MIC 105 Module 2

Mobile Genetic Elements (MGEs)

A type of DNA or RNA that can move around in the genome, or be transferred to a recipient organism

Mobile Genetic Element Examples

plasmids, viruses, transposable elements

Mechanism of MGE transfer

1. Conjugation

2. Transformation

3. Vesicles

4. Nanotubes

5. Capsids

Plasmid + types

term for any extrachromosomal heredity determinant

• can have broad or limited host ranges; range of low to high copy numbers

1. episome = non-essential genetic element replicating autonomously or integrated into the chromosome

2. conjugative = can transfer horizontally, carry transfer genes

3. mobilizable = can transfer horizontally, carry mobilization genes

Benefits of plasmids

may confer genes w/ beneficial traits to hosts

eg. bacterial produced toxins, antibiotic resistance, virulence, degradative enzymes

Plasmid replication

-rolling circle replication
-depends on a protein that guides DNA and assists replication in recipient
-ori determines the number of copies

Cloning Vector Features

1. origin of replication

2. selectable marker

3. multiple cloning site

4. insert

Phage Characteristics

structure: capsid, tail, proteases
genome replication: helicase, primase, dna pol
packaging: terminase, protal
regulation: repressor, anti-repressor
integration, excision: integrase, excisionase
tRNAs
*lots of "accessory" genes (bidirectional genome)

Phage lifecycle(s)

lytic cycle: productive phage infection --\> lysis of host
horizontal transmission: transmission of phage genome b/w contemporary hosts
lysogenic cycle: replication of the phage genome occurs with the replication (includes integration)
integration: physical insertion of the phage genome into the host
prophage \= integrated phage

Integration

• uses short pieces of homology recognized by integrase to cut and ligate

• maintained over generations

Phage-Host Arms Race

hosts: under constant and strong selection to inhibit phage infection

• phage adsorption + surface resistance

• restriction modification systems

• CRISPR/Cas9 phage: under constant and strong selection to infect hosts

Giant Viruses

• complex, mobile, monophyletic, broad host spectrum

• can reprogram host

• include virophages

Transposons

flanked by direct repeats and include terminal inverted repeats

Class I Transposons: Retrotransposons

- transpose by a copy-paste mechanism by copying as RNA transcripts and then using a reverse transcriptase --\> integrate (considered to be replicative)

Class II Transposons: DNA Transposons

• non-replicative cut and paste by excising themselves and integrating somewhere else

• have long terminal repeats on both ends

Conjugative Transposons

are able to transfer from one cell to another by conjugation (enable both mobilizable plasmids and nonconjugative transposons to be transferred)

Autotrophy vs Heterotrophy

auto: makes complex carbs from CO2 (need external source of energy)
hetero: makes energy from complex/organic carbon molecules --\> cellular carbon

Photoautotroph CO2 Fixation Pathways

1. Calvin Cycle

2. Reductive TCA Cycle

3. 3HP Bi-cycle (L8 Slide 7)

Calvin Cycle

a biochemical pathway of photosynthesis in which carbon dioxide is converted into glucose using ATP
("dark reactions" in cyanobacteria + plants)
essential enzymes: rubisco and phosphoribulokinase

Calvin Cycle Steps

1. CO2 fixation by rubisco

2. reduction to the oxidation state of carbohydrate + cell material

3. regeneration of CO2 acceptor by phosphoribulokinase

Bacterial Autotrophs

1. purple nonsulfur bacteria*

2. ammonia oxidizing bacteria*

3. purple sulfur bacteria*

4. sulfate reducing bacteria

5. green sulfur bacteria^

6. green nonsulfur bacteria"

7. some clostridia

Reverse/Reductive TCA Cycle

uses 4-5 ATPs to fix four molecules of CO2 and generate one oxaloacetate

• requires less energy

• same enzymes as in forward except there are three key enzymes that will send reaction into reverse (fumarate reductase, a-ketoglutarate synthase, citrate lyase)

