CMMB 343 - Lecture 8-9 Motility and Cell Growth

mobility

being able to be moved, by oneself or an external force

motility

being able to move independently on its own

not all bacteria are motile

types of motility

gas vesicles that allow vertical movement, swimming via flagellar rotation, gliding motility, twitching motility using pili

flagella

latin for whip, found on rod-shaped or curved bacteria

15-20 um in length, 15-20 nm in diameter

helical shape, made of a protein called flagellin

flagella structure

rotation of flagellum driven by PMF, H+ flowing into the cell to drive movement

L and P rings, C ring, Mot proteins, MS and rod

L and P rings

sleeves in the peptidoglycan and outer membrane

C ring

embedded in the cell membrane

Mot proteins

stator, fused with C, does not move

MS and rod

rotor, portions that move with the hook and flagellin

flagella biosynthesis

basal structures formed first, then the filament grows from the tip (not the base)

flagellin moves up through hollow core and attaches to terminal end via self assembly

flagellar rotation (peritrichous)

direction of rotation determines what happens to flagella

run and tumble movement

counterclockwise rotation

flagella form a trailing bundle and the bacterium will swim straight forwards

clockwise rotation

flagella fly apart and the bacterium tumbles

gliding and twitching motility

bacteria live at a very low Reynolds number (meaning the friction of water is massive compared to momentum)

as soon as a flagellated bacterium ceases driving its flagellum, it stops dead

at the mercy of currents, unless they can stay attached to a surface, there is an advantage to some bacteria in being able to move along a surface

slow motility but don’t lose surface contact

surface motility

bacteria adhesion proteins stick to surface like feet

extension (pilus) attaches to surface and pulls the bacterial cell towards the point of attachment

protein moves along helical tracks but stays stuck to surface (cell moves and twists)

adhesion proteins reassemble

directed movement

enhances access to resources or allows avoidance of damage/death

includes phototaxis, aerotaxis, thermotaxis, pH taxis, magnetotaxis, chemotaxis

phototaxis

movement towards certain wavelengths of light

aerotaxis

movement in response to O₂ or a gradient of O₂

thermotaxis

response to temperature or a temperature gradient

pH taxis

movement towards or away from acid/alkaline environments

magnetotaxis

directed movement along the geomagnetic lines of force which allows magnetotactic bacteria to seek microaerophilic environments necessary for growth

chemotaxis

directed movement in response to certain chemicals known as chemoeffectors

signal transduction

prokaryotes regulate cellular metabolism in response to environmental fluctuations

external signal is sense and a signal transmitted to regulatory machinery

most systems are two-component regulatory systems

otherwise, external signal is transmitted directly to a target

two-component regulatory systems

made up of two different proteins: sensor kinase + response regulator

also has a feedback loop to terminate the signal

most systems respond to the presence of a signal directly by expressing or repressing certain genes

sensor kinase

usually in the cytoplasmic membrane, detects environmental signal and autophosphorylates, transmits signal via phosphorylation of a response regulator

response regulator

in cytoplasm, usually a DNA-binding protein that regulates transcription

run and tumble behaviour

chemotaxis studied in E. coli

run - in smooth forward motion, flagellar motor rotates counterclockwise

tumble - stops and jiggles because flagellar motor rotates clockwise, flagellar bundle comes apart

after tumble, another run begins in a random direction

biased random walk

allows for directed movement, the length of the runs changes depending on whether conditions are becoming better or worse

bacteria respond to temporal, not spatial, difference in chemical concentration - alter the length of run

methyl-accepting chemotaxis proteins (MCPs)

usually at poles of cells, transmembrane orientation

directly or indirectly (ie. with periplasmic binding proteins) bind to attractants/repellents

they are the sensors, and they interact with the sensor kinase, sense temperature, various chemicals

CheA

the sensor kinase, between the MCPs and the CheY response regulator

becomes phosphorylated

CheW

link between CheA and MCP

CheY

a response regulator, signalling the flagellar rotation to change direction and tumble

CheZ

the OFF switch for CheY-phosphorylated (it dephosphorylates CheY)

CheB

a secondary response regulator, acts as and OFF switch for the MCPs by demethylating them

causes MCPs to be less active so that they stop phosphorylating CheY

will gradually return to inactive state naturally

CheR

the ON switch for the MCPs by methylating them, antagonistic with CheB

periplasmic binding proteins

bind to the attractant or repellant and interact with MCPs

attractant + PBP acts as an OFF switch to the MCPs

if MCPs are off, the length of the run increases, shuts off the normal cascade (tumble)

run

response regulators are inactive (non-phosphorylated) and the flagella are ON (swimming forward)

MCPs have a gradually increasing about of methylation due to CheR, making them increasingly more likely to signal a tumble

tumble

MCP signals CheA to be autophosphorylated to CheA-P

CheA-P activates CheY (CheY-P) and CheB (CheB-P)

CheY-P interacts with Fli proteins to reverse the flagellar motor and cell tumbles

reset

CheB-P demethylates MCPs, causing them to become less active and stop phosphorylating CheY

CheZ resets CheY-P to the inactive CheY state

run recommences

CheB-P gradually returns to inactive CheB and new cycle starts

constant level of attractant/repellant

a constant amount is bound to the MCPs and the run-rumble cycle repeats at roughly regular intervals

if attractant increases

MCPs bind more attractant and become less active (less likely to trigger cascade = tumble)

run phase becomes longer

eventually a tumble will occur because the CheR signal will override the attractant signal and cause a tumble

if attractant decreases

MCPs gradually bind less attractant, and become more likely to trigger the phosphorylation cascade to signal a tumble

run phase becomes shorter

bacterial growth medium

contains an energy source (organotrophs, lithotrophs, phototrophs), a carbon source (autotrophs, heterotrophs), macronutrients and micronutrients

different microorganisms may have vastly different nutritional requirements - need to understand physiology and nutritional requirements

macronutrients

nutrients required in large amounts

N, P, K, S, Ca, Na, Mg

micronutrients

nutrients required in minute amounts, including trace metals (B, Co, Cu, Fe, Mn), growth factors (organic), mostly vitamins (biotin, folic acid)

defined medium

exact chemical composition known

complex medium

composed of digests of microbial, animal or plant products, e.g. yeast and meat extracts

exact composition unknown

selective medium

contains compounds that selectively inhibit growth of some microbes but not others

e.g. antibiotics

differential medium

contains an indicator, usually a dye, that detects particular metabolic reactions during growth

lag phase

bacteria are put into the medium from a stock culture, could be exiting dormancy, producing new enzymes

adjusting metabolically

exponential phase

growth is exponential, growing at optimum rate in that particular medium

binary fission - double numbers each period

stationary phase

# of new cells = # of cells dying

depletion of nutrients, some resources have run out, e.g. energy source, O₂

acidification could be a limiting factor

new cells use old material from dead cells to persist

death phase

cells dying > new cells, not enough nutrients, death outpaces

culture may return to dormancy (endospores)

exponential growth formula

N_t = N₀ × 2ⁿ

N_t = cells at time t

N₀ = cells at time 0

n = number of generations

log exponential growth formula

logN_t = logN₀ + 0.30n

same formula as Nt = N0 × 2ⁿ but log

generation time formula

n = t/g

n = number of generations

g = generation time (h), the amount of time required for a population of cells to double (doubling time)