MICROBIOLOGY II

FS2019 Remo Bättig

1

Primary metabolism I (J. Vorholt)

Metabolic options for conserving energy Phototrophs use light as sole energy source, they are the starting point of the food chain.

Oxygenic and anoxygenic photosynthesis Photophosphorylation: light energy is used for electron transport → generates a proton motive force (pmf) which can be used to produce ATP. Under

standard conditions ∆G of ATP hydrolysis is ca. 30 kJ/mol. Under physiological conditions (in cell) it is ca. 50 kJ/mol, however since energy conversion is not 100% efficient it effectively takes 100 kJ/mol for the synthesis of 1 ATP. Two types of photosynthesis/phototrophy exist: anoxygenic (e-donor = H2S) and oxygenic photosynthesis (e-donor = H2O)

Phototrophs can be placed in 5 groups

• Green sulfur-bacteria (anoxygenic)

• Green nonsulfur-bacteria (anoxygenic)

• Heliobacteria (anoxygenic)

• Purple bacteria (anoxygenic) o Purple nonsulfur bacteria (alpha, beta) o Purple sulfur bacteria (gamma)

• Cyanobacteria (oxygenic) Key properties of phototrophic bacteria:

Oxygenic phototrophs Cyanobacteria

• Key genera: Synechococcus, Oscillatoria, Nostoc

• Contain heterocysts (not all): rounded enlarged cells with an anoxic environment inside → site for nitrogen fixation (nitrogenase is sensitive to oxygen)

• Gas vesicles are found in many cyanobacteria

• Most species are obligate phototrophs

• Widely distributed in terrestrial, freshwater, and marine habitats

Prochlorophytes Key genera: Prochlorococcus, the smallest (0.5-0.7 𝜇m) and most abundant photosynthetic microorganism on earth. Smallest genome of any free-living phototroph (1.65 Mbp, 1700 genes). Has Chla and Chlb (chlorophyll a&b). Most of the primary productivity in the open oceans is due to photosynthesis by Prochlorophytes.

Anoxygenic phototrophs • Possess two different types of photosynthetic reaction

centers: photosystem type I or II

• Depend on external electron donors. They may use anorganic compounds (H2, H2S, S or Fe2+) as well as organic compounds (e.g. fermentation products)

• Perform photosynthesis with the help of bacteriochlorophylls under anoxic conditions

Purple sulfur bacteria

• Key genera: Chromatium, Thiospirillum

• All gammaproteobacteria

• Obligate phototrophs

• Use hydrogen sulfide (H2S) as an electron donor for CO2 reduction in photosynthesis

• The hydrogen sulfide is oxidized to elemental sulfur that is stored as globules (S can be further oxidized to sulfate later)

Purple non-sulfur bacteria

• Key genera: Rhodobacter (alpha), Rhodocyclus (beta)

• Alpha- or betaproteobacteria

• Facultative phototrophs o Photolithoautotroph (H2 or H2S; only at low

concentration) in the light (anoxic) o Photoorganotroph (heterotroph) in the light

(anoxic) o Chemoorganotroph (fermentation, anaerobic

respiration) in the dark (anoxic) o Chemoorganotroph (aerobic respiration) in the

dark (oxic) o Chemolithotroph (H2) in the dark (oxic)

2

Green sulfur bacteria

• Key genera: Chlorobium, Chlorochromatium

• Non-motile, anoxygenic phototrophs

• Utilize H2S as an electron donor and oxidize it to SO42-

(inhabit anoxic environments rich in H2S)

• Autotrophy is supported using a reversal in the citric acid cycle

• Possess chlorosomes: bacteriochlorophyll-rich bodies bounded by a thin membrane

• Some green sulfur bacteria form consortia o Green sulfur bacterium + a chemo-

organotrophic bacterium o Phototrophic member is called epibiont.

Epibiont is physically attached to nonphototrophic cell

Green nonsulfur bacteria

• Key genera: Chloroflexus

• Chloroflexus o Thermophilic filamentous bacteria → form

thick microbial mats in neutral to alkaline hot springs

o Grows best phototrophically, can grow photoautotrophically

o Hydroxypropionate cycle for CO2 fixation Heliobacteria

• Gram positive bacteria

• Obligate anaerob; photoorganotroph or chemoorganotroph (fermentation)

• Produce bacteriochlorophyll g

• Ability to fix nitrogen

Pigments/reaction center Chlorophylls and Bacteriochlorophylls (Bacterio-)chorophylls necessary for an organism to be photosynthetic → part of the reaction center. Base structure is a porphyrin ring.

Carotenoids and Phycobilins Carotenoids: Accessory pigments, always found in phototrophic organism, prevent photo-oxidative damage to cells. Energy absorbed by carotenoids can be transferred to a reaction center.

Beta carotene

Phycobilins (cyanobacteria/red algae): accessory pigments, covalently bound to photosystem (II)? They can absorb at wavelengths that chlorophyll and carotene cannot (efficiently).

Tetrapyrrole ring structure that was cut open

Chlorosome: pigments in green sulfur bacteria Function as massive antenna complexes

LH = light harvesting molecules RC = reaction center

Electron flow in anoxygenic PS in a purple bacterium

3

Reverse electron flow from Q needed to reduce NAD+ to NADH → part of pmf is uded to bring Q to the energy lvl of NADH? Electrons of NADH then used to reduce CO2 to sugars. Arrangement of protein complexes in reaction center:

Is this arrangement true for oxygenic and/or oxygenic?? Or just for purple bacteria (P870)? Electron flow in purple, green sulfur and Heliobacteria (anoxygenic)

Green sulfur & heliobacteria reduce Fd (and not NADH), needed for reverse TCA… reduces NAD+ Electron flow in oxygenic PS

Unlike in anoxygenic PS there is no reverse electron flow needed to reduce NAD+ to NADH.

Zones within a lake

→ to enrich for e.g. purple / green sulfur bacteria take medium with H2S, for purple non-sulfur bacteria take organic compounds instead. Summary

• Photosynthesis is the most important biological process on Earth

• Most phototrophs are also autotrophs

• Photosynthesis requires light-sensitive pigments called (bacterio)chlorophyll

• Photoautotrophy requires ATP production and CO2 reduction

• Oxidation of H2O produces O2 (oxygenic photosynthesis)

• Oxygen not produced (anoxygenic photosynthesis)

• Anoxygenic photosynthesisis found in at least four phyla of Bacteria

• Electron transport reactions occur in the reaction center of anoxygenic phototrophs

• Reducing power for CO2 fixation comes from reductants present in the environment (i.e., H2S, Fe2+, or NO2

-)

• Requires reverse electron transport for NADH production in purple phototrophs

• In oxygenic photosynthesis light is used to generate ATP and NADPH

• The two light reactions are called photosystem I and photosystem II

• “Z scheme” of photosynthesis

• Photosystem II transfers energy to photosystem I

• ATP can also be produced by cyclic photophosphorylation

4

Vorholt II - phototrophy Aerobic anoxygenic phototrophs (AAP)

• Bacteria that use light energy but are no autotrophs

• Light is used for ATP synthesis via phosphorylation

• Photosynthetic light reactions in the presence of oxygen (in contrast to classical anoxygenic purple bacteria)

• Well known from coastal and ocean waters, but also occur in terrestrial systems. AAP found on leafes, & phyllosphere (methylobacteria)

• Alpha-, beta- and gammaproteobacteria

• E.g. Erythrobacter, Roseobacter, Bradyrhizobium Unlike classical photosynthesis, the light energy harvested by AAP bacteria does not fuel the CO2 fixation as in autotrophic cells. These AAP do not contain carbon fixation enzymes and use light as an auxiliary energy source for their mostly heterotrophic metabolism AAPBs are alphaproteobacteria and gammaproteobacteria that are obligate aerobes that capture energy from light by anoxygenic photosynthesis. Anoxygenic photosynthesis is the phototrophic process where light energy is captured and stored as ATP. The production of oxygen is non-existent and, therefore, water is not used as an electron donor (instead e.g. H2S).

