bacterial bioenergetics - kelly Flashcards
name 4 properties of the bacterial resp chain that is different from the mitochondrial respiration chain
modular - mix and match components
complexity - number of enzymes/e- acceptors that allow bacteria to grow in many envs
branching - branched chains
gene regulation - composition of chain changes in response to growth conditions
in order to couple e- transfer to proton translocation, does ΔE (redox potential change) have to be bigger or smaller than Δp?
give an equation to describe this relationship
ΔE > Δp if e- transport is coupled to proton movement
the efficiency of this is expressed by H+/e- = ΔE/Δp
give 4 reasons for oxygen being a good electron acceptor
high abundance
diffusible across membranes
reasonable water solubility
high midpoint redox potential of H2O/O2 make it an excellent e- acceptor
give 2 reasons for oxygen being a bad electron acceptor
incomplete reduction = ROS
Cells must produce enzymes to destroy ROSs
name 3 important anaerobic resp processes
methanogens
sulphur bac
nitrate reducers and denitrifiers
name 4 reductases and the cofactor they use
- Nitrate reductase – Molybdenum co-factor
- Nitrite reductase – C-type haem (Fe)
- Nitrous oxide reductase – Cu cofactor
- Fumarate reductase – FAD cofactor
why is menaquinone used instead of ubiquinone in e. coli anaerobic resp chains?
has lower redox potential - easier to find a suitable e- acceptor
name the 3 principles of fermentation
- The conversion of glucose to pyruvate by, for example glycolysis conserves energy by substrate level phosphorylation but is an incomplete catabolic pathway
- NADH generated during the process needs to be re-oxidised (redox balance)
- In the absence of an electron acceptor the microbe must re-oxidise NADH by the formation of fermentation products that are more reduced that the starting substrate
what is the impact on [ATP] [Biomass] in fermentation?
a large concentration of fermentation products means that there are low atp and biomass yields, thereby increasing flux to fermentation products
name 6 types of fermentation and what each pathway produces
alcoholic - ethanol homolactic - lactate heterolactic - lactate and ethanol mixed acid - formate acetate, ethanol acetone butanol - acetate, acetone, isopropanol, butyrate, butanol, ethanol malolactic - lactate from malate
describe alcoholic fermentation, state how ATP and NAD+ are generated and give key enzymes
• Occurs commonly in yeasts but also a few bacteria
• Glycolysis produces (2)pyruvate and ATP by SLP (substrate level P)
• (2)Pyruvate decarboxylated to acetylaldehyde by pyruvate decarboxylase (+2CO2)
• (2) Acetaldehyde reduced to ethanol by alcohol dehydrogenase enzyme, using (2)NADH as an e- donor, thus regenerating (2)NAD+
• Ethanol is sole fermentation end product
• At 12-15% ethanol yeast is killed
OVERALL: 2ATP/mol glucose
describe the difference between simple diffusion and carrier-mediated facilitated diffusion
• Simple diffusion
o Non-saturable – rate increases linearly with [solute]
o Not inhibited by energy coupling inhibitors
• Carrier-mediated
o Saturation kinetics – due to binding site for the solute within the carrier
o Active transport – requires cellular energy to accumulate the solute against its concentration gradient
o Inhibited by energy coupling inhibitors
define accumulation ratio
AR=[solute inside cell]/[solute outside cell]
diffusion AR = 1
what is the difference between primary active transport and secondary active transport?
primary:
- depends on direct hydrolysis of chemical bonds to release free energy for transport
secondary:
- using a pre-existing ion concentration gradient to transport substances across a membrane (commonly pmf)
name 4 primary active transporters
PEP driven (phosphotransferase systems)
ATP driven (ABC systems)
ATP driven solute-ATPases
decarboxylation driven
name 4 secondary active transporters
uniports
symports
antiports
TRAP transporters (bac only)
give a named example of a symport and its substrates
LacY permease
- lactose and H+
give 2 examples of an antiport and their substrates
DcuA (coupled with Frd) - succinate and fumarate
OxlA - oxalate(2-) and formate(1-)
name a TRAP transporter and its substrate
dctPQM
transports C4 dicarboxylates (malate, fumarate, succinate)
uses H+ (pmf)
give an example of a ABC transporter and its substrate
BtuCDF
transports b12
name the substrate of a decarboxylation driven transporter
oxaloacetate –> pyruvate + CO2
uses 2Na+ ions
name 2 p-type solute translocating ATPases and name their substrates
kdp K+ uptake system
K+ using ATP hydrolysis
E. coli Cu exporter
Cu+ using ATP hydrolysis
state the starting and end compound(s) of homolactic fermentation, key enzymes and where NADH/NAD+/ATP are evolved. which bacteria use this fermentation?
