Microbiology - Bacterial Uptake, Secretion, and Toxins

Bacterial uptake, secretion and toxins. Microorganism metabolism: scavenge nutrients from the surroundings require a central metabolism for: monomer synthesis, macromolecule polymerisation, biomass synthesis, and proliferation. Adapt and maintain metabolic fluxes for survival. Many bacteria are heterotrophic - they can thrive on many carbon sources. They do not express all transporters at the same time as it will consume valuable resources and occupy limited membrane space. In response to external stimuli, the microorganism will express some genes that will aid survival and express specific transporters. This helps to: conserve resources conserve membrane space co-ordinate nutrient availability with cellular functions Two component signalling system: kinase (phosphorylates proteins) transcription factor (binds DNA to allow controlled expression of genes) eg: NtrB and NtrC aid in nitrogen regulation. When a decrease in N is detected, NtrB is phosphorylated and this will act as an active kinase and phosphorylate NtrC, which can interact with the DNA and activate the genes involved with the uptake of N. In positive feedback loops, small triggers occur outside the cell and this triggers a large transcriptional response. Uptake mechanisms: pores/channels, PTS, uniport, symport, antiport. Methods to investigate uptake mechanisms: PROBING for uptake measure the uptake or export of substrates in intact cells can isolate the membrane vesicles and monitor the uptake of materials into these purification of the protein and incorporate it into the lipid bilayer to isolate its role in uptake structural biology of transporters eg electron microscopy, x-ray crystals and NMR introduce genetic mutations and see how it inhibits or enhances uptake biophysical approaches - structure, function and dynamics evolution and bioinformatics to look at similarities of DNA and protein sequences Carbon Sources - glucose, lactose, glucosamine, trehalose, fructose and maltose. Bacteria have a preferred carbon source so they will shut down transporters for alternative sources. The carbohydrate substrates are phosphorylated, and then released into the cell, and the net transport relies on movement of chemical groups in the PTS system (Phosphotransferase system). In gram-negative bacteria such as E. coli, there is a periplasmic space between the inner and outer membranes. For glucose to enter the bacteria, it passes through porins / Beta barrels in the outer membrane, into the periplasmic space. These are large, regulated, water-filled pores. Then there are specific transporters in the inner membrane. Glucose is transported in by E2C, and then phosphorylated by E2B to become Glucose-6-phosphate. The increase in unphosphorylated E2A causes it to bind and inhibit other transport proteins and enzymes. This pathway is dependent on the PEP:pyruvate ratio. FBP, a by-product of glycolysis, will turn the carrier protein HPr into its inactive form. Thus is there are many by-products being formed in the glycolysis pathway, this will inhibit the uptake of these alternative carbon sources. Lactose uptake is regulated by cAMP-activated global transcriptional regulator protein (Crp). This is activated by cyclic-AMP, and it promotes the expression of several carbon uptake/catabolism systems. In low glucose conditions, the phosphorylated E2A stimulates the activity of adenylate cyclase, which converts ATP to cAMP. This increase in cAMP ultimately leads to the allosteric activation of the Crp protein which stimulates the expression of lactose transporters and the conversion of allolactose to aloha-KG. LacI inhibits the expression of the lactose pathway. Cra = catabolite repressor/activator. This represses glycolytic enzymes and activates gluconeogenesis. when enough FBP is made, there is a feedback to stop stimulating this pathway and promotes the conversion of PEP to pyruvate. Nitrogen Sources The E. coli preferred nitrogen source is ammonium (NH4). The alternatives include putrescine and sialic acid. There will be a basal expression of transporters, and then there will be an internal detection of if there are sufficient source of nitrogen. This will lead to an up-regulation of the transport or catabolic mechanisms. Ammonia is transported into the cytoplasm via the Amt proteins, eg in E. coli the protein AmtB is a trimeric protein that transports ammonium, and it is regulated by GInk. When GInk is inactive AmtB is open, and if GInk binds to it, it closes. NtrC is a transcription factor which activates the transcription of N source uptake enzymes and N catabolism enzymes. When there is an abundance of NH4, it will go into the cell and bind with aKG to form the amino acids Glutamate and Glutamine. The presence of these amino acids stimulate a regulator called GlnB which prevents the uptake of other nitrogen sources. Not enough NH4 causes aKG to build up in the cell which inhibits GlnK and this stimulates the uptake of NH4, so it can form the amino acids. It will also inhibit GlnB activity so NtrC can take up alternative nitrogen sources. Amino acid uptake has a feedback mechanism of its own uptake - end-product-inhibition NtrC stimulates the uptake of Arginine and the breakdown of other nitrogen sources. Arginine itself can bind to ArgR (a transcription factor) which inhibits the uptake of Arginine and stimulates the breakdown of Arginine. If aKG not being used, it will stimulate NtrC which will stimulate uptake of Arginine. Some have intermediate activation. Eg- Cysteine is taken into the cell, and there is also a biosynthesis pathway from Ser -> Cys There in an intermediate by-product - CysB - which promotes the uptake and biosynthesis of Cys. When [Cys] is too high there is feedback inhibition where it prevents further biosynthesis and also prevents the expression of CysB. Iron is very insoluble. Bacteria first need to secrete siderophores outside the cell, which binds to iron 3+ and forms an iron-siderophore complex. This can then be transported by porins into the periplasm and the cytoplasm, where the iron is released (as a 2+ ion). The siderophore is then exported back into the extracellular region where it can bind to more iron. Secretion Protein secretion is needed to interact with the environment as the proteins can digest material which can then be taken up by the cell or it will carry a molecule which the cell can use. Also allows for motility eg flagella and pili allow the bacteria to move. Intercellular communication means it can interact with the host and provide an environment for it to grow. If it is a pathogen, proteins can degrade the host cell and cause further infection deeper in the body. DNA encoding transport mechanisms are genomic. Transposons are mobile elements of the genome that don’t have a set position. Plasmids are small circular bits of DNA that can carry antibiotic resistance. Gram-positive bacteria only have the inner membrane for the protein to be secreted across whereas gramnegative bacteria require secretion over both inner and outer membranes. Inner membrane transport: Sec system: co-translational translocation requires SRP and ribosomes post-translational translocation requires SecA and ATP. Tat (twin-arginine translocation): allows folded proteins to be transported substrate binds to protein in membrane (TatBC), and then TatA also associates, so that this complex allows the transport of the substrate across the membrane.