MCB301 Exam 4 Final Exam

Conserved regions located at the ends of the 16S rRNA gene can be used to synthesize a single pair of PCR primers that can amplify the whole 16S rRNA gene from different bacteria. The variable regions located throughout the 16S rRNA gene are unique to certain species, and can be sequenced to help identify the bacterial species.

Comparison of 16S rRNA sequences from different bacterial species reveal regions that are conserved and regions that are variable. How are these conserved regions and how are these variable regions useful in the Antibiotic Producing Bacteria Exercise?

CP buffer. DNA wash buffer Elution buffer

Following PCR (in the PCR cleanup stage), what was the order of buffers added to clean the DNA?

Actinomycetes

Gram positive bacteria High GC content 63-78% filamentous structure spore forming (sporophores) found in soil,water, plants

Bacteria - 30S and 50S Ribosomal subunits. Makes 70S Ribosome. Eukaryotes- 40S and 60S Ribosomal subunits. Makes 80S ribosome.

Protein synthesis in bacteria requires the ____ and ____ ribosomal subunits, while in eukaryotes it requires the ______ and ______ subunits

Bacitracin

This antibiotic binds bactoprenol and inhibits externalization of the NAG-NAM-peptide. This inhibits peptidoglycan synthesis.

Tetracycline

This antibiotic binds the 30S ribosomal subunit of bacteria and prevents bacterial translation.

Erythromycin

This antibiotic binds the 50S ribosomal subunit, preventing bacterial translation.

Quinolone

This antibiotic binds to DNA gyrase, prevents supercoiling and packaging of DNA during cell division, killing it.

Rifampin

This antibiotic binds to and inhibits bacterial RNA polymerase activity

Penicillin

This antibiotic blocks peptidoglycan synthesis. When PG synthesis is affected, the cell wall is not as strong, and the cell lyses due to osmosis. (It REQUIRES THE CELL TO BE GROWING).

Penicillins and cephalosporins (Beta-lactam antibiotics)

This antibiotic inhibits PG cross linking, weakening the cell membrane and causing cellular lysis.

Fosfomycin

This antibiotic inhibits the formation of the NAM-peptide complex, hence inhibiting peptidoglycan synthesis.

Kanamycin

This antibiotic normally binds the 30S ribosomal subunit, and can be resisted against by having a plasmid with the aph gene, which confers its resistance to the cell. (In Tn mutagenesis).

Cyclohexamide

This chemical is present on the AGS plate during the enrichment phase of Antibiotic producing bacteria. It inhibits growth of eukaryotic fungi, who have spores that can also survive desiccation.

Spin column

This contains a membrane that will bind DNA.

Transposition.

This is the movement of specific genetic elements from one locus to another, and requires three macromolecular elements: 1)DNA transfer 2) target DNA, a recipient of transferred DNA 3)Transposase, and enzyme catalyzing its transfer

Phage Typing

This is used to identify which phage (virus) a bacterium is susceptible to. This can be seen through plaques

Replicative Transposition

This mechanism of Transposition has a TE and a target site, and the TE is "copy and pasted" into the target site.

Conservative Transposition.

This mechanism of transposition has a TE and a target site, but the TE is CUT from the donor, and pasted into the target site. "Cut and paste method"

ELISA Enzyme Linked ImmunoSorbent Assay

This process can quantitate antigen or quantitate serum antibody depending on how you set it up.

Serology

This uses agglutination to test for identification. Often use blood tests to identify proteins made by immune system.

Glycerol

This was added during freezing process to act as a cryoprotectant to the culture, when it was stored at -80*C. It acts to bind intracellular water.

S. marcescens DAP+ plasmid transferred by conjugation, no pir gene, plasmid won't replicate. Kanr only if transposon jumps from plasmid to chromosome. Looking for strain where Tn5 derivative has jumped into pigment gene -> forms white colonies.

Transposant characteristics

parts of plasmid pRL27 image

parts of plasmid pRL27 image

Streptomyces

-Facultative aerobe -nutritionally versatile -growth factor requirements rare. -pigmented -produce 2ndary metabolites. Antibiotics, antifungal agents, antiviral, insecticides, herbicides.