Phytochromes

• In plants:

• Bacteria:

→ just the other way around than in plants. Bacterial phytochrome is important for the photosynthetic activity. Light-driven energy-generating mechanisms Two general biological systems are known to be capable of net energy conservation: one is based on chlorophylls and the other on retinal molecules. The two systems have very distinct characteristics! 1) Chlorophyll-dependent

• Complex: composed of proteins and pigments forming reaction centers, antenna complexes and photosystems

• Chlorophyll (tetrapyrrole, Mg2+; complex biosynthesis (16 metabolic reactions)

• Present in the domains of Bacteria and Eukarya

• Very efficient at transforming light energy into reducing power (NAD(P)H or ferredoxin), as well as ATP during photosynthetic electron flow

2) Retinal-dependent

• One protein: opsin (Type I: Bacteria, archaea, eukarya; Type II: animals, linked to G proteins)

• One chromophore: retinal (retinal biosynthesis one step from carotenoid beta-carotene)

• Present in all members of all three domains of life

• Modified to perform several different biological functions: Proton pumping, ion pumping, light sensing and gene regulation, phototaxis, vision

Microbial rhodopsins

• Proton pumping bacteriorhodopsins (Archaea; Halobacteria) and proteorhodopsins (Bacteria; large distribution and diversity)

• Light-driven rhodopsins that pump chloride or sodium ions

• Sensory rhodopsins Haloarchaea Some haloarchaea are capable of light-driven ATP-synthesis using microbial rhodopsin (bacteriorhodopsins)

• Key genera: Halobacterium o Extremely halophilic archaea → have a

requirement for high salt concentrations (1.5M)

Bacteriorhodopsin cytoplasmic membrane proteins that can absorb light energy and pump protons across the membrane (direct pump, no NADH produced)

Trans-cis conformational change upon ilumination is essential for H+ transport

Proteorhodopsin The most abundant marine heterotroph is Pelibacter, an oligotroph.

• Oligotroph: an organism that grows best at very low nutrient concentrations

• Pelibacter and other marine heterotrophs contain proteorhodopsin, a form of rhodopsin that allows cells to use light energy to drive ATP synthesis.

5

Vorholt III – Global C, N, S cycles The carbon cycle

• Phototrophic organisms are the foundation of the carbon cycle

• CO2 is fixed primarily by photosynthetic land plants and marine microorganisms

• CO2 is returned to the atmosphere by respiration of animals and chemoorganotrophic microbes as well as anthropogenic activities

o Microbial decomposition is the largest source of CO2 released to the atmosphere

• The carbon and oxygen cycles are intimately linked

• Plants dominate phototrophic organisms of terrestrial environments, bacteria dominate phototrophic organisms in the ocean

• The two major end products of decomposition are CH4 and CO2

Total carbon: 76*1015 tons

The nitrogen cycle

• N2 is the most stable from of nitrogen and is a major

reservoir o The ability to use N2 as a cellular nitrogen

source (through nitrogen fixation) is limited to only a few bacteria and archaea

• Ammonia produced by nitrogen fixation or ammonification can be assimilated into organic matter or oxidized to nitrate

• Denitrification is the reduction of nitrate to gaseous nitrogen products and is a primary mechanism by which N2 is produced biologically

• Anammox is the anaerobic oxidation of ammonia to N2 (with nitrite)

• Denitrification and anammox result in losses of nitrogen from the biosphere

6

Anaerobic respiration with nitrate

• Use of electron acceptors other than oxygen, e.g. NO3-,

Fe3+, SO42-, CO2, certain organic compounds

• Less energy released compared to aerobic respiration

• Energy released from redox reactions can be determined by comparing reduction potentials of each electron acceptor

• Dependent on electron transport, generation of a proton motive force, and ATPase activity.

Organisms: Enterobacteria (e.g. E. coli), Pseudomonas, Paracoccus…

Nitrate reduction

Denitrification

Chemolithotrophy

• Chemolithotrophs are organisms that obtain energy from the oxidation of inorganic compounds (e-acceptor = O2)

• Most chemolithotrophs obtain their carbon from CO2… but many other sources of reduced molecules exist. The oxidation of different reduced compounds yields varying amounts of energy

Nitrification (aerobic chemolithotrophs)

• NH3 and NO2- are oxidized by nitrifying bacteria (and

archaea) during the process of nitrification o Ammonia-oxidizing: Nitrosomonas europaeus o Nitrite-oxidizing: Nitrobacter vulgaris

• Two groups of organisms work in concert to fully oxidize ammonia to nitrate

• Nitrifiers are particularly active at oxic/anoxic interface of sediments and water bodies

• Only small energy yields from this reaction → growth of nitrifying bacteria is very slow

7

Nitrification: Oxidation of Ammonia O2 is not only an electron acceptor, but also a co-substrate

AMO: ammonia monooxygenase:

𝑁𝐻3 + 𝑂2 + 2𝐻+−→ 𝑁𝐻2𝑂𝐻 + 𝐻2𝑂 HAO: hydroxylamine oxidoreductase

𝑁𝐻2𝑂𝐻 + 𝐻2𝑂 → 𝑁𝑂2− + 5𝐻+ + 4𝑒−

Cyt aa3: terminal cyt c oxidase 1

2𝑂2 + 4𝐻+ + 2𝑒− → 𝐻2𝑂 + 2𝐻+(𝑜𝑢𝑡)

Nitrification: Oxidation of Nitrite to Nitrate

NXR: nitrite oxidoreductase 𝐻2𝑂 + 𝑁𝑂2

− → 𝑁𝑂3− + 2𝐻+

Cyt aa3: terminal cyt c oxidase Nitrification: Comammox Complete nitrification by single microorganism

Anammox bacteria : anaerobic chemolithoautotrophs

• Genera : Brocardia, Anammoxoglobus

• Anammox: anoxic ammonia oxidation (to N2) o Anammoxosome: specialized compartment

where anammox reaction occurs ▪ Protects cell from

reactions occurring during anammox →hydrazine (N2H4), an intermediate of anammox, is very reactive!

• Reaction: NO2- + NH4

+ -> N2 + 2 H2O (∆G0’ = -358 kJ/mol) Proposed process of nitrogen formation and ATP generation from ammonium and nitrite with nitric oxide and hydrazine as intermediates:

HZS: hydrazine synthase HDH: hydrazine dehydrogenase Nir: nitrite reductase The sulfur cycle

8

Organisms: Desulfovibrio, Desulfurococcus… Anaerobic respiration with sulfate

• Inorganic sulfur compounds (instead of O2) can be used as electron acceptors in anaerobic respiration

• Reduction of SO42- to H2S proceeds through several

intermediates and requires activation of sulfate by ATP

• During sulfate reduction, electron transport reactions lead to pmf formation (drives ATP synthesis by ATPase)

• Many different compounds can serve as electron donors in sulfate reduction, e.g. lactate, H2.

Sulfur-oxidizing bacteria (chemolithotrophs)

• Sulfur-oxidizing chemolithotrophs can oxidize sulfide and elemental sulfur at oxic/anoxic interfaces

• Bacteria (e.g. Beggiatoa) and archaea (e.g. Sulfolobus)

• Electrons from reduced sulfur compounds reach the electron transport systems and transported through the chain to O2.

• Generates a pmf that leads to ATP synthesis by ATPase

9

Piel – specialized metabolism I Primary metabolites

• Growth and development of organisms o Indispensable, removal kills cells, universal,

conserved among all/most organisms o E.g. lipids, sugars, amino acids

Specialized (secondary) metabolites

• Interaction between organisms and environment o Usually dispensible for growth and

development. Increase fitness and survival; unique, diverse, and often complex structures; adaptive

o E.g. natural antibiotics, pigments Biosynthetic relationship between primary and specialized metabolites

Relevance of secondary metabolites Almost 2/3 of all small-molecule drugs are based on natural products! Bacteria are main sources of known and new antibiotics, e.g.:

• Erythromycin (complex polyketide): inhibits protein biosynthesis by binding to the 50S ribosomal subunit

• Vancomycin (nonribosomal peptide): inhibition of cell wall biosynthesis → binds to D-Ala-D-ala, preventing binding of transpeptidase and thus cross-linking.

