Microbiology (A.F) Flashcards
Characteristics of Bacteria?
Single colonies of bacteria can be grown on agar surfaces
- Each colony is derived from a single cell
- A culture derived from a single colony is a pure culture
- All cells within a single colony are genetically identical
- Identical genetic material
Rules for naming prokaryotes?
genus species
Correct: Pseudomonas aeruginosa
Once the genus has been written out in full once, it can be abbreviated, provided it is unambiguous e.g. P. aeruginosa
- Always leave a space between genus and species names
- Always put names in italics (or underline if writing by hand)
- Always capitalise genus – never capitalise the species
- All the following are wrong:
E.COLI, E. Coli . Ecoli, ECOLI
What are the different types of bacterial shapes?
- Shape 2. Characteristic 3. Example
Rod –> Cylindrical –> Escherichia coli
Coccus (pl. cocci) –> Spherical or oval –> Staphylococcus aureus
Spirilla –> Curved rods –> Vibrio cholerae
What are the major elements, minor elements and trace elements needed by bacteria?
Major elements in a cell (Macronutrients): C (50%); H (8%); O (17%); N (13%) - required in large amounts
Minor elements (Macronutrients): P (2.5%); S (1.8%); K; Mg; Ca; Na - required in smaller amounts
Micronutrients (trace amounts): Fe and other metals
What are the different sources of energy that can be used by bacteria? What are the different pathways to produce energy (ATP)?
Chemtrophy –> oxidizing organic (also known heterotrophs) or inorganic chemicals (autotrophs)
Phototrophy –> photosynthesis.
Examples of different energy sources used –> Hint: not always carbon.
What is the generation time?
Time taken for the entire process of cell division –> Generation time (doubling time)
How to calculate generation time from a logarithmic graph?
Basically –> logarithmic graph which measures the absorbance of bacterial culture over time –> take an absorbance value at a particular point in time –> double the value to get a second absorbance value -> find the corresponding time for this absorbance value on the graph –> the difference between the two values for time is your generation time.
What is the equation to calculate the number of cells after a certain number of generations?
What are the two ways of calculating/estimating the number of cells?
Measuring cells:
- Turbidity (absorbance) –> includes dead cells.
- Evaluate how much viable bacteria is present by counting colonies (involves using dilutions otherwise there would be too many cell to count) –> more accurate cause only living cells are counted
What are the different phases in a bacterial growth curve?
- Lag
- Exponential
- Stationary
- Death
What happens in the lag phase?
Lag phase
- Minimal growth
- occurs because cells are adapting to a new environment –> metabolic adaption
- inoculum is usually depleted of certain nutrients/adapt to the nutrient source available.
- time is required for resynthesis
- time of lag phase varies greatly
What happens in the exponential phase?
Exponential phase
- rate of increase of cell numbers constantly rises
- cell numbers increase at the same rate as cell constituents
- growth rates of cultures vary and depend on: temperature, nutrients, pH, genetic factors, etc.
- doubling times can be ~20min to many hours
- the exponential growth phase is still limited by nutrients etc
What happens in the stationary phase?
Stationary phase
- Exponential growth cannot carry on indefinitely
- Growth is limited by either lack of an essential nutrient or builds up of a toxic waste product to an inhibitory level
- Cells are not dead. No net increase or decrease in cell numbers (although some cells may be growing and others dying)
- Certain genes are necessary for survival in stationary phase
- Sporulation commences in certain species
- Accumulation of storage products
Effect of increasing or decreasing temperature on cell growth?
Temperature
- one of the most important environmental factors
affecting the growth of micro-organisms
- incorrect temperature - no growth
Increasing temperature
- rates of chemical and enzyme reaction rates rise
- proteins denature at high temperatures
- membranes destabilise
Decreasing temperature
- membranes tend to “gel”
- transport through membranes becomes limiting
- enzymes become very inflexible
How is a viable cell count carried out?
