Practical 1. Differentiation of Bacteria and the Induction of Bacterial Gene Expression During Infection Flashcards
How can a rough phenotypic differentiation of bacteria be obtained ?
- The source of bacteria
- Cell shape and colony colour and morphology
- Gram status
- Metabolic features
- Phenotypic tests for presence of characteristic genes
6.
What test will identify if a bacteria is gram positive or gram negative ?
The potassium hydroxide test
What can potassium hydroxide do to bacteria ?
Quickly lyse gram negative cells as they have a weaker cell envelope and liberate DNA
What do gram positive cells have ?
- Wall technoic acids
- Lipotechnoic acids
- Capsular protein
What do gram negative cells have ?
- Lipopolysaharide
- Outer membrane
- Periplasm
- Outer membrane protein
What is the composition of LPS ?
- O-antigen
- Core saccharide
- Lipid A
What does a gram negative bacteria result of a potassium hydroxide test ?
Slimy stringy substance
What may gram negative bacteria be further characterised by ?
Whether they possess the ability to grow in the presence or absence of oxygen
What is the test used to show the presence of oxygen in gram negative bacteria ?
Oxidative fermentative test
What is used to seal the test tube in the oxidative fermentative test ?
Paraffin
What does paraffin prevent ?
Gas exchange with the environment
What does the growth medium in the oxidative fermentative test contain ?
A pH indicator
What will the pH indicator in the growth medium in the oxidative fermentative test do when bacteria have acidified/presence of oxygen ?
Will turn the colour of the growth medium yellow
What is the oxidase test used for ?
If a bacterium possesses certain cytochrome c oxidases
What does the possession of certain cytochrome c oxidases mean for bacteria ?
They can utilise oxygen for energy production via an electron transport chain
What does the oxidase test use as a redox indicator ?
N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD)
What happens when the reagent is oxidised is the oxidase test ?
Becomes dark blue
What colour is the reagent in the oxidase test in its initial reduced state ?
Colourless
What are many gram negative pathogens ?
Oxidase positive
What are some examples of gram negative, oxidase positive bacteria ?
- Pseudomonas aeruginosa
- Helicobacter pylori
- Vibrio cholera
- Campylobacter jejuni
What does the oxidase test show about the Enterobacteriaceae ?
They are oxidase negative
What do the results of the oxidase test show about staphylococci and streptococci ?
Oxidase negative
What will the catalase test do ?
Distinguish gram positive bacteria
What do most obligate aerobic and faculative anaerobic bacteria produce ?
Hydrogen peroxide as an oxidative end product of the aerobic breakdown of sugars
What do bacteria do to protect themselves from the toxic hydrogen peroxide ?
Produces catalase which converts hydrogen peroxide to harmless water and oxygen
What do you see if a bacteria possesses catalase ?
Bubbles of oxygen
What do bacterial infections occurring via the oral route involve ?
Bacteria passing through the stomach prior to entering the intestinal tract
What about the stomach kills most bacteria ?
The low pH (1.5-3.5)
What have bacteria like salmonella enterica serovar typhimurium evolved ?
The ability to survive acidic conditions for long enough to reach the intestine
What is the asr gene ?
highly acid-inducible and is up regulated during salmonella infection
What does the asr gene encode ?
A small basic periplasmic protein of 102 amino acids
What is the small basic periplasmic protein that the asr gene encodes required for ?
Growth of bacteria under acidic conditions
Where is the asr gene highly expressed ?
During the growth at acidic pH 4.5 and 4.0
What does the asr mRNA indicate ?
asr gene is not highly expressed at pH of 7.2 or 5.0
What have genetic tools been developed for ?
To enable scientists to visualise when a gene is expressed
What is a commonly used genetic tool ?
Green fluorescent protein
What were E.coli cells transformed with ?
A plasmid carrying the genetic reporter
What does the this plasmid carry ?
A genetic fusion between the aid responsive promoter of the asr gene of salmonella enterica
What does the genetic fusion between the acid-responsive promoter of the asr gene of salmonella control ?
The expression of the asr gene and the gene encoding green fluorescent protein
What happens when transcription is initiated from the asr promoter in response to acidic conditions ?
mRNA is transcribed from the downstream green fluorescent protein gene
What does the translation of green fluorescent protein mRNA lead to ?
Production of the fluorescent green fluorescent protien
What does the production of the fluorescent green fluorescent protein make ?
The bacteria fluoresce when exposed to light of the appropriate wavelength
What does the excitation of the green fluorescent protein protein with blue light lead to ?
The generation of green fluorescence
What does the origin of replication on plasmid required for ?
Maintenance and replication of the plasmid
What does the beta lactamase encoding gene provide the bacteria with ?
Ampicillin resistance
What is ampicillin ?
A beta lactam antibiotic
What is the beta lactamase encoding gene required for ?
To select for cells that carry the plasmid and for plasmid maintenance
What is the potassium hydroxide test an alternative to ?
Gram staining
What are some shapes of bacteria ?
- Coccus
- Coccoid
- Rod
- Comma shaped
- Spirillum
- Club shaped
- Rod with spore
How many genes does salmonella have ?
4500
How many salmonella genes can change in a few minutes to react to a new environment ?
Hundreds