Relevance for the microbial producer

• Chemical defense (antibiotics, cytotoxins)

• Developmental signals

• Pathogenicity factors Evolutionary playground for rapid adaptation: The Screening Hypothesis (Firn and Jones) Premise: bioactivity is a rare feature for any molecule to possess. Higher chemical diversity increases likelihood that active compound is encountered (“screening”). Implication 1: many natural products have no direct function (difficult to test, perhaps not true) Implication 2: mechanisms that increase diversity at low cost should be beneficial for organism, e.g. evolution of promiscuous or modular biosynthetic enzymes (true, see e.g. NRPS)

How to study natural products biosynthesis? 1) Educated guess (look for (peptide) bonds, aa residues…) 2) Feeding of isotopically labeled precursors (13C, 2H, 15N,

18O, 14C)

3) Generation of blocked mutants and chemical analysis

o Inactivate enzymes ▪ Random mutagenesis (UV / chemical /

transposon) ▪ Enzyme inhbitors ▪ Targeted gene deletion

o Observe intermediate or shunt products

4) Study of biosynthetic enzymes

• Bioinformatic prediction

• Targeted knock-out

• Heterologous expression and study in vivo / in vitro

• Structural studies by crystallography or NMR Genetic and biochemical basis of specialized metabolism: example nonribosomal peptides and complex polyketides Nonribosomal peptide synthetases (NRPSs) are giant multifunctional proteins that can assemble peptides from many nonstandard amino acids. They consist of modules, each of which incorporates one amino acid into the peptide chain. Peptides can carry D-amino acids. Adenylation (A) domain loads amino acids onto peptidyl carrier protein (T) domain. Peptide bonds are formed by condensation (C) domain and epimerized (=converted to D-amino acid) by epimerization (E) domain. A thioesterase (TE) domain releases peptide (e.g. penicillin) from NRPS.

The biosynthetic modularity of NRPSs allows for metabolic adaptation by module exchange (see screening hypothesis).

10

Polyketide synthases (PKS) Most biosynthetic gene clusters contain genes for biosynthesis, regulation, and resistance -> very large

Each PKS module elongates the polyketide chain by one building block

• Step 1: Acyltransferase (AT) loads acyl carrier protein (small circles) with new building block, e.g. a malonyl unit

• Step 2: Ketosynthase (KS) elongates chain by 2 carbon atoms

• Step 3 (optional): Ketoreduction by kedoreductase (KR)

• Step 4 (optional): elimination of water by dehydratase (DH)

• Step 5 (optional): Enoylreduction by enoylreductase (ER)

The modification of each building block depends on the architecture of the corresponding PKS. Like for NRPSs, the succession and architecture of PKS (Type I) modules is usually colinear with the succession of functional groups in the polyketide chain: “colinearity rule” Possible exam question: predict the PKS for a given polyketide:

(see lecture notes for solution)

11

Piel – specialized metabolism II Modern methods of natural product discovery:

• Genome mining o Find compounds that are predicted from gene

sequences

• Engineered biosynthesis

• New biological sources Engineered biosynthesis – strategies for redirecting biosynthesis

Precursor-directed synthesis – concept: Feed precursor analogue and hope that it gets incorporated

Examples of precursor-directed synthesis:

• Hormaomycin

• Avermectins Advantages

• Complex structures can be made that are not easily available by chemical synthesis

• No genetic modification necessary

• Screening of precursor analogues is fast Drawbacks

• Natural product is usually still made

• Yield of analogue often low

• Involves complex purification procedures

• Downstream enzyme(s) must exhibit low substrate specificity (not always observed)

• Often only few precursor analogues incorporate into the product

Mutasynthesis – concept Knock-out gene for precursor and add precursor analogue instead.

Example: Doramectin Advantages

• Complex structures can be made that are not easily available by synthesis

• Wild-type natural product is not made: higher yields, easier purification

• Screening of precursor analogues is fast Drawbacks

• Genetic modification is time-consuming and sometimes not possible

• Downstream enzyme(s) must exhibit relaxed substrate specificity (not always observed)

Combinatorial biosynthesis (Synthetic biology) – concept

Example: Mederrhodin Multimodular PKSs or NRPSs are ideal targets for combinatorial biosynthesis Challenges

• Some enzymes/domains are too specific to accept modified precursors → no / trace production

• Enzymology is often not well understood

• Huge PKS and NRPS genes are difficult to manipulate → not routine yet, but success rates increase New biological sources

• Anaerobic bacteria: genome sequences suggest rich secondary metabolism, but virtually unexplored

• Mutualists and pathogens: hypothesis – specialized ecology selects for distinct natural products

• Uncultivated bacteria (the great majority of all bacteria)

12

Fischer I – plant symbionts

Learning goals • You can describe characteristic features of the

phyllosphere and rhizosphere microbiota

• You know typical examples of microbe-plant interactions including rhizobia-legume symbioses

• You can describe cellular events during legume infection by rhizobia and structural-functional differences between determinate and indeterminate nodules

• You can describe the chemical dialogue underlying the host specificity of Rhizobium-legume interactions

• You can describe host-mediated mechanisms to control symbiont proliferation and differentiation

• You know specific physiological properties and challenges of symbiotic, N2-fixing bacteroids and strategies how they cope with them

Objectives

• Plant-microbe interactions

• rhizosphere and phyllosphere microbiota

• fungi

• bacteria

• N2-fixing bacteria

• Rhizobium-legume symbiosis

• phylogeny, diversity, genomics

• cell biology

• molecular signals and their perception

• symbiosis and plant innate immunity

• host-mediated control of bacteroid differentiation

• physiology of N2-fixing bacteroids Bacteria-plant interactions in the rhizosphere and phyllosphere

• Phyllosphere = above ground

• Rhizosphere = below ground

→ commensals, mutualists, (phyto)pathogens The bacterial plant microbiota (phyllo- and rhizosphere) is composed of only a few dominant phyla, mainly Proteobacteria, Actinobacteria, Bacteroidetes, and, to a lesser extent, Firmicutes. Host plant species and cultivar shape the bacterial community in the rhizosphere. Soil type and plant community composition are additional factors which determine rhizosphere microbiota

From soil to root (rhizosphere): factors contributing to reduction of community complexity

Influences on microbiome: 0. Soil type; environmental factors; vegetation history 1. General gradients: Carbon sources Photochemicals Oxygen pH Nutrient depletion 2. Plant genotype bacteria: motility/chemotaxis, pili, biofilms, adhesins 3. Plant genotype Bacteria: Flagella Twitching motility, LPS, ROS

detoxification, plant polymer degradation, quorum sensing, Type VI secretion system ( ?)

→ decrease in complexity in rhizosphere soil/root: selection taking place due to secreted nutrients, entry restriction etc. Examples of fungi-plant interactions: Mycorrhiza

Beneficial effects of arbuscular mycorrhizae (AM) AM increases “root” surface → better supply with e.g. phosphate for plant. The fungus on the other hand profits from sugars etc. produced by the plant.

13

→ Pathways of N, P, and C exchange between plant and AM fungi. Inorganic nitrogen (NH4

+ and NO3-) and phosphorous (Pi)

mined from the soil by the extraradical mycelia is translocated to the plant as arginine and polyphosphate (poly-P) through the mycelial network and delivered to the plant at the intraradical (plant cell-associated) mycelium. Ammonium and phosphate are regenerated in the intraradical mycelium for transfer to the plant cell. In exchange for the N and P, the plant provides organic carbon to the fungus.

N2-fixing bacteria-plant associations Biological nitrogen fixation is limited to prokaryotes, not found in eukaryotes!

• Actinorhizal plants: symbiosis with the nitrogen-fixing Frankia alni bacterium -> induces root nodules

• Associations with cyanobacteria: non-fixing vegetative cells and N2-fixing heterocysts of Nostoc sp. And Anabaena sp.

• Rhizobium – legume symbiosis Rhizobium – legume symbiosis

• Rhizobia: >70 species distributed among 12 genera belonging to α- or β-proteobacteria

• Formation of legume root nodules

• Bacteroids in infected plant cells: N2 → NH3 (nitrogenase)

Selected N2-fixing rhizobial species and their legume hosts Occurrence of nodulation within the Leguminosae (Fabaceae) family

• Caesalpinoiadeae

• Mimosoideae

• Papilionoideae

Types of nodules (host plant-determined)

• Root nodules

• Stem nodules (photosynthetic?)

• Determinate nodules (spherical shape): no persistent meristem.

• Indeterminate nodules (cylindrical shape): persistent meristem. Bacteroids in indeterminate nodules undergo genome endo- reduplication (genome amplification), enlarge and become non-viable (terminal differentiation)

o NCR-AMPs (nodule-specific cysteine-rich antimicrobial peptides) control bacteroid differentiation in indeterminate nodules.

NCR peptides (NCRs) have two (contradicting) functions: they are required for development and chronic infection of bacteroids but they also have antimicrobial activity. BacA of S. meliloti is essential for survival and sustained infection. In vitro, BacA protects S. meliloti from hypersensitivity to NCR AMPs possibly by exporting NCRs out of bacterial cells.

Genome structure of selected rhizobial model species

• Rhizobia have relatively large genomes (3.6-9.2 Mbp)

• Most rhizobial genomes are composed of several replicons, including large “megaplasmids”

• Many symbiotic genes are organized in species-specific clusters located on different replicons

• “symbiotic islands” can be transferred by HGT

• No general “symbiome” can be defined: no defined set of symbiotic genes, different from species to species.

