Molecular microbiology MAP prep

Anna's annotated slides

· Bacterial gene regulation: Sigma factors and transcription factors

· Bacterial signal transduction: Two component systems and second messenger signaling

· phase and the general stress response I: sigma S activity and sigma S-dependent
promoters and target genes- need to listen again

· Stationary phase and the general stress response II: signal input and regulation of the
master regulator sigma S

· Bacterial biofilms

· c-di-GMP signaling and the control of biofilm formation

· Signal input into c-di-GMP signaling in E. coli: RdcAB/DgcE and the CSS domain PDEs

· Protein secretion into and through the bacterial cell envelope

· Redox homeostasis in the bacterial cytoplasm and periplasm

· Bacterial pathogenicity

I dont know what this means

Missing information

Important for MAP

hdeB hdeA gadB osmY dps rpoS otsA sdhA ompF sdhD sdhC

Bacterial gene regulation: Sigma factors and transcription factors

DNA RNA Protein: code, sequence and function

DNA: the 1D sequence does not determine its 3D structure - DNA is a carrier of abstract digital
code (determining the structure of other molecules!)

RNA & protein: their 1D sequences determine their 3D structure - the code and its material
realization (folding into a biologically active structure) occur in the same molecule

Bacterial promoters are binding sites for RNA polymerase

Promoters have typical nucleotide sequences that are recognized by RNA polymerase: -35 and
-10 region, with a spacer of usually 17 bp in between Core RNAP consists of two alpha subunits
as well as one beta and one beta‘ subunits; Holo-RNAP consists of core + a sigma subunit

Initiation of transcription: The ‘transcription bubble ‘paradigm

When RNAP binds to a promoter, it separates the two DNA strands in the central part of the
promoter sequence (approximately between nucleotide positions -12 and +5)

The transcription cycle: Initiation, elongation, termination

RNA polymerase + sigma factor connect to the promoter region before the genes that need to
be transcribed. The sigma recognizes the promoter and initiation site. Transcription begins:
sigma released. RNA chain growth continues to the termination site. Termination site reached-
chain growth stops. Release of polymerase and RNA.

Bacterial transcription terminators

Free RNA emerging from RNAP can immediately form secondary structures.
Specific stem-loop structures followed by oligo-U are terminators, i.e. are recognized by RNAP
as a signal to stop further transcription and to release the transcript.

Structure of bacterial RNA polymerase

• Core RNAP consists of two alpha subunits as well as one beta and one beta‘ subunits;
sometimes also an omega subunit is present: elongation form of RNAP
• The RNA holoenzyme also contains a sigma subunit, which specifically binds to a promoter:
initiation form of RNAP
Holoenzymes are the active forms of enzymes.

Conserved regions of σ and their molecular functions

The bacterial core RNA polymerase complex, which consists of five subunits, is sufficient for
transcription elongation and termination but is unable to initiate transcription. Transcription
initiation from promoter elements requires a sixth, dissociable subunit called a sigma factor,
which reversibly associates with the core RNA polymerase complex to form a holoenzyme [1].
RNA polymerase recruits alternative sigma factors as a means of switching on specific
regulons. Most bacteria express a multiplicity of sigma factors. Two of these factors, sigma-70
(gene rpoD), generally known as the major or primary sigma factor, and sigma-54 (gene rpoN or
ntrA) direct the transcription of a wide variety of genes. The other sigma factors, known as
alternative sigma factors, are required for the transcription of specific subsets of gene s.

All plastid sigma factors belong to the superfamily of sigmaA/sigma70 and have sequences
homologous to the conserved regions 1.2, 2, 3, and 4 of bacterial sigma factors

Sigma factors are subunits of RNA polymerase. The alternative sigma factor σs, which is coded
by the rpoS gene, is the key regulator of the general stress response and thus of a complex
cellular network that regulates more than 10% of the Escherichia coli genome. The cellular σs
concentration is regulated by a large number of very different stress factors and takes place on
all three levels, but less transcriptionally and translationally, but mainly through proteolysis. In
exponentially growing cells, σs is rapidly degraded, in stressful situations, on the other hand, σs
is stabilized in the cell within a very short time and the cellular σs concentration increases
sharply.

The RNA-Polymerase DNA complex

After binding to the DNA, the RNA polymerase switches from a closed complex to an open
complex. This change involves the separation of the DNA strands to form an unwound section
of DNA of approximately 13 bp, referred to as the "transcription bubble".

ECF family:The extracytoplasmic function (ECF) sigma factors are small regulatory proteins that
are quite divergent in sequence relative to most other sigma factors. ... Upon receiving a
stimulus from the environment, the sigma factor is released and can bind to RNA polymerase to
stimulate transcription.

Global regulation of gene expression: Competition of sigma subunits for RNAP core
• Most bacteria have several sigma factors, which recognize different promoter sequences
• Different sigma factors compete for a limiting amount of RNAP core enzyme
• Large groups of genes can be coordinately up- or down-regulated when other sigma factors
‘take over‘ RNAP: e.g. σS controls >500 genes in stationary phase

Most bacteria do not only have one sigma factor subunit, but multiple ones. (so called
alternative sigma factors). When the environmental conditions change for example and the
bacteria has to react. They sometimes have a special sigma factor for this stress signal and that
activates specific genes that help the bacteria adapt to the stress conditions.

Most bacteria have a specific sigma factor flagellar for the building of flagella.(to become ??) to
become motile
E.coli actually has 7 sigma factors.(one is very specialized ? I don't know what this means) But
here are 6 (rpoD rpoH rpoE rpoS rpoF rpoN )

Regulation of sigma factor activity by anti sigma factors

Structure of the flagellar sigma FliA (σF) in a complex with its anti-sigma factor FlgM.
In this form FliA cannot be bound by the RNAP core and is thus inactive.

Anti-sigma factor: a protein that binds to sigma factors so it wouldn’t be able to bind to RNA
polymerase. (it’s packaging the sigma factor together in a very compact structure that cannot
bind to RNAP)

This is a very important sub unit when building the flagellum since it has many subunits that
have to be made in a certain order.

Regulation of gene expression: Transcription factors

Transcription factors: also proteins like sigma factors but they do not bind as strongly to RNAP,
their primary binding is to DNA.

Transcription factors may be activated (or deactivated) through their signal-sensing domain by a
number of mechanisms including:
1.ligand binding – Not only is ligand binding able to influence where a transcription factor is
located within a cell but ligand binding can also affect whether the transcription factor is in an
active state and capable of binding DNA or other cofactors (see, for example, nuclear
receptors).
2.phosphorylation– Many transcription factors such as STAT proteins must be phosphorylated
before they can bind DNA.
3. interaction with other transcription factors (e.g., homo- or hetero-dimerization) or coregulatory
proteins
The DNA sequence that a transcription factor binds to is called a transcription factor-binding site
or response element.

Palindromic inverted repeat sequence

Palindromes cause the joining of two DNA molecules. Complementary sequences are formed of
inverted repeats called palindromes. Secondary structure formed after pairing of complementary
strands shows inverted repeats in a single stem.

Transcription factors are DNA-binding proteins, which often form dimers and recognize
palindromic DNA sequences (inverted complementary repeats) in the major groove.

If you find such a palindromic inverted repeat, close to a promoter than you know you this is the
binding site of the transcription factor.

Helix-turn-helix proteins

HTH -a major structural motif capable of binding DNA. Each monomer incorporates two α
helices, joined by a short strand of amino acids, that bind to the major groove of DNA. The HTH
motif occurs in many proteins that regulate gene expression.

Positions of activator and repressor binding sites in promoter regions

Promoter= binding site of the polymerase

Operate= binding site of a transcription factor

They are both usually close to the promoter. (the position is different depending on whether a
transcription factor acts as an activator or repressor) Activator sites are more common right up
stream of the promoter. Repressor sites tend to overlap the promoter.

Binding positions of regulatory proteins: Adjacent to or remote from RNAP

Activator binds right up stream of the promoter in a position that allows it to interact directly with
the RNAP (and actually control the behavior of RNAP and help it bind to the promoter)

Repression of gene expression: A repressor does not allow RNAP to bind

There are different means of repression: the simplest one is

● Static hindrance: The repressor just sits between -10 and -35, overlapping the promoter,
so the RNAP can’t bind
● Looping: Sometimes there are multiple bindings one a bit up stream and one a bit
downstream and they bind and create a loop. (stronger repression!)
 ● Modulation of activator: the repression reacts to the activator and make it unsuitable to
bind to the RNAP. “repression by anti-activation”

Role of the α subunit of RNAP in promoter activation

Contacts by the two αCTDs to the up-element upstream of the promoter of highly expressed
genes (e.g. ribosomal genes)

2 identical alpha subunits sitting on the DNA.

UP element: exist upstream of the promoter, help to recruit RNAP so it will strongly bind into the
promoter.

Role of the alpha subunit CTD in the activation of gene expression

• The alpha subunit of RNAP has two domains connected with a flexible linker: NTD and CTD

• Alpha-CTD can specifically interact with many activators and mediates the contact to RNAP
core

Interaction between α-CTD and DNA

Uses positively charged amino acid

Class I activation: TF interacts with alpha-CTD

Interaction between at least 1 alpha CTD and one half of the dymeric transcription factor.