- Serial dilutions (10-1 , 10-2 , etc –> each time one part bacteria 9 part agar solution) are performed to get countable numbers on the agar plate.
- Sample taken –> spread an agar plate
- Leave bacteria to grow
- Calculate the number of colonies –> colony forming units (cfu) –> assume one bacteria = one colony
- From this, you can work back and calculate bacteria in the sample.
Have bacteria been able to adapt to different temperatures?
Yes, bacteria have evolved to function in a range of different temperatures –> developed proteins that can withstand high or low temperatures.
Why do we need to stain bacteria when viewing them under the microscope?
Reason for staining –> cells are not too small with the current microscopes but the contrast between cell and surroundings is the problem —> staining solves this –> basic dyes that are positively charged are useful cause it interacts with the many negatively charged polysaccharides.
What is the procedure for staining?
Before step 1 on the picture
- Get a microscope slide
- Get a sample of bacteria and smear it on the slide
- Heat it –> dry it out
What colour do gram-positive and gram-negative bacteria appear after staining?
Gram-positive –> Purple
Gram-negative –> Pink
Why do some bacteria retain the dye while others don’t?
- Blue membrane –> cytoplasmic membrane
- Gram-positive –> thick layer of peptidoglycan –> treated with alcohol –> becomes impermeable –> dye remains.
- Gram-negative –> Thin layer of peptidoglycan –> permeable to the dye –> instead they have an outer membrane (second membrane) –> made of phospholipids and LPS.
Why do the differences between gram-positive and gram-negative occur when staining?
Structure of the membrane of gram-positive bacteria?
- Plasma membrane
- Thick peptidoglycan layer
Structure of the membrane of gram-negative bacteria?
- Plasma membrane (inner)
- Thin peptidoglycan layer
- Plasma membrane (Outer)
Note –> The composition of the inner and outer membranes are different.
How is the peptidoglycan layer formed?
Peptidoglycan layers are similar between most bacteria.
Composed of two sugar derivatives and a small number of amino acids:
Sugar derivatives:
N-acetyl glucosamine (NAcGlc)
N-acetyl muramic acid (NAcMur)
Amino acids:
L-alanine
D-alanine
D-glutamic acid
How are the components of the peptidoglycan layer arranged?
- Glycan chain –> sugars linked by Beta 1-4 linkage –> sensitive to cleavage by lysosome.
- Peptides branch off from these glycan chains.
How are two glycan chains linked to each other? (Difference between gram-positive and negative)
Left gram negative –> Connect multiple glycan chains via peptide bonds between amino acids.
Right gram-positive –> uses an inter-bridge using more complex peptides to connect two glycan chains –> nature of inter bridge differs
What molecules/proteins are found in the peptidoglycan layer of gram-positive bacteria?
Wall-associated proteins –> linked to the peptidoglycan
Two types of Teichoic acid:
- Teichoic acid –> covalently connect to proteoglycan layer.
- Lipoteichoic acid –> attached to the cytoplasmic membrane –> negative charged –> reinforces organisation.
Structure of the outer membrane in gram-negative bacteria?
- Lipoprotein connects the outer membrane to the proteoglycan layer
- Outer membrane –> still impermeable
- Outer membrane –> inner leaflet is made of phospholipids the outer leaflet is made lipopolysaccharides –> many porins in the outer membrane used to transport substances in and out.
What is the structure of lipopolysaccharide in gram-negative bacteria?
LPS found on the outer leaflet of the outer membrane.
What are the different configurations of flagella?
Polar –> can be monotrichous (one pole) or amphitrichous (both poles)
What is the structure of the flagella?
Flagella is possible on both gram-positive and negative bacteria but the structure would be different as the membrane is not the same
Example –> gram-negative
- Basal body (within the membrane), hook and filament region
- Basal body –> L-ring attached to the outer membrane, P-ring attached to peptidoglycan, S-M ring attached to the inner membrane –> Yellow tube is called the rod, orange motor (provide propulsion)
- Important to remember that there is a proton gradient –> one of the driving force for the motor
A more detailed structure of flagellum –> proteins included. Give an overview of the different pathways that lead to the production of the flagellum.
