MCB3020 - Microbial Growth and Metabolism - SP26

V. natriegens is unique in that

it replicates in less than 10 minutes

Constraints of replication

Intrinsic constraints and extrinsic restraints

Intrinsic constraints

inherent to all members of a species

ex. genome size, metabolism, cell physiology

extrinsic constraints

extragenic, not inherent to genes

biotic extrinsic constraints

microbe interactions, antiobiotics

abiotic

temperature, pH, salinity, nutrients, water

STP

the narrow range of extrinsic pressure that most microbes live in

- 20-40 C

-6.5-7.5 pH

-0.5-1% salinity

- 1 atm (0.1 MPa)

- 0.9-1.0

Extremophiles

can grow in the extremes of extrinsic pressure

hyperthermophiles

extremophiles for temperature above 80 degrees C

ex. T. kodakarensis

thermophile

grows between 50-80 degrees C

hydrophobic amino acids in enzymes

structurally more salt bridges, ionic bonds

Mesophile

grows between 15-45 degrees C

Psychrophile

growth below 15 degrees C

can use CSPs, AFP, INP, membrane flexiblity, structurally less salt bridges and more weak bonds

ex. C. tohnii

alkalophile

growth above pH 9

uses things such as Na+/H+ antiporter system, CPAs, cell wall modifications

neutralophile

growth between pH 5 and pH 8

Acidophile

growth below pH 3

Halophile

growth in high salt, >2 M NaCl

uses K+ uniport mechanism

low-slat, organic solute-in strats

increased negative of enzymes

Aerobe

grows only in oxygen

facultative

grows with or without oxygen

microaerophile

growth only in small amounts of oxygen

barotolerant

growth between 10 and 495 atm

Anaerobe

growth only without oxygen

barophile

growth at high pressure, greater than 380 atm

Tolerance

microbes that can survive extrinsic pressures

How does c. cohnii adapt to its environment through lipid chemistry

uses docosohexanoic acid (DHAs) in its membrane, which is hyperfluidizing, containing >60% at 5C

full of cis-double bonds (unsaturated) making it highly kinked and fluid,

how does T. kodakarensis adapt to its environment via lipid chemistry

glycerol alkyl tetraethers (GDGTs) create hyper-rigid membranes monolayers, containing >90% concentration at 85C

Full of alkyl groups, which lock the molecules together

How does t. kodakarensis adapt to heat via protein chemistry?

amino acids are adapted for more intramolecular bonding, such as salt bridges, h-bonding, and covalent bonds

How does c. cohnii adapt to its environment via protein chemistry

its amino acids are adapted for thermal lability and increased flexibility.

How do chaperones help with protein stability in extreme temperatures

Hot and cold-adapted chaperones fold that back into their native shape

Piezophiles

live in pressure extremes of deep ocean, >1000 meters deep, >10 MPa hydrostatic pressure

ex. Shewanella benthica with >90% DHA and other fluidizing unsaturated lipids at >30,000 ft and 11,000 PSI

What are piezophiles most similar to?

psychrophiles, they both use DHA and other fluidizing lipids to survive high pressures

radiophile adaptations

DNA repair enzymes

extremolytes

DNA damage response protein C , DdrC

Xerophiles

adapt to low Aw/low water

concentrates on osmoprotectants

ex. uses dormancy induction, eps secretion, membrane modifications

ex. aspergillus adapting to Aw of 0.5

metalophiles

adapting to using metals

ex. EPS production, metal bisorption, enzyme detoxification, ion efflux

metalophile example

Metallosphaera, >50 uM Hg, using mercuric reductase, MerA

halophile example

Halobacterium, adapting to 10-35% NaCl

concentrat osmoprotectants, such as trehalose, betaine, glycine

alkalophile example

Acidothiobacillus adapting to PH 1-2, using F0F1 ATPase

psychrophile example

crypthecodinium, adapting to 5 degrees C using DHAs

Thermophile example

Thermococcus, using GDGTs

what bacteriaq is used in DHA supplement production

C. cohnii and schizochytrium spp.

What is used in metal biomining?

Metallosphaera sedula is used by Mintek

What microbe is used for cosmetics

D. radiodurans by Deinove

Thermus aquaticus

thermophilic bacteria, isolated in the 1970s from hot springs in yellowstone NP

Taq

thermostable DNA pol III found in T. aquaticus, that is commercialized for use in PCR assays

feast

rapid growth phase in natural systems for microbes, usually caused by excess of nutrients

famine

non-growth cycles in natural systems, caused by a lack of nutrients

exponential growth

population doubles every generation, as fast as possible

ex. in vitro cultivation, alcohol fermentation, microbial blooms, infectious disease

g

generation time, length of time from one generation to the next

varies in natural systems

assumptions of exponential growth model

1) constant generation time

2) unlimited resources

3) cytokinesis by binary fission

k

growth rate (k)

inversely proportional to growth rate

number of generations per unit time

k = log10(Nt/N0)/0.301t

phases of growth

1. Lag

2. Log

3. Stat.

4. Death

lag

cells are preparing for growth

detecting environment

expressing genes

synthesizing components

log

cells are growing at maximum k possible under specified growth conditions

low g

stationary

growth rate (k) = death rate (d)

determined by loss of resources, accumulation of metabolic wastes, etc.