Class II activation: TF interacts with Sigma, alpha-NTD or alpha-CTD

A different location of the transcription, much closer to the RNAP, at -41.5 (exact). Can make a
few different contacts. Need examples !

Class II activation by FIS at the proP2 promoter

Activation by conformation change: MerR

Simultaneous and independent contacts of two activators
For example - if a gene as to be activated by 2 conditions - a combination of conditions. The
only the combination of both conditions will result in activation of the gene

Indirect ways to activate genes:

If you have 2 activators , the first one can push the second one into position where it then can
activate the RNAP.
Another example would be if the first activator is far from the RNAP but the second activator is a
strong bending protein that creates a U loop that helps the first activator interact with RNAP

Co-operative binding of two activators

For example when the activator closest to the RNAP is weak and will not stay bind to the DNA
but the second and stronger activator helps it stay connected to the DNA.

Anti-repression :
“One activator counteracts the activity of a repressor, such that the second activator can
activate RNAP.” so if there is a repressor one of the 2 activators can block the repressor while
the other activator does its job.

Dynamic changes of the gene expression

The activity of activators and repressors has to be controlled in response to the environment
and cellular signals: Signal transduction.

Control mechanisms:
1. De-novo expression of the regulator
2. Binding of a small ligand- allostery (second messenger)
3. Binding of another protein for example antagonist or anti-factor
4. Chemical modification of the regulator (by phosphorylation two component systems or di-
sulfide bond formation)
5. Proteolytic processing of the regulator
6. Sequestration of the regulator (interaction with membrane proteins)
7. Export of a regulator (of the anti-sigma factor FigM)

Repressor activation by a corepressor: the Arg operon

Required for repression , without it transcription will happen. If there is enough Arg around the
gene will not be expressed - True for most amino acid operons!
Repressor inactivation by an inducer: the lac operon
The inducer is the small molecule/nutrient. When the repressor is connected and the inducer
binds to it the repression detaches from the DNA and the transcription proceeds.

Activation of the genes: the maltose operon

This kind of ligand control activation not only for repressors but also for most activators.
Activator protein that is sensitive to an inducer will bind to it and it will “sit down” on the DNA and
activate the transcription of the maltose genes.

Structure of cAMP-CRP (transcription factor) bound to DNA

+CPR is a helix-turn-helix type transcription factor dimer
+CPR forms dimers
+CPR binds the second messenger cAMP
+cAMP-CPR is the active form that binds to the operator sites on the DNA
+Consensus binding site: TGTGA-6bp-TCACA
+cAMP-CPR is globally acting TF it controls >100 genes

Complex regulation: Catabolite repression and the lac operon

-Hierarchy of carbon sources: Glucose is used first, genes for lactose uptake and metabolism
are activated only when glucose is exhausted: Diauxic growth
-Catabolite repression: Glucose “represses” the lac operon

Mechanism of the catabolite repression:

Activation (via CPR, activated by cAMP)
Derepression (via LacI, inactivated by lactose)
Activation of the lac operon follows AND gate logics: no glucose (cAMP levels high, formation of
cAMP-CPR) and lactose (LacI inactive)

Second messenger: signaling molecule that is made inside the cell as a response to some
change happening outside of the cell.

Two component system: a transcription factor becomes phosphorylated - it can bind to the
DNA to a specific binding site (mostly upstream)

Stimulus response networks: stimulus- sensor-signal- regulator - operons- regulon proteins-

response
Something changes inside or outside the cells=stimulus
sensors= notices the change
signal= for example second messenger
regulator= becomes active or inactive
operon=
Protein production=
response= the proper response to the specific stimulus, stops by negative feedback

Signal transduction between sensor and regulator

One component systems: The regulator has a sensory domain that reacts to a molecule that
enters the cell.
Two component systems: The sensor phosphorylates the regulatory protein. (the two proteins
interact with each other but they are separate proteins)
Second messenger signaling: the sensor generates a small molecule(=second messenger)
that binds to the regulator (=effector)

Two component systems (TCS)- signal transduction through the cytoplasmic membrane
Most bacteria have multiple TCS which sense very different signals: small molecules, state of
distinct protein in the periplasm, membrane alterations
2 component= sensor kinase and response regulator(usually a transcription factor that can bind
to DNA but not always), many of these systems have a 3rd component which is a phosphatase
Output of two component systems: transcriptional regulation, enzyme activity, chemotaxis

Physiological functions of two-component systems:

Two-component systems (TCSs) are ubiquitous signaling units found in prokaryotes. A TCS
consists of a sensor histidine kinase and a response regulator protein as signal transducers.
These regulatory systems mediate acclimation to various environmental changes by coupling
environmental cues to gene expression.

Two-component systems are involved in sensing for instance:

• nutrient limitation (e.g. phosphate, nitrogen)
• changes in osmolarity
• redox state of the respiratory chain
• small molecules, including quorum sensing molecules (‘socio-microbiology‘)
• in plants: e.g. ethylene and cytokinin (developmental signal molecules)

Physiological outputs of two-component systems :

• stress responses and adaptation
• chemotaxis
• development
• virulence

Molecular function of two-component systems Highly conserved domains:

• transmitter domains (SK)
• receiver domains (RR)

Variable domains:
• sensor domains (SK)
• output domains (RR)

Sensor domains are often membrane-inserted and can have extracellular subdomains
Output domains act as:
• transcription factors
• enzymes (some can produce second messengers)
• control factor for flagellar motor
• proteolytic targeting factor

Two-component systems and phosphorelay systems

• Phosphorelay systems use a serial phosphotransfer - often in hybrid sensor kinases
• Phosphotransfer in phosphorelays is often reversible
• Phosphorelays react more slowly and show sigmoid time and stimulus-response curves
• Phosphorelays are ideally suited for noise suppression and regulatory switches

Structure and function of His sensor kinases

Domains of a typical SK:
• membrane-inserted sensor domain
• linker/HAMP domain
• transmitter domain
A transmitter domain consist of:
• a phosphorylation site: the conserved His (H-box)
• a conserved ATP binding site

Conformational changes in His sensor kinases

HAMP domains: Histidine kinases, adenylyl cyclase methyl-accepting proteins, and other
prokaryotic signaling proteins

CA: catalytic domain

H: histidine

Models for signal transduction via the HAMP domain.

(A) In the inactivated state, HAMP domain helices bind intramolecular to the anti-parallel helices
of the dimerization domain forming a four-helix bundle. Ligand binding alters the conformation of
HKs allowing dissociation of the HAMP domain helices and subsequent alignment of the four-
helix bundle within the dimerization domain. This in turn leads to the alignment of the conserved
histidine residue with the ATP binding site of the CA domain required for catalysis. The
alignment could be achieved through the opening and closing of the kinase core as depicted in
the illustration, or by sliding of the histidine containing helix of the dimerization domain relative
to the CA domain.
(B) In the inactivated state, the HAMP domain helices lie parallel to the plane of the membrane
surface and transiently associate with one another. Ligand binding releases helix I from the
membrane and changes HAMP helical association. This causes conformational changes that
result in activation of the kinase core as discussed in (A)

Modular architecture of response regulators

CheY Spo0F CheB PleD VieA OpmR ArcA NarL NtrC HoxA
Structure and function of response regulators

Domains of a typical RR:

• highly conserved receiver domain
• linker
• output domain - usually with a DNA-binding HTH motif

A receiver domain: (always look the same in all response regulators)

• consists of 5 α helices and 5 β strands alternating with each other (about 120 aa)
• contains Asp (around position 54-58), which together with several other highly conserved aa
forms an acidic pocket where Mg++ binds
• catalyzes the phosphotransfer from SK to its own Asp residue
• alters its conformation upon phosphorylation

Communication between the receiver and output domains

• Phosphorylation induces a conformational change on the surface comprising α3-β4-α4-β5
• this surface area changes from acidic to hydrophobic
• the hydrophobic surface changes the interactions with other domains or proteins

NarL: a response regulator involved in anaerobic respiration (NO3 - as e- acceptor) HTH motif
NtrC: a DNA-binding RR that is phosphorylated and oligomerizes upon nitrogen starvation.

Control of output domain activity by phosphorylation of the receiver domain

Altered surface properties of the receiver domain induced by phosphorylation change the
interactions with other domains or proteins:
• an intramolecular inhibition of the output domain can be relieved (e.g. CheB)
• dimerization/oligomerization can stabilize DNA binding (e.g. NtrC)
• interaction with RNA polymerase to activate transcription
• ‘stand-alone‘ receiver domain proteins: interaction with other proteins (e.g. CheY)
If the receiver domains are transcription factors-
Initially in the non-phosphorylated form the protein would just be a monomer, and a monomer is
not active as a transcription factor (usually transcription factors have to at least dimerize to bind
to the DNA). The phosphorylation produces a hydrophobic surface and the 2 receiver domains
come together and this also brings together the DNA binding domains. And then you have a
dimer that can actually sit on the DNA and bind properly.

