Bacterial Sensing and Motility

Bacterial Sensing

Learning Objectives

• Two Component Systems

  • General characteristics

• Second Messengers

  • Synthesis and activity

• Alternative Sigma factors

  • Why have them and how do you control them

  • Sporulation

    • Characteristics of spores

    • Sigma cascade

Two Component Regulatory System

• Found in all three domains of life

• Two proteins govern pathway

  • Sensor kinase

    • Extracellular receptor for metabolite

    • Intracellular communication pathway

  • Response-regulator protein

    • Activated by sensor kinase

    • DNA binding protein

      • Activator - enhances transcription needed

      • Repressor - inhibits transcription unless needed

Sensor Kinases

• Located in cell membrane

• Acts as kinase and sometimes phosphatase

• Normally dimerizes to autophosphorylate itself

  • At conserved histidine residue

  • Uses ATP

Response Regulators

• Cytoplasmic

• Accepts phosphate from sensor kinase to conserved aspartate

  • Can mutate Asp to Glu to mimic phosphorylated state

• Output

  • Can bind DNA to control transcription

  • Can bind proteins to control activity

TCS Examples

• EnvZ/OmpR

  • Senses osmolarity changes

  • Represses ompF and activates ompC

• ArcBA

  • O2 sensor

  • Controls ~9% of genes in E. coli

• NtrBC

  • Response to N2 starvation

  • Controls gene for importing N or recycling N compounds

Second Messengers

• Small molecules produced in response to a signal

• cAMP

  • Cyclic AMP

  • Talked about with Lac operon

• C-di-GMP

  • Cyclic dimeric GMP

• ppGpp

  • Guanosine tetraphosphate

cAMP Reminder

• Used in catabolite repression

• Made when glucose is low by adenyl cyclase

• Ensure cells only make enzymes for sugars when that sugar is present AND glucose is not

C-di-GMP

• 2 GMP molecules linked by their phosphates

• Control many functions

  • Motility, biofilm production, etc.

• Act through effector proteins

  • Control transcription

  • Control enzyme activity

Synthesis/Destruction

• Diguanylate cyclases

  • Synthesize

  • GGDEF domain

• Phosphodiesterases

  • Destroy

  • EAL domain

• Both contain a sensory domain

  • Interacts with a molecule for activation

ppGpp

• Guanosine tetraphosphate

• Controls stringent response

  • Amino acid starvation

Stringent Response

• Amino acid starvation

• Protein synthesis cannot proceed

• Cell decreases production of tRNA and rRNA to conserve energy

• Cell increases production of genes needed for biosynthesis of amino acids

ppGpp Synthesis

• Empty tRNA enters A site

  • Sensed by RelA

• RelA makes ppGpp

  • Uses ATP and GDP

  • ATP+GTP=pppGpp

• Quickly converted to ppGpp

Effects of ppGpp and DksA on Transcription

• ppGpp interacts with RNApol

  • Stop transcription of tRNA and rRNA genes

  • Upregulates transcription of amino acid biosynthesis genes

• DksA + ppGpp

  • Destabilize open complex during transcription of downregulated genes

  • Enhances open complex during transcription of upregulated genes

Which promoters to chose

• Region between -10 and + 1 nucleotide is important

• Downregulated genes have GC rich region

  • Helps form unstable open complexes

  • DskA+ppGpp make them even more unstable

• Upregulated have AT rich

  • Immune to DskA

Other roles of ppGpp

• Stress response

• Made by SpoT instead of RelA

  • Senses fatty acids depletion, iron, and carbon

  • Can hydrolyze ppGpp

• Can affect protein stability or activity

  • Targets GuaB

• Involved in GTP synthesis

RNA Polymerase

• The sigma factor has no catalytic activity but helps the core enzyme recognize the start of genes

