Week 3

• In liquid media, bacteria are often found as freely swimming individuals (referred to as planktonic). Upon encountering a surface, some bacteria will use surface appendages such as pili to adhere to the surface

  
• Bacterial biofilm: A population of bacteria encased in self-produced extracellular polymeric substances (EPS).

  
• After successful surface colonization, many species of bacteria will start to produce a slime-like coating comprised of extracellular polymeric substances (EPS). The combination of bacterial cells and their surrounding EPS is called the biofilm, which can be advantageous to bacterial survival.

  
• The biofilm matrix (EPS) acts as a scaffold and fulfills many beneficial roles for the bacteria inhabiting the biofilm. The EPS scaffold contains openings and channels that allow for the flow of nutrients and waste

  
• Biofilms facilitate bacterial interactions. In contrast to aqueous environments where bacterial cell-cell interactions are transient, biofilm-embedded bacteria have prolonged interactions with other microbes. Consequently, many different types of microbial interactions occur in biofilms: quorum sensing, metabolic sharing and microbial competition.
• Existing in biofilm increases bacterial survivability in hostile environments, which in the case of human pathogens, makes them difficult to treat with antibiotics.
• Stages of the biofilm life cycle: initial adhesion, early development, mature biofilms, biofilm dispersal.

  
• stages of biofilm life cycle [initial adhesion]: many bacteria use pili to facilitate the initial stages of adhesion to solid surfaces

  
• stages of the biofilm life cycle [early development]: Build up of the biofilm matrix, usually through the biosynthesis and export of exopolysaccharides.

  
• Stages of the biofilm life cycle [mature biofilms]: Many cells adopt a dormant state, which during infection, makes them harder to kill because many antibiotics target growing cells.

  
• Stages of the biofilm life cycle [Biofilm dispersal]: released biofilm fragments are able to diffuse to new locations and the biofilm life cycle can start anew

  
• WspA: mechano receptor that detects growth on solid surfaces

  
• WspE: is a demethylase

  
• WspE: is a histidine kinase that phosphorylates WspR an WspF

  
• WspC: a methylase of WspA

  
• WspR: is a GGDEF protein that becomes enzymatically active upon phosphorylation of its receiver (domain). This enzyme domain synthesizes the nucleotide second messenger c-di-GMP.

  
• c-di-GMP binds and inactivates FleQ, which is a positive regulator of flagella motility and negative regulator o biofilm

  
• Each monomer of WspR contributes a half active site that binds one molecule of GTP.

  
• inactivating DGCs [phosphorylation-dependent inhibition] --> When a bacterium no longer senses a surface, WspR can be dephosphorylated resulting in a return to a planktonic lifestyle. This can happen spontaneously as no more signals are coming from WspA

  
• Inactivating DGCs [phosphorylation-independent inhibition] --> Even when high c-di-GMP levels are desired, unrestrained c-di-GMP production can cause GTP depletion, which is toxic to cells. Thus, a DGC inhibition mechanism is required during biofilm growth. the c-di-GMP will dimerize and bind allosterically to WspR and pull on the DGC domain changing its conformation to its monomeric conformation (inactive form).

  
• Bacteria form biofilms using a self-produced extracellular matrix. bacterial biofilms can form on biotic or abiotic surfaces and can promote positive and negative interactions. It is highly regulated and has distinct lifestyle stages.
• The Wsp system (TCS) influences biofilm formation by controlling c-di-GMP synthesis. WspR cyclase makes c-di-GMP, and are regulated by phosphorylation and dimeric c-di-GMP
• Biofilms are made of extracellular polymeric substances. Biological polymers such as polysaccharides, proteins and DNA act as a structural scaffold that holds together the biofilm matrix. These polymers are secreted by bacteria upon switching to a biofilm lifestyle

  
• Exopolysaccharides (coating around a bacteria in biofilm) are synthesized and exported by dedicated molecular mechanisms that are embedded in the cell envelope. All exopolysaccharide systems are turned on by c-di-GMP.

  
• Cellulase is made up of beta1,4 linked glucose polymer.

  
• similar to LPS and peptidoglycan, biofilm polysaccharides are built from activated sugar-nucleotide precursors. In the case of cellulose it's UDP-glucose

  
• Cellulose (and other biofilm polysaccharides) posses many hydrogen bond donors, which results in high water-binding capacity

  
• the biosynthesis and export of cellulose is facilitated by BcsQ which can be split into 4 parts: BcsA, BcsB, BcsZ, and BcsC.
• BcsA: this component has 2 domains: one domain binds to c-di-GMP while the other is a glycosyl transferase that takes the glucose molecule of UDP-Glucose

  
• BcsB: is a membrane transporter for cellulose, it is also a periplasmic scaffold

  
• BcsZ: it is a cellulase that digests improper cellulose production. It regulates the length of cellulose.
• BcsC: is an outer membrane porin
• A complex of the BcsA and BcsB proteins catalyzes the simultaneous polymerization and translocation of cellulose across the inner membrane

  
• BcsA, which contains the cellulose synthase active site, requires c-di-GMP binding to adopt a catalytically active conformation

  
• Exopolysaccharides are a major component of bacterial biofilms. Cellulose is an exopolysaccharide that is the most abundant biopolymer on earth
• c-di-GMP activates cellulose synthesis by binding to BcsA, opening up the active site (R580)
• Different TCSs can influence cellulose synthesis (and biofilm formation) by directly influencing c-di-GMP production.