Mix the vector and the gene together with a ligase enzyme which seals the DNA together. Use various techniques to insert the vector +gene into a new cell. Tools of the Lab: Producing Recombinant DNA Grow cell on selective or differential media to find out which cells possess the recombinant plasmid. Tools made in the Lab: Transgenic Bacteria Pseudomonas syringae Natural bacteria that grow on plants but promote frost crystals Alteration of the normal frost gene now prevents frost crystals from forming on plants (bacteria applied with crop duster to compete with the natural bacteria in the field) Pseudomonas fluorescens This bacteria was engineered to contain an insecticide gene. The bacteria is sprayed on fields with crop dusting planes. The bacteria grow on the plants and when the insects start to eat the plant they will also eat some bacteria with the insecticide. The ingestion of insecticide kills the insects. Tools of the Lab: Transgenic Animals Nkx2.2 KO Pharmaceutical production Knockout mouse Tailor made genetic defects Cystic fibrosis Gaucher s disease Alzheimer s disease Sickle cell anemia Allow us to research cures to these diseases 7
6/28/2016 Genotypic Methods of Identifying Bacteria: Nucleic Acid Sequencing Best way to identify a bacterial species is to determine the 16s rrna sequence of that bacteria 16s rrna is part of the 30s subunit of the bacterial ribosome 16s rrna is highly conserved across species and evolutionary time perfectly suited for bacterial identification and diagnosis of infection Diagnosis Infections: Immunologic Methods (15.5) Characteristics of antibodies such as their quantity or specificity can reveal the history of a patient s contact with microorganisms or other antigens Serology Involves in vitro testing of serum Based on the principle that antibodies have an extreme specificity for antigens Visualization of the interaction of antigens and antibodies provides a powerful tool for detecting, identifying, and quantifying antibodies or antigens Basic Principles of Serological Testing Using Antibodies and Antigens Genotypic Methods of Identifying Bacteria: Fluorescent in situ hybridization Fluorescent in situ hybridization (FISH) rapidly identifies 16s RNA sequences without first culturing the organism Relies on dyes to emit visible light in response to UV radiation Turnaround time for identifying suspect pathogens present in blood cultures has been reduced from 24 hours to 90 minutes Basic Principles of Serological Testing Using Antibodies and Antigens -Serological tests were developed to produce an endpoint reaction visible to the naked eye or with light microscopy General Features of Immune Testing Specificity: property of a test to focus only on a certain antibody or antigen, and not react with an unrelated or distantly related antigen Sensitivity: detection of even minute quantities of antibodies or antigens in a specimen; reflects the degree to which a test will detect every positive person 8
Agglutination Tests In both reactions, one antigen is interlinked by several antibodies to form insoluble aggregates Agglutination: antigens are whole cells or organisms such as red blood cells, bacteria, or viruses - If antigen is present visible clumps of cells form Hemagglutination Agglutination of red blood cells Can be used to determine blood type Antibody Titers Titer: concentration of antibodies in a sample Determined by serially diluting patient serum into test tubes or wells of a microtiter plate containing equal amounts of bacterial cells Reflection of the highest dilution of serum that still produces agglutination The more a serum sample can be diluted and still react with antigen, the greater the concentration of antibodies and thus its titer Used to diagnose autoimmune disorders and determine past exposure to certain diseases Use different concentrations of patients serum Immunofluorescence Testing Fluorescent antibodies (FAbs) Monoclonal antibodies labeled by a fluorescent dye Use Fabs to label cells so that they can be visualized Immunochromatography Testing Very rapid Antigen solution flows through a porous strip and encounters labeled antibody Visible line produced when antigen antibody immune complexes encounter antibody against them Used for pregnancy testing and rapid identification of infectious agents Immunoassays Alternative methods that employ monoclonal antibodies and permit rapid, accurate measurement of trace antigen or antibody levels Radioimmunoassay (RIA) Antibodies or antigens labeled with a radioactive isotope used to pinpoint minute quantities of a corresponding antigen or antibody Used to detect hormone levels in samples and diagnose allergies Same idea as Fabs but rather than a fluorescent dye a radioactive one is used. 9
Immunoassays ELISA Enzyme linked immunosorbent assay Uses an enzyme as the label Reaction of enzyme with its substrate produces colored product Commonly used to detect presence of antibodies in serum Immunoassays: ELISA Antibody sandwich ELISA Modification of the ELISA technique Commonly used to detect antigen Antigen being tested for is sandwiched between two antibody molecules Immunoassays: ELISA Advantages of the ELISA Can detect either antibody or antigen Can quantify amounts of antigen or antibody Easy to perform and can test many samples quickly Plates coated with antigen and gelatin can be stored for later testing 10