14

How is symbiosis established? Overview of early symbiotic events

Roots secrete (iso)flavones which induce Nod factor synthesis in bacteria (Rhizobia attach to the tip of root hairs). The Nod factors then induces a developmental program in plants which leads to root hair curling, invasion via infection threads and stimulation of (inner) cortical cell division. Root epidermis infection by arbuscular mycorrhizae and rhizobia Both types of microbial symbionts are housed within membrane-surrounded compartments which develop from exocytic membrane vesicles as symbionts enter host cells. Arbuscular mycorrhizae symbiosis:

Rhizobial symbiosis

The symbiotic dialogue between hosts and rhizobia

Molecular determinants of the dialogue

Inducers secreted by host plants

Nod and Myc factors synthesized by rhizobia and AM fungi, respectively

15

Biosynthesis of the S. meliloti Nod factor

NodA, B, C: synthesis of the GlcNAc backbone NodE, F: synthesis of the N-acyl chain NodH, P, Q: O-sulfation NodL: O-acetylation Other rhizobia NodS: methylation NodU: carbamoylation NodZ: glycosylation Nod factor perception by LysM receptor-like kinases (LysM-RLKs)

LysM domain: responsible for recognition of Nod factor

• Binds β-1-4 glucans that have N-acetylglucosamine (GlcNAc) residues such as peptidoglycan, Nod factors, Myc factor, or chitin.

• LysM-RLKs are also involved in perception of mycorrhizal lipo-chitooligosaccharides (Myc-LCOs) and chitooligosaccharides (COs) derived from fungal cell walls or insects → defense reaction

Current model of Nod- and Myc-factor perception and signaling – common featuers

Nod and Myc factors are both perceived by heterocomplexes of LysM receptor-like proteins:

• Lotus japonicus: NFR1/LYK3 + SYMRK

• Medicago truncatula: NFR5/SYM + DMI2

As mentioned LysM-RLKs can recognize Nod/Myc factors, but also PAMPs or MAMPs and induce a corresponding response.

PTI: PAMP-Triggered plant Immunity MTI: MAMP-Triggered plant Immunity PAMPS: Pathogen-Associated Molecular Patterns MAMPS: Microbe-Associated Molecular Patterns Plants perceive bacterial molecules (pathogen associated molecular patterns, PAMPs) using pattern recognition receptors (PPRs) that activate mitogen activated kinase (MAPK) cascades that trigger host defense responses. Adapted pathogens use the type III secretion system (T3SS) to deliver effector proteins into the cytosol of host cells. Bacterial effectors can inhibit the MAPK cascade, leading to suppression of host defenses. In some plants varieties, these effectors are recognized by nucleotide binding site leucine rich repeat domains (NBS-LRR) receptors, which trigger a second tier of host defense responses. Recognition of Nod factors produced by compatible rhizobia by specific receptors (NFR) (triggers a signaling cascade leading to nodulation (NF-Pathway). Rhizobial effectors can also promote nodulation by directly activating the NF Pathway. The symbiosis receptor like kinase (SYMRK) is also necessary for nodule formation, but the nature of its putative ligand is unknown In a second stage of rhizobia recognition, exopolysaccharides (EPS) produced by rhizobia are perceived by exopolysaccharide protein receptor 3 (EPR3) inactivating the defense signaling pathway through unknown mechanisms.

16

Proposed factors to limit PTI of rhizobial hosts

• PAMP divergence in rhizobia (no binding recognition)

• PRR (Pathogen/Pattern Recognition Receptor) absent or with repressed expression

• Bacterial factors repressing plant defenses

• Active plant repression of defense signaling pathways Rhizobial exopolysaccharides contribute to host specifity

3 layers of specificity: (1) plant inducer, (2) Nod factor, (3) EPS fragment

• A given legume host plant may secrete not only one flavonoid but a mixture of flavonoids

• Chemotactic response of rhizobia to flavonoids is variable

• Rhizobia may contain different NodD homologs with disparate recognition specificities for flavonoids and nod gene promoters

• As a result of the above, efficiency of nod gene induction is variable, leading to great differences in the quantity and quality of Nod factor production

• Host plants possess a family of Nod factor receptors, where each member may have different recognition specificity for Nod factor and its variants

• Host plants may differ in the organization and function of signal transduction pathways which may result in disparate efficiency of expression of nodule development genes

• Apart from the canonical Nod factor-dependent nodule formation, certain rhizobia use alternative, Nod factor-independent nodulation strategies which depend on effectors secreted via a type three secretion system (T3SS) or yet undefined factors.

Nitrogenase: the key player of N2 fixation NifDK:

• “nitrogenase”

• α2β2 heterotetramer

• FeMo cofactor in active site

• 8Fe-7S P-cluster at interface NifH:

• “nitrogenase reductase”

• α2 homodimer

• ATP binding and hydrolysis

• 4Fe-4S cluster at interface

Bacteroids have an oxygen dilemma: • Bacteroid respiration depends on oxygen but…

• Fe-S cofactors of nitrogenase are oxygen-sensitive! Solution:

• Oxygen diffusion barrier around bacteroids

• Leghemoglobin: reduces the amount of free O2 drastically (<25 nM vs. 250 μM)! KDiss[O2] = 40 nM

• High-affinity cbb3-type cytochrome oxidase (FixNOQP): KM[O2] = 10 nM

17

• Multiple systems for transport of nutrients, cofactors and fixed nitrogen across symbiosome and bacteroid membrane

• Diverse metabolic interactions and interdependences of host and symbiont

Low oxygen is a major regulatory signal for symbiotic genes!

Green arrow: S. meliloti, red arrow: B. japonicum (two rhizobial species) Take home messages

• Microbes can interact with plants in the rhizosphere and phyllosphere

• Root microbiota comprises ectophytic, endophytic and endosymbiotic microorganisms

• Phyllosphere and rhizosphere microbiota are generally dominated by proteobacteria

• N2-fixing legume root nodule proteobacteria include “α-and β-rhizobia”

• Rhizobial host specificity is based on host-secreted flavonoids, rhizobial Nod proteins involved Nod factor synthesis, and host-specific nod factor receptor-like kinases

• In addition to Nod factor-mediated signaling, bacterial access to legume roots is regulated by recognition of an exopolysaccharide (EPS) signal which is recognized by a specific receptor-like kinase

• Signal perception by plants of rhizobial or mycorrhizal symbionts and microbial or insect pathogens involves structurally-functionally related receptor-like kinases

• Host plant immunity is suppressed by various mechanism to avoid defense reactions during Rhizobium legume symbiosis

• Unlike host plants forming determinate nodules, plants with indeterminate nodules control bacteroid differentiation and survival via nodule-specific cysteine-rich antimicrobial peptides

• Symbiotic N2 fixation by rhizobia not only requires regulated synthesis of oxygen-sensitive nitrogenase but also the spatially and temporal control of numerous additional functions that are essential for effective symbiosis

• Low free-oxygen concentrations prevailing inside nodules are critical but not sufficient for induction of symbiotic

18

19

Plant pathogens – Vorholt Plants lack adaptive immunity, however plant disease-resistance and mammalian innate immunity share similarities:

• Preformed (passive) defense

• Induced (active) defense Plants are continuously exposed to a large number of microorganisms, including pathogens such as:

• Bacteria

• Fungi

• Oomycetes

• Viruses

• Nematodes Entry routes of plant pathogens

Bacterial plant pathogens and the type III secretion system Type III secretion system (T3SS)

• hrp gene organization

• hrp gene expression

• Structure and function of T3SS

• Effectors (formerly designated as avirulence proteins) o Identification o Plant receptors o Function

Type II secretion:

• Export of extracellular cell-wall degrading enzymes Interactions between plants and phytopathogenic bacteria 1. Plants detect bacteria and trigger BASAL DEFENSE

• Recognition of PAMP/MAMP: LPS, Flagellin, EF-Tu, cold shock proteins

• Basal defense: cell wall reinforcement, ROS, Callose deposition, lytic enzymes, Anti-microbial proteins (defensins), anti-microbial metabolites (phytoalexins)

2. Bacteria inject effectors that suppress BASAL DEFENSE 3. Plant resistance proteins recognize type III effectors and

trigger gene-specific defense 4. Bacteria inject type III effectors suppressing gene-

specific defense

20

The hrp type III secretion system

Hrp: hypersensitive response and pathogenicity Principles of plant immunity and models of direct and indirect recognition:

Model for the evolution of bacterial resistance in plants

Innate immunity

Agrobacterium tumefaciens Overall, species of the genus Agrobacterium can transfer T-DNA to a broad group of plants. Individual Agrobacterium strains however have a limited host range.

