Bacterial Growth and Genetics Flashcards
What is an organism that requires higher CO2 concentrations than is in room air called?
capnophile
What is fastidious nutrition?
The need of a cell to have numerous essential nutrients for growth
(they will not grow on a normal agar plate)
What is fermentation?
Anaerobic breakdown/oxidation of organic compounds (sugars, carbons) to form alchohols or acids
What type of bacteria would Pasteurization destroy?
It is a process where dairy is exposed to temperatures for a time to destroy pathogenic NON-SPORE FORMING bacteria
To produce overt illness, the bacteria must multiply to a point where they ______________________ and the host response is ___________________________.
they need to disrupt normal cell function and get to a point where the host response is sufficient to form disease
What are abortive, subclinical and inapparent infections?
How are they diagnosed?
These are infections that are not able to mount a sufficient host response to cause overt illness.
Seroconversion (circulating antibodies) should still show evidence of infection
What determines the ability of a bacteria to reach a level where they cause overt illness?
inoculum size/ infection dose - this is difficult to diagnose in humans because of person-to-person variation in host defenses and individual variations
How does generation time differ between growth in a host and growth in pure culture?
Apparent growth in a host is generally longer because the environment is more dynamic
For bacteria, what is the definition of “growth”?
Multiplication in cell number NOT an increase in size of the existing cell or components of the bacteria
Which bacteria use O2 as their terminal electron acceptor?
What are the respiratory products?
How does the amount of ATP produced compare to fermentation?
Aerobic and facultative aerobes are able to use O2 as the final electron acceptor.
They generate CO2 and H2O
Compared to fermentation, they make a lot of ATP
What bacteria are not able to utilize O2 as their final e- receptor?
What is their final electron acceptor?
What are the products?
How does the amount of ATP produced compare to respiratory oxidation?
Facultative anaerobes, strict anaerobes and microaerophiles use metabolic intermediates as their final e- acceptor
They produce organic compounds with or without gas
Low levels of ATP are produced comparatively
What is fermentation?
How many pathways exist? How are they distinguished from each other?
It is the utilization of carbohydrates as the final e- acceptor.
There are six pathways and they are differentiated by their end products (organic acids, neutral solvents, gas, in varying proportions)
What is the simplest fermentation pathway?
Homolactic pathway (glucose +2ADP–> 2 lactic acid + 2ATP)
What two toxic substances are produced during bacterial growth in the presence of O2?
What enzymes are used to break them down?
h2o2 is produced and is broken down by catalase to H20 and 02
O2- (superoxide) is produces and is broken down by superoxide dismutase (SOD) to for H202 and O2)
What are the four classifications for O2 use in bacteria? Which utilize catalase? Which utilize SOD?
Which can ferment?
- Aerobes- catalase/SOD, no fermentation
- Anaerobes- no catalase/SOD, fermentation only
- Facultative anaerobes- catalase/SOD, oxidative and fermentation
- Microaerophilic- SOD, no catalase, fermentation
What percent of bacteria in the colon are anaerobic?
99.99%
What is the difference between an autotroph and heterotroph?
What are all medically important bacteria?
Autotrophs derive energy from oxidizing inorganic substrates or sunlight
Heterotrophs require one or more organic carbon component as energy
All medically important bacteria are heterotrophs
If a bacteria requires an exogenous source of AA, B vitamins and/or nucleic acid components, what kind of growth is it said to have?
Fastidious growth
Bacteria grow optimally at certain temperatures. What are bacteria called that grow at:
- low temp
- medium temp
- high temp
and what are the temperature ranges?
Most bacteria that grow in humans are considered what type?
- psychrophiles- 0 to 25 degrees C (10-15 optimum)
- mesophiles- 15 to 45 degrees C (30-37 optimal)
- Thermophiles- 35 to 70 degrees C (55 optimal)
Most bacteria in humans are mesophiles
At what pH do most bacteria grow?
What lead to some exceptions?
6-7.4 (optimal body pH)
Some have pH for certain niches though. Ex. acidic pH in urine allows UTI pathogens to grow
Growth in alkaline or acidic environments have diagnostic value
What osmotic pressure do bacteria grow best at?
What food storage practice utilizes this information?
They grow best at a pressure equivalent to physiological saline
Conditions with high osmotic pressure (canned goods because of salt/.sugar content) limit the growth
How do bacteria grow in pure culture?
Bacteria multiply by binary fission where each parental cell produces two identical daughter cells (there can be spontaneous mutations however that change the daughter cells from each other slightly)
What are liquid and solid media usually composed of?
partial digests or animal and plant extracts
Liquid- extracts and extra elements required for growth
Solid- extracts, extra elements for growth, agar
On solid media, each colony that grows is derived from _______________.and is ___________.
