Lecture 3: Genetic approach to studying bacterial pathogenesis Flashcards
In Figure 4-3, what occurs in step 7?
There are E. Coli that invade the mammalian cells
In Figure 4-3, what occurs in “gentle lysis” in step 6?
To liberate E. Coli cells that are present inside mammalian cells
In Figure 4-3, what is gentamicin used in step 5?
To kill E. Coli cells that are present outside of mammalian cells
In Figure 4-3, does step 7 have a complete E. Coli genome and contain more than 1 Y. pseudo
gene?
Yes
In Figure 4-3, does step 7 have bacteria that can be killed if exposed to gentamycin?
Yes
In figure 4-3, does step 7 have bacteria that contain an insertion mutation?
No
Generate DNA sequence =
Inv gene
Deduce protein coding region =
Invasin protein
Suicide plasmid in Figures 4-4 and 4-5 contains what kind of mutation?
Inv Loss-of-function mutation
An F+ plasmid has
F plasmid
An F- plasmid lacks
F plasmid
Insertion of a transposon is a gene that most often creates a
Loss-of-function mutation
Transposons help mark the site of the
Mutation
These encode a periplasmic phosphatase
phoA gene
The transposon is inserted where?
Between the ORF
Engineered phoA genes lack what?
N-terminus
Engineered phoA gene lacks an N-terminus so expression depends on ________
Fusion after transposition
What are the most important properties of transposon insertion in bacteria in a dark colony at step 2 in the Tn-phoA mutagenesis scheme in Figure 4-7?
a. Inserted between ORF
b. Inserted downstream of a promoter for a periplasmic protein
What is a correct statement related to the white colonies at step 2 in the Tn-phoA scheme in Figure 4-7?
The pho’ gene is NOT FUSED to a vibrio cholera gene that encodes a secreted protein
What is a correct statement related to the blue (dark) colonies at step 2 in the Tn-phoA scheme in Figure 4-7?
The pho’ gene is FUSED to a vibrio cholera gene that encodes a secreted protein
In the phoA enzyme, the engineered transposon is present within cells in what colony?
White colony
Is the phoA enzyme expressed outside of the bacteria cells?
No
What is an INcorrect statement related to the white colonies at step 2 in the Tn-phoA mutagenesis scheme depicted in Figure 4-7?
The PhoA enzyme is synthesizing a colored molecule inside bacterial cells
In the Tn-phoA scheme used to identify virulence factors for Vibrio cholera, why did researchers choose pH activity at 6.5 AND high osmolarity and low pH activity at 8 and low osmolarity?
a. These are some known vibrio cholera virulence factors that are expressed under these conditions
Vibrio cholera virulence genes are maximally expressed at pH what? And osmolarity?
a. 6.5 pH
b. High osmolarity
PhoA+ colonies turn what color?
Blue
This is examining individual bacteria for desirable traits
Genetic screen
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the lacZY gene on the plasmid?
To help identify candidate virulence genes that are NOT expressed during laboratory growth (lac-minus)
This is selecting only bacteria with desirable trait growth
Genetic Selection
In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the DARK spots in the recovered pool blot?
Dark spots represent salmonella mutants that survived in mice
In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the BLANK spots in the recovered pool blot? +++++
The blank spots represent salmonella mutants that did not grow in mice and, therefore, these mutants represent specific genes necessary for virulence
The DNA sequence tags in (signature-tagged) Figure 4-8 are used to compare ++++
DNA sequence tags are used to compare the Input and recovered pools of bacteria
In step 5 of Figure 4-10, why are bacterial cells reintroduced into macrophages and run through
the fluorescence-activated cell sorter?
To identify bacterial cells that fluoresce within macrophages from the bacterial cell population that did not fluoresce when growing on their own in a lab environment, since these are bacterial of interest
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the purA gene on the plasmid?
To identify candidate virulence genes that are transcriptionally active in the mouse (lac-plus)
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the ampicillin resistance gene on the plasmid?
To select for salmonella bacteria that contain the plasmid inserted within their genome
In DFI or Figure 4-10, what protein do they contain that helps them fluoresce?
GFP
In step 4 of Figure 4-10, why are bacterial cells run through the fluorescence-activated cell
sorter? +++++
To identify bacterial cells that do not fluoresce when growing on their own in a lab environment
In step 3 of figure 4-10, what is true about the macrophages that fluorescence?
Some contain bacteria with GFP fused to genes that are specifically turned on only in macrophages
In IVIAT, figure 4-11, what would occur if step 3 were NOT performed and the original, unmodified patient serum sample was used for step 4?
Antibodies would bind the entire replica filter, generating a signal from the entire filter surface, making individual phage plaques indistinguishable
In IVIAT, figure 4-11, what would occur if the patient had never been exposed to M. tuberculosis?
Antibodies would not bind and no signal would be generated from any of the phage plaques replicates
In IVIAT, figure 4-11, what would ideally occur in step 4 when using serum from a tuberculosis patient, processed as outlined in the figure?