Glucose –> 2 pyruvate –> 2 lactate
pyruvate –> lactate by lactate dehydrogenase (regen of 2NAD+)
aldolase
2NADH used GAP–>1,3BP
lactic acid bacteria gram +ve aerotolerant
OVERALL: 2 ATP made
state the starting and end compound(s) of heterolactic fermentation, key enzymes and where NADH/NAD+/ATP are evolved. which bacteria use this fermentation?
glucose –> 2pyruvate –> lactate + ethanol
pyruvate—>lactate = LDH enz
phosphoketolase does SLP
lactate formation arm = 2ATP regen, NADH use and NAD+ regen
ethanol formation arm = 2NAD+ regen
2NADH used in main pathway (before branchpoint)
lactic acid bacteria
OVERALL: 1ATP/mol glucose
state the starting and end compound(s) of mixed acid fermentation, key enzymes and where NADH/NAD+/ATP are evolved. which bacteria use this fermentation?
glucose ---> 2 pyruvate ---> 2 formate + ethanol + acetate enzymes: phosphotransacetylase (PTA) Acetate kinase - ACK Formate hydrogen lyase (FHL) Pyruvate formate lyase (PFL) ADH 2NADH used 2NAD+ regen 3ATP formed 2SLP (by Pta and Ack) E. coli OVERALL: 3ATP/1 mol glucose
state the starting and end compound(s) of ABE fermentation, the key enzymes and where NADH/NAD+/ATP are evolved. which bacteria use this fermentation?
glucose ---> 2pyruvate ---> 1ethanol + 3acetone + 6butanol (biofuel) (+3 others) anaerobic clostridia bacteria 5 NAD+ regen 2 NADP+ regen 4 ATP made ADH and BDH used
state the starting and end compound(s) of malolactic fermentation, key enzymes and where NADH/NAD+/ATP are evolved. which bacteria use this fermentation?
malate ---> lactate lactic acid bacteria MleA catalyses no redox balancing/ATP gen uses antiport malate2-/lactate1-
name the 2 systems for protein translocation across the inner membrane, state simply how they work
SEC - protein threaded through then folds in periplasm
TAT - protein folded in cytoplasm, passes through TAT folded
describe the SEC mechanism of protein translocation across IM
- Chaperone protein SecB binds nascent protein – prevents folding
- Signal sequence directs protein to SecYE – forms pore that protein is threaded through
- SecA = ATPase, drives transport process (with help from Δψ)
- After export, N-terminal signal sequence is removed by periplasmic signal peptidase
- Requires accessory protein SecG
name and explain the 3 regions of the sec signal sequence
o N-terminus: Positively charged (K/R) - Stop signal
o Hydrophobic α-helical region (18-21 a/a) - This inserts into membrane (is cleaved off)
o C terminus: signal peptidase cleavage site (AXA)
state how proteins with cofactors are transported across IM
• Many electron transport proteins are located in the cytoplasm
• A simple threading mechanism would mean the cofactor was lost
• Cytochromes c:
o Cytochrome c protein enters periplasm via Sec system
o Haem (cofactor) exported separately
o Ligase enzyme binds haem to cytochrome c (at CXXCH motif) after translocation
• All other redox proteins with cofactors are exported via a totally different system, as fully folded proteins
describe when the TAT (twin arginine translocase) system is used and in which kingdoms it is found
• Found in bacteria, archaea, and plant chloroplasts
• Often used for proteins that contain complex cofactors
o Cofactor inserted in protein as it folds in cytoplasm
• Proteins that are exported via TAT have 2 Arg residues next to each other in the signal sequence
describe the TAT mechanism
• Signal peptide of TAT substrate recognised and bound by TatBC complex
• TatA polymerises to create a pore (variable diameter to suit that of translocated protein)
o Protein goes through TatA
o Driven by pmf (no ATP)
o Pore size modified by addition/loss of subunits
• After translocation signal sequence cleaved
• TatA and TatBC complexes dissociate
• Tat system mainly used for e- transport proteins, allows proteins to function in periplasm
name and explain the 3 regions of the TAT signal sequence
n-terminal region: contains RR motif Hydrophobic region doesnt really do much, may help tatA and tatBC bind c-region AXA cleavage region
why do bacteria need to secrete proteins beyond the periplasm?