Derived from a single progenitor cell and are clonal
What are indirect measures of growth?
What is the pro? What is the con?
Measurements from parameters related to cell mass.
In liquid media this would be:
1. Turbidity- density of the liquid prep/bacteria
2. dry weight
3. bacterial nitrogen
Pro- it is faster/easier. Con- don’t know whether the bacteria are alive or dead
What are direct measurements of bacterial growth?
Utilize total or viable counting techniques.
- Hemocytometer- gives total count
- CFU (viable plate count)- needs dilution of sample so after incubation you can count colonies)
What is bacterial viability?
The ability of a bacteria to grow on media when it is plated and incubated on suitable media
What is the preferred method for testing bacteria growth and is used to test pools, milk, drinking water, etc?
Viable plate counting
What are the four phases of bacterial growth?
- Lag phase
- Logarithmic growth phase
- Stationary phase
- Death/decline phase
In theory, all bacteria go through the same growth stages. They only differ in what three aspects?
- duration of the phase
- maximum growth rate
- max number of bacteria obtained
What is different in every phase of bacterial growth?What controls this change?
- type of proteins in the phase
- different amounts of the same protein in each phase
Protein synthesis and expression is governed by sigma factor of the RNA polymerase. Different sigma factors= different proteins
In the lag phase, what happens to the number of cells, the metabolic activity of the cells and the cell mass?
number of cells- no increase
cell mass- increase
metabolic activity - increase
What determines the duration of the lag phase?
- inoculum size
- metabolic state
- whether or not the medium is optimal for growth
What phase of bacterial growth do most antibiotics target?
They target the logarithmic stage because they will target aspects of bacterial replication and this is where bacteria are replicating the most
What happens to the cell number, cell mass, and metabolic activity during the logarithmic phase of bacterial growth?
cell number- increase rapidly
Cell mass- increase at the same rate as cell number
metabolic rate- max logarithmic rate (doubling each replication)
Describe a bacterial cell that is in the logarithmic growth phase.
It will be large, with lots of ribosomes and will be very metabolically active
What causes the growth rate to slow down and forces the bacteria into stationary phase?
The metabolic products produced in log phase accumulate and energy/nutrients are depleted so growth rate slows and viable count is constant
What happens to the viable cell count during the stationary phase?
It becomes constant
Does division occur during stationary phase?
Yes, but it is balanced out by cell death
Why are bacterial cells in stationary phase more resistant to antibiotics?
They are smaller and less metabolically active.
Antibiotics target replication machinery
What is cellular activity like during the death phase? what is the cell size?
Cells are small and activity is practically absent
What is the difference between sterilization and disinfection?
Sterilization is the complete absence of life and is achieved by physical and chemical methods.
Disinfection implies the killing or removal of pathogenic or potentially pathogenic microorganisms
All methods of bacterial killing are ________ dependent and require a sufficient ________ so that resistant bacteria can be killed.
Time-dependent; time
What are the four physical methods used to disinfect and/or sterilize?
- Temperature
- Radiation
- Filtration
- Asepsis
For sterilization ____ heat is better than _____ heat.
Moist is better than dry
What is the mostly widely used sterilization technique?
Autoclaving- put tools in a pressure chamber with 15lbs pressure (121degrees C) for 15 minutes
How long does autoclaving (moist heat) take to kill bacteria and at what temperature?
How long do ovens (dry heat) take and at what temperature?
Autoclaving- 121degrees for 15 minutes
Oven- 160-180 degrees for 2 hours
Why does refrigeration not work as a sterilization technique?
It does not kill the bacteria, only slows/stops the growth
What is UV radiation typically used to sterilize? Why?
It sterilizes surfaces and air masses because it has little penetrating power
For selective removal of bacteria, what technique is used?
What is an important consideration when selecting this technique?
Filtration- typically cellulose ester
It is important to consider the pores size (bacteria are 0.2-2 microns so you want the pore to be smaller)
What is filtration usually used to sterilize?
Biologicals and other substances that are denatured at high temps
What is asepsis and how is it different from the other sterilizers?
It is a technique to maintain sterilization, but it can not sterilize itself.
Asepsis provides a barrier impervious to bacteria (plugs, wraps, closures)
What are the 8 chemical methods for disinfection, germicisdes, antiseptics, etc?
- alcohols
- detergents
- phenols
- halogens
- heavy metal
- h2o2
- formaldehyde and glutaraldehyde
- ethylene oxid
What are detergents and how are they similar to alcohols in mechanism?