• core enzyme + sigma factor = holoenzyme

• Number of sigma factor vary by organism

  • E. coli has 7 sigma factors

  • Mycoplasma genitalium has 1

  • Streptomyces coelicolor has 60

    • is a intracellular pathogen

Alternate Sigma Factors Affecting Bacterial Transcription

• σ70: General house keeping/ exponential growth

• σS: Stress and stationary phase

• σE: Restore membrane integrity and proper folding of membrane proteins

• σH: Heat shock and other stress

• σF (σ28): Flagella synthesis

• σ60: Nitrogen metabolism

• FecI σ: Iron starvation

• The 7 sigmas of E. coli

Controlling Alternative Sigma Factors

• Sigma competition for core pol

  • Concentration + affinity determine who wins

• Concentration

  • Have transcriptional control

  • mRNA stability

  • Anti-sigma factors

• Affinity

  • σ70 has the greatest affinity

  • Also has the greatest concentration

  • ppGpp can reduce σ70 affinity

Sporulation

• Relies on a sigma cascade

• Spore- differentiated cells that are highly resistant

  • Difficult to destroy by heat, radiation, and chemicals

• Are metabolically dormant

Endospore Structure

• Spore surrounded by thin covering called exosporium

• Thick layers of protein form the spore coat

  • >70 proteins make the coat, which are highly crosslinked

• Cortex, beneath the coat, thick peptidoglycan

Core Properties

• Contains high levels of dipicolinic acid and calcium

  • Helps to reduce water

• Dehydrates spore resulting in gel-like consistency

  • Gives resistance to heat and chemicals and inactivates proteins

  • Stabilizes DNA with help from small acid-soluble DNA-binding proteins (SASPs)

• SASPs

  • Protect DNA from UV, desiccation, and heat

  • Are carbon and energy source during germination

• Low pH

  • Drops from 7 to 5.5-6

Sporulation Cycle

• Cell division

• Free spore

• Lysis of sporangium, spore liberation

• Completion of coat synthesis, increase in refractility and heat resistance

• Coat synthesis

• Cortex

• Cortex formation

• Axial filament formation

• Plasma membrane

• DNA

• Septum formation and forespore development

• Engulfment of forespore

• Asymmetric cell division

• Wall

Formation of Vegetative Cell

• Activation

  • Prepares spores for germination

  • Often results from treatments like heating

• Germination

  • Environmental nutrients are detected

  • Spore swelling and rupture of absorption of spore coat

  • Increased metabolic activity

• Outgrowth

  • Emergence of vegetative cell

Sporulation in Bacillus subtilis

• Two-component systems are also important

• Sensor kinases activate (KinA)

  • Senses low nutrients passes phosphate to Spo0F passes phosphate to Spo0B

• Activates response regulator (Spo0A) controls >500 genes

Sporulation sigma cascade

• During normal growth cells use σH and σA

• Starvation signals activate other sigma

• σE is made by Spo0A as in an inactive form must be cleaved by protease to be active

Motility

Learning Objectives

• Swimming motility

  • Flagella organization

  • Flagella synthesis

  • Flagella movement

• Chemotaxis

  • Signaling pathway

  • Adaptation

• Twitching motility

  • Pili assembly

  • Movement

Swimming motility

• Flagella based

• Cells swim through liquid

Flagella Organization

• Monotrichous

  • Single polar flagellum

• Amphitrichous

  • Single flagellum on each side

• Lophotrichous

  • Tuft of polar flagella

  • 1 or both sides

• Peritrichous

  • Many flagella around the body

Flagella

• Motility appendage

• 20nm thick and up to 20μm long

  • Can’t see them with light microscopy

• Type III secretion needle complex

• Can be virulence factor

Bacterial Flagella

• Ultrastructure composed of three parts

  • filament extends from cell surface to the tip

    • hollow, rigid cylinder of flagellin protein

  • hook links filament to basal body

  • basal body is series of rings that drive flagellar motor

Flagellar Synthesis

• 3 tiered regulatory system

  • Class I genes are transcriptional regulator for class II

  • Class II genes make basal body, rod, and hook

    • Include sigma factor (sigma 28) for class III genes and anti-sigma factor (FlgM)