- FliG, FliM, FliN –> form C ring –> known as the motor of flagella.
- The stator of the motor is MotA and MotB –> covalently attached to peptidoglycan layer.
- Middle of the motor –> you find FliOPQR, FLhB, FLhA, FlK and ATPase complex (FliH, FliL, FliJ) –> secretion apparatus (Type 3 secretion system) transports the subunits required to make the first rod, second hook, third filament.
- Rings are transported via a different pathway (sec pathway) –> assembled at the place that they need to be assembled.
What do Cap proteins do in the assembly of the flagellum?
Cap proteins –> allow for sequential assembly –> assemble rod with rod cap –> cap removed –> assemble hook with hook cap –> hook cap removed –> etc…
Outline the sequential assembly of the flagellum.
- M-S ring with secretion apparatus
- Rod subunits move through –> rod assembled with rod cap.
- Other rings are added via sec pathway
- Rod cap removed and hook cap added
- Hook subunits move through –> hook is assembled
- Hook cap removed –> filament cap added
- Filament subunits move through –> filament assembled.
Outline how the flagellum uses the proton gradient to generate movement.
Proton flow from the periplasm to the cytoplasm drives the proton motor
MotB and MotA (stator) –> you find Negatively charged aspartate –> H+ can bind –> induce a conformational change in the protein –> change the conformation of the whole set –> produces a power stroke –> H+ released à conformation changes back –> 2nd power stroke –> piston-like mechanism.
Apart from the swimming motility, what is the other type of motility is the flagellum used for?
Swarming –> more social –> no individual movement
How do bacteria change direction?
Changing direction –> mechanism changes depending on the position of the flagellum
- Polar flagellum –> forward –> counter-clockwise /backwards –> clockwise rotation –> sometimes the cell stops and the orientation changes and then start again
- Peritrichous –> form bundle and rotate –> counter-clockwise results in forward motion. But when the bacteria rapidly changes to clockwise –> flagellum spread and bacteria tumbles –> orientation changes –> once again counter-clockwise results in forward motion in a new direction.
- Movement can be random or biased –> depends on whether there is an attractant or repellant –> chemotaxis
How to test whether something is an attractant or repellant?
A solution in a capillary tube with attractant and repellant is placed into liquid culture.
Outline the assembly of type 4 pili.
Same principle as flagella –> this case you have platforms instead of rings –> one in inner and one in the outer membrane –> they will help to bring the subunits through for assembly.
Energy provided by ATPase –> two types –> B and T –> B ATPase results in assembly but T ATPase results in disassembly.
What are the three types of motility?
Outline the general structure of the fimbriae.
Note –> sec pathway is used to transport the subunits –> reach the extracellular compartment.
- Usher
- Major subunit –> A
- Minor subunits –> pilus
- Last subunit –> adhesin.
Subunits brought to usher in a sequential order which gets pushed through to be assembled.
How does catabolite repression work?
Basically –> when I have glucose I don’t want to express any other genes related to other sugars (lactose maltose, etc.)
Involves control of transcription (RNA from DNA) by an activator protein.
Example –> glucose/lactose
Transcription of particular genes uses RNA polymerase –> RNA polymerase can only act if the activator protein CAP has bonded to the DNA at the promoter region –> but CAP will only bind when cAMP has bonded to CAP.
Glucose represses adenylate kinase –> represses cAMP production –> so when glucose is present cAMP production is repressed –> transcription of genes related to other enzymes for metabolism of other sugars doesn’t occur.
How is Beta-galactosidase production inhibited if there is no glucose and no lactose present?
Absence of glucose –> cAMP is high –> binds to CAP –> binds to promoter region –> allows transcription.
What happens if lactose is not present?