Detergents are quarternary ammonium compounds
Alcohol and detergents disrupt cell membranes
How do phenols disinfect?
They disrupt the cell membrane and denature proteins
How do halogens disinfect?
They are oxidizing agents effective against sulfhydryl groups
How do heavy metals disinfect?
They bind to sulfhydryl groups and block enzyme activity
How does hydrogen peroxide disinfect? What is the drawback?
It is an oxidizing agent but it is not effective against catalase + bacteria because they will break down the H2O2
How do formaldehyde and glutaraldehyde disinfect?
They alkylate NH2 and OH groups on proteins and nucleic acids to disrupt function
How does ethelyne oxide disinfect?
It is an alkylating agent in gas form.
It is used 2nd to autoclaving but for heat sensitive material
It you want to sterilize a plastic tube, you cannot use autoclaving because the plastic will melt. What is your second choice for sterilization?
Ethelyne oxide- a gas form alkylating agent
What are three ways bacteria are able to confer resistance to host defenses?
- their rapid generation time -> increased mutation
- horizontal gene transfer via transposons/plasmids
- Adapt to conditions
What is the central dogma of the genetic code?
DNA->mRNA-> protein
Codons are read in triplets and are:
1. redundant - multiple triplets encode the same AA
2. non-overlapping -> each triplet only encodes one AA
What is the start codon sequence and what does it encode?
ATG encodes methionine
What are the three stop codons?>
TAA, TAG, TGA
What is the benefit of a bacteria having a haploid genome?
Since there is only a single chromosome, a mutation can confer a direct phenotypic change so it helps them adapt faster
What is merodiploid?
When a second gene is introduced on a plasmid to mask the phenotypic expression of another allele
- it is like being diploid but only for one specific gene
Bacteria are normally __________. They become ______ for a very short time prior to cell separation. If an allele is introduced via plasmid they become ________.
haploid
Diploid
merodiploid
What is the mutation rate?
How do mutations arise in bacteria?
the specific rate that correlates to spontaneous mutation due to replication errors and other forms of mutagenesis
Mutations arise randomly and in the absence of antimicrobial agents (selection)
Resistance to the drugs ______ and ______ require only a single base pair change in the bacterial chromosome.
Streptomycin and Rifampicin
If you plate a bacteria on a plate lacking antibiotics, it results in ________________________.
If you grow bacteria on a plate with antibiotics, it willl ________________________.
No antibiotics = non-selective growth of all cells
Antibiotics= growth of only those bacterial strains resistant to the antibiotic
___________do NOT cause resistance. ________cause colonies that are resistant to antibiotics.
Antimicrobials do not cause resistance.
Random mutations cause colonies resistant to antibiotics
What would be a low rate of bacteria mutation?
A high rate?
Low - 10^8 to 10^9
High- 10^6
What are silent, missense and nonsense mutations?
Silent- single bp change that does not change the AA
Missense- single bp change that changes the AA
Nonsense- single bp change that inserts a stop codon
What is a reversion?
When a second point mutation happens and converts the stop codon back to the original AA
What is a suppression?
When a second point mutation happens and converts the stop codon to a new and different AA
What is a frameshift mutation?
What is the most common outcome?
What sterilization technique tends to cause frameshift?
Where a base pair or codon is inserted or deleted from the mRNA changing the reading frame
the most common outcome is a truncated protein that may be non-functional.
UV radiation tends to cause frameshift
What is the difference between spontaneous mutations and induced mutations?
Spontaneous require no external stimuli. Ex:
1. imperfect replication
2. insertion of transposon
Induced requires external stimuli. Ex.
1. physical mutagen- UV
2. Chemical mutagen - alcohol/ethylene oxide/sterilizers
3. site-directed mutagenesis- research
What are the components of a composite transposon?
- host gene (ex. antibiotic resistance gene)
2. insertion sequence
In terms of bacterial genetics, what is the definition of competence?
The ability of a bacteria to take up and recombine DNA into their chromosomes
What is conjugation?
Exchange of genetic material between two cells through cell-to-cell contact
What is an episome?
plasmid that can integrate itself into the host genome
What is an insertion sequence and how does it differ from a composite transposon?
An insertion sequence is a transposase gene flanked by sequences that are necessary for movement.
The composite transposon doesnt have sequences for movement but have insertion sequences and host genes (like antibiotic-resistance genes)
What is lysogeny?
When bacterial and viral genomes repcate in synchrony and the virus genome gets integrated into the bacterial genome
What is a replicon?
What are three examples?
Any form of DNA with the elements necessary for replication.