Key events in the formation of crown gall tumors 1. a) chemotaxis and specific attachment of bacterium to

plant cell wall b) recognition of plant-derived signals

2. induction of vir genes 3. processing of T-DNA 4. transport of T-DNA to the plant cell 5. T-complex assembly and transfer to nucleus 6. T-DNA integration into the host genome 7. a) plant hormones synthesis, responsible for tumor

growth b) formation of opines (novel aa-sugar conjugates)

Structure of a Ti plasmid

T-region

Note: T-DNA encoded genes have eukaryotic regulatory sequences. Thus, they are not expressed in Agrobacterium but only in plants!

21

Octopine and nopaline synthesis:

Virulence region of Ti plasmid: organization and functions of vir genes

Note: Tumorgenesis requires additional, chromosomal genes (e.g. chv) Signal integration and activity by the ChvE/VirA signal transducting proteins

General model of Agrobacterium-mediated transformation Read: McCullen and Binns, 2006, Annu. Rev. Cell Dev. Biol. 22:101

Agrobacterium as a tool for plant genetic engineering Natural features provided by Agrobacterium

• T-DNA is permanently integrated into the host cell genome

• Only the T-DNA borders and the vir genes are required for transformation

Tools in plant biotechnology: binary vectors Binary vector with modified T-DNA: > Multiple cloning site (MCS) > no opine-or phytohormone synthesis genes > ORI for E. coli and Agrobacterium > Selection marker > mobilizable > relatively small, facilitates cloning

Production of transgenic plants using a binary vector system in Agrobacterium tumefaciens

1. Binary vector system: Cloning of gene(s) of interest in E. coli into small cloning vector between border sequences

2. Transformation / conjugation into Agrobacterium containing disarmed Ti plasmid 3. Infection of plant cells 4. Selection of transformed plant cells 5. Regeneration of transformed plant

22

Eberl – Biofilms & cell-cell

communication “Chemical languages”

• Gram-negative o N-Acylhomoserine lactones o PQS o DSF o C18-FME

• Gram-positive o γ-Butyrolactones o cyclic peptides o CSP-1

Quorum-sensing: the lux gene cluster of Vibrio fischeri

Quorum sensing leads to bioluminescence. LuxR = family of transcriptional regulators AHL signal molecules

Functions regulated by quorum sensing

• Bioluminescence

• Virulence factors

• Conjugal plasmid transfer

• Antibiotic production

• Motility

• Cell aggregation

• Symbiosis with plants

• Biofilm formation: o On prosthetic devices o Cystic fibrosis (P. aeruginosa)

P. aeruginosa The quorum sensing cascade of P. aeruginosa

Regulators induce lasR expression, LasR induces production of LasI, regulates multiple genes (Exoenzymes, Exotoxin A, secretion apparatus, biofilms, catalase, haemolysin, siderophore) and also induces rhlR expression. RhlR regulates multiple genes (Exoenzymes, lectins, hydrogen cyanide, rhamnolipids, siderophore, secretion apparatus) The GAC system network in P. aeruginosa controls the reversible transition from acute to chronic infection.

The small regulatory protein RsmA binds to the promoters of multiple genes, enhancing bacterial motility and activating the production of several acute virulence factors while repressing the production of virulence factors associated with chronic infections. GacA phosphorylation via GacS

23

stimulates the production of the small RNAs RsmZ and RsmY, which bind to the RsmA protein, releasing the repression of virulence factors associated with chronic infections and repressing the production of acute infection-associated factors. Biofilms:

• P. aeruginosa readily attaches to the accumulated tracheobronchial mucin in CF patients where it forms thick biofilms

• Up to 1000x more resistant to antibiotics than free-living “planktonic cells”

• Very effectively evade clearance by the immune system

• Are responsible for chronic P. aeruginosa infections in the lung

In most biofilms, the microorganisms account for less than 10% of the dry mass, whereas the matrix can account for over 90%. The matrix is the extracellular material, mostly produced by the organisms themselves, in which the biofilm cells are embedded. It consists of a conglomeration of different types of biopolymers — known as extracellular polymeric substances (EPS) — that forms the scaffold for the three-dimensional architecture of the biofilm and is responsible for adhesion to surfaces and for cohesion in the biofilm.

a | A model of a bacterial biofilm attached to a solid surface. Biofilm formation starts with the attachment of a cell to a surface. A microcolony forms through division of the bacterium, and production of the biofilm matrix is initiated. Other bacteria can then be recruited as the biofilm expands owing to cell division and the further production of matrix components. b | The major matrix components — polysaccharides, proteins and DNA — are distributed between the cells in a non-homogeneous pattern, setting up differences between regions of the matrix. c | The classes of weak physicochemical interactions and the entanglement of biopolymers that dominate the stability of the EPS matrix. https://www.nature.com/articles/nrmicro2415

Regulation of biofilm formation – cyclic di-GMP as signal molecule

Left: input, right: output The biofilm matrix – proteins (RbmA, RbmC, Bap1) (I) When a single planktonic cell encounters a surface, (II) initial attachment occurs and RbmA (blue) accumulates on several sites on the cell surface. (III) As the founder cell divides, RbmA continues to accumulate and Bap1(green) appears at the cell-surface interface at the initial division site, ensuring that the daughter cell adheres to the surface. (IV) As the cells further divide, a cell cluster gradually forms, encased in a flexible envelope (red) containing RbmC, Bap1, and VPS (exopolysaccharide). Cluster formation is accompanied by more production and accumulation of RbmA (on individual cell surfaces) and Bap1 (on the cell-surface interface). (V) The mature biofilm forms as individual cell clusters expand and contact other clusters.

24

The biofilm matrix – polysaccharides (P. aeruginosa)

• Alginate: negatively charged polymer consisting of guluronic acid and mannuronic acid. Overexpression gives rise to mucoid strains in the cystic fibrosis clinic

• Psl: neutral polysaccharide consisting of a pentasaccharide repeat containing glucose, mannose and rhamnose

• Pel: positively charged polysaccharide consisting of α1,4-linked partially acetylated galactosamine and glucosamine sugars. Protection against aminoglycoside antibiotics.

Either carbohydrate-rich matrix component (PsI or PeI) appears to be sufficient for mature biofilm formation, and at least one of them is required for mature biofilm formation in P. aeruginosa strains (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC438632/) All P. aeruginosa strains (tested so far) carry the pel genes, but expression of these genes varies greatly among strains. psl genes on the other hand are not present in all strains. → strain-to-strain variation in both gene content and expression level might in part account for the great diversity of biofilm phenotypes.

• cellulose

• (VPS: see previous page) The biofilm matrix – DNA DNase I prevents biofilm formation → there is extracellular DNA in biofilms P. aeruginosa produces outer membrane vesicles

Vesicle biogenesis in bacteria

Model of type IV pili assembly and retraction.

Prepilin leader sequences are cleaved (and the pilin N-methylated) by PilD. Processed PilA is assembled on a base of minor pilins (PilE, V, W, X, and FimU) by the action of the cytoplasmic membrane protein PilC and the NTP-binding protein PilB, and the pilus extruded through the outer membrane via a pore composed of multimeric PilQ, stabilized by the lipoprotein PilP. Pili are retracted by the action of PilT (aided by PilU).

Model for the development of the heterogeneous P. aeruginosa biofilm

25

26

Vorholt – bacillus sporulation Cellular differentiation during development

• Morphology o Shape and size o Symmetry, polarity o Surfaces, structures, appendices o Cell communities, multicellularity, different cell

types

• Physiology: loss of general functions in favour of gain of specific functions

Mechanisms controlling developmental events

• Transcriptional cascades

• Regulated phosphorylation of signal transduction proteins

• Proteolysis of signal transduction proteins

• Transient genetic asymmetry

• Gradients – temporal and spatial

• Cell-to-cell signaling Bacterial sporulation Endospore formation by members of the genus Bacillus and Clostridium (Pathogens: B. anthracis, C. tetani, C. botulinum). Endospores survive treatments including

• High temperatures (100°C)

• Ionizing radiation

• Chemical solvents

• Detergents

• Hydrolytic enzymes → sporulation = last resort Objectives

• Survive long periods of stress

• Dissemination through the environment Best-studied endospore-forming bacterium: B. subtilis

Initiation of sporulation:

• key (positive) regulators o σH, Spo0A

• How does a cell know when to sporulate? o Nutritional depletion (GTP↓) o Cell density – Quorum sensing (oligopept. ↑) o DNA synthesis (signal = ?)

