Topic 4 Flashcards
Most microbial cells divide by
binary fission: DNA replicated, cell grows, cytoplasm evenly divided, producing two ~identical daughter cells
What happens after replication in binary fission?
segregation: DNA to opposite sides of the cell so that each daughter gets one copy
What happens after segregation?
cells elongate and a septum begins to form to separate the cell into two
once this is complete (formation of cell walls causes cell separation), the division site constricts and the cells are pinched off from one another
DNA replication is controlled by
binding of protein DnaA to oriC
ONE important mechanism for controlling binding of DnaA to oriC
via a protein called SeqA (competes with DnaA for oriC binding site)
For some bacteria, generation time is shorter than amount of time it takes to replicate chromosome. How ???
multiple DNA replication forks can be active at once; essentially, OriC region starts getting replicated again before the first fork is finished
-> occurs in E. coli and Bacillus subtilis
Divisomes
multi-protein complexes that help identify cell centers to build a septum
FtsZ
- acts as a divisome; a central protein
- polymerizes to form a ring around circumference of cell at the mid-cell where division will occur
- constricts to help division occur
- recruits proteins to help build cell envelope (SEPTUM)
- depolymerizes at the end
nucleoid occlusion
when chromosome is at mid-cell, prevents divisome from forming there!
-> push nucleoid to the side to resolve this!
Factor in finding the mid-cell besides occlusion
FtsZ (Z-ring) inhibitors
- clustered at cell pole and lowest concentration at mid-cell
- EX: “min” system - MinC/MinD at poles
T or F. Cell wall must be remodelled but remain intact during cell division to prevent cell from bursting
T
Cell wall growth - peptidoglycan precursors brought across cytoplasmic membrane by
bactoprenol (hydrophobic - lipid)
Autolysin
break glycolytic bonds for insertion of new monomers for cell wall growth
Cell wall growth: transglycosylases and transpeptidase
Transglycosylase enzymes form new bonds in sugar backbone; new beta-1,4- linkage
transpeptidase enzymes form new peptide crosslinks; seal up peptides (built by D-alanine)
What dictates shape?
How cell wall is built during cell growth
Rod vs Spherical shaped cells
Rod - actin-like cytoskeleton protein MreB ensures that new cell wall is added along its long axis – rod elongates
Spherical - lack MreB; new cell wall added only at midcell; spherical shape is default
Alternatives to binary fission (assymetrical)
- budding division: new cells frow from old cells by budding off from a particular site on mother cell
- multiple fission: new cells bud off from long extensions called hyphae and then some instances occurs where long hyphal filaments will form multiple septa at once and break off into many new cells
- sometimes cells grow only from one poly to produce cells built from new and old material
Growth or culture media
(medium, singular) can be highly variable depending on the microbe… and for a given microbe
Defined media
media prepared by adding precise/known quantities of chemicals to water; know the exact composition
Complex media
contain extracts or digested organic material with an unknown composition (eg: yeast extract, casein (milk protein) digests)
Advantage of defined medium
you know what you’re working with
The more common type of media
Complex media; cheaper, easier, work for a broader array of different microbes
Some microbes can make most or all of the organic molecules they need
- many microbes that live in nutrient-poor environments
- certain ‘flexible’ microbes that adapt to many different environments (such as E. coli)
Other microbes can require a great number of growth factors – things like vitamins, amino acids, purine/pyrimidines, etc.
- many organisms have an obligate symbiotic lifestyle
- organisms that live in nutrient-rich environments (such as many lactic acid bacteria)
Auxotrophy vs Prototrophy
A - inability to produce a molecule you need for your growth
P - opposite; can produce molecule
Media used to isolate out a specific microbe or determine which or if a species of microbe is present
Selective, differential, enrichment
Selective media
used to isolate a limited range of microbes (often bacteria) — this could be a single species; often a combination of positive (nutrients few organisms can grown on) and negative (substances that kill most microbes) selection
Differential media
contain some sort of an indicator (eg. a dye that changes colour) when particular organisms are present
Enrichment culture
similar idea to selective media … but less selective and richer medium; promotes growth – increase numbers from isolates to make it easier to isolate a particular microbe; usually somewhat selective
Solid vs Liquid cultures
Solid - useful for isolating single colonies; can identify contamination
Liquid - should generally be started using isolated single cultures streaked on agar plates; came from single colonies on plates
The great plate count anomaly
When ‘natural’ samples are plated on genetic growth plates (mean to be ‘non-selective’) … 99.9% of microbes don’t grow.
T or F. We cannot culture the vast majority of microbes
T! One big reason is syntrophy (microbes feeding off one another =; microbes require other community members to grow)
Counting microbial cell numbers
- Direct count: cells in the sample are counted using a microscope
- Visible plate counts: a small sample of cells are spread on agar plates and incubated – colonies counted
- Turbidimetric: absorbance of light by a microbe growing in a liquid culture is measured in a spec
- other indirect methods: O2 consumption, CO2 production, metabolic activity; quantitative PCR to determine the number of ‘genome equivalents’
Much more convenient than colony counting (no dilutions, just OD measurement
Turbidity measurement - creates a standard curve, linear range, etc
Significant advantage of turbidity measurements
- not labor intensive
- growth can be continuously or regularly measured over time.
- growth can be visually observed ; “cloudiness”
Microbial “growth”
refers to increase in population size – cell division resulting in multiplying in numbers
Generation time
AKA doubling time (exponential phase) - amount of time it takes for one cell to become two (double in numbers and mass); varies greatly depending on microbe and growth conditions
Batch cultures
cultures in a fixed volume in a closed (closed system) container like a flask or a test tube; finite amount of time - reach a saturation point where cells cannot grow anymore
Continuous cultures
cultures within systems where waste product are being removed and new media fed in; environment can be held constant for a long period of time to allow for extended cultures to take place
In _____ cultures, microbes typically exhibit a growth curve that reflects the different stages of growth in this environment
batch
Lag phase
period of slow (or no) growth as microbes adjust after they are inoculated into this new environment
Exponential phase
growing population doubles at regular intervals; nutrients not yet exhausted, waste products not slowing growth
Stationary phase
nutrients begin to be exhausted (limiting) and/or accumulating waste products inhibit growth; little to no net growth (still some growth and death)
Decline phase
if you leave cultures long enough, cells will start to die (net decline in cell numbers)… this phase often isn’t all relevant
Phase used for studies; bacteria are all in a physiologically identical phase
Exponential phase
