second messengers, intracellular communication, and biofilms

What is the role of second messengers in bacteria?

They transmit and amplify intracellular signals in response to environmental stimuli.

what is quorum sensing

Cell-density-dependent regulation of gene expression via signaling molecules.

what is an autoinducer

Diffusible molecule that accumulates with increasing cell density

what is quorum quenching?

Interfering with quorum sensing to disrupt bacterial coordination

Name four major bacterial second messengers.

(p)ppGpp, cAMP, c-di-GMP, c-di-AMP

What does (p)ppGpp regulate?

The stringent response to amino acid and nutrient starvation.

Which enzyme synthesizes (p)ppGpp during amino acid starvation?

RelA

-strong synthase activity

Which molecule accumulates when glucose is absent?

cAMP

Which second messenger is most associated with biofilm formation?

c-di-GMP

What cyclic dinucleotide is essential for cell wall homeostasis in some bacteria?

c-di-AMP

What enzyme synthesizes cAMP?

Adenylate cyclase

Which second messenger regulates the transition from motile to a nonmotile state?

c-di-GMP

Which second messenger is involved in the stringent response and growth arrest?

(p)ppGpp

How does RelA detect amino acid starvation to synthesize (p)ppGpp?

RelA is activated when uncharged tRNA binds to the A-site of the ribosome.

Explanation: This signal indicates amino acid scarcity and initiates the stringent response

What dual function does SpoT serve in (p)ppGpp metabolism?

SpoT can both synthesize and degrade (p)ppGpp, responding to fatty acid starvation and other stress.

Explanation: Its activity is regulated by interactions with ACP and other metabolites

What happens to rRNA and tRNA transcription when (p)ppGpp levels are high?

rRNA synthesis is repressed.

Explanation: This conserves resources and slows growth during starvation.

How does the cAMP-CRP complex influence gene expression?

cAMP binds CRP, enabling it to bind DNA and activate genes for alternate carbon sources.

Explanation: This is crucial during glucose starvation (catabolite repression).

What domains are responsible for c-di-GMP synthesis and degradation?

GGDEF synthesizes c-di-GMP; EAL and HD-GYP degrade it.

Explanation: These domain-containing proteins respond to environmental and cellular cues.

What role do PilZ domain proteins play in c-di-GMP signaling?

They bind c-di-GMP and regulate motility, adhesion, and biofilm formation.

Explanation: PilZ is one of several effector types.

What is the 'I-site' in GGDEF domain proteins?

A feedback inhibition site (RxxD motif) that binds c-di-GMP allosterically (non-covalent).

Explanation: When bound, it inhibits further cyclase activity.

How does c-di-AMP contribute to bacterial physiology?

It regulates osmotic stress, DNA integrity, and cell wall homeostasis.

Explanation: Often essential in Gram-positives.

What is the significance of local vs. global pools of c-di-GMP?

Local pools ensure signaling specificity by compartmentalizing responses.

Explanation: Avoids crosstalk between pathways in cells with many DGCs/PDEs.

Which second messenger is responsible for catabolite repression

cAMP

explain the cAMP-CRP system when glucose is present

glucose present --> low adenylate cyclase activity --> low cAMP --> CRP inactive --> no transcription of alternative metabolic pathway genes

explain the cAMP-CRP system when glucose is absent

glucose absent--> high adenylate cyclase activity --> high cAMP --> cAMP binds CRP --> transcription of alternative metabolic pathway genes

c-di-AMP is synthesized by what enzyme?

deadenylate cyclase (DAC)

c-di-AMP is degraded by what enzyme?

phosphodiesterase

when is c-di-AMP necessary?

in lab media growth conditions

when levels of c-di-GMP are high, you would expect motility to ___, biofilm formation to ____, and pathogenicity to ____

decrease, increase, increase

The GGDEF domain is activated by environmental signals, triggering synthesis of ___ from ___

c-di-GMP , 2 GTP

what are the regulatory mechanisms of c-di-GMP

transcriptional (control DGC/PDE gene expression)

post transcriptional (sRNAs or riboswitch)

translational (regulatory proteins)

Allosteric (feedback inhibition by I-site)

What molecules are used by Gram-negative bacteria in quorum sensing?

Acyl-homoserine lactones (AHLs)

What signaling molecules are used by Gram-positive bacteria?

Autoinducing peptides (AIPs)

What is a universal quorum sensing molecule used by many species?

Autoinducer-2 (AI-2)

What does LuxI do?

