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Microbiology > Lecture 3: Genetic approach to studying bacterial pathogenesis > Flashcards

Lecture 3: Genetic approach to studying bacterial pathogenesis Flashcards

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Biology 101 flashcards
1
Q

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

A

There are E. Coli that invade the mammalian cells

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2
Q

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

A

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

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3
Q

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

A

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

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4
Q

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

A

Yes

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5
Q

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

A

Yes

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6
Q

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

A

No

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7
Q

Generate DNA sequence =

A

Inv gene

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8
Q

Deduce protein coding region =

A

Invasin protein

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9
Q

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

A

Inv Loss-of-function mutation

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10
Q

An F+ plasmid has

A

F plasmid

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11
Q

An F- plasmid lacks

A

F plasmid

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11
Q

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

A

Loss-of-function mutation

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12
Q

Transposons help mark the site of the

A

Mutation

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13
Q

These encode a periplasmic phosphatase

A

phoA gene

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14
Q

The transposon is inserted where?

A

Between the ORF

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14
Q

Engineered phoA genes lack what?

A

N-terminus

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15
Q

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

A

Fusion after transposition

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16
Q

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

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

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17
Q

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

A

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

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18
Q

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

A

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

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19
Q

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

A

White colony

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20
Q

Is the phoA enzyme expressed outside of the bacteria cells?

A

No

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21
Q

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

A

The PhoA enzyme is synthesizing a colored molecule inside bacterial cells

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22
Q

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

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

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23
Q

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

A

a. 6.5 pH
b. High osmolarity

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24
Q

PhoA+ colonies turn what color?

A

Blue

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24
Q

This is examining individual bacteria for desirable traits

A

Genetic screen

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25
Q

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?

A

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

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25
Q

This is selecting only bacteria with desirable trait growth

A

Genetic Selection

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26
Q

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

A

Dark spots represent salmonella mutants that survived in mice

26
Q

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

A

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

26
Q

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

A

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

27
Q

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

A

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

27
Q

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?

A

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

27
Q

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?

A

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

28
Q

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

A

GFP

29
Q

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

A

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

29
Q

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

A

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

30
Q

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?

A

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

31
Q

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

A

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

32
Q

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?

A

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

33
Q

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

A

Mycobacterial antigens that are expressed in the patient

34
Q

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

A

Green - higher Cy3 levels of expression

35
Q

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?

A

Red - higher Cy5 levels of expression

36
Q

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

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

37
Q

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

A

RNA sequencing

38
Q

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

A

Yes

39
Q

What is not true regarding RNA sequencing?

A

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

40
Q

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

A

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

41
Q

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

A

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

42
Q

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

A

they are bacteria that contain an insertion mutation

43
Q

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

A

yes

44
Q

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

A

Yes

45
Q

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

A

There are E. Coli that invade the mammalian cells

45
Q

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

A

No

46
Q

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

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

47
Q

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

A

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

48
Q

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

A

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

49
Q

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

A

White colony

49
Q

Is the phoA enzyme expressed outside of the bacteria cells?

A

no

49
Q

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?

A

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

49
Q

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?

A

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

49
Q

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

A

Dark spots represent salmonella mutants that survived in mice

49
Q

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

A

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

49
Q

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

A

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

50
Q

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?

A

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

51
Q

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

A

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

52
Q

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

A

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

53
Q

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

A

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

54
Q

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.

A

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

55
Q

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?

A

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

55
Q

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.

A

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

56
Q
A

Microbiology (6 decks)

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