Streptomyces

50% of soil Actinomycetes -> geosmins produced give the soil their earthy odor. >500 species Genome x2 size Ecoli, 8 Mbp. 69-73% GC content Type of Actinomycete Septation at end of sporophore produce uninucleave cells that develop into spores called conidia (triggered by nutrient depletion).

An efflux pump that transports the antibiotic out of the cell A decreased permeability or entry of the antibiotic into the cell through the membrane A change in a ribosomal component or ribosome structure (e.g., due to a mutation in a gene encoding a ribosomal protein or rRNA) so it no longer interacts with the antibiotic

A bacterial cell can become resistant to kanamycin by several different mechanisms. One way is how pRL27 confers resistance to kanamycin to its host cell. What are two other general mechanisms of kanamycin resistance?

Beta-lactamase (or penicillinase) (1 pt) The enzyme cleaves the beta-lactam ring of penicillin to inactivate it. (1 pt)

According to the assigned readings, the initial cultures of Penicillium that were grown to extract penicillin were of low antibiotic activity. This was due to the culture being contaminated with E. coli. Although not actually stated in those readings, what is the name of the enzyme that was probably produced by E. coli that affected penicillin? Specifically, how does this enzyme work?

The "phagocyte receptor-binding site" is part of the constant domain of the heavy chains of certain antibody classes (e.g., IgG). The antibody binds to an antigen such as a pathogen, and then a phagocyte can recognize and bind to the "phagocyte receptor-binding site" on the antibody so that the pathogen can be ingested and killed by the phagocyte (this is called opsonization).

As described in the ELISA exercise, where is the "phagocyte receptor-binding site" located and how does it function in host defense?

The presence and identification of enterics (or coliforms, or Escherichia coli) is usually performed on water. The presence of enterics in water indicates that the water is contaminated with stool or feces (which is composed of enterics). Stool could also contain certain pathogens (especially those that cause diarrhea) and thus the contaminated water would be unsafe to drink.

As discussed in lecture, water from a water treatment plant is usually analyzed for what type of microorganism, and why?

A plaque is a clear zone formed in a lawn of bacterial growth where cells have lysed because they became infected by a virus. This would indicate that the bacterium is susceptible to infection by that particular virus (the bacterial cell must possess the appropriate receptor recognized by the phage to infect that cell). Each bacterial species is susceptible to specific phage, so an unknown bacterium may be identified if it can be shown to be susceptibility to the same set of specific phage as a known bacterium.

As discussed in the Identification lecture, what is a plaque and how can the formation of a plaque be used in the identification of a bacterium? Note: in this question, the term "plaque" does not refer to growth of a biofilm on a tooth surface

The tube contains several individual compartments, each with a different selective/differential medium. A wire runs through the middle of each compartment so all of the media can be inoculated quickly. This is performed by touching one end of the wire to a bacterial colony and pulling and then pushing the wire through the tube The tube is incubated to allow growth, and then the tests are scored (usually by looking for color changes; some compartments require reagents to be added). The test results are compared to a database of results from known bacteria to identify the microorganism

Describe how an Enterotube (Enteropluri Tube) is inoculated and used to help identify a bacterial organism

Each of the following: They are non-human antibodies (e.g., a rabbit or goat). They bind to human antibodies. They are conjugated or linked to an enzyme

Describe the secondary antibodies used in an HIV ELISA? Do not describe their structure (e.g., 2 heavy chains and 2 light chains connected by disulfide bonds, etc), but describe three other unique characteristics important for the HIV ELISA

End result conjugation

End result conjugation

The person was infected recently so they have not produced enough antibodies to produce a positive result (i.e., they have not seroconverted). or The person's immune system has been compromised by HIV, so that they do not produce a strong HIV antibody response

How could a person who is infected with HIV produce a negative HIV ELISA result (i.e., a false negative result)? Describe one possibility. Assume the HIV ELISA procedure was performed correctly

The classical Approach: -morphological characteristics -differential staining : Gram stain -Biochemical tests: Selective+differential media enzymatic tests

How did we in the lab try to do the identification of bacteria?

Cross linking involves cleaving of D-ala-D-ala in peptides of PG by the PBPs. D-ala-D-ala looks like B lactam ring. PBPs bind irreversibly to antibiotic, inactivating it, so it no longer interacts with the peptides.