Switching off two component systems

When the stimulus is no longer present, sensor kinases and response regulators are
dephosphorylated:
• SKs switch to autophosphatase activity in the absence of a stimulus
• RR receiver domains have autophosphatase activity which can also be stimulated by
interaction with the cognate SK (in the autophosphatase state)
• some RRs have their specific phosphatases to accelerate dephosphorylation (e.g. CheZ for
CheY)
• phosphorelays: reverse phospho-flow back to the primary transmitter domain, which then
dephosphorylates
• dephosphorylation can show very different kinetics - between msec and hours in different RRs!

Regulation of porin (purine?) expression in E. coli by EnvZ-OmpR in response to

osmolarity signals
Low osmolarity: [OmpF] > > [OmpC]
High osmolarity: [OmpF] < [OmpC]

Phosphorylated OmpR is carrying the expression of both genes (OmpF and Ompc?) even
though their regulation is inverse (if one is high the other one is low) but both of them are
activated by the same transcription factor (both are positively controlled by the OmpR-P)
At low osmolarity there is very little OmpR phosphate in the cell, but this low concentration is
enough to bind to two high affinity sites , so OmpF is activated.
Now for the gene OmpC , it has only low affinity sites, which means the OmpR-P binds only
when there is a lot of it in the cell (high osmolarity). This high osmolarity actually blocks OmpF
because it has 2 low affinity sites on both ends of the high affinity sites which create a loop and
block the transcription.

Regulated by:
• EnvZ (membrane-inserted SK)
• OmpR (DNA-binding and transcription -activating RR

The ArcB-ArcA phosphorelay system

• ArcB responds to the redox state of the respiratory chain in the cytoplasmic membrane - a
parameter that integrates energy supply and oxygen supply
• ArcA is a transcription factor that controls genes involved in aerobic/ anaerobic switching and
in stationary phase
The Arc system consists of a membrane-associated histidine kinase, ArcB, and a response
regulator, ArcA. Mutations in the arc genes cause derepression of several genes and repression
of a relatively short list of others, indicating that phosphorylated ArcA is both a repressor and
activator of gene transcription under anaerobic conditions.

ArcB senses the redox status of the respiratory chain

The redox state of the components in the RC depends on:
• e- influx into the RC (i.e. on energy supply, reflected by the NADH/NAD+ ratio)
• e- efflux from the RC onto O2 (depending on availability of O2)

ArcB is inactivated by oxidized quinones, i.e. when:

• either energy supply is low (stationary phase)
• or when O2 supply is high (aerobic conditions)

Respiratory chain RC :
NADH H - Chinon - Cyt b - cyt c -cytochrom oxidase - oxygen

Oxidized quinones oxidize and inactivate ArcB

• Oxidized quinones oxidize ArcB: formation of di-sulfide bonds between the two ArcB
molecules in the dimer
• covalent crosslinking by these di-sulfide bonds changes the conformation of the ArcB dimer -
kinase activity is inhibited and ArcA remains unphosphorylated • this happens during energy
starvation or in the presence of high oxygen
• ArcA-P controls genes in growing cells and under anaerobic conditions

Crosstalk between different two-component systems?

• Purified SKs and RRs show phosphotransfer in specific pairs
• Some SKs can also phosphorylate other RRs - usually this is ineffective and slow in vitro and
physiologically not relevant in vivo
• However: some SKs have more than one physiological RR - e.g. ArcB phosphorylates ArcA
and RssB in vivo
• Some RRs can interfere with phosphotransfer in distinct SK/RR pairs

Signal transduction between sensor and regulator One-component systems: The regulator has
a sensory domain that reacts to a molecule that enters the cell Two-component systems: The
sensor phosphorylates the regulatory protein Second messenger signaling: The sensor
generates a small molecule (= second messenger) that binds to the regulator (= effector)

Basic mechanism second messenger signaling

• Cellular levels of nucleotide second messengers are in a dynamic equilibrium between
synthesis and degradation
• Specific synthases produce distinct second messengers in response to specific signals
• Specific phosphodiesterases (PDE) degrade second messengers in response to specific
signals
• Effectors are second messengers binding proteins or RNAs: transcription factors, regulatory
subunits of enzyme; riboswitches
• Targets can be promoters (TF binding sites), catalytic subunits of enzymes, complex cellular
structures (e.g. flagella, exopolysaccharide secretion systems); mRNAs

riboswitch-a regulatory segment of a messenger RNA molecule that binds a small molecule,
resulting in a change in production of the proteins encoded by the mRNA.

What two genes are regulated by riboswitches? Virulence gene, Flagella gene expression

cAMP synthesis upon exhaustion of glucose in the growth medium

• cells growing on glucose do not produce cAMP
• upon glucose exhaustion cAMP synthesis is rapidly and strongly stimulated
• during stationary phase cAMP is slowly excreted

The transcription factor CRP is the cAMP effector

• ‘cAMP receptor protein‘ (CRP) is a helix-turn-helix-type transcription factor dimer
• CRP forms dimers • CRP occurs in two conformations, i.e. +/- cAMP bound
• cAMP-CRP is the active form that binds to operator sites on the DNA
• Consensus binding site: TGTGA-6bp-TCACA
• cAMP-CRP controls >100 genes in E. coli: nutrient scavenging systems, flagella, etc

Complex regulation: Catabolite repression and the lac operon

• the lac operon encodes β-galactosidase (LacZ

), lactose permease (LacY); LacI is the lac-repressor

• Hierarchy of carbon sources: Glucose is used first, genes for lactose uptake and metabolism
are activated only when glucose is exhausted: Diauxic growth
• Catabolite repression: Glucose ‘represses‘ the lac operon
• lac operon expression requires: (1) presence of lactose (2) high cAMP, i.e. cAMP-CRP

Mechanism of catabolite repression:

• activation (via CRP, activated by cAMP)
• derepression (via LacI, inactivated by lactose)

Activation of the lac operon follows AND gate logics: No glucose (cAMP levels high,
formation of cAMP-CRP) AND lactose (LacI inactive)

ppGpp: a general stress signaling molecule

• In E. coli (p)ppGpp is produced by two synthases: RelA and SpoT
• RelA is ribosome-associated and is activated by amino acid starvation (uncharged tRNA in the
A-site)
• SpoT is soluble and is activated by diverse starvation and stress conditions
• (p)ppGpp levels are inversely correlated with growth rate
Molecular structure of RelA and SpoT
• RelA and SpoT are paralogs, and have the same multidomain structure
• Both have (p)ppGpp hydrolase (HD) and synthase domains
• However, RelA only synthesizes (p)ppGpp
• SpoT synthesizes or degrades (p)ppGpp - depending on the conditions

(p)ppGpp binds to RNA polymerase and other proteins

• ppGpp binds to the ‘back‘ side of RNAP, close to the alpha-NTD and has an allosteric effect on
transcription initiation and sigma factor binding
• (p)ppGpp also binds to and affects the activity of other effector proteins: certain GTPases,
decarboxylases, and enzymes involved in nucleotide and lipid metabolism

The ‘stringent response‘: (p)ppGpp downregulates ribosomes

• (p)ppGpp bound to RNAP reduces open complex formation at ribosomal promoters (GC rich
‘disriminator‘ region downstream of -10 region) - ribosomal gene expression is reduced
• (p)ppGpp inhibits exopolyphosphatase (PPX), polyphosphate accumulates and binds to free
ribosomes and Lon protease - free ribosomes are degraded as an internal source of amino
acids

Activation of genes by (p)ppGpp

• Stage 1: release of σ70-RNAP from ribosomal promoters allows increased expression of other
vegetative genes (with AT-rich ‘discriminator‘ regions; e.g. amino acid biosynthetic genes; rpoS
which encodes the general stress sigma factor σS)
• Stage 2: (p)ppGpp promotes the binding of alternative sigma factors to RNAP (σS, σE) -
expression of stress resistance and survival genes

Alarmone regulate the gene expression at transcription level

The general stress response in E. coli: σS(RpoS)-controlled promoters and target genes

Bacterial stress responses

Role of sigma factor competition for core RNAP in global readjustment of gene expression in
response to stresses

rpoD rpoH rpoE rpoS rpoF rpoN

Two fundamentally different physiological states: growth versus maintenance/survival

A Exponential or log phase (until OD=0.3): most resources go into ribosome synthesis, cells
are non-motile
B Post-exponential phase (0.3<OD<3): gene expression becomes more diverse; cells produce
flagella; master regulators: σ70, σFliA, CRP, FlhDC low c-di-GMP
C Stationary phase (OD>3): cells stop to grow and become small and ovoid; cells become
multiple stress-resistant; produce amyloid curli fibres and cellulose; master regulator: σS high c-
di-GMP

Both states occur in liquid cultures as well as in biofilms!

A two-layer architecture of E. coli K-12 colony biofilms
Top layer: Stationary phase σS-dependent gene expression in small starving cells, including
genes required for synthesis, secretion and assembly of amyloid curli fibers (stained with
thioflavin S)
Bottom layer: Post-exponential growth Vegetative gene expression (σ70, σFliA) in growing cells,
including genes required for synthesis, secretion and assembly of flagella

Agar/Nutrients

The temporal succession of flagella and curli synthesis in liquid culture translates into a spatio-
temporal pattern of expression in a macrocolony biofilm that reflects gradients of nutrients,
oxygen, waste products, signaling compounds, etc.