• reverse glycolysis supports biomass building

• only pathway used by both bacteria and archaea

3-hydroxypropionate bi-cycle

A carbon fixation process that condenses hydrated CO2 and acetyl-CoA to form 3-hydroxy propionate (5ATP)
Key enzymes: acetyl-CoA + proprionyl-CoA carboxylases
(anaerobic as one step is O2 sensitive)

The 3-Hydroxypropionate/4-Hydroxybutyrate Pathway

(thermoacidophilic crenarchaeota)
same intermediates different enzymes

Dicarboxylate/4-hydroxybutyrate cycle

an autotrophic pathway found in thermophilic crenarchaeota
-patchwork pathway from rTCA and 3HP/4HB cycle

Reductive Acetyl-CoA Pathway

• Used by anaerobic soil bacteria, autotrophic sulfate reducers, and methanogens

• Two CO2 molecules are condensed through converging pathways to form the acetyl group of acetyl-CoA.

• Carbon monoxide is an intermediate.

• Reducing agent is H2 instead of NADPH.

Why so many CO2 fixation pathways

• energy requirements

• oxygen sensitivity of enzymes

• temp

• requirements of metals

• products formed

Phototrophy

involves light capture by chlorophyll, usually coupled to splitting of H2S or H2O or organic molecules

chlorophyll-based (auto + heterotrophs) vs retinal-based (heterotrophs)

Chlorophyll-based phototrophy

1. photopigments = absorb different wavelengths of light

2. reaction centers = PS I (Fe-S protein) or II (mobile quinone)

3. membranes = house pigments, rc, + photosystems --> connect to ETC

Light-Harvesting Pigments

1. Chlorophylls = tetrapyrrole + Mg2+ (cyanobacteria)

2. Bacteriochlorophylls = side groups differ (purple,green phototrophs)

Accessory Light Harvesting Pigments

carotenoids \= long chain hydrocarbons w/ extensive conjugated double bonds
- determine wavelengths absorbed

Cyanobacteria

monophyletic w/ similar physiologies
metabolism: oxygenic photoautotrophs (some organoheterotrophs in the dark)
habitats: anywhere there's light
carboxysome: contains rubisco --\> sequesters CO2 for efficiency and to protect rubisco from O2

spp. prochlorococcus \= most abundant photosynthetic organism; fix 50 billion tons of CO2; large pangenome

Cyanobacteria pigments

chlorophyll a, carotenoids, phycobilins
phycobilin \= phycocyanin (dark blue-green)

Electron Transport (oxygenic phototrophy)

PS I and PSII involved
(L9 slide 50)

Purple Phototrophs

proteobacteria
metabolism: anoxygenic photoauto/heterotrophs

Electron Flow (anoxygenic phototrophy)

PSII; reverse electron flow needed to generate NADH
(L9 slide 61)

Purple Nonsulfur Bacteria vs Purple Sulfur Bacteria

PNS: only phototrophic under anaerobic conditions

PS: mostly obligate photoautotrophs using H2S --\> S ; also use S as e- source; variety of metabolisms
habitats: anaerobic environments w H2S and light

both can use a variety of electron donors

Green Sulfur Bacteria (Chlorobi)

physiology: anoxygenic photoautotrophs (some photoheterotrophs)
- use H2S or H2
Habitat: freshwater + marine sedimens
Enzyme: PSI (efficient light gatherers)
CO2 Fixation: reductive TCA cycle

Green Nonsulfur Bacteria (Chloroflexi)

physio: photoheterotrophs (sugars, organic acids or aa); photoautotrophs w/ H2 or H2S; can grow as aerobic organoheterotrophs
habitat: freshwater and marine environments; alkaline hot springs
Enzyme: PSII
CO2 Fixation: 3 HP Bi-Cycle