Antibodies would only bind to phage replicates that express mycobacterial proteins expressed on the bacterial cell surface within the patient, generating a signal from just a few spots
In step 5 of Figure 4-11, what do the black dots represent?
Mycobacterial antigens that are expressed in the patient
In the microarray scheme (Figure 4-12), what would be the signal for a gene that is expressed at increased levels in V. cholera during infection compared to during growth in the laboratory
Green - higher Cy3 levels of expression
In the microarray scheme (Figure 4-12), what would be the signal for a gene that is expressed at increased levels in V. cholera during growth in a laboratory compared to during infection?
Red - higher Cy5 levels of expression
In the microarray scheme (Figure 4-12), what would be the signal for a “housekeeping” gene that is expressed at the same level in V. cholerae during infection and during growth in the
laboratory?
A yellow fluorescent signal (equal levels of Cy3 and Cy5)
Are RNA or microarray sequencing more sensitive and have a wider range?
RNA sequencing
Can RNA sequencing be performed on impure samples that contain multiple bacterial species?
Yes
What is not true regarding RNA sequencing?
They can only be performed on PURE samples that contain a single bacterial species
What is gentamicin used for in step 5 of the scheme depicted in figure 4-3?
a. to kill E. coli cells that are present outside of mammalian cells
Why is “gentle lysis” used for in step 6 of the scheme depicted in figure 4-3?
c. to liberate E. coli cells that are present inside of mammalian cells
What is false about the cells growing on the petri dish in step 7 of the scheme depicted in figure 4-3?
they are bacteria that contain an insertion mutation
In Figure 4-3, does step 7 have a complete E. Coli genome and contain more than 1 Y. pseudo
gene?
yes
In Figure 4-3, does step 7 have bacteria that can be killed if exposed to gentamycin?
Yes
In Figure 4-3, what occurs in step 7?
There are E. Coli that invade the mammalian cells
In figure 4-3, does step 7 have bacteria that contain an insertion mutation?
No
What are the most important properties of transposon insertion in bacteria in a dark colony at
step 2 in the Tn-phoA mutagenesis scheme in Figure 4-7?
a. Inserted between ORF
b. Inserted downstream of a promoter for a periplasmic protein
What is a correct statement related to the white colonies at step 2 in the Tn-phoA scheme in Figure 4-7?
The pho’ gene is NOT FUSED to a vibrio cholera gene that encodes a secreted protein
What is a correct statement related to the blue (dark) colonies at step 2 in the Tn-phoA scheme in Figure 4-7?
The pho’ gene is FUSED to a vibrio cholera gene that encodes a secreted protein
In the phoA enzyme, the engineered transposon is present within cells in what colony?
White colony
Is the phoA enzyme expressed outside of the bacteria cells?
no
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the ampicillin resistance gene on the plasmid?
To select salmonella bacteria that contain the plasmid inserted within their genome
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the lacZY gene on the plasmid?
To help identify candidate virulence genes that are NOT expressed during laboratory growth (lac-minus)
In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the DARK spots in the recovered pool blot?
Dark spots represent salmonella mutants that survived in mice
In signature-tagged mutagenesis (figure 4-8), which of the following is a correct statement about the blank spots in the recovered pool blot?
the blank spots represent Salmonella mutants that lost the kanamycin resistance gene during growth in the mouse
Which of the following is a correct statement about signature-tagged mutagenesis (figure 4-8)?
the DNA sequence tags (variable region) are used to compare the input and recovered pools of bacteria
In the vivo expression technology scheme to identify virulence genes (IVET, figure 4-9), what is
the function of the purA gene on the plasmid?
To identify candidate virulence genes that are transcriptionally active in the mouse (lac-plus)
In step 5 of Figure 4-10, why are bacterial cells reintroduced into macrophages and run through
the fluorescence-activated cell sorter?
To identify bacterial cells that fluoresce within macrophages from the bacterial cell population that did not fluoresce when growing on their own in a lab environment, since these are bacterial of interest
In step 3 of figure 4-10, what is true about the macrophages that fluorescence?
Some contain bacteria with GFP fused to genes that are specifically turned on only in macrophages
In step 4 of Figure 4-10, why are bacterial cells run through the fluorescence-activated cell sorter?
to identify bacterial cells that do not fluoresce when growing on their own in a lab environment
In the in vivo induced antigen technology scheme (IVIAT, figure 4-11), what would occur if the patient had never been exposed to M. tuberculosis.
Antibodies would not bind and no signal would be generated from any of the phage plaque replicates
In the in vivo induced antigen technology scheme (IVIAT, figure 4-11), what would ideally occur in step 4 when using serum from a tuberculosis patient, processed as outline in the figure?
Antibodies would only bind to phage plaque replicates that express mycobacterial
proteins expressed on the bacterial cell surface within the patient, generating signal from just a few spots
In the in vivo induced antigen technology scheme (IVIAT, figure 4-11), what would occur if step 3 were not performed and the original, unmodified patient serum sample was used for step 4.
Antibodies would bind to the entire replica filter, generating signal from the entire filter surface, making individual phage plaques indistinguishable