→ no single effect acts as trigger Activation of Spo0A

Prespore chromosome segregation

The spo0A regulon:

27

Establishment of cell-specific transcription Formation of an asymmetric septum is a key event in development. This triggers a cascade of changes in gene expression, involving different programmes of gene expression in the mother cell and prespore. Sigma factors play an integral role in this process

• Mother cell o σE o σK

• Prespore/spore o σF o σG

Regulatory network during B. subtilis sporulation

Sigma factor cascade σF and σE are setting the cell specific programs of gene expression. Both are synthesized before the septum is formed, but are held in inactive state until the septum forms. Morphogenesis and gene regulation during spore formation. (a) Activation of Spo0A and σH in the predivisional cell leads to asymmetric division (b) and early compartmentalized gene expression with σF becoming active in the prespore and σE in the mother

cell. (c) A series of proteins produced in the mother cell degrade the asymmetric septum and trigger migration of the membrane around the prespore, a process called engulfment, represented here by red arrows. (d) When the membranes fuse at the pole of the cell, the prespore is released as a protoplast in the mother cell, and a second round of compartmentalized gene expression occurs, with σG becoming active in the prespore and σK in the mother cell. These late factors activate transcription of genes that build the structural components of the spore that provide its resistance qualities. σF activation in the prespore σF is regulated by two distinct mechanisms which work together to ensure that σF is ONLY activated in the newly formed prespore compartment 1) chain of regulators: cosynthesized with an inhibitor

protein called SpoIIAB 2) instability of SpoIIAB and transient exclusion from

prespore

Regulation of σF activation (I) Chain of regulators: cosynthesized with an inhibitor protein called SpoIIAB

• SpoIIAB = anti-σ factor of σF and kinase

• SpoIIAA = anti-anti-σ factor (antagonist) (regulated by (de)phosphorylation)

• SpoIIE = phosphatase that dephosphorylates SpoIIAA; enriched in the prespore

SpoIIE associated with septal membrane recruited to FtsZ rings enriched in prespore

Cell-specific activation of σF. Two distinct mechanisms help to ensure the correct compartmentalization of σF activity in the prespore. a–c | The SpoIIE protein (blue spots) has a phosphatase activity that can overcome the negative regulation of σF by the SpoIIAB protein. SpoIIE is also required for the correct formation of the prespore septum. SpoIIE is recruited to the FtsZ rings by a direct interaction with FtsZ. The development of the two rings is asymmetrical and the ring that contains the most SpoIIE protein (upper in this case) usually achieves division first. During or following division, it seems that the SpoIIE protein becomes enriched in the prespore compartment, greatly enhancing the likelihood of σF activation in that compartment. SpoIIE phosphatase activity also seems to be regulated and it is possible that this regulation responds in some way to formation of the septum. Regulation of σF activation (II) Instability of SpoIIAB and transient exclusion from prespore. spoIIAB: one of the last genes to be transferred to the prespore by SpoIIE. Delayed chromosome translocation → crucial factor for σF regulation.

28

d–f | A chromosome position effect operates in a quite different way to compartmentalize the release of σF activity. The SpoIIAB anti-σ factor (green circles) is an unstable protein that is encoded by a gene that is located at an oriC-distal part of the chromosome (green boxes). During the period immediately after formation of the polar septum (e), and before the spoIIAB gene is translocated into the prespore (f), SpoIIAB protein concentrations decrease, allowing the more stable σF protein to become active in the small compartment. σE activation – proteolsysis σE:

• Prevents asymmetric division in the mother cell

• Triggers engulfment of prespore (by mother cell)

• Initiates spore coat assembly

• Directs transcription of σK

Regulation of σE activation. Parallel vertical lines separating the prespore (right) from the mother cell (left) represent the asymmetric septum. Broken arrows represent transcriptional activation, and solid arrows represent posttranslational regulation. Spo0APO4 is present in the predivisional cell as well as both compartments and therefore is represented above the sporulation septum. Pro σE is synthesized in a Spo0A-PO4-dependent manner and therefore is present in both compartments; however, recent study has indicated that Spo0A-PO4-dependent transcription is largely confined to the mother cell after asymmetric division. This distinction is represented here by a thick line, indicating a high level of expression, in the mother cell and a thin line, indicating a low level of expression, in the prespore. Pro-σE, which is membrane bound, is processed into the active form, σE, by the inferred membrane-bound protease SpoIIGA. SpoIIGA becomes active in response to SpoIIR, whose expression is activated by σF. SpoIIGA is presumably present in both compartments, but σE becomes active only in the mother cell, at least in part because of the higher concentration of pro-σE in this compartment, as well as because of degradation of pro- σE in the prespore. The prespore specificity of SpoIIR expression may contribute to but is not critical for mother cell-specific activation of σE.

σG - Regulation of spore development (prespore) Activation:

• SpoIIAB anti σ factor → inhibits σG

• Activated only after completion of engulfment Role of σG dependent genes:

• Couple late prespore and mother cell gene expression

• Protect spore from hazardous conditions

• Prepare the spores for germination σK – regulation of spore development (mother cell) Activation:

• Controlled by σE

• sigK (encoding σK) contains prophage that needs to be excised (site specific recombinase, under σE control).

Role of σK dependent genes:

• activation of genes involved in the formation of spore coat and spore maturation

Spore morphogenesis – core, cortex and coat The interior of the spore undergoes marked changes in physicochemical properties:

• Synthesis of large amounts of low-molecular weight proteins

o Coat and protect the DNA o Source of amino acids during germination

• Synthesis of large amounts of dipicolinic acid (in the mother cell and taken up by the prespore) and Ca2+

o Leads to the dehydration and mineralization of the spore

• The spore cortex, a modified cell wall, is synthesized outside the spore protoplast membrane

• Assembly of a multilayered proteinaceous coat outside the cortex

Sporulation is a bistable developmental process, only a subpopulation of cells differentiate into endospores. Bistability: occurrence of two distinct subpopulations that exhibit different phenotypes within an isogenic population-

29

Vorholt: Caulobacter/Myxobacteria Developmental biology in bacteria

• Endospore formation in Bacillus

• Swarmer and stalk cells in Caulobacter

• Fruiting body formation in Myxobacteria Mechanisms controlling developmental events

• Transcriptional cascades/ circuits

• Regulated phosphorylation of signal-transduction proteins

• Proteolysis of signal-transduction proteins

• Transient genetic asymmetry

• Gradients - temporal and spatial

• Cell-to-cell signaling

• "Noise" -> bistability Asymmetric cell division in bacteria

Caulobacter crescentus

• Dimorphic polarized bacterium (Alphaproteobacterium)

• Model organism for cell-cycle-dependent differentiation

Cell cycle in Caulobacter crescentus

Screening for factor(s) involved in transcriptional regulation

• Observation: Chromosome replication is required for flagellum formation → link between cell cycle and developmental changes → common regulator for initiation of DNA

replication (essential) and flagellum biosynthesis?

• Genetic screen:

• Look for ts (temperatur sensitive) mutants which grow at 28°C but not at 37°C and are defective in the regulation of the early flagellar gene fliF.

The response regulator CtrA

• Cell-cycle transcription regulator

• Coordinates the timing of cellular events with chromosome replication

• Essential for viability

• CtrA is a master regulator that controls multiple cell-cycle processes throughout the cell cycle

o Activation (66 genes activated by CtrA-P: polar morphogenesis, among them flagellum, pilum biogenesis, holdfast synthesis)

o Repression (29 genes repressed by CtrA-P)

• CtrA activity during the cell cycle is controlled on at least

3 levels 1. Transcription 2. Phosphorylation 3. Proteolysis

• CtrA is asymmetrically restricted to the non-replicating swarmer cell, not found in stalked cells!

30

CtrA regulation occurs throughout the cell cycle

Control of CtrA: 1) Transcription The two CtrA promoters P1 and P2 are active at different times during the predivisional stage, they are regulated by CtrA itself.

Control of CtrA: 2) Phosphorylation

Two -component system: CtrA: response regulator CckA: histidine kinase

RD = CtrA is phosphorylated on Aspartate

Control of CtrA: 3) Proteolysis CtrA is degraded by the ClpXP protease. Proteolysis occurs in the swarmer to stalk cell transition and in the stalked half of predivisional cell only after septum formation ensures asymmetric distribution of CtrA-P. 4 master regulators of the cell cycle control

• CtrA

• DnaA

• GcrA

• CcrM

Cyclical genetic circuit that drives cell cycle progression

Key points

• Two daughter cells produced that are morphologically and physiologically distinct

• Inherit a different developmental program

• Two cellular programs: the cell cycle and the development of polar organelles

• Utilizes signaling mechanisms that communicate spatial and temporal information. Activity of the key master regulator CtrA is controlled by phosphorylation and localized degradation!