Synthesizes AHL quorum sensing molecules.

What does LuxR do?

Binds AHL and activates transcription of target genes.

Why does quorum sensing require high cell density?

Autoinducers must accumulate to a threshold to activate receptors.

name the essential components of a quorum sensing system

signal molecules (autoinducer)

synthase

receptor/regulator

gene targets (ex. biofilm, virulence, motility)

signal decay molecules

How does the agr system in Staphylococcus aureus regulate virulence factor expression?

AgrD is processed and secreted as AIP; AIP binds AgrC (histidine kinase), which phosphorylates AgrA. AgrA activates RNAIII expression, which regulates target genes.

Explanation: This is a TCS-mediated quorum sensing circuit using peptides.

Describe the LuxI/LuxR system in Gram-negative quorum sensing.

LuxI synthesizes AHL. AHL diffuses out and back in at high density, where it binds LuxR. LuxR-AHL activates target gene transcription (e.g., bioluminescence in Vibrio).

Explanation: AHL concentration reflects cell density.

What is quorum quenching and give an example of a quorum quencher.

Interference with quorum sensing by degrading or blocking signal molecules.

Example: AiiA is a lactonase that degrades AHLs.

How does quorum sensing contribute to biofilm formation?

QS regulates expression of EPS synthesis, surface adhesins, and dispersal factors.

Explanation: This coordination ensures biofilm is formed at high density.

What is the role of AI-2 and why is it considered a 'universal' signal?

AI-2 is recognized by diverse bacteria for interspecies communication.

Explanation: It enables coordination across different species in polymicrobial communities.

How do Gram-positive bacteria typically detect their autoinducing peptides (AIPs)?

Via membrane-bound histidine kinases in two-component systems.

Explanation: This allows extracellular sensing of small peptides.

What is the purpose of using quorum sensing inhibitors in clinical or agricultural settings?

To block virulence and biofilm formation without killing the bacteria.

Explanation: This reduces selective pressure for resistance.

How do Gram-negative bacteria typically detect their acyl homoserine lactones (AHLs)

LuxI-LuxR like system

LuxI synthesizes AHL

LuxR is activated by AHL, triggers gene expression

Gram-negative bacteria rely on ____ for synthesis of AHLs while Gram-Positive bacteria rely on ___ for synthesis of AIPs

themselves; synthase molecule, ribosome dependent

what do lactonases do in the context of quorum quenching

reversibly (in low pH) cleave the HSL ring of an AHL molecule

what do acylases do in the context of quorum quenching

irreversible cleave fatty acyl chain of an AHL molecule

what do molecular antagonists do in the context of quorum quenching

stabilize the closed receptor confirmation which inhibits receptors ability to transcribe genes

What is a biofilm?

A structured community of bacteria encased in a self-produced matrix attached to a surface.

What are the main stages of biofilm development?

Attachment, microcolony formation, maturation, and dispersal.

What is the major component of the biofilm matrix?

Exopolysaccharides (EPS)

How do biofilms compare to planktonic cells in antibiotic tolerance?

Biofilm cells are up to 1000× more tolerant to antibiotics.

What second messenger promotes biofilm formation?

c-di-GMP

What triggers dispersal from a biofilm?

Environmental cues such as nutrient depletion or QS signals.

what is in a biofilm matrix?

exopolysaccharides (EPS), proteins, extracellular DNA, lipids

How does c-di-GMP regulate biofilm formation and motility?

High c-di-GMP promotes EPS production and biofilm formation; low c-di-GMP promotes motility.

Explanation: This switch allows bacteria to transition between sessile and motile states.

How do biofilms resist antibiotics despite no genetic resistance?

Slow growth, limited penetration, efflux pumps, and metabolic heterogeneity.

Explanation: Biofilm structure creates gradients and protects inner cells from drug exposure.

What is the function of eDNA in biofilm matrices?

Provides structural support and promotes horizontal gene transfer.

Describe the physiological heterogeneity within a mature biofilm.

Outer layers are metabolically active, while deeper layers are dormant and stress-tolerant.

Explanation: Nutrient and oxygen gradients drive distinct physiological zones.

What clinical condition is associated with chronic biofilms of P. aeruginosa?

Cystic fibrosis lung infections.

Explanation: Thick mucus and immune evasion allow long-term colonization.

how can we disrupt biofilms?

surface modification to prevent adhesion, enzymatic degradation, interfere with quorum sensing, target eDNA