How do PBPs bind penicillin?

1) Digest DNA into smaller fragments with Restriction Enzymes. 2) Separate fragments by size ie run on gel. 3) visualize bands 4) compare unknown bands to known bands to identify bacteria.

How do you do DNA fingerprinting?

Asks if DNA from organism X can bind with DNA from organism Y. If so they may be similar. southern blot.

How does Nucleic Acid Hybridization work?

pRL27 without an oriT site would be unable to transfer itself by conjugation from the donor cell to the recipient cell Therefore, the recipient cell would not receive the transposable element needed to create the insertion mutations

If pRL27 does not have an oriT site, then our Transposon Mutagenesis Experiment would be unsuccessful in generating the proper mutants. How would the lack of an oriT site prevent generation of the proper mutants?

Lysozyme, 20mM tris, 2mM EDTA, Triton X-100 mix. lysozyme degrades thick PG cell wall in Gram positive bacteria. Triton-detergent disrupts membrane integrity Tris buffers the pH EDTA chelates divalent metal ions, which are required by nucleases to function, limiting spontaneous cleavage of DNA.

In PCR, the lysis buffer contains?

Ethanol

In PCR, what helps to precipitate nucleic acids in preparation for purifying by spin columns?

Proteinase K. Helps degrade proteins released from the cells when you lyse them.

In PCR, what is added to degrade proteins released from the cells when you lyse them?

BDL buffer

In PCR, what is the alkaline solution that rapidly lyses the cells?

E.coli donor S. marcescens recipient

In Tn mutagenesis, _______ was our donor containing the PRL27 plasmid, and ________ was the recipient of the plasmid via conjugation.

Negative pole, they move neg to positive bc the DNA is negatively charged.

In gel electrophoresis, DNA wells are placed on the _____ pole

Yes, because the extracted C antigen must bind to at least two antibodies at the same time for agglutination to occur. Since each antibody has two antigen binding sites (if it is IgG), many C antigens can be joined in a chain or cluster by the antibodies to form a visible clumping or agglutination. if the C antigen only had one epitope, then IgG would only bind to two C antigens and not result in a visible clumping of many antigens.

In order for the Streptex test to work, does the C antigen have to have multiple epitopes (i.e., the same epitope present multiple times on the C antigen) that is recognized by the antibody used in the test? If yes, why? If no, why not?

Positive control- streptomyces sp. negative control- extraction buffer

In the PCR portion of antibiotic producing bacteria, what was the positive and negative controls?

Elution Buffer

In the PCR wash steps, this is added to each spin column prior to a 5 minute incubation to unbind the DNA from the membrane in the spin column.

DNA wash buffer

In the PCR wash steps, this is added to remove most of the RNA from the DNA preparation.

HBC Buffer

In the PCR wash steps, this is added to the spin columns to remove protein from the DNA preparation.

We plated to LB-Kan. Kan kills the original recipient cells that didn't get a Tn, bc they don't have kanamycin resistance gene from Tn. (selection) Counter selection: Donor cells are killed because there is No DAP, and they need DAP to grow.

In the Tn experiment, what kills cells?

Genes that encode the sex pilus, mating bridge, nicking enzyme, and DNA polymerase

In the Transposon Mutagenesis Exercise, what four genes important for the conjugation process are located on the chromosome of the donor strain?

The medium used as a negative control (i.e., LB Kan) does not contain DAP Since the donor cell cannot synthesize its own DAP it needs it preformed in the medium. Otherwise, when the bacterium grows it cannot form a strong cell wall and it will be killed by osmotic lysis.

In the Transposon Mutagenesis Exercise, why can't the donor cells grow on the medium serving as a negative control?

1492R and 27F

In the antibiotic experiment, the two primers used were?

Just Know this

Just Know this

Just know this

Just know this

pig gene cluster produces red pigment, (prodigiosin). White colonies are what we look for. Signals for disruption in the pigment gene. Restreak to confirm. Biochemical tests on white colonies to further compare.

Mutant Screening, what produces red pigment? and what are we looking for in our colonies on the LB-Kan plates?