31

Myxobacteria

• Typical soil bacteria

• Deltaproteobacteria

• Specialized in the degradation of macromolecules

• Produce exoenzymes with hydrolytic activities

• Lyse dead and living bacteria and fungi

• Forms fruiting bodies

• Genome sequence M. xanthus 9.5 MBp Myxobacterial fruiting bodies

Sporulation in Myxobacteria

• Complete differentiation of the entire vegetative cell ≠ Bacillus sporulation (through asymmetric septation)

• Within the fruiting body, rod shaped vegetative cells of Myxococcus turn into almost spherical spores that contain specific spore proteins and show a high degree of cross linking in their peptidoglycan

• The resulting myxospores are resistant to desiccation, heat, and detergent treatment, and can survive in soil for long periods with little metabolic activity

Gliding motility on solid surfaces – the A and S gliding engines

• Engine S (“puller”)

o Mechanism based on type IV pili retraction o PilA: pilus filament o PilT: involved in Pilus disassembly and thus

retraction o Presence of fibril-network important

• Engine A (“pusher”) o Mechanism based on slime production

Alternative model: Gliding A-motility The Myxococcus motility complex moves directionally along a helical path Assessing starvation If a colony is derived from nutrients (C, N, P) this results in

a) Slower growth b) Fruit body development and sporulation → new

proteins required ➔ Capacity to synthesize proteins is needed during

sporulation phase → cells must opt for development before any nutrient essential for protein synthesis is absent.

Intracellular signaling

• Precedes aggregation and fruiting body formation

• Evidence for extracellular factors Identification of different complementation classes (A-E) of non-autonomous developmental mutants:

• asg

• bsg

• csg

• dsg

• esg (short for A-E-signal)

32

A-Signal

• Needed prior to aggregation

• Required for synthesis of the C-signal

• Two fractions: o Heat-labile fraction: 2 different proteases (10

and 27 kDa) with distinct substrate specificities → produce extracellular aa’s and polypeptides

o Heat-stable fraction: mixture of polypeptides that mainly consist of certain aa’s (Pro, Tyr, Phe, Trp, Leu, Ile, Ala) → induces gene expression in the asg mutants at the micromolar lvl → indicator of the nutritional status of environment → indicator of the cell density “quorum sensing”

C-Signal

• Needed to complete aggregation and to initiate sporulation

• Induces aggregation, sporulation and gene expression after 6h

• Cell-surface associated

• C-signal transmission occurs by contact-dependent mechanism (end-to-end)

• 17 kDa protein (processed from a 25 kDa precursor encoded by csgA)

• Anchored in the outer membrane

• Nanomolar concentrations of purified C-factor from fruiting bodies restores aggregation and sporulation in csgA mutants

• C-factor is tightly associated with the producer cells

• C-signal orchestrates morphogenesis of the fruiting body by modifying the movement behavior of cells. It also induces the differentiation into spores.

How can such a tightly bound protein act as a signaling factor? Observation: motility required for C-signal transmission

The C-signal transduction pathway

33

Hardt – molecular basis of infection I Terminology

• disease: deviation from state of „good health“

• infectious disease: disease caused by presence / replication of microbe / parasite / virus

• symbiosis: both partners profit from the interaction

• commensalism: only one partner profits; „neutral“ for the other partner

• parasitism: one partner profits, the other one suffers

• pathogenicity: ability to cause disease (species comparison)

• virulence: „quantitative“, ability to cause disease (strain comparison)

• endemic: constant occurence in population

• epidemic: sudden high incidence; local

• pandemic: sudden high incidence; widespread

Koch’s postulates (1870s) 1. identify 2. Isolate 3. infect 4. re-identify + re-isolate

Shortcomings of Koch’s postulates:

• different strains (same species) have different virulence characteristics

• role of the host (immunocompetence, susceptible species)

• evolution of infectious agents not explained → shift to molecular biology of infection (Falkow): Virulence factor

• gene/factor which is encoded/expressed in pathogenic strains of a bacterium

• disruption of gene must reduce virulence

• complementation of the mutant must restore virulence

Concepts:

• non-virulent strains lack virulence factors

• virulent strains emerge by acquisition of virulence factors

• quantitative analysis of bacterial infections When do virulence factors act?

• Exposure/Entry

• Colonization

• Time of incubation

• Prodromal state (= first symptoms)

• Invasive state

• Regression

• Convalescence →7 stages of infection, virulence factors can act at all levels The landmark paper: Isberg & Falkow – virulence factor of Yersinia pestis:

• Experiment: Y. pseudotuberculosis DNA cloned into plasmids and transformation into E. coli. E. coli were added to model host cell. Gentamicin was used to kill extracellular bacteria, then the host cell was lysed and E. coli (which managed to enter cells) were reisolated → invasin gene (from Y. pseudotuberculosis) found to be responsible for virulence

Three classes of bacterial virulence factors:

• Offensive

• non-specific

• defensive

1.

2.

3.

4.

34

Non-specific virulence factors

• Trivial: all essential genes (ribosomes, RNAP…)

• Nutrient uptake (C, P, N, S, Fe, micronutrients, vitamins)

• Terminal electron acceptor

• biofilm formation Note: biofilm formation can be considered as a non-specific

and as a defensive virulence factor. By facilitating permanent

adhesion to a given target site, possibly even as an offensive

virulence factor in some cases.

• E.g. trehalose utilization by Clostridium difficile

Certain Clostridium difficile ribotypes/strains can use trehalose (disaccharide (2 Gluc)) more efficiently than others

(due to a mutation, TreR (regulator of trehalose uptake systems) is already active at low trehalose concentrations) → increased pathogenicity https://courses.cit.cornell.edu/biomi290/microscopycases/methods/ribotype.htm H2-fuelled growth of Salmonelly typhimurium in the gut

S. typhimurium can grown on H2 rather than sugar (feeds electrons from H2 into respiratory chain, fumarate to succinate → Hyb is crucial). There are three take home messages: 1. The gut colonization really comprises two distinct stages 2. During niche invasion, S.Tm uses microbiota-derived

hydrogen to grow up; Thus H2 can be considered as an achilles heel of microbiota metabolism

3. The interactions with the microbiota is far more complex than previously anticipated (CR…. Now also fostering).

Salmonella can use gut inflammation to their advantage/to outcompete microbiota → Salmonella survives granulocyte attack (= immune response to inflammation) better than microbiota. Salmonella iron uptake Salmochelin S2 (virulence factor) is not inhibited by host defenses.

Defensive virulence factors

• General protection o Spore formation o Biofilm formation o Tolerance o Surface masking:

capsules LPS modifications outer layers fibrin deposition (coagulase)

• Disabeling innate/adaptive immune defenses o Catalase, superoxide dismutase o IgA proteases o Protein A o Toxins suppressing NFkB gene expression

programs Tolerance: survive antibiotics without genetic change

Tolerant subpopulations (“persisters” ≠mutants; phenotypic adaption, not genetic !).

35

Tolerance is inducible:

• Slow growth (S. Typhimurium cells in monocytes: toxin antitoxin (TA) systems: toxin catalyzed modifications → translation blocked → no/slow growth)

• Antibiotics

• Toxin-antitoxin

• Nutrient limitation

• Quorum sensing

• Oxidative stress Biofilms

• Colonization (environment, heart valves, catheters)

• Resistance to phagocytosis

• Antibiotic resistance Capsules, mucous layers

• Definition: loose, unstructured surface polymers

• Composition/Structure: o negatively charged polysaccharides o linear or branched o 1 or several different sugar monomers

▪ Hyaluronic acid; sialic acid → mimicry of e.g. mammalian sugars (host)

• Present in extracellular pathogens

• Effects o Adherence o Inhibit phagocytosis o Biofilm formation o Interfere with antibody recognition of LPS

(serotyping) o Inihibition of C3b deposition (no destruction by

MAC) → see also “serum resistance”

• Capsule as virulence factor in E. coli meningitis: capsule of K1 (= α(2,8)-N-acetylneuramate) strain facilitates brain infection

