College 4+5 (Biocontrole + QS)

microbial growth phases

• lag: no pop growth

• log: exponential pop growth

• stationary phase: no pop growth

• death phase: decrease pop

primary vs secondary metabolites

• primary

  • necessary for survival eg amino acids

• secondary

  • response to environment eg toxins, anti MO compounds, pigments

metabolism

network of metabolic reactions of a cell

Fields of interest in microbiology

• agriculture (symbiosis)

• medical microbiology (pathogenic)

• biotechnology (beneficial)

human interpretations of microorganisms

• pathogen

• saprophytes

• beneficials

Where can MOs be found (plants)

• leaf and roots

  • rhizosphere

• soil biology

How do MOs move

Chemotaxis: movement to compounds (flagella)

Biofertilizers

• nitrogen fixation N2>NH4

  • rhizobium nodules

• P solubilizers

  • po4- forms insoluble salts with ca2+ Al Fe

• Mycorrhizae

  • uptake PO4 and other ions

phytostimulators

• auxin production IAA

  • trp nodig om dit te produceren

• trp exuded by root but varies per plant

• phytohormones produced by MOs

vorming van nodules

• plant scheidt flavonoides uit

  • induceren expressie knot factoren

• knotfactoren worden geproduceerd door MO en worden richting wortel verplaatst

bioremediators

• clean up of polluted soil

  • use things like DDT and benzen as carbon sources

• xenobiotic degrading microbes

  • kunnen tegen hele slechte omgevingen

biocontrol

control of phytopathogens by beneficial MOs

biocontole waarom?

• environmentally friendly

• locally produced

• biodegradable

• no residue in drinking water

• not harmful

• negative perception against use of chemicals

advantages over GMO plants of bio control

• no yield decrease

• not expensive

• flexible

• resistant plants not always available

• no negative perception

disadvantages

• unstable: live organism

• vegetative cells difficult to formulate (shelf life problem)

• registration of agents cost/time consuming

bio control agents

• production anti MO compounds

• induced system resistance (ISR)

• predation and parasitism

• competition for niches and nutrients

• disruption quorum sensing

ISR induced systemic resistance

• induced after contact with no pathogenic rhizobacteria

  • salicylic acid

  • flagella

• detection depends on ethylene and jasmonic acis

• physical barries

• comparible to the innate immune system of mammals

bio control agent: predation and parasitism

• disrupt cell wall

• production of lytic enzymes

niches and nutrients

• root colonisation

• essential for biocontrol

• establish on the root

• competition with endogenous population

• deliver anti MO compounds

root colonisation traits

• motility/chemotaxis towards root system

• synthesis amino acids and B1

• attachment

• growth rate in root system

Characteristics of a good biocontrol agent

• good root colonisation

• makes a substance which actively stops sickness

industrial production

• bioreactor

  • vessel for controlled MO growth

  • no contact with outside world

  • follow/measure over time

artificial growth

• MO growth influenced by surroundings

• two types: allrounder

  • thrive in diverse environments

• sprinter

  • highyl specialsed for specific environmetal conditions

artificial conditions

• strain managemment

  • DNA typing

  • clean test

• growth

  • clean test/culture

formulation

• collection biomass

• mycelium and spores separated

• spores concentrated using in line centrifuge

• spore concentration checked: counting

• contamination checked

quorum sensing

regulation of cell to cell communication by allowing bacteria to share info about cell density and encvi etc and adjust gene expression accordingly (global and cell cell communication)

MOs can detect signals from each other

two component system

• sensor kinase on membrane gets signal

• phosphorylation cascade

• response regulator in the cell changes gene expression

quorum sensing in high and low population densities

• low

  • basal production level of signal molecule

  • low signal concentration

• high

  • high signal concentration

autoinducers

secretion of extracellular signal molecules

discovering quorum sensing

• bacterial bioluminescence

  • checked the growth curve

  • took late log cells and put them with early log cells

  • instant bioluminescence

• compound accumulates in culture media during growth of luminescent bacteria under conditions when it was unexpected > quorum must be reached before induction

signaling molecules

• intraspecies

  • interspecies: gram pos and negative bac

addition of exogenous homoserine lactons to media (AHL)

• inducers used by gram negative bacteria to communicatie

specificiteit AHL

determined by the chain length

diffusion/secretion AHL

• amphipathic character can diffuse freely through membrane

• AHL short fatty acid tails diffuse easier than longer

breakdown AHL

• enzymatic (cut through lacton ring and fatty tail)

• non enzymatic (high pH)

advantages of symbiosis (vibrio fisheri)

• light: highly energetic costs

• exchange of nutrients

• host

  • squid: mislead predators

quorum sensing systems

• symbiosis

• virulence

• swarming

• biofilms

• root colonisation

what is a biofilm

population of motile bacteria attached to a surface enclosed in a layer of extracellular polysaccharides produced by the bacteria themselves

where are biofilms formed

• solid moist surfaces

• soft tissue in living organisms

• interfaces between air and liquid

stages of a biofilm

1. attachment

2. irreversible attachment

3. growth and division of bacteria

4. biofilm formation + prod exopolysacch

5. (attachment other organisms)

6. cells escaping from biofilm to start one somewhere else

biofilm shapes

• planktonic cells

• mushroom structures

interest in biofilms why?

• bacterial biofilms huge problem

  • lung tissue

  • medical devices

• multi resistant

development of new therapeutics for biofilms

antimicrobial peptides (AMP_

• natural (broad spectrum of killing microbes)

• synthetic

biosurfactants what are they

amphipathic molecules accumulating at interfaces and can form micelles eg PCL1445, inhibit biofilm formation

biosurfactants functions

• less toxic

• biodegradable

• better activity under environmental conditions

• enhance swarming motility + bioavailability of hydrophobic pollutants (bioremediation)

four classes of biosurfactants

• glycolipids

• lipopeptides and lipoproteins

• fatty acids

• polymeric surfactants

natural peptides

• lactoferrine: anti MO peptide in milk > high conc in colustrum (improve immune system calf)

• anti inflammatory

• sequestering of iron ions (none for pathogen, inhibition)

lactoferrine and biofilms

• inhibition biofilm formation

• anti tumor activity

• inhibition proteinases etc

derivatives lactoferrine

• lactoferricine

  • degraded in the stomach to smaller fragments of peptides

• lactoferrampin

  • active again e coli etc but not against fermenting bacteria

mode of action of natural peptides

• positively charged AMP targets negatively charged outer leaflet of bac membrane

• barrel stave model

  • holes in the membrane > cel lyseert

• carpet model

  • peptiden cover the membrane like a carpet > gaten weer en cel lyseert