extracellular enzymes for digestion
virulence factors
which secretion systems transport across the OM only and which transport across the OM and into another cell?
• Secretion across the OM only TSS5 TSS2 TSS1 • Secretion across the OM and into another cell TSS3 TSS4 TSS6
describe the TSS1 system
• Used to secrete HlyA (haemolysin) only
o Bacteria use HlyA to release haem for Fe collection
• 3 components:
o ABC-transporter (HlyB) which is connected to (in IM),
o HlyD – multidomain protein which interacts with (in IM),
o TolC – an outer membrane porin-like protein (in OM)
• C-term of HlyA is recognised by HlyB
o HlyA is transported as un unfolded protein (driven by ATP hydrolysis)
• Entire protein is translocated (no signal sequence)
describe the TSS2 system
• General transporter
• Secretory protein loaded onto platform in periplasm
o Platform pushed out of cell by polymerisation
o Imagine syringe plunging – similar to this
o Requires ATP
• Protein delivered to periplasm by TAT/SEC
describe the TSS5 system
• Autotransporter – transports itself
• 1 protein
C-terminal part of the secreted protein forms a β-barrel (porin like) in OM
Rest of protein transported through itself (passenger domain)
• Passenger domain is cleaved off and translocator domain (β-barrel) is left then degraded
• Initial transfer to the periplasm is via Sec system
• Used for some bacterial virulence factors
• Structure of protein domains:
N-terminus: signal sequence
Middle: Passenger domain (exported protein)
C terminus: translocator domain (β-barrel)
describe the TSS3 system
• Acts like needle
Tip injects proteins into eukaryotic cells
• Goes through IM, OM and into another cell
• Structure resembles flagella
Evolutionary link
Found that some flagella export proteins
• ATP driven
• Effector proteins (proteins that disrupt the host cell functioning in some way) and toxins secreted
describe the TSS4 system
• ATP hydrolysis in cytoplasmic domain
• Transports protein/DNA
Involved in conjugation
Found in some pathogens for protein secretion into host cells
- Eg Agrobacterium tumefaciens (vir plasmid) and Helicobacter pylori (cag protein – disrupts cytoskeleton)
• Genes for this protein encoded on ‘pathogenicity island’
• Related to pilus proteins
describe the TSS6 system
• Recently discovered in various gram -ve cells
• Syringe-like system related to phage injection apparatus
Maybe phage derived system
• ATP driven
• One example ‘’scratches’’ OM of another bacteria and delivers enzymes that dissolve peptidoglycan
Doesn’t have to target OM of another bacteria
which transport systems transport proteins folded
TAT
TSS3
TSS4
TSS6
which transport systems transport proteins unfolded
SEC
TSS1
TSS2 (can do folded via TAT)
TSS5 (can do folded via TAT)
which protein transport systems use ATP as an energy source?
SEC (uses pmf too) TSS1 TSS2 TSS3 TSS4 TSS5 TSS6
which protein transport systems use pmf as an energy source?
SEC (uses ATP too)
TAT
give an example of a (specific) molecule that is transported via each protein translocation system
sec - periplasmic tat - cofactor proteins TSS1 - haemolysin TSS2 - extracellular enzymes TSS5 - IgA protease TSS3 - Toxins/effectors TSS4 - effectors/DNA TSS6 - effectors/lytic enzymes