  • Class III genes include flagellin and motor proteins

Flagellar Synthesis

• 3 tiered regulatory system

  • Class I genes are transcriptional regulator for class II

  • Class II genes make basal body, rod, and hook

    • Include sigma factor ( sigma 28) for class III genes and anti sigma factor (FlgM)

    • Exported by Sec system

  • Class III genes include flagellin, motor proteins

    • Flagella forms Type III-like secretion systems

      • When functional FlgM is excreted allowing sigma 28 to work

      • Flagellin subunits are made and exported through

Flagellar Synthesis

• 3 tiered regulatory system

  • Class I genes are transcriptional regulator for class II

  • Class II genes make basal body, rod, and hook

    • Include sigma factor ( sigma 28) for class III genes and anti sigma factor (FlgM)

    • Exported by Sec system

  • Class III genes include flagellin, motor proteins

    • Flagella forms Type III-like secretion systems

      • When functional FlgM is excreted allowing sigma 28 to work

      • Flagellin subunits are made and exported through

    • Filament subunits self-assemble with the help of the filament cap at the tip, not base

Mechanism of Flagellar Movement

• 4 rings

  • C-ring: controls direction

  • MS ring: turns and anchors

  • P-ring: interact with PG

  • L-ring: interact with OM

• Stator - Mot A and Mot B proteins

  • form a channel through the plasma membrane

  • protons move through Mot A and Mot B channels using the energy of proton motive force

  • MotAB don’t move, the MS ring does

Multi-Flagella Swimming

• Flagella can spin both clockwise or counterclockwise

• CCW

  • Flagella bundle

  • Cell moves forward

• CW

  • Individual filaments unbundle

  • Cells tumble

• Reorients the swimming direction

Monotrichous swimming

• CCW rotation makes cells move forward

• CW rotation

  • Cells reverse

  • Stop and reorient randomly

Chemotaxis

• Chem = chemical; taxis = movement

• The movement of a cell corresponding to a gradient of concentration of a substance

• In E. coli leads to random walk

  • Run/tumble phenotype

Chemotaxis in E. coli

• Methyl-accepting chemotaxis proteins (MCPs) are chemoreceptors in the membrane

  • binds environmental chemicals

  • initiates a series of interactions with cytoplasmic proteins that affect flagellar rotation

• Activates sensor kinase CheA (with help of CheW) which autophosphorylates

• CheA phosphorylates the response regulator CheY

• CheY governs the rotation of flagella

  • Default direction is CCW rotation (run)

Methyl-Accepting Chemotaxis Proteins

• In the inner membrane

  • At the poles

• Forms a trimer of dimers

  • Act cooperatively

  • Can integrate signals from all trimers

• Bind chemicals and transduce signal into cells

• Activity is modulated by methylation

Chemotaxis is a Two-Component System

• CheA – Sensor Kinase

• CheY – Response Regulator

• End result is not change in gene expression

Activation of system leads to tumble

• Moving down good gradient

• Moving up bad gradient

  1. CheA autophosphorylates

  2. Phosphate transferred to CheY

  3. CheY bind motor causing CW rotation (tumble)

  4. CheZ removes P from CheY acts all the time

How does the cell know the concentration is changing?

• Adaptation

• Cell senses over time

  • Too small to use distance

• Trying to balance cytoplasmic methylation with periplasmic attractant binding

• CheR is constantly adding Methyl groups

• CheB removes methyl groups

  • Only when phosphorylated

  • Activated by CheA

• CheA will auto - phosphorylate if methylation exceeds attractant binding

Twitching Motility

• Pili based

• On surfaces

Type IV pili

• Similar to Type II secretion

• 5-7nm thick

• Made of PilA

Pilus Assembly

• PilA has a leader sequence

• Cleaved and secreted by PilD

• PilQ is a pore

• Built from the inside and pushes out

• PilB is ATPase

Pilus retraction powers movement

• Cells move because of pili retraction

• Powered by PilT ATPase

Cells can move against the flow

Upstream twitching increases spread