- chromosomal DNA
- plasmids
- bacteriophages
TRANSPOSONS ARE NOT REPLICONS
What is transduction? What is the difference between generalized and specialized?
Transduction is when foreign DNA enters a cell using viruses as vectors
Generalized- any host gene has an equal chance of being transfered
Specialized- only the host gene next to the phage DNA will be tranduced
What are three examples of events that change bacterial DNA?
What are three examples of new DNA incorporation?
Change DNA: 1. mutation 2. insertion of transposons 3. recombination New DNA incorporation 1.transformation 2. conjugation 3. transduction
What is recombination?
What are the three major benefits?
The breakage and rejoining of DNA into new combinations
- adaptation
- change genome order
- aid in DNA repair of large chromosome breaks
What are the four kinds of recombination? What degree of homology is required for each?
- Homologous recombination - large degree of homology
- Site-specific recombination- small region of homology
- Insertion sequence and transposons- very small homology
- Illegitimate - no homology necessary
Which types of homolgy require RecA?
Homologous recombination
Which types of homology require sequence specific enzymes?
- site specific homology
2. insertion sequences and transposons
What two things are homologous recombination specifically useful for?
- DNA repair
2. integration of linear DNA during transformation
What is a RecA protein?
It is a protein that aligns sequences in DNA but does NOT recognize specific sequences. (it can match ABCD to abcd but it doesnt know that it is looking for a specific sequence like the antibiotic resistance sequence for example)
In homologous recombination, the insertion of linear DNA requires __________ while the incorporation of a plasmid or circular DNA only requires _________.
Linear requires 2 crossing over events while circular DNA only requires a single crossover
What happens to the chromosomal DNA that is excised during homologous recombination with linear DNA?
It is lost during further bacteria growth unless it has a replicon that will allow it to act as a plasmid
Homologous recombination results in ________ meaning there is no net gain of DNA in the genome
reciprocal exchange
Describe the process of nonhomologous recombination (illegitimate)
There is no homology and therefore no use for Rec A. It does require enzymes that recognize specific DNA sequences
Describe site specific recombination.
Recombination of two specific sequences that share some sequences but not enough for homologous recombination.
No RecA
Requires recombinase- an enzyme that recognizes the site in DNA for the crossover
What is recombinase? What type of recombination utilizes it?
It is a site specific enzyme that recognizes the site in DNA and initiates crossover
If site specific recombination involves a plasmid or bacteriophage, what can result?
Net increase in chromosomal DNA
What are transposons?
Genetic elements that can hop from one location in the DNA to another.
It can duplicate and appear in different locations on the same and different genetic elements
What is required for the movement of transposons?
Transposase- a site specific recombinase enzyme
All transposons identified so far have __________________ at their ends.
Inverted repeats
Composite transposons have _________ that can move between different bacteria through genetic transfer.
What are Tn5 and Tn3?
antibiotic resistance
Tn5 is kanamyocin resistance and Tn3 is ampicillin resistance
How do we know that pathogenicity islands originated from different organisms?
How are they recombined into the new chromosome?
The CG content of the genome is different.
Pathogenicity islands are inserted via site specific recombination at a common site
What is bacterial transformation?
The insertion of “naked DNA’ into a recipient bacterial cell followed by its introduction into the chromosome or existence as extrachromosomal plasmid
How was bacterial transformation discovered?
Griffith did experiments where he noted that rough bacteria were avirulent and smooth were virulent.
If a mouse was injected with rough bacteria it survived, and with smooth bacteria it died.
It rough bacteria was incubated with dead smooth bacteria, the DNA is transferred and now the rough bacteria converts to smooth and is virulent
Because transformation used naked DNA, what is it sensitive to?
The presence of DNAase or other enzymes that could degrade DNA
What are the two major types of transformation?
Natural transformation- Certain bacteria take up DNA naturally and are naturally competent
Induced transformation- bacteria can be manipulated to take up naked DNA by increasing salt concentration or electroporation
What are three examples of bacteria that are competent for natural transformation?
- Strep pneumonia
- Haemophilus influenza
- N. meningitidis
What are two ways transformation can be induced?
- High salt concentration
2. electroporation
What is required when naked DNA is taken up by competent bacteria?
homologous recombination between the donor and the recipient is required to form a stable genome
The process of _______ requires direct contact between the donor and recipient bacterial cell via an appendage called a _______________.
Conjugation; pilus
Conjugation is almost exclusively mediated by __________.
plasmids
A plasmid is a DNA molecule that replicates _________ of the chromosome and controls its own _______.
it replicates independently of the chromosome and controls its own copy number
What is the typical shape of a plasmid?
what is the notable exception?
