Lecture 3: Genetic approach to studying bacterial pathogenesis

Question 1

In Figure 4-3, what occurs in step 7?

Answer 1

There are E. Coli that invade the mammalian cells

Question 2

In Figure 4-3, what occurs in “gentle lysis” in step 6?

Answer 2

To liberate E. Coli cells that are present inside mammalian cells

Question 3

In Figure 4-3, what is gentamicin used in step 5?

Answer 3

To kill E. Coli cells that are present outside of mammalian cells

Question 4

In Figure 4-3, does step 7 have a complete E. Coli genome and contain more than 1 Y. pseudo
gene?

Answer 4

Yes

Question 5

In Figure 4-3, does step 7 have bacteria that can be killed if exposed to gentamycin?

Answer 5

Yes

Question 6

In figure 4-3, does step 7 have bacteria that contain an insertion mutation?

Answer 6

No

Question 7

Generate DNA sequence =

Answer 7

Inv gene

Question 8

Deduce protein coding region =

Answer 8

Invasin protein

Question 9

Suicide plasmid in Figures 4-4 and 4-5 contains what kind of mutation?

Answer 9

Inv Loss-of-function mutation

Question 10

An F+ plasmid has

Answer 10

F plasmid

Question 11

An F- plasmid lacks

Answer 11

F plasmid

Question 11

Insertion of a transposon is a gene that most often creates a

Answer 11

Loss-of-function mutation

Question 12

Transposons help mark the site of the

Answer 12

Mutation

Question 13

These encode a periplasmic phosphatase

Answer 13

phoA gene

Question 14

The transposon is inserted where?

Answer 14

Between the ORF

Question 14

Engineered phoA genes lack what?

Answer 14

N-terminus

Question 15

Engineered phoA gene lacks an N-terminus so expression depends on ________

Answer 15

Fusion after transposition

Question 16

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?

Answer 16

a. Inserted between ORF
b. Inserted downstream of a promoter for a periplasmic protein

Question 17

What is a correct statement related to the white colonies at step 2 in the Tn-phoA scheme in Figure 4-7?

Answer 17

The pho’ gene is NOT FUSED to a vibrio cholera gene that encodes a secreted protein

Question 18

What is a correct statement related to the blue (dark) colonies at step 2 in the Tn-phoA scheme in Figure 4-7?

Answer 18

The pho’ gene is FUSED to a vibrio cholera gene that encodes a secreted protein

Question 19

In the phoA enzyme, the engineered transposon is present within cells in what colony?

Answer 19

White colony

Question 20

Is the phoA enzyme expressed outside of the bacteria cells?

Answer 20

No

Question 21

What is an INcorrect statement related to the white colonies at step 2 in the Tn-phoA mutagenesis scheme depicted in Figure 4-7?

Answer 21

The PhoA enzyme is synthesizing a colored molecule inside bacterial cells

Question 22

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?

Answer 22

a. These are some known vibrio cholera virulence factors that are expressed under these conditions

Question 23

Vibrio cholera virulence genes are maximally expressed at pH what? And osmolarity?

Answer 23

a. 6.5 pH b. High osmolarity

Question 24

PhoA+ colonies turn what color?

Answer 24

Blue

Question 24

This is examining individual bacteria for desirable traits

Answer 24

Genetic screen

Question 25

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?

Answer 25

To help identify candidate virulence genes that are NOT expressed during laboratory growth (lac-minus)

Question 25

This is selecting only bacteria with desirable trait growth

Answer 25

Genetic Selection

Question 26

In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the DARK spots in the recovered pool blot?

Answer 26

Dark spots represent salmonella mutants that survived in mice

Question 26

In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the BLANK spots in the recovered pool blot? +++++

Answer 26

The blank spots represent salmonella mutants that did not grow in mice and, therefore, these mutants represent specific genes necessary for virulence

Question 26

The DNA sequence tags in (signature-tagged) Figure 4-8 are used to compare ++++

Answer 26

DNA sequence tags are used to compare the Input and recovered pools of bacteria

Question 27

In step 5 of Figure 4-10, why are bacterial cells reintroduced into macrophages and run through the fluorescence-activated cell sorter?

Answer 27

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

Question 27

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?

Answer 27

To identify candidate virulence genes that are transcriptionally active in the mouse (lac-plus)

Question 27

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?

Answer 27

To select for salmonella bacteria that contain the plasmid inserted within their genome

Question 28

In DFI or Figure 4-10, what protein do they contain that helps them fluoresce?

Answer 28

GFP

Question 29

In step 4 of Figure 4-10, why are bacterial cells run through the fluorescence-activated cell sorter? +++++

Answer 29

To identify bacterial cells that do not fluoresce when growing on their own in a lab environment

Question 29

In step 3 of figure 4-10, what is true about the macrophages that fluorescence?