Heliobacteria

anoygenic photoheterotrophic endosporeformers (can fix nitrogen); chemoheterotrophs in dark
Enzyme: PSI w/ unique bCHLg
Substrates: organic acids
Habitats: anaerobic alkaline soils, rice paddies

Aerobic anoxygenic phototrophs

marine a-proteobacteria
pigments: bchl a, carotenoids
enzyme: PSII w/ cyclic photophosphorylation
habitats: variety but mostly in marine environments
- use light as a supplement until conditions improve

*candidatus chloracidobacterium containing PSI and chlorosomes; very small genome

Retinal-based phototrophy

Light driven proton pump

Bacteriorhodospin - light sensitive pigment retinal

Halorhodopsin - pumps Cl- in

Use lights, and protons pumped out to create ATP

Haloarchaea

most obligate aerobic organoheterotrophs (proteins + aa)

• require high NaCl concentrations for membrane stability

• use light to suppement energy when nutrients/O2 are low -bacteriorhodopsin synthesized under low O2 conditions --> absorbs green and reflects blue and red; makes cells buoyant so they can get to the surface for maximal light exposure

Proteorhodopsin

A bacterial membrane-embedded protein that contains retinal and acts as a light-driven proton pump; it is homologous to the archaeal protein bacteriorhodopsin

• widespread in many marine bacteria and archaea

• apparently horizontal gene transfer from haloarchaea

Acetogens

-Anaerobic bacteria, most are firmicutes
-Use carbon dioxide as electron acceptor. reduces to acetate
-Multiple donors are possible but H2 is common for reducing CO2
-Pathway is called Reductive Acetyl-CoA pathway
habitats: anoxic (soils, termite hindgut, cow rumen)

Reductive Acetyl-CoA Pathway (Acetogenesis)

C1 Carrier \= Tetrahydrofolate (THF)
Key enzymes: CO dehydrogenase/acetyl-CoA synthase; electron bifurcating hydrogenase

Methanogenesis

• Methane from CO2, acetate, or methanol

• Produces Coenzyme M, Coenzyme F420, Coenzyme F430, MP, MF, CoM, CoB (C1 careers)

• Creates a sodium motive pump for ATP production

• **H is e- donor and CO2 is e- acceptor + carbon source

• Strictly anaerobic process

• Obligate anaerobes

• thermophiles, mesophiles, etc Habitats: anoxic enviro, wetlands, animal digestive tracts, landfills, wastewater, hydrothermal vents

Methanogenesis Pathway

• like reductive acetyl-coa pathway

• energy conserved by converting CO2 --> CH4 coupled to H+ or Na+ transport

Reductive Acetyl-CoA Pathway (Methanogens)

formation of acetyl-coa from two CO2 molecules that are independently reduced by two different mechanism
key enzymes: acetyl-coa synthase (bifunctional)

Methylotrophic Pathway

(L10 slide 23)
4 CH3OH --\> 3 CH4 + CO2 + 2H2O

Acetoclastic Pathway

Acetate + H + --\> CO2 + CH4

Methanogens + Syntrophy

methanogens + secondary fermenters

• common fermentation end products: CO2, H2, acetate, ethanol

• allows for interspecies hydrogen transfer as it 'pulls' the fermentation reactions (L10 slide 28)

Filamentous Methanogens (wastewater treatment)

• bacterial waste products from aerobic respiration + fermentation --> methane

• bacteria are packed together by filamentous methanogens (settle out of liquid)

Methane Hydrates

Small bubbles or individual molecules of methane (natural gas) produced by psychrophilic methanogens trapped in a crystalline matrix of frozen water

Methylotrophy

The metabolic oxidation of single-carbon compounds such as methanol, methylamine, or methane to yield energy
(all C-C bonds synthesized de novo)
- facultative and obligate

Methanotrophy

located at oxic/anoxic interface in aquatic sediments and soil environments
obligate anaerobes

Methane monooxygenase (MMO)

- introduces oxygen into methane to produce methanol

two forms: solume and membrane bound (most common)