Plasmids are usually circular except spirochetes have linear DNA
What is the difference between a conjugative and non-conjugative plasmid?
Conjugative- have F factor and other genes that allow transfer to other bacteria cells. They are low in copy number (1-3)
Non-conjugative- cannot transfer themselves alone and have high copy number (over 10)
What is a strategy for preventing conjugation?
Because conjugation requires cell to cell contact, it can be prevented by separating bacteria with a filter or other physical barrier
What are the four types of conjugation?
- Sexduction
- Hfr chromosome transfer
- nonconjugative plasmid mobilization
- conjugative transposons
What is sexduction?
When a donor bacteria extends a pilus to the recipient bacteria and allows transfer of the plasmid.
What if F factor?
fertility factor- F+ cells are donors of the F plasmid which encodes the pilis and F- cells are recipients.
After the interaction, they are both F+ and have F plasmid to encode a pilus
What is Hfr chromosome transfer?
F plasmid integrates into the bacterial chromosome using site-specific recombination to produce high frequency recombination (Hfr) donor strains.
Hfr donor strains can :
1. excise the plasmid back out to form F plasmid again
2. conjugate into an F- cell directly from the chromosome
When F plasmids are excised back out the Hfr chromosome, what can potentially happen?
- it is excised perfectly and reforms the F plasmid
- it is excised imperfectly thus carrying a small piece of chromosomal DNA on the plasmid (F’ element) and leaving a small piece of plasmid in the chromosome
What is F’ element?
if there is imperfect excision of the F plasmid from the Hfr chromosome which allows a small amount of genetic transfer to F- bacteria
If the Hfr conjugates into a recipient F cell directly from the chromosome, the transfer will be time-dependent based on the __________. It results in ________________________________.
original insertion point.
It results in the exchange of large amounts of DNA
What is mobilization of nonconjugative plasmids?
They lack F+ but they do have an origin of transfer (oriT) so transfer factors can be given by a helper plasmid
What are the two ways conjugative transposons can get into new bacterial cells?
- Self-transfer where the transposon (which has transposase) is able to move itself to the recipient via conjugation and integrate into the new chromosome
- Mobilization- there is a co-resident plasmid and the transposon can donate its conjugation machinery to the plasmid or can insert into the plasmid
What is R plasmid?
resistant plasmid that
- carries antibiotic resistance markers
- produces proteins that kill other bacteria and promote self immunity
What are the 6 major plasmid-mediated virulence factors?
- antibiotic resistance
- colonization factors
- toxins
- virulence
- invasions
- secretions
Bacterial viruses destroy cells by ______ . If this occurs on a lawn of bacteria on an agar plate, you will see ___________________.
Viruses kill bacteria by lysis
They will form a cleared zone called a plaque
What is transduction?
Virus phage carries chromosomal DNA from one bacteria to a newly infected recipient.
Transducing phages must package________.
DNA although normal phages can package RNA or DNA.
To tranduce–> must be DNA
What type of phage interacts with E. coli like a lock and key (conferring specificity)?
T4 phage
How do phages initiate infection?
By binding to specific receptors on the surface of the bacteria like conjugation pili, teichoic acid, flagella
Once phage genome is injected into the host what occurs?
It takes over cell machinery, produces progeny genomes and phage structural components, reassembles and lyse the host.
What occurs if there is aberrant packing of the DNA into the phage?
large fragments of donor chromosomal DNA gets packaged instead of the viral phage genome.
“transducing particles” are not lytic and are used to inject recombinant DNA into a new host.
The DNA can then be incorporated into the recipient cells genome via homologous recombination
What are the two life cycles a temperate phage can enter? Describe each.
- lytic- rapid accumulation of new phage particles and lysis of the cell for release
- lysogenic where phage genome incorporates into bacterial genome (prophage -> lysogen). The phage remains part of the lysogen until stress, then it converts to lytic cycle and kills the cell
A ___________ is when phage DNA incorporates into host DNA during lysogenic phase. It is immune from _________________________.
lysogen is immune from second or superinfection with another similar phage
How is the lysogen converted back to lytic cycle?
Stress produces bacterial proteases that cleave repressor molecules.
How does specialized transduction differ from generalized?
Only certain temperate phages will mediate the phenomenon and only a few specific donor genes will be transmitted
What is phage conversion?
if a temperate phage carries a virulence factor, then formation of a lysogen can convert a non-pathogenic bacteria into a pathogenic bacteria
What are three examples of bacteria that do phage conversion?
- Vibrio cholera
- Clostridium botulinum
- Strep pyogenes