In cytoplasm 1) NAM Linked to Peptide 2) NAM-Peptide binds bactoprenol in CM. 3)NAG linked to Nam-peptide 4) NAG-NAM-Peptide moved across CM by bactoprenol outside of membrane/outside of cytoplasm. 5) NAG-NAM-Peptides linked together by NAG-NAM contacts 6) Glycan Strands cross linked together

Name the Steps of Peptidoglycan synthesis.

Tryptone is a digest of casein (milk ptn) comprised of short chain peptides, source of nitrogen, carbon, energy. Soytone - papain digest of soybean, rich in AAs, also contains carbohydrates.

On Tryptic soy agar we used to plate our antibiotic bacteria + 4 others on, what is the tryptone and soytone used for?

The genomes of various strains of bacteria were each screened for a specific gene required for the synthesis of phosphonic acid products. This was performed by PCR using DNA primers to conserved regions of that gene. Generation of the amplification fragment by PCR indicated that the bacterial strain possessed that gene and thus made a type of phosphonic acid product.

One of the assigned readings described genome mining and its use to discover potentially new phosphonic acid products. Briefly describe how PCR was used in genome mining described in the reading.

Doubles, after 30 cycles over 1billion copies, theoretically but not actually.

PCR Starts with 1 copy of ds DNA, and does what after each cycle?

DNA-DNA hybridization of genomes -> greater than or equal to 70% hybridization. 16S rRNA gene sequences -> 97% similarity.

Species defined by nucleic acid similarities. What percents are needed in DNA-DNA hybridization of genomes? What about in 16S rRNA gene sequences?

True

T/F Antibiotic production is linked to sporulation.

True

T/F Streptomyces are resistant to their own antibiotics produced, but are susceptible to other antibiotics.

Classification - domain, phylum, class, order, family, genus, species. Nomenclature- Binomial system (genus and species). e.g. Escherichia coli.

Taxonomy both classification types and nomenclature =?

The 16S rRNA gene is found in all bacteria and contains conserved sequences so that it can be amplified from all of those bacterial species (using "universal" PCR primers). (1 pt) Note: it is also only about 1,500 bp long and can be amplified easily The 16S rRNA gene also contains variable sequences unique to each bacterial species. So sequencing of the gene and searching databases of 16S rRNA gene sequences from known bacteria, can help determine the identity of the microorganism.

The 16S rRNA gene is ideal for PCR amplification and determination of bacterial species. Explain how it is ideal

9 Variable regions

The 16S rRNA we sequenced is 1540 nucleotides and has _____ hypervariable regions.

Arginine

The AGS plate contains __________, to test for nitrogen utilization in the antibiotic producing bacteria.

The recipient bacterium is a facultative aerobe because it can also perform fermentation

The recipient cell used in the transposon mutagenesis experiment can do aerobic respiration. Based on further information about the recipient cell that you were given, is the recipient an obligate aerobe, obligate anaerobe, facultative aerobe, microaerophile, or aerotolerant anaerobe, and why?

Those that interact with the 50S ribosomal subunit, those that interact with the 30S ribosomal subunit, and those that interact with tRNA.

There are several different antibiotics that inhibit translation. They can be categorized into different general groups based on how they function. Based on the Lab Manual, what are three of those groups?

Cross-linking enzymes (bound by b-lactam antibiotics).

These enzymes were called PBPs, penicillin binding proteins, before their real function was known in the cytoplasmic membrane. What are these?

Bactericidal (kill bacteria) Bacteriostatic (inhibit their growth doesn't kill them).

What are effects of antibiotics?

Amino Acid sequencing Fatty acid analysis by Gas chromatography Base composition of Nucleic acids -> determine entire sequence of bases in organism's DNA. - DNA fingerprinting, hybridization, PCR, microarry

What are some other identification methods for bacteria?

E. coli DAP- (needs diaminopimelic acid), used to make peptidoglycan of cell wall. Has pir gene (pi protein -> replicates plasmid) carries pRL27 plasmid Kanr (has aph gene on plasmid)

What are the donor strain characteristics in the Tn experiment?

oriR6K- origin of replication aph- gene that confers Kanr oriT- site of transfer tnp- transposase gene tetAp- promoter originally associated with TetR gene often several transfer genes (tra) are on the plasmid to encode pilus, mating bridge, nicking enzyme, etc. but we used one on the chromosome of the donor in our case.

