Microm Midterm 2

Catabolism, anabolism, oxidation, reduction, donor, acceptor definitions

• Catabolism: releases energy

• Anabolism: consumes energy to make amino acids, nucleotides, etc

• Oxidation: loss of electrons

• Reduction: gain of electrons

• Donor: is oxidized

• Acceptor: is reduced

Function of NAD⁺/NADH/FAD/FADH₂, ATP, and PMF

• NAD⁺/NADH/FAD/FADH₂: transfer electrons

• ATP: carries usable chemical energy

• PMF: stores ECG energy

Glycolysis pathway

Glucose → 2 pyruvate; consumes 2 ATP, produces 4 ATP and 2 NADH (net 2 ATP)

Pentose phosphate pathway (PPP)

A central pathway supplying e- for reducing and makes building blocks for nucleotides

TCA cycle

Oxidizes acetyl-CoA and generates reduced electron carriers and intermediate metabolites

2 central metabolites pathway functions

• Help w/ energy and ATP

• Provide the building blocks for biosynthesis,

  • including in phototrophs and lithotrophs

Function of F₁F₀ ATP synthase and PMF

• F₁F₀ uses proton flow to drive ATP synthesis

• PMF supports flagellar rotation, nutrient uptake, and efflux

Fermentation, Aerobic respiration, Anaerobic respiration:

- ETC needed

- Terminal electron acceptor

- How ATP is made

- Redox balance regeneration

- Energy generated

Fermentation: no, internal organic metabolite, substrate level phosphorylation, regen NAD+, 2 ATP/glucose

Aerobic: yes, O2, oxidative phosphorylation, ETC, 38 ATP/glucose

Anaerobic: yes, inorganic non O2 acceptor, oxidative phosphorylation, ETC, 3-30 ATP/glucose

Chemoorganotroph, chemolithotroph, phototroph

• Chemoorganotroph: organic chemical electron donors

• Chemolithotroph: inorganic electron donors (H2, H2s)

• Phototroph: light energy

What is BOD and what can high BOD lead to

• Biological oxygen demand is the amount of oxygen needed for microbial decomposition of organic matter

• High BOD can lead to oxygen depleted aquatic dead zones

What can facultative anaerobes do if oxygen is becoming depleted and how can pili be used

• Facultative anaerobes can shift respiratory pathways as oxygen becomes depleted

• Some microbes use pili as conductive wires to transfer electrons

4 environmental controls on microbial growth

• Temperature: freezing/refrigeration can prevent/slow growth; heating kills cells but may not spores/toxins

• Oxygen: removing O2 stops many aerobes, but some can live without

• Salt/water activity: salt lowers available water and causes osmotic stress

• pH: acidification limits many pathogens

Process and purpose of food fermentation

• Regens NAD+ by transferring e- to organic metabolites w/o ETC → ATP made by substrate phosphorylation

• Preserves food by limiting microbial growth

• Improve vitamin content and digestibility

• Flavor

Fermentation lactic acid bacteria: gram type, O2 type, fermentation type, tolerances

• Lactobacillus, lactococcus, leuconostoc

• G+

• Aerotolerant anaerobes

• Obligate fermenters

• Salt and acid tolerant

Difference between homolactic and heterolactic fermentation

• Homolactic: makes primarily lactic acid

• Heterolactic: makes lactic acid, ethanol, CO2

Staph aureus: illness mechanism, gram type, tolerance, food prevalence, cooking effect

• Intoxication, preformed enterotoxin

• G+

• Salt/desiccation tolerant

• Improperly prepared/handled foods like meats/dairy

• Cooking kills cells but enterotoxin can remain active

Listeria monocytogenes: illness mechanism, gram type, tolerance, food prevalence, cooking effect

• Infection, ingest live bacteria

• G+

• Cold tolerant

• Refrigerated foods

• Cooking kills vegetative cells

Clostridium botulinum: illness mechanism, gram type, tolerance, food prevalence, cooking effect

• Intoxication, preformed neurotoxin

• G+

• Heat resistant endospores

• Canned foods

• Cooking may not kill spores

3 things phage requires host cell machinery for

• Energy & nutrients

• Nucleotides and amino acids

• Protein synthesis

Lytic phage replication cycle

• Phage attaches to host via receptor(s) that recognize surface molecules

  • Capsule can impact adsorption so some phages have capsule degrading enzymes

• Genome delivery via tail sheath penetration to release genome; high pressure moves DNA to cytosol

• Replication and assembly

  • Early genes: DNAse degrades host DNA, DNA replication machinery

  • Late genes: capsid, DNA packaging, lysins & holins (break cell wall and escape)