Another gene (Laci) not under control of CAP protein -> Laci protein will bind to the operator region which is between Lac genes of lactose operon and promotor region –> Laci is present bind to operator –> prevents transcription
When lactose is present?
WIll bind to repressor (laci) –> conformational change –> no longer binds to operator region.
How does OmpR control the expression of OmpC and OmpF?
Low osmolarity –> Low levels of signal is detected by EnvZ (sensor) –> low levels of EnvZ is phosphorylated –> Low levels of Phosphorylated OmpR –> Low levels of OmpR will preferentially bind to high-affinity binding sites –> as there is a high-affinity binding site by the promoter region of OmpF –> OmpR-P will bind there preferentially –> promote activity of RNA poly –> increases OmpF.
High osmolarity –> more signal detected –> a lot more Omp-P –> Both high and low-affinity binding are gonna be occupied by OmpR –> since OmpF has a low-affinity binding site as well which is located closer to the gene –> interfere with RNA poly binding –> stops expression of OmpF –> BUT –> by OmpC there is only a low-affinity binding site –> RNA poly binds –> increases expression of OmpC.
How is the chemotaxis regulated?
Also regulated in a similar way to the two-component regulatory system.
Extracellular receptors –> sensing domains –> called MCP –> Clusters of receptors –> used to perceive a gradient.
Che-A domain –> kinase domain (note –> not permanently bonded to receptors –> uses Che-W (adaptor protein) to bind to a receptor.
- Binding to receptors (MCP)
- Binding of adaptor protein Che-W allows Che-A to bind to receptors –> receptors trigger certain level of phosphorylated (autophosphorylation) of Che-A.
- Receiver domain (Che-Y) –> phosphorylated on its aspartic residue from the phosphates from Che-A.
- Che-Y-P interacts with the motor of flagellum –> converts counter-clockwise to clockwise –> tumbling.
Repellants –> increases Che-A phosphorylation –> increased tumbling.
Attractants –> decreases Che-A phosphorylation –> increases in straight travel.
Mechanisms that tells the frequency of straights and tumbles.
How does MCP regularly evaluate the gradient?
Important to constant evaluate gradient –> changes as the bacteria moves.
You have to reset the system –> allow for re-evaluation
Two different options
- Dephosphorylate Che-A –> using a phosphatase called Che-Y.
- Two proteins Che-R and Che-B –> Che-R is a methylase –> methylation of MCP at glutamic acid residue –> once MCP is fully methylated –> no longer sense environment –> Too much methylation will results in no more sensing –> Che-B is responsible for demethylation –> Che-B is activated by phosphorylation by Che-A –> Demethylation resets the system.
Do different types of MCP’s exist?
Yes, different MCPs probe the environment for different molecules.
What is the lux operon?
Found in Vibrio bacteria
Codes for enzyme luciferase –> essential for light generation.
All the genes in the operon are regulated by 2 regulatory genes –> luxR and luxl –> transcriptional activator.
LuxI gene –> codes for an autoinducer synthase –> involved in autoinduction.
Explain the mechanism of Q. sensing using the lux operon.
LuxI synthase –> makes an acyl-Homoserine lactone (AHL) –> Acyl –> varies –> Lactone is conserved
- Build up of the molecule inside and outside of cell –> diffuse in and out –> hydrophobic membrane.
- Lux-R is only active when bound to AHL –> when it is sufficient concentration of activated Lux-R –> it will bind to DNA –> Activate Lux operon.
- Since LuxI is the first gene in the operon –> increased Lux operon expression –> make more AHL –> positive feedback loop.
Describe the expression of AHL throughout the cell cycle.
- Basal expression initially –> low levels of AHL.
- Reach threshold –> all Lux-R become activated –> more LuxI –> positive feedback loop.
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Does Q. Sensing occur with other genes?
YES.
remember Q.Sensing is the ability of bacteria to sense a population density and to respond by an alteration in gene expression.