CH4 + O2 + NAD(P)H + H --\> CH3OH + NAD(P) + H2O

Type I Methanotrophs

• gamma proteobacteria

• obligate methylotrophs, no complete TCA cycle

• assimilate C1 via ribulose monophosphate pathway (key enzyme = hexulose-P synthase)

Type II Methanotrophs

• alpha proteobacteria

• assimilate C1 via serine pathway (THF) (key enzyme = serine transhydroxymethylase)

• complete TCA cycle

• many are facultative and can utilize simple organic compound

Nonproteobacterial methanotroph (verrumicrobia)

• live at oxic/anoxic interface of geothermally released methane

• hyperthermophilic, acidophilic

• obligate methylotroph

• use serine pathway

Methane to formaldehyde

• occurs in methano or methylotrophs

• CH3OH oxidized by methanol dehydrogenase

Anaerobic methane oxidation

• members of euryarchaeota in syntrophic association w/ sulfate reducing bacteria

• reverse methanogenesis with oxidization of methane in anoxic ocean sediments

• not energetically favorable --> syntrophy required

• ANME oxidizes CH4 as electron donor for SRB

• slow growth but important for capturing greenhouse gas

• MMO for conversion + reduces nitrite to produce O2 (candidatus methylomirabilis oxyfera) habitat: anoxic freshwater and marine sediments, soils, peat bogs, wastewater treatment plants

Respiration

oxidation of organic compounds coupled to transfer of electrons to an external electron acceptor
(38 ATP/glucose)
reoxidizes NADH, coupled to energy conservation
(L11 slide 5)

Fermentation

internally balanced oxidation-reduction reactions
excretion of products (some C wasted)
energy conserved via substrate level phosphorylation
*requires reversed function of ATPase to have an energized membrane/generate PMF
(2 ATP/ glucose)
(L11 slide 8 + 9 + 13)

Anaerobic Respiration

• variety of alternative anaerobic respiration

• carried out by specific microorganisms

• higher energy yielding respirations are used preferentially through gene regulation --> depends on O2 presence, presence of alternative electron acceptor *e coli (O2 > nitrate > fumarate > fermentation)

Nitrate Reduction

The process by which nitrate (NO3-) is reduced to ammonia (NH3) using membrane bound or periplasmic enzymes (also generates PMF)

Degradation of Polymers

degradation of polymers into utilizable compounds by exoenzymes that are secreted or attached to the surface of bacteria (production carefully regulated)

Degradation of plant biomass

degradation of cellulose by cellulases (relatively rare among bacteria) that are secreted or bound --\> starch, xylans, pectin which have their own exoenzymes
*cellulosomes in clostridium thermocellum

Degradation of proteins

degradation of proteins by proteases --\> deaminated to form organic acids for TCA cycle or fermentation

Degradation of lipids

cleavage of lipids by lipases (secreted) to release fatty acids --\> degrated by B-oxidation

Degradation of lignin

polymer of aromatic compounds

• extremely hydrophobic

• low aqueous solubility

• tend to partition into membranes

• very stable --> funnel into common intermediates *catechol is key intermediate

Aerobic Aromatic Degradation Pathways

addition of oxygen to the ring for destabilization and ring cleavage by mono or dioxygenases and ring cleavage dioxygenases --\> convert to compounds that enter central metabolis

Aromatic Ring Cleavage

Cleavage of Catechol

• intradiol (ortho) = between hydroxylated carbons

• extradiol (meta) = adjacent to one hydroxylated carbon eg P Putida benzoate degradation uses oxygen as a substrate

Xenobiotic Chemicals

unnatural or synthetic chemicals such as herbicides, pesticides, refrigerants, solvents, organic compounds

Bacterial Degradation of Xenobiotic Aromatic Compounds

• reactions are catalyzed by monooxygenases or dioxygenases that use oxygen as a substrate w/ catechol as a key intermediate

