Lecture 1.3: Genetic Approaches to Studying Bacterial Pathogenesis

What is the primary goal of identifying bacterial genes that encode virulence factors?

To develop new and better ways of preventing or treating infections

Which specific organism's genes are identified using genetic complementation in the lecture notes?

$$Yersinia\,pseudotuberculosis$$

On which organism does the genetic complementation exercise confer invasiveness?

$$E.\,coli$$

What is the first step in creating a genomic library?

Digest the genome of the organism (chromosomal DNA) with a restriction endonuclease

What are the fragments labeled in Figure 4-2 during genomic library creation?

Fragments $A$ through $E$

In the context of ligation, what must the plasmid and the restriction fragments share?

The same "sticky ends"

What process is used to join restriction fragments with a plasmid?

Ligation

What is the product of ligating restriction fragments into a plasmid?

Recombinant plasmids (DNA with cloned donor DNA)

Following ligation, what happens to the recombinant plasmids?

They are transformed into $E.\,coli$

Which antibiotic kills $E.\,coli$ that do not invade mammalian cells?

Gentamycin

Why does Gentamycin fail to kill $E.\,coli$ that have invaded mammalian cells?

Gentamycin does not penetrate mammalian cells

What type of selection is used in the Gentamycin enrichment protocol?

Positive selection

In the enrichment protocol for genetic complementation, what is done after Gentamycin selection?

Bacteria are picked and tested individually

After identifying an invasion factor gene, what is the next logical step?

Manipulate the gene further to prove it really promotes cell invasion

What is the name of the gene sequence identified for the invasion factor?

$inv$ gene

What is the name of the protein coding region deduced from the $inv$ gene?

Invasin protein

What term refers to a mutation that disables the function of a gene?

Loss-of-function mutation

Where can the suicide plasmid used for identifying the $inv$ gene replicate?

In $E.\,coli$

Where can the suicide plasmid NOT replicate during the identification of the $inv$ gene?

In $Y.\,pseudotuberculosis$

How is the suicide plasmid containing the $inv$ loss-of-function mutation transferred?

From $E.\,coli$ to $Y.\,pseudotuberculosis$

How many recombination events are required to replace the native $inv$ gene?

$2$ recombination events

What is replaced during the recombination events in $Y.\,pseudotuberculosis$?

The native $inv$ gene is replaced with the loss-of-function $inv$ allele

What is the expected result when testing $Y.\,pseudotuberculosis$ $inv$ mutants?

They do not invade cells

What generally happens to a gene when a transposon is inserted into it?

It creates a loss-of-function mutation

What does a transposon 'mark' once it is inserted?

The site of the mutation (via sequence and antibiotic resistance)

What are the two general types of transposons shown in the diagrams?

Simple transposon and composite transposon

What does the $phoA$ gene encode?

A periplasmic phosphatase

What is missing from the engineered $phoA$ gene used in transposon mutagenesis?

The $N$-terminus

Because it lacks an $N$-terminus, what does the expression of the engineered $phoA$ gene depend on?

Fusion to an adjacent gene after transposition

What color do $PhoA^{+}$ colonies turn in the presence of $X-P$ dye?

Blue

What effect does $Tn-phoA$ mutagenesis have on $Vibrio\,cholerae$?

Decreased virulence

At what $pH$ are $Vibrio\,cholerae$ virulence genes maximally expressed?

$$pH\,6.5$$

What osmolarity level maximizes expression of $Vibrio\,cholerae$ virulence genes?

High osmolarity

What involves examining individual bacteria for a desirable trait?

Genetic screen

What involves a process where only bacteria with the desirable trait are able to grow?

Genetic selection

The Gentamycin assay in genetic complementation is an example of what?

Selection

What disease model is used in the example for Signature-tagged mutagenesis (STM)?

Mouse model for typhoid fever

In the STM typhoid fever model, what negative trait is screened for?

Inability to grow in the spleen

What organism's mutant library is created for Signature-tagged mutagenesis?

$$S.\,typhimurium$$

What specific transposon is used in the creation of the $S.\,typhimurium$ mutant library?

$$mini-Tn5$$

How is the $mini-Tn5$ moved from $E.\,coli$ to $S.\,typhimurium$?

Via a suicide plasmid

What is unique to each transposon in Signature-tagged mutagenesis?

A unique marker or tag

What does IVET stand for?

In vitro expression technology

What is the purpose of IVET (Promoter-trapping)?

To identify genes expressed during infection but not in the laboratory

What does DFI stand for?

Differential fluorescence induction

In DFI, what happens to the chromosome of $Salmonella$ initially?

Partial digestion

What plasmid is the $Salmonella$ DNA ligated into during DFI?

A $gfp$-fusion plasmid

What reporter gene is used in the DFI technique?

$gfp$ (Green Fluorescent Protein)

In DFI, what are the pooled, transformed $Salmonella$ used to infect?

Macrophages

What technology is used in DFI to separate bacteria and macrophages?