What are the parts of plasmid pRL27?

S. marcescens pigment genes (red) can conjugate with donor strain to receive plasmid No pir gene (can't replicate plasmid) Kans (no aph gene) DAP+

What are the recipient strain characteristics?

1) Contact between D/R by sex pilus, which retracts and brings cells together. 2) Mating bridge forms. 3) Activation of DNA transfer by cleaving of one DNA strand at OriT. 4) ssDNA plasmid transfer as DNA replicated in the Donor 5) synthesis of functional plasmid inside recipient cell "2nd strand synthesis"

What are the steps of Conjugation?

Chromagen or substrate added (Tetramethylbenzidine or TMB) interacts with enzyme on 2ndary enzyme resulting in color production. Darker color=higher titer

What are the substrate(s) types and names that are added after 2ndary antibody usage in ELISA?

Natural pdts- fungi/bacteria synthetic (from chemists) semi-synthetic, natural pdts modified

What are the types of Antibiotics?

Constant characteristics of the cell- Morphology: shape, size, arrangement, presence of spores, flagella. (limited however). Metabolic properties: enzymatic activities, utilization of carbon/nitrogen substrates, waste pdts.

What are things we look for in the identification process?

The 2ndary antibody binds the H constant regions (or Fc) of 1primary antibody

What does the 2ndary antibody bind in the ELISA?

Genes to make sex pillus. genes to make mating bridge plasmid with OriT (site nicked) genes needed to make enzyme that nicks oriT enzymes and substrates to synthesize DNA.

What elements are needed for transfer of DNA in conjugation?

The best fit method.

What identification method involves using several biochemical tests at once using an enterotube which allows you to innoculate many tests simultaneously?

Transpeptidase. Has OH, binds one Ala, Glycine from peptide chain can attack this back, linking the peptides and releasing the free OH again.

What is a cross linking enzyme that finds the D-Ala-D-Ala and links peptides.

Need to know the sequences at the ends of the DNA to make 2 PCR primers.

What is a problem with PCR?

The variable regions of the Heavy and light chains bind an epitope on the HIV antigen (or any antigen it is specific for for that matter).

What part of the antibody contains the antigen binding sites?

DNA synthesis RNA synthesis Protein Synthesis Peptidoglycan synthesis

What process might antibiotics target?

IgM because they are normally composed of 5 IgM monomers joined together and thus have 10 antigen binding sites, whereas IgG exists as a monomer with 2 antigen binding sites. The more binding sites present, the more cells (with antigen) can be bound together (i.e., agglutinated). (2 pts) Note: students may need to find out information about IgM which was not discussed in this course

Which class of antibody would be better at agglutination reactions: IgM or IgG? Explain why?

Sulfa drugs inhibit the activity of an enzyme that makes a vitamin (i.e., folic acid) required for the growth of both bacteria and humans. However, bacteria make this enzyme, and humans do not (Note: humans therefore require the vitamin preformed in their diet). Consequently, the drug affects the growth of bacteria and not human cells (i.e., it has selective toxicity)

Why do sulfa drugs (like sulfanilamide) have selective toxicity? Include in your answer how sulfa drugs work. Note: The additional assigned readings may be helpful.

A mutation could change the result of a single biochemical test so that it results in going down the wrong pathway of the key and misidentification of the bacterium will occur. However, with the "best fit" method, several biochemical tests are done at once to find a bacterium that most likely possesses those characteristics (a perfect match may not have to occur to find a likely candidate)

Why would a mutation in a bacterial genome more likely cause a misidentification of a bacterium when using a dichotomous key than a "best fit" method?

50%

______% of streptomyces isolates produce antibiotics (a single isolate may produce more than one type)

DNA microarrays

_______ are used to test for gene expression in mass number

Transduction

_________ is DNA transfer by bacteriophages

Transformation

_________ is the uptake of free DNA from the environment

Conjugation

____________ is direct cell to cell transfer of DNA. This is a one-way or unidirectional process where DNA is transferred from donor to recipient.

Requires Pi protein, encoded by pir gene Pi protein

oriR6K requires _________ which is encoded by the _________ gene which binds at the ori site. Hosts with this _________ protein can only replicate pRL27