• enzymes for different aromatic compounds are often homologous to other oxygenases

• selection allows for utilization of new carbon, nitrogen

• compounds may be cooxidized but not provide anything

• compounds are funneled to common intermediates and pathways are modular

• pathways are STRAIN specific not species specific

BTEX

Benzene, Toluene, Ethylbenzene, Xylene
most soluble aromatic hydrocarbons in gasoline

Nitroaromatic compounds

used in tnt, dyes, pesticides ,polymers

• synthetic

• high stability

• toxic, mutagenic, carcinogenic

Anaerobic Aromatic Funneling Pathways

central intermediate = benzoyl-CoA

• occurs under denitrifying, sulfate-reducing, photoheterotrophic conditions

• involves ring reduction and B-oxidation like reactions (addition of CoA to destabilize)

• much slower than aerobic and very oxygen sensitive eg fumarate addition to toluene --> benzylsuccinate

Sulfate/Sulfur Reducing Bacteria

• inorganic sulfur compounds serve as electron acceptors in anaerobic respiration

• end product of respiratory reduction is H2S (excreted)

Sulfate Reducing Bacteria + Archaea

many are gamma-proteobacteria, thermophiles
(endosporefomers)
all can also respire with sulfur
archaeoglobus \= hyperthermophilic euryarchaeote, oxidize organic carbon sources w/ SO4 as terminal electron acceptor (anoxic conditions)
bacteria \= obligate anaerobes; H2 (lithoautotrophs) lactate or pyruvate, other fermentation products as electron donors; most use reductive acetyl-CoA pathway; compete for H2 with methanogens but conserve more energy and use a wider range of substrates

Sulfate-Reduction

habitat: anoxic aquatic and terrestrial enviro w/ sufficient sulfur sources

• can be enriched in anoxic medium containing lactate and ferrous iron

• less energetically favorable than more common respiration or fermentation

• ATP is invested to activate sulfate and drive reaction

• reduction requires 8 electrons; form of anaerobic respiration (1 ATP/sulfate) key enzymes = ATP sulfurylase, APS reductase, sulfite reductase, hydrogenase (L12 S17)

Sulfur/sulfide oxidizing bacteria

anaerobes: PS and GS use H2S/S as e donor

"colorless" sulfur oxidizing bacteria

aerobic sulfur oxidizing bacteria that oxidize reduced sulfur compounds H2S and S as energy sources

chemolithotrophic w reduced sulfur compounds while most are obligate chemolithoautotrophs using inorganic compounds + TCA cycle

habitat: soil, sediments, freshwater, and marine enviro (typically in narrow zones where sulfide + oxygen coexist); symbionts w/ riftia at hydrothermal vents

'Large' colorless sulfur oxidizing bacteria

gamma-proteobacteria
large cell size + internal sulfur deposits (sparkly inclusions)
habitat: both freshwater and marine (difficult to culture)
some are lithoautotrophs
eg thiomargita nambiensis: large vacuole containing nitrate

colorless sulfur bacteria examples

beggiatoa: filamentous gliding, typically in microoxic water enviros @ surface of sediments; chemotactic + repellent to O2 and H2S; can form thick mats

thioploca: large filamentous sulfur bacteria, filaments in polysaccharide sheaths; motile by gliding w/ positive chemotaxis to nitrate + negative taxis to oxygen; -response to high sulfide; oxidize H2S --\> S --\> SO4

sulfolobus: hyperthermophilic, acidophilic sulfur-oxidizing chrenarchaeote; aerobic lithoautotroph that oxidizes H2S or S to H2SO4 + fixes CO2 w/ 3HP/4HB cycle