FACS (Fluorescence-Activated Cell Sorting)

In DFI step 5, what must macrophages contain to be isolated by sorting?

Fluorescent bacteria

What is done to macrophages after they are sorted for containing fluorescent bacteria?

They are lysed to release the bacteria

In DFI, where must the clones NOT be fluorescent to be analyzed?

When grown on media

In DFI, where MUST the cloned sequences be fluorescent for analysis?

When grown in macrophages

What does IVIAT stand for?

In vivo-induced antigen technology

What is the underlying approach type for IVIAT?

Antibody-based approach

What specific library is created for IVIAT?

A $\lambda gt11$ expression library

What inducer is used to replicate the IVIAT expression library?

$$IPTG$$

What is the source of antibodies used to screen the IVIAT library?

Patient serum

In IVIAT, why is the antiserum absorbed?

To remove antibodies that bind to factors from organisms grown on medium

What method is used to perform the immunoassay on the IVIAT expression library?

In situ immunoassay

What organism is the example for IVIAT antigens?

$$M.\,tuberculosis$$

What does transcription analysis 1 primarily utilize?

Microarrays

In the microarray scheme, what is compared to $Vibrio\,cholerae$ grown in vitro?

Stool from cholera patients (in vivo)

What is synthesized from extracted RNA in the microarray protocol?

Labeled DNA (cDNA)

What is the fluorescent label for in vitro $V.\,cholerae$ DNA in the microarray diagram?

$$Cy5$$

What is the fluorescent label for in vivo (stool) $V.\,cholerae$ DNA in the microarray diagram?

$$Cy3$$

What is hybridized to the microarray in the transcription analysis?

Labled DNA (derived from RNA)

What approach compares nonpathogenic and pathogenic strains using DNA?

Whole genome sequencing

Which pathogenic strain of $E.\,coli$ is compared to the non-pathogenic strain in genome analysis?

$$O157:H7$$

Which non-pathogenic strain of $E.\,coli$ is compared to strain $O157:H7$?

$$K-12$$

In Figure 4-13, what do the red regions of the genome signify?

Segments unique to $E.\,coli$ strain $O157:H7$

What is generally contained within the unique red regions of the $O157:H7$ genome?

Genes important for pathogenicity

According to Nature Reviews Microbiology 10:618, what is Transcription analysis 2?

RNA sequencing

What is being sequenced in RNA sequencing?

The total RNA population of the pathogen

Why is RNA sequencing considered superior to microarray analysis for sensitivity?

It is more sensitive and has a wider dynamic range

What is unique about the RNA sequencing approach mentioned on Page 16?

Sequencing of total RNA populations of pathogen and host together

What does dual RNA sequencing compare (Page 16)?

Total RNA of bacteria within the host versus microarray analysis of separated bacterial and host RNA

What does the term 'transcriptome' refer to?

The collection of all RNA transcripts in a cell or population

What type of DNA is used as the starting point for a genomic library?

Chromosomal DNA

In Fig 4-2, what generates the sticky ends?

Restriction endonuclease

Identify the bacterium in which invasion factor genes were sought?

$$Yersinia\,pseudotuberculosis$$

What does the gentamycin protection assay identify?

Invasive bacteria

What refers to using a functional gene to restore a phenotype in a mutant?

Genetic complementation

What is the function of the periplasmic phosphatase in $phoA$ mutagenesis?

To cleave the $X-P$ dye and turn colonies blue

What determines if the engineered $phoA$ gene is expressed?

Fusion to a promoter of an adjacent gene via transposition

What is the effect of $Vibrio\,cholerae$ virulence gene expression at $pH\,6.5$?

Maximal expression

What is the purpose of the unique market in STM?

To identify each specific mutant in a pool

Where does the $S.\,typhimurium$ library grow in the STM screen?

In the spleen (or lacks the ability to grow there)

Is IVET used to find genes expressed in vitro?

No, it finds genes expressed in infection (in vivo) but NOT in vitro

What kind of sorting does FACS perform?

Fluorescence-Activated Cell Sorting

What is the indicator of gene expression in the DFI method?

Fluorescence (from $gfp$)

What is the first step of IVIAT?

Create a genomic DNA fragment library in $\lambda gt11$

What is removed from patient serum during IVIAT processing?

Antibodies against bacterial factors produced in laboratory media

What does the stool from cholera patients provide in Fig 4-12?

In vivo transcriptomes of $V.\,cholerae$

What color label does $Cy3$ typically represent in the context of the diagram?

The in vivo/patient sample DNA

What color label does $Cy5$ typically represent in the context of the diagram?

The in vitro/laboratory sample DNA

What is the specific name of the $E.\,coli$ K-12 variant?

Non-pathogenic $E.\,coli$ strain $K-12$

What is the specific name of the $E.\,coli$ O157:H7 variant?

Pathogenic $E.\,coli$ strain $O157:H7$

What is a 'suicide plasmid'?

A plasmid designed to not replicate in a specific host organism after transfer