Explain the regulation sigma 32 synthesis

Regulation of synthesis of sigma32 (rpoH)

First mechanism of regulation: occurs at the level of rpoH transcription which only has a minor effect on
sigma32 synthesis. (rpoH transcription = minor effect of sigma32)
HOWEVER, rpoH CONTAINS A REGULATORY REGION THAT CONTAINS AT LEAST FOUR
PROMOTERS, THREE ARE SIGMA70 DEPENDENT.
Sigma32 is responsible for EXPRESSION OF GENES THAT GET TURNED ON WHEN TEMPERATURE
IS HIGH. (some gene examples: FkpA and OmpK which are protein folding catalysts).
Different temperature = use of different promoters = different genes turned on.
SECOND MECHANISM MEDIATING STRESS CHANGE IN SIGMA 32 AFFECTS THE TRANSLATION
OF rpoH mRNA.
During temperature shift, there is a 12-fold higher translation level compared to PRE-HEAT SHOCK
conditions.
Translation becomes increasingly repressed during the "shut-off" phase of the heat shock phase of the
heat shock response to reach a new steady level state.

σS (RpoS) is the master regulator of the general stress response in E.coli

cause for stress: Slow growth, high cell density. Starvation for C,N,P or amino acids, high or low
temperature, high osmolarity, acidic pH
Response trigger of sigma S: multiple stress resistance, small round cell morphology(egg
shaped), storage compounds(glycogen, polyphosphate), protective compounds
(trehalose),adhesive fimbriae, extracellular matrix (biofilm), metabolic alterations (maintenance)

The σS-dependent general stress response in E.coli:

• Structure and function of the stress RNA polymerase EσS and σS dependent genes: EσS/
Eσ70 selectivity of promoters
• Architecture of the σS controlled transcriptional network: structure, function and regulation of
modules
• Regulation of the cellular σS-level: Transcriptional and post-transcriptional regulation in
response to environmental signals
The EσS/Eσ70 promoter selectivity paradox- So what makes a promoter sigma s
specific ?
EσS and Eσ70 control different genes in vivo, but they recognize the same promoters in
vitro …
Promoters that are EσS-dependent in vivo (as known until about 2004)
• the -10 region seems rather similar to that of a vegetative promoter • there seems to be a
strong preference for a C(-13) and a T(-6) • sequence and position of the -35 region is unclear •
length of the spacer is unclear
Sequence of a promoter evolved in vitro (SELEX) under selection for optimal EσS binding
A promoter optimized for binding σS-containing RNAP (EσS) is essentially identical to the
vegetative consensus promoter bound by Eσ70! EσS-selective promoters must deviate from this
consensus promoter or must have additional elements!

Identification of σS controlled genes in Escherichia coli by genome-wide microarray analysis:

rpoS+ vs. rpoS::Tn10
“Microarray analysis techniques are used in interpreting the data generated from experiments
on DNA (Gene chip analysis), RNA, and protein microarrays, which allow researchers to
investigate the expression state of a large number of genes - in many cases, an organism's
entire genome - in a single experiment.”

σ S-dependent genes 483 positively σ S-controlled genes have been identified in E. coli K12
(i.e. approximately 10 % of all genes in the E.coli genome) Shown by microarray technology of
rpoS + vs. rpoS - under three different growth/stress conditions that induce σ S: • LB /
OD578=3.0 • 20 min after +0.3 M NaCl • 20 min after shift to pH5

Total mRNA from rpoS+ and rpoS::Tn10 strains was differentially labeled and co-hybridized on
whole-genome E.coli microarrays (σS dependency is defined as more than 2fold difference
between rpoS+/rpoS- in three completely independent experiments for each condition tested).
Among a total of 483 genes there is a core set of 140 σS-dependent genes
- they concluded that those 140 genes not only depend on sigma s but probably don’t have any
other specific mechanisms on top of sigma s control

Functions of σS-dependent genes in E.coli

How do these minor differences contribute to EσS selectivity?

Allele-specific suppression is a genetic indicator for direct interaction between two

components
How can you prove that two factors actually interact with each other ?(prove genetically)
allele specific suppression method- A mutation in one gene is suppressed by a particular set of
mutations in a second gene, whereas other mutations in the first gene are suppressed by a
different set of mutations in the second gene

Systematic variation of spacer length in a synthetic σS dependent promoter

In the presence of a -35 region, C(-13) and TAA (downstream of the -10 region) produce
moderate EσS selectivity
However, with non-optimal spacers, EσS selectivity increases, since Eσ70-dependent
expression is more strongly reduced than EσS-mediated expression

Role of a distal UP element half-site in EσS selectivity of a promoter

A synthetic promoter (synP13) fused to lacZ was combined to either half or full UP-elements
and activity was tested in rpoS+ and rpoS::Tn10 strains.
A distal half-site of an UP-element strongly favours EσS (if a -35 region is present), whereas a
full or proximal half-site of an UP-element favour Eσ70

Additional transcription factors and EσS selectivity : Role of the position of the CRP box
at the csiD promoter
At the csiD promoter, the CRP box has an non-typical class I posititon (-68.5) - when shifted to
the classical position (-60.5), EσS selectivity of csiDp is entirely lost and Eσ70 takes over!

Additional transcription factors and EσS selectivity : Role of the IHF, Lrp and CRP binding sites
at the osmY promoter
At the osmY promoter, cAMP-CRP and the histone-like proteins IHF and Lrp interfere more
strongly with Eσ70-dependent expression than with activity of EσS (in vitro transcription data
shown): EσS binds very strongly to the -10 region and the spacer (18bp) is nonoptimal for Eσ70.

Summary: EσS selectivity generating features of a promoter

Multi-input control in the general stress response
regulatory network
Combinatorial input (positive and/or negative) results in highly
differential gene expression!

Sigma factors in Escherichia coli that are relevant during

entry into stationary phase
A Exponential or log phase (until OD=0.6-0.8): σ70
B Post-exponential phase (0.8<OD<3): σ70, σFliA, σS
C Stationary phase (OD>3): σ70, σS, σE
σS has the lowest affinity for RNAP core and its level reaches
only about 1/3 of σ70 - σS needs help to form EσS !

Induction of σS and formation of σS-RNAP during entry

into stationary phase
• σS levels begin to increase during post-exponential phase
• σS-containing RNAP holoenzyme (EσS) is formed with a delay, resulting in massive switching
to σS dependent gene expression during entry into stationary phase only
During the increase in the cellular EσS content, σS-dependent genes (which do not require
additional regulatory factors) are induced in a temporal order that reflects the affinities of their
promoters for EσS!
(Promoters with -35 and -10 region have higher affinity, those with an extended -10 only have
lower affinity)

Factors that control EσS activity at its cognate promoters

Crl (+) binds to σS (in domain 2) and stimulates EσS formation during entry into stationary
phase
Rsd (+) an anti-σ70 factor that binds a fraction of the cellular σ70, so that it no longer
participates in competition for core RNAP; Rsd expression is stimulated during entry into
stationary phase
ppGpp (+) binds to RNAP core; not only induces rpoS transcription, but also seems to influence
sigma factor competition in favour of alternative sigmas
FliZ (-) abundant nucleoid-binding protein under flagellar control (FlhDC) that interferes with σS-
dependent gene expression by specifically binding to σS-dependent promoters

The general stress response in E. coli: σS (RpoS) regulation in response to cellular and
environmental signals

Regulation of the cellular σS level in E.coli

Sigma S is an unstable protein when there’s no traumatic stress because it is degraded by a
protease (rapidly degraded)
We cannot see sigma S in a western blot because it's rapidly degraded.
Inhibition of sigma s (what keeps it going and delays proteolysis): high osmolarity ,carbon
salvation high temperature , low pH
EσS E sigma S- the E stands for enzyme . the ability to bind to RNAP. the active form of sigma
S and leads to sigma S dependent genes (about 10% of E. coli genes)

Regulation of rpoS transcription in E.coli

Starting with transcription control of rpos - E sigma 70 is a promoter that responds to increase in
ppGpp (which you find when nutrient sources become of lower quality) and it responds in a
positive way. (ribosomal genes are down-regulated by ppGpp but these vegitative promoters
tend to be up-regulated) so there is a positive regulation by ppGpp. That means the reduced
growth rate actually stimulates rpoS transcription.
In E. coli, rpoS transcription is controlled by a two-component system and by the cAMP receptor
protein (CRP) somehow through the signaling of ppGpp and polyphosphate

“In Escherichia coli, the stationary phase alternative sigma factor σs controls the expression of
genes involved in cell survival in response to cessation of growth (stationary phase) and
provides cross-protection to various stresses. Levels of σs increase dramatically at the onset of
the stationary phase and are regulated at the transcriptional, post-transcriptional and post-
translational level, making this one of the most complex regulatory systems in bacteria.”

Regulation of rpoS mRNA translation

Not many genes are controlled in the translation part but rpoS is. The stimulation of translation
of preexisting messenger RNA

Regulation of rpoS mRNA translation: role of regulatory proteins and small RNAs
• Activating factors: Hfq, HU, ArcZ-RNA, DsrA-RNA, RprA-RNA
• Inhibitory factors: H-NS, LeuO, OxyS-RNA
We need one small RNA and one protein. The small RNA is transcribed under certain stress
conditions
small RNA represses rpoS at a post-transcriptional level. We also discovered that OxyS
repression of rpoS translation requires Hfq and that the OxyS RNA binds the Hfq protein

Structural rearrangement of rpoS mRNA : Role of Hfq and small RNAs

Regulation of σS proteolysis

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids.