Answer 29

Some contain bacteria with GFP fused to genes that are specifically turned on only in macrophages

Question 30

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?

Answer 30

Antibodies would bind the entire replica filter, generating a signal from the entire filter surface, making individual phage plaques indistinguishable

Question 31

In IVIAT, figure 4-11, what would occur if the patient had never been exposed to M. tuberculosis?

Answer 31

Antibodies would not bind and no signal would be generated from any of the phage plaques replicates

Question 32

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?

Answer 32

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

Question 33

In step 5 of Figure 4-11, what do the black dots represent?

Answer 33

Mycobacterial antigens that are expressed in the patient

Question 34

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

Answer 34

Green - higher Cy3 levels of expression

Question 35

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?

Answer 35

Red - higher Cy5 levels of expression

Question 36

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?

Answer 36

A yellow fluorescent signal (equal levels of Cy3 and Cy5)

Question 37

Are RNA or microarray sequencing more sensitive and have a wider range?

Answer 37

RNA sequencing

Question 38

Can RNA sequencing be performed on impure samples that contain multiple bacterial species?

Answer 38

Yes

Question 39

What is not true regarding RNA sequencing?

Answer 39

They can only be performed on PURE samples that contain a single bacterial species

Question 40

What is gentamicin used for in step 5 of the scheme depicted in figure 4-3?

Answer 40

a. to kill E. coli cells that are present outside of mammalian cells

Question 41

Why is “gentle lysis” used for in step 6 of the scheme depicted in figure 4-3?

Answer 41

c. to liberate E. coli cells that are present inside of mammalian cells

Question 42

What is false about the cells growing on the petri dish in step 7 of the scheme depicted in figure 4-3?

Answer 42

they are bacteria that contain an insertion mutation

Question 43

In Figure 4-3, does step 7 have a complete E. Coli genome and contain more than 1 Y. pseudo gene?

Answer 43

yes

Question 44

In Figure 4-3, does step 7 have bacteria that can be killed if exposed to gentamycin?

Answer 44

Yes

Question 45

In Figure 4-3, what occurs in step 7?

Answer 45

There are E. Coli that invade the mammalian cells

Question 45

In figure 4-3, does step 7 have bacteria that contain an insertion mutation?

Answer 45

No

Question 46

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?

Answer 46

a. Inserted between ORF b. Inserted downstream of a promoter for a periplasmic protein

Question 47

What is a correct statement related to the white colonies at step 2 in the Tn-phoA scheme in Figure 4-7?

Answer 47

The pho’ gene is NOT FUSED to a vibrio cholera gene that encodes a secreted protein

Question 48

What is a correct statement related to the blue (dark) colonies at step 2 in the Tn-phoA scheme in Figure 4-7?

Answer 48

The pho’ gene is FUSED to a vibrio cholera gene that encodes a secreted protein

Question 49

In the phoA enzyme, the engineered transposon is present within cells in what colony?

Answer 49

White colony

Question 49

Is the phoA enzyme expressed outside of the bacteria cells?

Answer 49

no

Question 49

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?

Answer 49

To select salmonella bacteria that contain the plasmid inserted within their genome

Question 49

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?

Answer 49

To help identify candidate virulence genes that are NOT expressed during laboratory growth (lac-minus)

Question 49

In signature-tagged mutagenesis, Figure 4-8, what is a correct statement regarding the DARK spots in the recovered pool blot?

Answer 49

Dark spots represent salmonella mutants that survived in mice

Question 49

In signature-tagged mutagenesis (figure 4-8), which of the following is a correct statement about the blank spots in the recovered pool blot?

Answer 49

the blank spots represent Salmonella mutants that lost the kanamycin resistance gene during growth in the mouse

Question 49

Which of the following is a correct statement about signature-tagged mutagenesis (figure 4-8)?

Answer 49

the DNA sequence tags (variable region) are used to compare the input and recovered pools of bacteria

Question 50

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?

Answer 50

To identify candidate virulence genes that are transcriptionally active in the mouse (lac-plus)

Question 51

In step 5 of Figure 4-10, why are bacterial cells reintroduced into macrophages and run through the fluorescence-activated cell sorter?

Answer 51

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

Question 52

In step 3 of figure 4-10, what is true about the macrophages that fluorescence?

Answer 52

Some contain bacteria with GFP fused to genes that are specifically turned on only in macrophages

Question 53

In step 4 of Figure 4-10, why are bacterial cells run through the fluorescence-activated cell sorter?

Answer 53

to identify bacterial cells that do not fluoresce when growing on their own in a lab environment

Question 54

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.

Answer 54

Antibodies would not bind and no signal would be generated from any of the phage plaque replicates

Question 55

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?

Answer 55

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

Question 55

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.

Answer 55

Antibodies would bind to the entire replica filter, generating signal from the entire filter surface, making individual phage plaques indistinguishable