Defense against superoxide of neutrophilic granulocytes

Detoxification of 𝑂2

− and 𝐻2𝑂2

• Catalase: 2 𝐻2𝑂2 → 2 𝐻2𝑂 + 𝑂2

• Peroxidase: 𝐻2𝑂2 + 𝑁𝐴𝐷𝐻 + 𝐻+ → 2 𝐻2𝑂 + 𝑁𝐴𝐷+

• Superoxide dismutase: 2 𝑂2− + 2 𝐻+ → 𝐻2𝑂2 + 𝑂2

• Superoxide dismutase/catalase in combination: 4 𝑂2

− + 4 𝐻+ → 2 𝐻2𝑂 + 3 𝑂2

S. Typhimurium has 1 catalase and 3 superoxide-dismutases

• sodC1(perimplasmatic)

• sodA (cytosolic)

• sodB (cytosolic)

Staphylococcus aureus peptidoglycan is lysozyme resistant The acetyl-transferase OatA modifies the sugar-backbone of peptidoglycan, as a result lysozyme can’t bind anymore. Serum resistance

• Definition: ability to resist killing by membrane attack complex (MAC) → Capsules interfere with C3b deposition (central to all complement pathways)

• LPS modifications o Attachment of sialic acid (host molecule) o Long O-antigen side chains keeps MAC at

distance

36

Offensive virulence factors

• Bacterial toxins Protein toxins

o Pore forming o Internalized (AB-toxins)

▪ ADP ribosylation ▪ N-glycosides ▪ Metalloproteases ▪ Adenylate cyclases

o Enzymatic membrane disruption o Surface acting

▪ Superantigens ▪ “small” toxins”

• Non-protein toxins o LPS, LOS, peptidoglycan

• Invasins

• Adhesins

Example – pore forming toxins α-toxin/Aerolysin-family of β-barrel pore forming proteins

S. aureus α-toxin

In vivo the toxin concentration is usually too low to kill cells by punching holes into their membranes → “killing potential” must come from another mechanism → aerolysin activates Caspase-1, and somehow as a result the cells start punching holes in their own membrane → death?

Example – internalized (AB-toxins) AB-toxins are taken up by the host cell. A = active part, B = binding part AB-toxins bind to their target cells via B-subunit, which catalyzes the translocation (across cytoplasma/vesicle membrane) of A. The A-subunit acts in the cytosol of the host cell: ▪ ADP ribosylation ▪ N-glycosides ▪ Metalloproteases ▪ Glycosyl transferase

Examples of AB-toxins: Tetanus toxin, Botulinus toxin (inhibits stable SNARE complex formation)

„AB5“ „AB“

37

C. botulinum neurotoxin

Botulinum neurotoxins A and B bind to SV2 and synaptotagmin and are internalized by endocytosis into clathrin-coated vesicles. Acidification of the vesicles triggers membrane insertion of the B chains and translocation of the A chains across the membrane into the cytoplasm. The A chains then cleave SNAP-25 or synaptobrevin, cleavage of either SNARE protein interferes with the formation of stable SNARE complexes that are required for exocytotic fusion of synaptic vesicles with the plasma-membrane. Example: Superantigens (Surface actin toxins) Overstimulation of immune system E.g. Toxic-Shock Syndrome Toxin 1 (TSST-1) from S. aureus.

Example non-protein toxins - MAMP

Adhesins (offensive virulence factors) Role of adhesion in bacterial pathogenesis

Colonization/adhesion factors of pathogenic bacteria – classification according to structure/morphology

• Fimbrial adhesins (multi-subunit adhesion organelle)

• Non-fimbrial adhesins (multi-subunit)

• Polysaccharides

• Biofilm formation

• Invasion-adhesins

38

Fimbrial adhesins: Three types of adhesion organelle assembly – classification based on assembly 1. Chaperone/usher dependent (Type I fimbriae) 2. General secretion pathway (Type IV fimbriae) 3. Extracellular nucleation/precipitation

Type 1 fimbriae

• Virulence factor of uropathogenic E. coli (attachment to bladder epithelium)

• Biogenesis: Donor strand complementation – the N-terminal extension of one subunit completes the lg fold of its neighbor in a canonical manner, as the N-terminal extension runs antiparallel to the F strand

Invasion-Adhesins – three different mechanisms

• Tandem beta-zipper mechanisms

• Zipper mechanism

• Trigger mechanism

Zipper-mechanism Invasin allows host cell invasion by Yersinia spp. Invasin mimics fibronectin. Rho-GTPase Signaling is involved in wrapping membrane around Yersinia → Rac1 self-association is stimulated

39

Trigger mechanism: Salmonella host cell manipulation by the TTSS-1

SipA: Actin SipC: Actin, translocon SopB: PI-Phosphatase SopE: RhoGTPases SopE2: RhoGTPases → involved in actin cytoskeleton rearrangement. Activating RhoGTPases increases actin de-novo polymerization

SopE: molecular mimicry

SopE cooperates with host GEF ARNO.

WRC = WAVE regulatory complex SopE activates Rac, SopB produces PIP3 which activates ARNO (=GEF for Arf1) →activation of Arf1. Now that both Rac and Arf1 are active, WRC can promote actin assembly

40

Hardt – Microbiota 1. Evolution → co-evolution of gut <-> microbes 2. Community structure 3. Methods for analysis 4. Functions

1. Metabolism 2. Colonization resistance 3. Maturation of the GALT

5. Host barriers 6. Role in diseases: IBD (also other diseases?) 7. Future directions Human gut microbiota:

• 3*1013 – 4*1014

• > 500 species

• > 5’000’000 genes Gut microbiota: “Enterotypes” There are three predominant enterotypes in humans

• Bacteroides

• Prevotella

• Ruminococcus

Enterotypes differ in metabolic gene abundance: Four enzyme in the biotin biosynthesis pathway are overrepresented in enterotype 1, four in the thiamine biosynthesis in enterotype 2 and six enzymes in the haem biosynthesis pathway in enterotype 3.

Open question: Do these differences manifest in different health outcomes?

Microbiota are associated with several diseases

• Clostridium difficile diarrhea

• Bacterial vaginosis

• Obesity

• Type 2 diabetes

• Liver diseases:

• Colorectal cancer

• Allergies?

• Psychiatric diseases? Open question: Cause, consequence or side effect ? Microbiota analytics

• Microbiota composition changes with age

• Many therapeutic agents affect microbiota growth

• Culture techniques (pure culture, mixtures)

• Animal models (germ free, gnotobiotic, complex SPF)

• Metabolomics (pure culture, mixtures, host tissues)

• DNA sequencing o 16S rRNA o Genome o Metagenome

Small ribosomal subunit RNA sequence (16S rRNA)

• Ubiquitous and essential

• Ancient

• Easy RNA isolation

• Conserved and variable regions

• Sufficiently long

→ study «long distance» relationships

16S community sequencing - State of the art:

• complete 16S-Sequence: o Gold standard for species identification o too expensive and time-consuming for routine

microbiota analyses

• 250-500 nt of the 16S Sequence (454, Ion torrent, Illumina):

o reduced resolution (→ OTU) o Strain/species not well resolved (e.g.

Salmonella <-> E. coli) o variability between publications o no functional information o cost-effective o Standard for rough estimate of microbiota

community composition

Enterotype 2

Enterotype 1

Enterotype 3

41

Microbiota function in health & disease

The composition of the microbiome can determine how effective certain drugs work! E.g Gardnerella can degrade a HIV-preventive drug whereas drug treatment in women with a Lactobacillus dominant vaginal bacterial community is more successful. Microbiato also influence maturation of gut immune system Microbiota metabolism

• Specialized on compounds we (humans) can’t digest, e.g. complex carbohydrates

• Several 100 genes involved in carbohydrate degradation! (only 9 in humans)

• Primary & secondary fermentations → our gut epithelial cells are nurtured by SCFA, up to 10% of food energy from them?)

SCFA (short chain fatty acids), e.g. acetate, butyrate

• Produced through bacteria fermentation of dietary fiber

• Prevent inflammation: SCFAs promote regulatory T cell (Treg) expansion and de novo generation

• Source of energy

Colonization resistance: protection against entero-pathogenic bacteria Enteropathogenic bacteria:

• Salmonella spp.

• V. cholerae

• Yersinia spp.

• Shigella spp. Protection by means of:

• Nutrient limitation → no food left for pathogen

• Binding sites → pathogens have problem to adhere to cells

• Inhibitors… → antibiotics also affect/weaken or gut microbiome, which increases the risk of conolization (antibiotics associated diarrhea (C. difficile) Physical barrier: Mucus layer Chemical barrier: antimicrobial peptides Microbiota → maturation of gut immune system

Metabolic axes between host and the gut microbiota

Colibactin – producing E. coli strands lead to colon carcinoma?