Stopping proteolysis is much more rapid than starting the transcription and translation of sigma
s.

Recognition and degradation of σS by the RssB/ClpXP proteolytic machinery

ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to
perform mechanical work during targeted protein degradation within cells. ClpXP
consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP).

Proteases with substrate-processing internal cavities

They are huge complexes that have many subunits
A protease is an enzyme that catalyzes (increases reaction rate or "speeds up")
proteolysis, breaking down proteins into smaller polypeptides or single amino acids.

Domain structure of complex ATP-dependent proteases (E. coli)

Sequence of events in the molecular recognition of σS for proteolysis

1. σS binds to RssB
2. σS undergoes a change of conformation that exposes its ClpX6-binding site near the N-
terminus
3. σS interacts with ClpX 6, i.e. a RssB- σS-ClpX6 complex is formed
4. ClpP14 joins the complex
5. RssB plays its second role , either in unfolding of σS or in threading σS into the ClpXP pore;
RssB is not dephosphorylated
6. Degradation of σS begins
7. RssB is released

The main components of proteolysis are the response regulator RssB, a specific σs recognition
factor, and the protease complex ClpXP, through which σs is degraded. After RssB has been
activated by phosphorylation (RssB-P), proteolysis is initiated by 1: 1 binding of RssB-P to the
turnover element, a helix in region 3 in σs. The binding catalyzes a conformational change in σs
which exposes the N-terminal ClpX binding site. After binding to ClpX, RssB-P is released and
σs is degraded by ClpXP in an ATP-dependent manner.
σS levels in RssB, arcA and arcB mutants
ArcB stimulates σS proteolysis in an ArcA-independent manner.
ArcB phosphorylates two response regulators: RssB as well as ArcA
ArcA competes with RssB for phosphorylation by ArcB (but reverse phosphate flow from
prephosphorylated RssB via ArcB to ArcA does not occur)
RssB is phosphorylated by ArcB with slower kinetics than ArcA.
ArcA represses rpoS transcription (but ArcB plays almost no role)
Two binding sites for ArcA-P were identified by Dnase I footprinting analysis: Site 1 overlaps
with a class I-activating cAMP-CRP site, site 2 is located just downstream of the transcriptional
start site ArcA may act as an anti-activator (at site 1) as well as a classical repressor (at site 2)
and may even form a ArcA-tetramer-bound DNA loop

ArcA is a transcription factor that binds to DNA and has 2 binding sites in the promoter region
for rpoS. So ArcA acts on transcription of rpoS.

High energy supply/ low oxygen: low rpoS transcription, rapid sigma s proteolysis

Low energy supply/ high oxygen: high rpoS transcription, reduced sigma s proteolysis

The histidine sensor kinase ArcB not only phosphorylates ArcA, but also the sigma(S) proteolytic
targeting factor RssB, and thereby stimulates sigma(S) proteolysis. Thus, ArcB/ArcA/RssB constitute
a branched "three-component system", which coordinates rpoS transcription and sigma(S) proteolysis
and thereby maintains low sigma(S) levels in rapidly growing cells.

Role of the ArcB/ArcA/ArcZ system in σS control

ArcB/ArcA/RssB downregulate σS (via proteolysis and transcriptional repression, respectively)
ArcZ is a small RNA that is mutually antagonistic with the ArcB/ArcA system (double negative
FBL produces switch-like behavior) and it also up-regulates σS (by stimulating rpoS mRNA
translation)
Regulation of σS expression, proteolysis and activity

Feedforward loops (FFLs) consist of three genes which code for three different transcription
factors A, B and C where B regulates C and A regulates both B and C.
Feedforward loops in the control of σS can be either transcriptional only, or combine different
levels of control (e.g. transcription and proteolysis)
Multiple feedback loops in the control of σS combine regulation at all possible levels and include
negative (homeostatic) as well as positive (switch-stabilizing) feedback

The reaction product of RssA, LPG, interferes with phosphorylation of ArcB and other SKs, and
thereby slows down σS proteolysis. RssA also is the first signaling phospholipase in bacteria!

ppGpp- has two functions in the regulation system: it binds to RNAP but by doing so it affects
the affinity for sigma s to bind to RNAP and also has an effect on the transcription or RpoS.

Planktonic, forms of microorganisms: Single cells High motility and low attachment

Biofilm, or sessile, forms of microorganisms: Aggregates Low motility and high attachment
Biofilm is a complex structure of microbiome having different bacterial colonies or single type of
cells in a group.

MIC= minimal inhibition concentration The lowest concentration of an antimicrobial capable of

stopping the growth of the tested microbial strain
MBC= minimal bactericidal concentration The lowest concentration of an antimicrobial capable
of killing the tested strain
MBEC= minimal biofilm eradicating concentration The lowest concentration of an antimicrobial
capable of killing the strain growing as a biofilm

• Sub inhibitory levels of antibiotics accelerate the emergence and spread of antibiotic-resistant
bacteria by selecting for resistance
Phage therapy
• Phage ability to disrupt biofilms where traditional antibiotics fail is a significant advantage
• Slow growth rate has been recognized as one of the major contributing factors to antibiotic
resistance of biofilm-associated bacteria
• Slow growing or non-dividing metabolically inactive cells give rise to persistent colonization

The ribosomal silencing factor RsfS slows cell growth by inhibiting protein synthesis during
periods of diminished nutrient availability.

The development of a biofilm is usually divided into five stages:

1. Reversible adhesion of planktonic cells to a surface with the help of fimbriae and surface
adhesions
2. Transition from reversible to irreversible adhesion
3. early stage of maturity
4. Development of microcolonies into a mature biofilm
5. Dispersion of cells from the biofilm back to the planktonic way of life

The biofilm matrix also offers protection against dehydration, oxidizing biocides, UV light and
protozoa as predators

Biofilm is natural way of living for bacteria

Biofilm are complex structures containing different components (i.e. polysaccharides, eDNA,
proteins)
Biofilm cells distinguishable from those of planktonic cells
Biofilm are more tolerant and resistant to antibiotics and the immune cells
“Biofilm” not virulence factor but a factor of virulence
We need new techniques to better understand the relationship between biofilm and
pathogenicity

Cyclic-di-GMP signaling and bacterial biofilm formation

Like many other bacterial species, E.coli cells can assemble flagella which provide the
structural basis for the motile single cellular planktonic life-style.

On the other hand, E. coli can express adhesins, amyloid curli fibers, and various
exopolysaccharides, e.g. cellulose, colanic acid and poly-GlcNAc (PGA), which provide the
basis for the sedentary and multicellular biofilm life-style.

• How is c-di-GMP produced and degraded?

Cyclic-di-GMP (c-di-GMP) is synthesized from two GTP molecules by diguanylate cyclase,
which contains a “GGDEF” protein domain. Cyclic-di-GMP is degraded by phosphodiesterases
that contain the “EAL” or “HD-GYP” domains.

Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle,
differentiation

Motility: expression and/or activity of flagella, swimming, swarming - Planktonic/single cellular

‘life-style‘
Virulence: expression of acute virulence genes (e.g. in Vibrio cholerae)
Adhesion/biofilm formation: synthesis of fimbriae (e.g. curli) and exopolysaccharides -
Sessile/multicellular ‘lifestyle‘ - also associated with chronic disease (e.g. in P. aeruginosa)
Cell cycle progression: Proteolysis of a replication inhibitor (C. crescentus)

Modular structure of GGDEF/EAL proteins

GGDEF and EAL domains can occur alone or in combination, and often are linked to N-
terminally located sensory domains (e.g. receiver, PAS, GAF, BLUF and other domains).
A majority of GGDEF/EAL proteins also carry transmembrane regions, i.e. are membrane-
located.

Structure and regulation of DGCs (diguanylate cyclase)

• GGDEF domains have to dimerize to synthesize c-di-GMP
• Dimerization can be stimulated by the sensory domain receiving a signal (e.g. PleD or WspR:
a REC is phosphorylated)
• c-di-GMP binding to the I-site triggers structural rearrangements that inhibit DGC activity
• An intercalated c-di-GMP dimer binds at the I-site

Structure of PDEs with EAL domains

Monomers of EAL domains bind a single c-di-GMP molecule
• EAL domains can dimerize
• Whether dimerization is essential for activity as a PDE is unclear

Multiplicity of GGDEF/EAL domain proteins

E. coli K-12 has 29 proteins with GGDEF and/or EAL domains with many different sensor
domains: • 12 DGCs • 13 PDEs • 4 ‘degenerate‘ proteins 19 are membrane associated
c-di-GMP binds as:
• an extended monomer: in degenerate EAL domain proteins
• an intercalated dimer: in some PilZ domain proteins and at I-sites of degenerate GGDEF
domains
• a bent monomer: in some PilZ domain proteins and in CRP-like transcription factors

Cellulose synthase: the BcsA subunit carries the active center and a C-terminal PilZ
domain
• Cellulose synthase consist of 2 membrane-integrated subunits: BcsA and BcsB
• BcsA contains 2 cytoplasmic loops: the GT active center and a PilZ domain
• Inhibition of the GT domain by PilZ is relieved upon c-di-GMP binding

What attracts c-di-GMP to PilZ?