Oxidation States of Nitrogen

NH3, NH4 = incorporated into cells

N2 = most abundant form

HNO3, NO3- = oxidized to

Nitrogen Cycle Reactions

Assimilatory Nitrate Reduction = NO3- → NH4+

Denitrification = NO3- → N2

Nitrogen Fixation = N2 → NH4+

\
Nitrification/Comammox = NH4+ → NO3-

Ammamox = NH4+ + NO2- → N2

\

N2 Fixation

widespread among bacteria and archaea; NN bond very stable

\
fixers: proteobacteria, firmicutes, cyanobacteria, archaae, chlorobi, actinobacteria, spirochetes

N2 + 8e- + 8H+ + \`16ATP → 2NH3 + H2 + 16 ADP + 16 Pi

Key enzyme = nitrogenase which reduces N2 → NH3 (very oxygen sensitive; irreversibily inactivated)

\*H2 always a byproduct

Nitrogenase

complex enzyme with \~20 genes required for its production (only 3 make up the components)

homologous in all orgs as it evolved once

Dealing with O2 while fixing N2

Control expression of nitrogenase genes: when NH4+ is limiting and O2 absent

\
microaerophiles + obligate aerobes: high respiration rate that is often faster or as fast as O2 diffusion to protect nitrogenase (azotobacter have polysaccharide capsule slows O2 diffusion + harbors aerobes)

Separation of O2 and N2

temporal separation of photosynthesis from N2 fixation based on cycles with N2 fixation in dark

\
spatial separation of photosynth + N2 fixation: filamentous cyanobacteria have development of heterocysts when N2 is limiting (5-10%); heterocysts limit diffusion of O2 w/ thick cell wall

Symbiotic Nitrogen Fixers

Rhizobia = gram- proteobacteria that are symbiotic with eukaryotes (aerobic heterotrophs)

\
form root nodules and are significant N2 fixers

* nitrogen is provided to the plant + rhizobium gets C-source and protected environment

different species form specific associations with certain plants

Root Nodule Formation

1. Recognition of correct partner and attachment of the bacterium to the root hair

   1. secrete flavonoids (chemoattractants) that induce Nod factor production (host-microbe specificity)

2. Invasion of the rhizobia into the plant root

   1. nod factors stimulate production of infection thread → rhizobia colonize root cells through infection thread

3. Differentiation of plant cells into nodule tissues and bacterial differentiation into bacteroids

   1. nod factors stimulate nodule formation (O2 binding protein leghemoglobin controls O2 levels)

   2. sym plasmid expressed for nif/fix gene

Diversity in symbiotic nitrogen fixation

Frankia = actinomycete alder symbiote

• can fix nitrogen at full oxygen tension + when in symbiosis

• sequesters nitrogenase in vesicles (protect from O2)

Nitrification

oxidation of ammonia to nitrate: carried out by ammonia oxidizers and nitrite oxidizers

\
aerobic chemolithoautotrophs that use calvin cycle

Ammonia oxidizing bacteria (AOB)

beta and gamma proteobacteria + nitrospira

NH3 + 1/2 O2 → NO2- + H2O (energetically favorable)

Key enzymes:

1. ammonia monooxygenase (converts ammonia to hydroxylamine; related to MMO)

2. hydroxylamine oxidoreductase (catalyzes hydroxylamine to nitrite + coupled to ETC)

Nitrite oxidizing bacteria (NOB)

alpha, beta, gamma, and delta proteobacteria + nitrospira

NO2- + H2O → NO3- + 2H+ + 2e- (energetically favorable)

\
Key enzyme: nitrite oxidoreductase (catalyzed nitrite to nitrate; coupled to ETC w/ PMF)

AOB + NOB

obligate aerobes in a tight mutualistic symbiosis

\
habitats: soil + water where ammonia is high, sewage treatment plants

Ammonia oxidizing archaea

chemolithoautotrophic ammonia oxidizing marine archaea that can grow on low NH3 concentrations and also solely on ammonia as energy source

\
fix CO2 using 3HP/4HB cycle

Comammox

nitrospira has genes for all the key enzymes and can completely oxidize ammonia to nitrate by itself as the reactions are energetically favorable