death

death rate exceeds growth rate d>k

Coupling

Catabolism and anabolism depend on each other

uncoupling = death

KCN

cyanide

uncouples electron flow, causing ATP depletion and cell death

Entropy

disorder

microbes need low metabolic entropy

2nd law of thermodynamics

entropy is absolute

how is entropy decreased

cells use metabolic building blocks to increase cellular order

ordered states

value of entropy

higher order = low entropy (ex. lacl dimer)

lower order/increased system disorder = high entropy (ex. lacI monomer)

metabolic reactions that increase order

require energy

energy carriers

energy for energy-requiring reactions is provided by energy carriers by coupling their energy-producing reactions with the energy-requiring reactions

Microbial growth requirements

- metabolic coupling (ex. catabolism and anabolism)

- low metabolic entropy via energy

Macronutrients

Requirements of minerals for growth

Big 6 nutrients

C, N, H, O, S, P

Miscellaneous metal macronutrients

Fe, Mg, Co, K, Ca

Carbon

controls growth

50% of globular cellular biomass

Why does carbon control growth

it has properties of tetravalency and catenation

how is carbon added to biomass

heterotrophy and autotrophy

heterotrophy

acquiring carbon from eating other living things

the carbon is broken down then added to central metabolic pathways

autotroph

acquires carbon from CO2

uses CO2 and water to make glucose through calvin cycle, which is then added to CMPs

How is growth powered?

large amounts of energy

chemotroph

microbes that gain energy from chemical compounds

Phototroph

an organism that gets its energy from sunlight

where can energy from metabolism go?

work or stored in energy carriers and membrane potentials

high energy molecules

ATP, GTP, NADH, NADPH, FADH2, PEP

how many substrates can microbes metabolize?

virtually any, such as glyphosate herbicide or PAHs

P. aeruginosa

capable of metabolizing >100 substrates

ranked order of substrate metabolic preference

1. carbs

2. fats

3. proteins

4. nucleic acids

5. aromatics and xenobiotics

biopolymers

how substrates are largely organized into

need to be hydrolyzed for assimilation

hydrolases

used by microbes to digest biopolymers

ex. glycosidases, proteases, lipases, nucleases, esterases

what does substrate breakdown need

hydroxylation from water of covalent chemistries

glycosidases

catalyze glycosidic bond hydrolysis in complex carbs, glycoproteins, and glycolipids

glycosidase examples

cellulase hydrolyzing cellulose to glucose in C. thermocellulum

chitinase hydrolyzing chitin to N-acetylglucosamine (NAG) in actinomycetes spp.

lipase

hydrolyze ester bonds in complex lipids and glycolipids

lipase example

ex. tributyrin hydrolysis by tributyrase to glycerols and fats in lactic acid bacteria

proteases

amide bond hydrolysis in protein and glycoproteins

protease examples

hydrolysis of gelatin by gelatinase to amino acids in bacillus sp.

hydrolysis of casien by casienase to amino acids in bacillus cereus

how are hydrolases useful for diagnostics

hydrolase production is conserved

N. gonorrohoeae

tested for using carbohydrate utilization profile, which reads for ara, gly, glc, suc, man, xyl, and sor

This bacteria tests positive for glucose and sucrose metabolism, and negative for the others.

what is C. thermocellulum used for

bioprocessing of cellulosic wastes in biofuel

high Kmax prevents contamination during fermentation

how are microbial lipases used as biocatalysts

they are used as biocatalysts in the creation of detergents like Tide and in environmental bioremediation (pseudomonas spp.)

CMPs

- central metabolic pathway

- assimilate carbon and make energy, building blocks, and waste

- generally conserved

EMP

- aka Glycolysis,

- assimilates carbon from carbohydrate substrates

- anaerobic

- evolved to metabolize glucose, but is still highly versatile

- confirmed in bacteria in the 1940s and in archaea by Danson et al in 1986

Where is oxygen depleted

- greater than 200m underwater in oceans

- greater than 30cm deep in soi

- gets lower the more deeper in the intestine and colon

Cellulose

50% of earth's biomass

emp path

Glc + 2 ADP + 2 NAD+ --EMP--> Energy (2 ATP + 2 NADH) + 2 Pyruvate

Salvaging

non-glc substrates converted into EMP intermediates (Glc-6p, Fru-6P, Gly-3p) by salvage pathways

Chitin salvage pathway

NAG =NAGK=> NAG-6P =Deactylase AGM-1=> GlcN-6P =Deaminase NagB=> Fru-6P =EMP=> 2 ATP + 2 NADH + 2 pyruvate

lactose salvage pathway 1

Lac =LacZ=> Glc =EMP=> 2 ATP + 2 NADH + 2 Pyruvate