The negatively charged phosphates of c-di-GMP and the positively charged Arginine residues

BcsA is the catalytic subunit that synthesizes cellulose and forms the TM pore across the inner
membrane and BcsB is a large periplasmic protein that is anchored to the inner membrane via a
single C-terminal TM helix

PelD: a degenerate GGDEF protein that still binds c-di-GMP

• PelD binds c-di-GMP at its still intact I-site
• PelD is membrane-inserted and controls the synthesis and secretion of the Pel
exopolysaccharide in Pseudomonas aeruginosa

c-di-GMP-regulated exopolysaccharide synthesis and secretion machineries

• Pel exopolysaccharide: PelD binds c-di-GMP via the intact I-site of a C-terminal degenerate
GGDEF domain
• Alginate: Alg8 is activated by Alg44, a separate c-di-GMP-binding protein with a PilZ domain
• Cellulose: BcsA ist activating by c-di-GMP binding to its C-terminal PilZ domain

• Poly-GlcNAc (PGA) synthase consists of membrane-integrated PgaC and PgaD

• A single c-di-GMP molecule ‘bridges‘ PgaC and PgaD and stimulates enzymatic activity and
formation of the PGA secretion channel

c-di-GMP control of a transcription factor: BldD in Streptomyces

• BldD dimerizes via two intercalated c-di-GMPdimers that connect the two C-terminal domains
• c-di-GMP-BldD is a global regulator that acts as a repressor for numerous sporulation genes
• c-di-GMP-BldD inhibits premature sporulation during vegetative growth

Role of ppGpp in the curli&cellulose control network

• reduces FlhDC expression
• stimulates σS expression
• favors σS activity by promoting binding of alternative sigma factors to RNAP core

Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator
CsgD.

c-di-GMP signaling in the curli & cellulose control network

• c-di-GMP inhibits flagellar rotation
• stimulates csgD transcription
• stimulates the activity of cellulose synthase
• E. coli K-12: 29 GGDEF/EAL proteins - 12 DGCs & 13 PDEs
-> blocks flagellar and stimulates c&c

• The transcription factor cascade σS/MlrA/CsgD drives production of curli subunits and DgcC
• c-di-GMP module A: PdeH and DgcE/PdeR/DgcM control MirA activity = switch to expression
of CsgD
• c-di-GMP module B: DgcC/PdeK locally control (i) cellulose synthase (BcsA) (ii) cellulose
modification (BcsEFG)

Control of curli & cellulose: • DgcE, DgcM • PdeH, PdeR

Control of cellulose: • DgcC
PdeR is the switch and acts as a trigger enzyme: • it directly interacts with and inhibits DgcM
(YdaM) and MlrA • it has enzymatic activity: binding and degradation of c-diGMP releases DgcM
& MlrA, which activate CsgD expression

• Spatial regulation of matrix production generates the complex supracellular architecture and
macroscopic morphogenesis of macrocolony biofilms
• Regulatory role of nutrient gradients, σS and c-di-GMP
• Local signaling I: In the core c-di-GMP switch the trigger PDE PdeR (YciR) acts as a c-di-GMP
sensitive inhibitor of a complex that activates csgD transcription and thereby turns on matrix
production (heterogeneous)
• Local signaling II: DgcC & PdeK in a complex with BcsA/BcsB control local c-di-GMP
concentration in close vicinity of the PilZ domain of BcsA

Role of transcription factor cascades in the curli&cellulose control network

• vegetative layer: FlhDC/σFliA flagellar cascade flagella production
• stationary phase layer: σS/MlrA/CsgD cascade curli & cellulose synthesis

Role of sigma factor competition for RNAP in the curli&cellulose control network
• vegetative layer: σ70/σFliA dominate RNAP activity (fellagela / nutrient)
• stationary phase layer: σS dominates RNAP activity (c&c / oxygen)

PDE
• major PDE of E. coli that maintains c-di-GMP levels low

PdeH
Involved in the control of the switch from cell motility to adhesion via regulation of cellular levels
of cyclic-di-GMP.
Part of a signaling cascade that regulates curli biosynthesis.
PdeH allows PdeR to inhibit DgcM/MlrA

DgcE: Signal input DgcE: Signal input into CsgD/matrix control

• DgcE is the top level DGC in the control of CsgD and matrix production
• DgcE is the antagonist of PdeH - thus, the trigger of matrix production
• DgcE is σS-dependent and thus stationary phase-induced
DgcE is a GGDEF/EAL protein that is involved in regulation of the switch from flagellar motility
to sessile behavior and curli expression

What controls DgcE activity?

gcE activity is controlled by an interaction of the RdcA GTPase with the MASE1 domain
• DgcE shows rapid turnover - regulation is highly dynamic

DgcE structure and functions of domains

• MASE1: membrane-intrinsic, essential for activity of DgcE
• PAS3: essential for activity
• GGDEF: produces c-di-GMP, essential for activity
• EALdeg: no PDE activity, inhibitory function for DgcE

Deleting the inhibitory EAL domain promotes oligomerization and stabilization of DgcE - these
oligomers are probably the active form of DgcE
Mutation in any domain besides EAL causes a null phenotype= like a deletion of the entire gene.
When eliminating EALdeg there's even more matrix ,the colony is stiff

PDE activity is responsible for degradation of mRNA after it had been translated to protein and
of DNA during apoptosis.

Biofilm formation is exquisitely controlled by c-di-GMP in E. coli (and most bacteria)

The system in the lab is called macrocolonies Biofilm (that grow on agar plates). It produces an
extracellular matrix, and this matrix is arranged in a very complex architecture which has a
functional role.

“dense brickwork“ (curli&cellulose) most oxygen least nutrients

“vertical pillars“ (cellulose)
“loose horizontal network“ (curli)
Flagella - most nutrients least oxygen

C-di-GMP signaling:
Basic principles principles and its role its role in building building the 3D matrix
architecture
???

There are two kinds of enzymes that kind that makes c-di-GMP from 2 GTP (GGDEF domain
proteins) and the one that degrades… those enzymes are controlled by the N terminal sensor
domains.these sensor domains are very different, while the other domains (GGDEF EAL HD-
GYP) are highly conserved, the input signal domains are very… those enzymes are controlled
at the level of expiration and at level of activity. It’s very dynamic because it's always produced
and degraded at the same time.

The matrix production is under control of the stationary stage sigma factor - sigma s, including
transcription factor MirA

GTP binding to RdcA inhibits its interaction with DgcE

RdcAB and DgcE link GTP and GTP and c-di-GMP signaling
Model:
• High GTP in growing cells interferes with DgcE activation by RdcA/RdcB
• RdcA acts as a GTP sensor and GTP binding inhibits RdcA/DgcE interaction
• RdcB may be a GTPase-activating factor
• Kd(GTP) and GTPase activity may be fine-tuned to differentially sense 0.1-1 mM GTP

The DsbA/DsbB system promotes DSB formation in PdeC

What are the natural reducing/activating conditions for CSS-PDEs? Low O2?
“It could be conditions where we have low oxygen because oxygen helps to withdraw
electrons (and the dsb system is connected to the respiratory chain). So if there's a lot
of oxygen then the elimination of electrons from the periplasm works much more
efficiently.”

Proteins are made in the cytoplasm - how do they reach extracytoplasmic

compartments?
All of these proteins are made inside the cell, many of them are hydrophilic and they
have to cross at least one membrane (which is a lipid membrane) so they would never
go through spontaneously. (The hydrophobic proteins could go through spontaneously)

Exported proteins have signal sequences

The bacterial protein export machinery

I. Membrane-integrated system with cytoplasmic targeting chaperones
II. Soluble Targeting system

bacteria faces the triple barrier of transporting the polypeptide first across the inner membrane
(IM), then through the periplasmic space, and finally across the outer membrane (OM). The
general secretory pathway (GSP) exports proteins carrying an amino-terminal signal sequence in
a stepwise manner, across the IM first, and then across the OM. Proteins secreted via the
different terminal branches of the GSP require the Sec system to cross the IM but use different
approaches to get through the OM. The Sec-independent pathways are able to transfer proteins
directly from the cytoplasm to the outside of the bacteria.

Identification of sec mutations:

I. Selections based on LacZ fusion proteins
II. Selections based on regulation of secA

Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal
peptide from proteins that are secreted across the bacterial cytoplasmic membrane. It is an
essential membrane bound enzyme whose serine/lysine catalytic dyad resides on the exo-
cytoplasmic surface of the bacterial membrane.

SecB- small protein in the cytoplasm (not the membrane) and forms a tetramer, binds into the
protein backbone
SecA- large protein that has several domains , it has 2 binding sites for ATP, it’s ATP
depended. It binds at the signal squence

SecA cotranslationally recognises nascent Sec substrate proteins as they emerge from the
ribosome by virtue of an internally encoded targeting signal. SecA may then recruit SecB to the
substrate protein or deliver the protein directly to SecYEG. Alternatively, a subset of substrate
proteins may be recognised by SecB and delivered to the Sec machinery through the interaction
of SecB with SecA , which ultimately delivers the protein to SecYEG . Incorporation of the signal
sequence into the lateral gate of SecYEG may serve as a final quality control step to prevent the
translocation of proteins with signal-sequence-like regions in their primary structure .