Difference with temperate phages

• Temperate phages integrate into the host genome → is vertically transmitted to bacterial progeny (prophage) → environmental signals cause lysogen/lysis conversion

Transduction definition, types, mechanism

• Transduction: bacteriophages carrying host DNA from one cell to another

  • Generalized: transfer any gene; lytic and lysogenic

    • host DNA not fully degraded → packaged into capsid → transducing particle

  • Specialized: transfer a few closely linked to phage insertion site; lysogenic

    • prophage excision error → host DNA packaged in phage → transducing particle

Corynebacterium lysogenized effect and potential usage in food

• Lysogenized creates diphtheria toxin

• Spraying food w/ listeria bacteriophage decreases contamination for listeria

Definition of silent, missense, nonsense, and frameshift mutation

• Silent: no genomic impact

• Missense: change amino acid code

• Nonsense: premature stop codon

• Frameshift: insert/delete nucleotides which changes reading frame

Sources of spontaneous mutation

• Abnormal base pairing; tautomerization

• Water; deamination (de amine, remove NH2)

• Oxygen; toxic intermediates

Mobile genetic elements gene and mechanism

• Jumping genes/transposable elements have transposase gene

• Transposase inserts transposon seq which can disrupt gene/promoter

3 induced mutations

• UV light, reactive O2 species, ionizing radiation

DNA repairing methods for modified bases, replication, UV damage

• Base excision repair, mismatch repair, nucleotide excision repair

Relevance of Deinococcus radiodurans

• Can survive 1000x radioactivity vs humans; very radioresistant

Homologous recombination process and double cross over

• Double strand break → nuclease digests 5’ → RecA binds 3’ DNA overhangs → strand invasion via base pairing → repair polymerases synthesize DNA → invading strand released

• Double cross over: 2 regions switch locations

3 types of horizontal gene transfer

• Transformation: uptake of naked DNA

• Transduction: phage mediated transfer

• Conjugation: vertical transmission of DNA by F-factor

What did Griffith experiment show

• Transforming principle: dead encapsulated bacteria transformed unencapsulated bacteria into live encapsulated

Plasmid definition, origins of replication, transfer mechanism between donor and recipient

• Extrachromosomal DNA that can have genes for toxins, resistance etc

• oriV: vertical inheritance of plasmid

• oriT: transmission between bacteria

• Type IV secretion system pilus that transfers DNA from F+ to F-

Agrobacterium tumefaciens plasmid mechanism and effect

• Ti plasmid encodes T pilus to transfer DNA through into plants

• Causes uncontrolled plant cell growth

Function, mechanism, location of activators and repressors

• Activator: turns genes on by binding inducer which allows binding to activator site → recruits RNA poly to promoter via α subunit

  • Upstream of promoters

• Repressor: turns genes off by binding operator site

  • Bind co-repressor and turn gene off

  • Inducer releases repressor from operator

  • Downstream/overlap promoter

LacY/Z/A/I and allolactose functions

• LacY: permease for transport

• LacZ: β-galactosidase to cleave lactose in allolactose and others

• LacA: transacetylase

• LacI: repressor that recognizes 2 operators, lacO/lacOi

  • Binds both operators and acts as repressor; autoregulates itself

• Allolactose: inducer for LacI → LacI releases operator and stops repression (still needs activator for transcription)

CR(A)P, adenylate cyclase function and what type of growth is lactose

• CRP/CAP: transcriptional activator for lac operon that requires cAMP

• Adenylate cyclase: enzyme that produces cAMP

• Grows on glucose and lactose: diauxic growth relying on PTS of glucose transport

Function of EIIA and relationship with glucose

• Glucose present: EIIA unphosphorylated → binds and blocks LacY (inducer exclusion) and can’t activate adenylate cyclase

• Glucose absent: EIIA phosphorylated → doesn’t bind LacY and activates adenylate cyclase

Catabolic and anabolic regulation

• Catabolic: substrate present → yes, induction | other preferred source → yes, catabolite repression

• Anabolic: end product present → yes, feedback inhibition

How is the trp operon regulated

• Trp operon under negative regulation using trp as co-repressor

Trp operon length, # regions, unique aspects of region 1/1-2/4

• trpL leader sequence has 14 amino acid peptide

• 4 regions in RNA

• 2 trp codons in region 1, stop codon between 1-2, many Us after 4

3 stem and loop structures of trp operon

• 1-2: pause loop + 3-4: terminator

• 2-3: anti terminator

Transcription and translation process for trp operon (transcriptional attenuation)

• RNA poly transcribes regions 1-2 and stalls at 1-2 pause loop

• Ribosome starts translation and RNA poly resumes

• W/ trp:

  • ribosome translates 2 trp codons and stalls at 1-2 stop codon → 3-4 form terminator loop → no structural trp genes reached

• W/o trp:

  • ribosome stalls at 2 trp codons → 2-3 form anti terminator loop → terminator loop can’t be formed → transcription of operon continues

What are the 3 types of regulatory RNAs and their function

• Riboswitches: in 5’ UTR of mRNA

  • Regulate transcription: binding ligand causes mRNA structure change → prevents/promotes anti terminator/terminator loops

  • Regulate translation: binding ligand causes mRNA structure change → block/allow ribosome binding site

  • Regulate uncharged aminoacyl tRNA synthetases via transcriptional attenuation

  • Alter mRNA structure in response to temperature: thermosensor

• Cis encoded RNA: separate transcript from own promoter

  • Use sense strand to make anti sense RNA to block translation via dsRNA

  • Only regulates gene from which its transcribed

• Trans encoded RNA: separate transcript from own promoter

  • Can regulate multiple genes w/ Hfq

Overall function of sRNAs and examples

• Separate RNA molecules that regulate translation, regulate mRNA degradation

• Cis/trans encoded RNA

Iron regulation under high and low conditions

High:

• Fur uses Fe as co-repressor to repress ent and sRNA ryhB

• Free iron scavenged by siderophore enterochelin

• Genes for iron-requiring proteins are actively expressed

Low:

• Fur releases operator sites → express enterochelin and sRNA ryhB

• ryhB binds iron-requiring protein and targets them for degradation

Elements of environmental sensing functions: stimulus, sensor, transducer, response regulator, feedback mechanism

• Stimulus: environmental signal

• Sensor: receptor to detect stimulus

• Transducer: converts environmental signal into internal signal

• Response regulator: responds to internal signal by altering gene expression

• Feedback mechanism: shuts system off as further events warrant

Stimulus, sensor, transducer, response regulator, feedback for Chemotaxis

• Stimulus: chemoattractant

• Receptor: MCP

• Transducer: CheA/W

• Response regulator: CheY

• Feedback mechanism: CheZ

2 parts of two-component regulatory system and base mechanism

• Sensor transducer protein called histidine kinase (HK) that’s usually transmembrane protein

• Cytoplasmic response regulator regulator (RR) that’s usually DNA binding protein

• Mechanism: HK auto phosphorylates a histidine and transfers phosphate to RR

Chemotaxis two-component regulatory system pieces and effect of separate sensor

• HK: CheA

• RR: CheY

• Separate sensor allows response to variety of different chemical signals

OmpF and OmpC structure difference, osmolarity preference

• OmpF has larger bore/channel than OmpC

• OmpF is free living, prefers low osmolarity (solute in cell > out)

• OmpC: enteric, prefers high osmolarity (solute out cell > in)

EnvZ and OmpR functions

• EnvZ is sensor kinase that responds to turgor pressure

• OmpR is RR that regulates OmpF/C transcription via 3 binding sites

EnvZ and OmpR system in high and low turgor pressure

High:

• Low EnvZ autophosphorylation → low OmpR

• OmpR binds high affinity OmpF site, no others

  • OmpF activated

Low:

• High EnvZ autophosphorylation → high OmpR

• OmpR binds high and low affinity OmpF site (rerpessed) and low affinity OmpC site (activated)

• Activates sRNA micF (represses OmpF w/ HFQ)

Biofilms definition and characteristics

• Surface associated microbial community embedded in extracellular matrix

• Highly ordered, resistant to antibiotics, different genes vs planktonic growth

Cyclic di-GMP function, production/degradation, exponential vs stationary phase, bacterial effect

• Cyclic di-GMP is second messenger associated with biofilms

• Production: DGCs Degradation: PDEs

• Exponential phase: more PDEs Stationary phase: more DGCs

• Represses flagellar genes and promotes pilus

LuxR, LuxI, luciferase functions

• LuxR: sensor transducer and RR

• LuxI: signal generator

• Luciferase: catalyzes light creation

Lux operon mechanism and goal

• Autoinducer made by LuxI → more cells = more autoinducer → autoinducer binds LuxR protein → autoinducer-LuxR complex binds operon as activator

• Do not express until cell density is high enough to make light visible since light requires lots of ATP and reducing

Bacterial functions regulated by quorum sensing/population density

• Genetic exchange, colonial behavior, extracellular functions, multicellular organization

How do AHL metabolites differ across G- bacteria and purpose of having multiple signals

• Modifications of 3rd carbon, presence of unsaturated bonds

• Multiple signals allows different regulation for different target genes