What do signal recognition particles recognize?

The signal recognition particle (SRP) is an abundant, cytosolic, universally conserved

ribonucleoprotein (protein-RNA complex) that recognizes and targets specific proteins to the
plasma membrane.
SRP targets hydrophobic proteins into the cytoplasmic membrane

The Sec system utilizes two different pathways for secretion: the SecA and signal recognition
particle (SRP) pathways. SecA is an ATPase motor protein and has many related proteins
including SecD, SecE, SecF, SegG, SecM, and SecY. SRP is a ribonucleoprotein (protein-RNA
complex) that recognizes and targets specific proteins to the endoplasmic reticulum in
eukaryotes and to the cell membrane in prokaryotes.
The two pathways require different molecular chaperones and ultimately use a protein-transporting
channel SecYEG for transporting the proteins across the inner cell membrane In the SecA pathway,
SecB acts as a chaperone, helping protein transport to the periplasm after complete synthesis of the
peptide chains. Whereas in the SRP pathway, YidC is the chaperone, and transports proteins to the
cell membrane while they are still undergoing peptide synthesis.
SRP pathway
SRP competes with TF and binds to the N-terminal signal sequence. Proteins from the inner
membrane stops the process of chain elongation. The SRP then binds to a membrane receptor,
FtsY. The peptide chain-SRP-FtsY complex is then transported to SecY, where peptide elongation
resumes.

SecA or post-translational pathway

Proteins are synthesized in ribosomes by a process of serially adding amino acids, called
translation. In the SecA pathway, a chaperone trigger factor (TF) first binds to the exposed N-
terminal signal sequence of the peptide chain. As elongation of the peptide chain continues, TF is
replaced by SecB. SecB specifically maintains the peptide in an unfolded state, and aids in the
binding of SecA. The complex can then bind to SecYEG, by which SecA is activated by binding with
ATP. Driven by ATP energy, SecA pushes the protein through the secYEG channel. SecD/F
complex also helps in the pulling of the protein from the other side of the cell membrane

Bacterial protein excretion systems

Type I secretion: the TolC-MacAB complex,• Via specialized inner membrane components,
secretory proteins are delivered to TolC.A TolC trimer ‘bridges‘ the periplasm acting as a protein
channeling ‘cage‘
Type III secretion: produces external structures that deliver proteins - flagella and infection
needles (Gram-negative pathogens)
Type II secretion: secretion and release of exoenzymes (which may go over the inner
membrane via the Sec or the Tat system)
Type IV secretion: produces pili of diverse functions (e.g. DNA transfer; twitching pili, etc.)

Autotransporters (type V)
‘Autotransporting‘ proteins are transferred over the cytoplasmic membrane by the Sec
system
They have C-terminal domains that fold into beta-barrels in the outer membrane and transfer
the N-terminal part of the protein to the cell surface
The N-terminal part can be cleaved off or remain attached
The domain is always located at the C-terminal end of the protein and forms a beta-barrel
structure.
‘Autotransporting‘ proteins often are adhesins (in pathogenic bacteria) Example: Invasin in
Yersinia

The reduction potential Eo‘

Reduction potential (also known as redox potential, oxidation/reduction potential, or Eh)
measures the tendency of a chemical species to acquire electrons and thereby be reduced.
Reduction potential is measured in volts (V) or millivolts (mV).

• The reduction potential Eo indicates the tendency of a compound to release electrons relative
to 2H+/H2 at standard conditions (1M)
• Biologically relevant: Eo‘ (at pH 7, i.e. with [H+]=10-7 M)
• More negative Eo‘: good electron donor (e.g. H2 at pH7: Eo‘ = -0.42 V)
• More positive Eo‘: good electron acceptor (e.g. O2: Eo‘ = +0.82 V) • Unit of Eo‘: Vol

Reduction potential and the electron ‘tower’

• The ‘tower‘ represents the range of reduction potentials possible for redox couples in nature.
• Redox couples with the most negative E0' are at the top, those with the most positive E0' at
the bottom.
• Energy released by a redox reaction: ΔGo = -nF(Eo acceptor - Eo donor)

Transfer of electrons and protons in the RC

• The components of the RC are arranged in a series in/at the cytoplasmic membrane that
corresponds to their reduction potential
• The components of the RC that take up electrons and protons (flavoproteins, quinones),
transfer these protons across the membrane and generate a H+ gradient
• Cytochrome oxidase is also a proton pump
• high nutrient/low O2: RC is reduced
• low nutrient/high O2: RC is oxidized
the redox state of the RC can be sensed by specific redox sensors!

Electron carriers in the RC: role of FAD/FADH2

Flavoproteins contain flavins as electron carrier:
FMN: Flavin mononucleotide
FAD: Flavin adenine dinucleotide
Flavins take up or release both electrons and protons FAD is also a common cofactor of other
redoxactive enzymes

Electron carriers in the RC: iron-sulfur proteins

• There are Fe2S2 as well as Fe4S4 clusters, linked to cys side chains (-SH) of the proteins
harboring the Fe/S clusters
• Fe/S clusters take up or release electrons only
• Fe/S clusters also occur in many other redox sensing or redoxactive proteins

Electron carriers in the RC: quinones

• Quinones take up or release both electrons and protons
• Quinones are relatively small molecules with hydrophobic side chains
• Quinones are freely diffusible in the cytoplasmic membrane
• Bacteria express different quinones with different reduction potential, e.g. ubiquinone or
menaquinone
• Quinones participate in many redox-signaling processes

Quinones function as electron transport cofactors in photosynthesis and cellular respiration.

Oxygen can generate toxic radicals

Cellular problem: Macromolecules (DNA, proteins, lipids, etc.) get oxidized and functionally
damaged

• Oxygen radicals are formed as side products of aerobic respiration (incomplete reduction of
oxygen)
• Superoxide anione is generated by redox cycling compounds (paraquat, plumbagin)
• Hydroxyl radical is generated from hydrogen peroxide by the Fenton reaction: Fe++ + H2O2 +
H+ Fe+++ + OH° + H2O

H2O2-dependent oxidation of proteins at cysteine

• 1. step: formation of sulfenic acid as a reactive group
• formation of intraor intermolecular DSBs
• formation of glutathion adducts
• further oxidation to sulfinic and sulfonic acid
• formation of sulfenamide in the protein backbone

When proteins get oxidized in the cytoplasm, they are reduced again by the GRX and TRX
systems

DsbA/DsbB oxidize periplasmic proteins

• DsbA and DsbB introduce DSBs into periplasmic proteins
• involves a series of thiols/DSBs in DsbA and DsbB themselves
• electrons are transferred to the RC via the quinones (ubiquinon)
The periplasm is an oxidizing environment
The cytoplasm is a reducing environment

DSB generating reaction center of DsbB

Electrons are successively transferred from Cys44 and Cys41 in DsbB to a quinone bound into
the adjacent cleft with the help of the positive charge of Arg48

DsbD/DsbC/DsbG can reduce periplasmic proteins

• DsbA/DsbB can also introduce wrong DSBs into periplasmic proteins
• additional DSBs or sulfenic/sulfinic/sulfonic groups are also generated by oxidative stress
(ROS)
A reducing ‚repair‘ system is required: DsbD/DsbC/DsbG
• DsbD reduces either DsbC or DsbG to free thiol cysteins, which then reduce distinct groups of
mis-oxidized proteins

Structure and function of DsbD

• DsbD has three domains (α, β, γ) - all have cysteins that serve as a transmembrane electron
shuttle
• TRXred reduces C163 and C285 in the membran-inserted β domain of DsbD
• these free thiols donate electrons to the C461/C464 in the periplasmic γ domain, from which
electrons are transferred to either DsbC or DsbG

Structure and function of DsbC and DsbG

• DsbC and DsbG are homologs with very similar dimeric structures and redox-active CxxC
motifs at the same positions
• DsbC and DsbG have large clefts of different size between their monomers, which may play a
role in recognition of different subsets of substrate proteins

Structural and sensory/regulatory DSBs in proteins

• Redox potentials of DSBs/free thiol pairs in proteins range from -0.45 V to approx. -0.10 V
• Structural DSBs in proteins arise from very negative redox potentials and therefore are very
stable
• Sensory/regulatory DSBs/free thiols in proteins have less negative redox potentials and
therefore are reversible, i.e. can switch between oxidized and reduced state
DSB/free thiol-based redox sensing: OxyR
• OxyR is a H2O2 sensor that gets oxidized by H2O2
• Oxidation changes the conformation of the OxyR tetramer and its binding at the DNA (note:
both reduced and oxidized conformation do bind to its specific binding sites, but differently)
• Only OxyRox can activate target genes (e.g. catalase genes)

Although functioning as an activator, E. coli OxyR proteins in both oxidized and reduced forms
possess DNA binding activity for a conserved binding motif comprising four regularly spaced
ATAG elements OxyR is composed of a helix-turn-helix DNA binging domain (DBD) in the N-
terminus and a regulatory domain in the C-terminus where lies the two conservative cysteine
residues .

DSB/free thiol-based redox sensing: ArcB

• ArcB is a membraneanchored sensor kinase that phosphorylates ArcA and RssB response
regulators
• ArcB gets oxidized by oxidized quinones • intermolecular DSB formation distorts the ArcB
dimer and inhibits its kinase activity
• oxidized quinones accumulate at low energy and/or high oxygen levels
ArcA and RssB are not phosphorylated, i.e. inactive

Fe/S cluster-based redox sensing: SoxR

• SoxR is a ‘One-component regulators‘: sensor and regulator function in one protein
• Its 2Fe/2S cluster becomes oxidized by redox cycling compounds (e.g. paraquat), which in
parallel also generate superoxide anion
• SoxRox can activate transcription of soxS (and other genes) • SoxS then activates expression
of many oxidative stress response genes

Transmembrane signaling by CSS domain PDEs: a novel periplasmic redox sensor controls
cytoplasmic enzymatic activity
PDE activity of CSS-PDEs regulated by the redox state of the CSS domain

The ligand binds to a site on the repressor or activator thus activating or deactivating itHow
does SPoT regulate PPPGPP
ACP accumulation (Fatty acid synthase stall?) -> ACP binds to SPoT to cause conformational
change, allowing synthase domain to become active makes and accumulates PPPGPP.
No starvation, regulatory domain blocks synthase domain.

When is SPoT hydrolase domain active?

When there is no starvation, degrades pppgpp

How does RelA synthesize PPPGPP

Amino acid starvation-> uncharged tRNA-> stalling ribosome-> Signals RelA to bind to
ribosome-> transforms to active state for synthesis and accumulation of pppgpp-> once
accumulated, RelA is released

How does PPPGPP directly affect transcription?

PPPGPP interacts with RNA pol directly with help of DKSA to further destabilize open complex
at GC rich discriminator regions, down regulating sigma70 binding. Sigma70 binding stabilized
at AT rich regions for synthesis of amino acids.

How does PPPGPP indirectly affect transcription?

PPPGPP promotes alternate sigma factors to interact with RNApol. Alt sigma factors can out
compete sig70s.

How is RpoS regulated during translation?

The 5'-UTR of its mRNA folds back on itself to create a stem loop. The folded mRNA blocks the
RBS and is a target degredation by RNaseIII. DsrA,sRNA,and Hfq can reopen the stem loop to
prevent degredation. OxyR sRNA can sequester Hfq.

How is degradation of RpoS regulated?

In stationary phase and in stress conditions, stable Rpos can bind free core RNA pol and is
protected of degradation-> transcription of RpoS dependent genes.
In exponential phase, RSSB delivers RpoS to ClpPX protease. ArcA/B phosphorylate RSSB for
interaction with RpoS.

How does PPPGPPP help RpoS outcompete sig70?

PPPGPP can increase transcription of RSD which sequesters sigma 70

Describe the RNAP switch model

During stringent response, RNApol is redirected to transcribe genes of RpoS regulon. During
growth, PPPGPPP is absent and does not interact with RNApol.

gene expression regulation:

Alterations of DNA sequence,Control of transcription,Control of mRNA stability, Translational
control, Post-translational control

How does a cell regulate sigma factors? What is the purpose of sigma factor regulation?
Sigma factor regulation is more effective in regulating transcription than using activators and
repressors. Transcription occurs when a sigma factor recognizes a promoter. If there is no
sigma factor present to recognize a promoter, then transcription cannot occur. Because there
are specific sigma factors that recognize specific promoters that lead to the synthesis of specific
genes, controlling the presence of certain sigma factors can regulate transcription of certain
genes. Temperature (and the heat shock stress response) is one example of how the cell
regulates sigma factors

DgcE activity
At high cellular GTP levels, the RdcA/RdcB complex is predominantly in the GTP-bound form
that is unable to interact with the MASE1 domain of DgcE, whose GGDEF domain remains in
the monomeric and enzymatically inactive form since the C-terminal EAL domain counteracts
dimerization via the PAS3 region. B: During entry into stationary phase, the cellular GTP level is
decreased substantially, the RdcA/RdcB complex is predominantly in the nucleotide-free form
that binds to the MASE1 domains of two DgcE molecules, which promotes an alignment of the
PAS3 and GGDEF domains, thereby overcoming the anti-oligomerizing effect of the EAL
domain of DgcE. For simplicity, the c-di-GMP production-promoting forms of both the
RdcA/RdcB complex as well as DgcE are depicted here as dimers, but these may also form
higher order oligomers (as suggested by immunoblot data in Fig 3). C-di-GMP produced by the
GGDEF domains is bound and degraded by the trigger PDE PdeR, thereby relieving inhibition
imposed by PdeR onto DgcM and the transcription factor MlrA, which results in the initiation of
csgD transcription by the DgcM/MlrA complex.

TATA box is a sequence of DNA found in the core promoter region of genes in archaea and
eukaryotes. The bacterial homolog of the TATA box is called the Pribnow box which has a shorter
consensus sequence. The TATA box is considered a non-coding DNA sequence

Phosphorylation- Chemical reaction in which phosphate is transferred from ATP (or other donor)
to another molecule.

Three components of Sec pathway

1) SecYEG
2) Cytoplasmic chaperone Sec B
3) ATPase Sec A

SecYEG: heterotrimer (conserved in eukaryotes), forms pore conducting channel (8.5nm

diameter)

Sec B: Cytoplasmic chaperone - binds to proteins post-translationally - protect hydrophobic from

the aqueous environment
-can't aggregate
-can't fold otherwise wouldn't ft through pore
Targeting function to SecYEg with help of SecA

SecA
Unique to bacteria
helps recruit proteins to SecYEG
ATPase - provides energy to push the polypeptide through the translocon by ATP hydrolysis

The twin-arginine translocation pathway (TAT pathway) is a protein export, or secretion pathway
found in plants, bacteria, and archaea. In contrast to the Sec pathway which transports proteins
in an unfolded manner, the Tat pathway serves to actively translocate folded proteins across a
lipid membrane bilayer

σS turnover requires ClpXP and the response regulator RssB, whose phosphorylated form
exhibits high affinity for σS. the RssB/ClpXP system involves two distinct regions in σS. Region
2.5 of σS (a long α-helix) is sufficient for binding of phosphorylated RssB. However, this
interaction alone is not sufficient to trigger proteolysis. A second region located in the N-terminal
part of σS, which is exposed only upon RssB–σS interaction, serves as a binding site for the
ClpX chaperone. RssB plays a second role in the initiation of σS proteolysis that goes beyond
targeting of σS to ClpX, and suggest a model for the sequence of events in the initiation of σS
proteolysis.
The N-terminal region of σS contains an element that interacts with ClpX.

Cellular levels of c-di-GMP are controlled by enzyme classes that have antagonistic activities,
diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that degrade c-di-GMP.
c-di-GMP controls cellular processes at the transcriptional, translational and post-translational
level, and through an increasing number of c-di-GMP-binding proteins and riboswitches.
During biofilm formation, c-di-GMP levels increase as a result of σS (also known as RpoS)-
induced expression of DgcE (formerly known as YegE)42 and other DGCs, and the consecutive
downregulation of the PDE PdeH

since most cellular compartments are reducing environments, in general, disulfide bonds are
unstable in the cytosol. Disulfide bonds in proteins are formed between the thiol groups of
cysteine residues by the process of oxidative folding. The other sulfur-containing amino acid,
methionine, cannot form disulfide bonds.

The disulfide bond stabilizes the folded form of a protein in several ways:
1. It holds two portions of the protein together
2. The disulfide bond may form the nucleus of a hydrophobic core of the folded protein
3. increases the effective local concentration of protein residues, and lowers the effective local
concentration of water molecules.
Disulfide bonds play an important protective role for bacteria as a reversible switch that turns a
protein on or off when bacterial cells are exposed to oxidation reactions. Hydrogen peroxide
(H2O2) in particular could severely damage DNA and kill the bacterium at low concentrations if
not for the protective action of the SS-bond.
As disulfide bonds can be reversibly reduced and re-oxidized, the redox state of these bonds
has evolved into a signaling element.
disulfides play a significant role on redox state regulation of Two-component systems (TCSs)

In Gram-negative bacteria, proteins that require disulfide bonds for their final folded state are
translocated across the cytoplasmic membrane into the periplasm, where enzymes that catalyze
the formation and isomerization of disulfide bonds reside.

The oxidation of a cysteine pair into a disulfide bond in the substrate protein by DsbA results in
the consequent reduction of the redox-active site cysteines in DsbA. The active site cysteines in
DsbA are restored to their oxidized active state by the inner membrane protein, DsbB, that
channels electrons to the respiratory chain via its interaction with the quinone pool.

The analysis of PdeC as one of five CSS-PDE in Escherichia coli K-12 showed that the
formation of a disulfide bridge between the conserved cysteines is catalyzed by the oxidizing
DsbA/DsbB system and leads to a reduction in the enzymatic activity of the EAL domain. In
contrast, the reduced free thiol form of the CSS domain results in greatly increased PDE activity
associated with dimerization across TM2. The reduction of the CSS domain also leads to
processing by the HtrA proteases DegP and DegQ into a membrane-bound fragment of
TM2+EAL domain with high enzymatic activity. Degradation by DegP and DegQ in the
periplasm is very efficient.

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