INDIAN AGRICULTURAL RESEARCH INSTITUTE

NEW DELHI-110012
DIVISION OF MICROBIOLOGY

SEMINAR
ON
Properties and functions of Bacterial protein networks

SEMINAR LEADER SUBMITTED BY

Dr.RAJEEV KAUSHIK ABIRAAMI.T.V(20700)
Introduction:

 In the past two decades, molecular cell biology has experienced a dramatic
paradigm shift.
 Whereas the focus had traditionally been on single proteins, it has now
become clear that most cellular functions in bacteria, as in eukaryotes, are
executed by multiprotein complexes or groups of dynamically interacting
proteins.
 Protein interactions within and between these complexes or groups can be
holistically viewed as protein networks.
 As bacterial protein networks are less complex but more diverse than their
plant or animal counterparts at the cellular level, studying their structure and
properties can reveal both conserved and unique principles of network
organization.

Structure and properties of protein networks

Overall connectivity:

 Cellular networks can be represented and visualized in many different ways.

 In protein networks, nodes depict proteins, and edges represent
interactions, be they direct (physical) or indirect (genetic or functional).
 Most networks share basic structural properties across organisms, such as a
large number of nodes with few connections, and a relatively small number
of highly connected nodes — so-called hubs.
 Hubs tend to be conserved and are often essential for the stability of the
protein network and the survival of the organism, which makes them
attractive drug targets.
 Two classes of hubs have been proposed: 'party' and 'date' hubs.
 Party hubs have multiple docking domains or binding interfaces to
simultaneously interact with their binding partners and organize multiprotein
complexes.
 By contrast, date hubs have mutually exclusive partners, which allows them
to be involved in different processes. Such exclusion can occur at a temporal
level
Modularity:
 An integral property of biological networks is modularity.

 Within protein networks, functional modules are well-connected groups of

proteins acting as units that execute specific autonomous functions.

 Typically, functional modules are only weakly connected with each other.
Nevertheless, modularity can be seen at multiple levels, and some modules
are further organized into higher-order modules or divided into sub-modules
(Wagner, G et.al.2013).
 Modularity is prominent in bacterial protein networks and may be a natural
consequence of evolution.
 Bacterial protein networks evolve primarily through gene duplication and
diversification, or through horizontal gene transfer and subsequent
adaptation of gene function in the new genomic context.
 Consistent with this, functional modules tend to be conserved across species,
but the wiring between them changes(Hartwell, L. H et.al. 1999).
 The maintenance of modularity in prokaryotes is further enhanced by the
organization of genes into operons that can be transferred to a new host as an
entire unit.

Crosstalk and sharing components.

 Despite the modular structure of protein networks and the tendency to sup-
press undesirable crosstalk between modules, individual modules are not
completely insulated from each other.
 A certain degree of interconnection is necessary for coordinating cellular
functions .
 An example of this trade-off between specificity and crosstalk can be seen in
bacterial two-component signalling systems.
 Bacteria usually possess multiple homologous two-componentsystems,
creating a potential for crosstalk, during which a sensory kinase of one
system could phosphorylate not only its cognate response regulator but also
response regulators of other systems.
 Such nonspecific crosstalk is prevented in vivo through several mechanisms,
including the specificity of interactions between a kinase and its response
 regulator, substrate competition, dephosphorylation of the response
regulator, and the physical coupling of kinase and response regulator within
the same protein( Goulian, M.2010)
 In addition to crosstalk via signalling or PPIs, distinct network modules can
be interconnected by the sharing of individual components.
 This increases network complexity without increasing the number of
constituent proteins. (Rajagopala, S. V. et al.2014)
 Sharing can be used to ensure the spatial or temporal coordination of two
processes (see below about cell cycle protein machineries) and/or to
facilitate resource management and co-regulation — for example, all iron
forms that are chelated by siderophores are recruited by different outer-
membrane receptors on the cell surface of E. coli, but a shared complex in
the inner membrane (the ExbBD–TonB complex) is used for siderophore
import into the cell.
 Finally, sharing also enables the reuse of the same core components for non-
overlapping physiological processes; this is exemplified by the use of a
common motor (termed Agl) in Myxococcus xanthus for both sporulation
and gliding motility .

Dynamics and scaffolding.

 Network modules can include stable and transient PPIs.

 Protein network hubs seem to be typically stable complexes (that is, party
hubs).
 Although this observation is partially due to the biases of mapping
techniques (which are better suited to the identification of stable
interactions), some of the most conserved protein machineries, such as DNA
and RNA polymerases and the ribosome, have stable core components.
Stable multiprotein complexes often contain extensive interfaces, which
make them robust to genetic variation and environmental fluctuations.
 In some cases, stable complexes are scaffolded by specialized proteins or by
cytoskeletal elements.
 An extreme case of scaffolding is the emergence of specialized subcellular
compartments in bacterial cells, such as carboxysomes
 In addition to stable cores, protein machineries contain transiently associated
regulatory components, and the association and dissociation of these
 components is often controlled in a spatiotemporal manner by small
molecules or post-translational modifications.
 Such dynamic PPIs facilitate crosstalk between functional modules, allowing
for plasticity in the network.
 Dynamic associations can also make the function of the module less
sensitive to fluctuations in the levels of its constituents, as is the case for the
essential peptidoglycan synthase, penicillin-binding protein 2 (PBP2), in E.
coli cell elongation: E. coli can maintain its growth rate even with only half
of the PBP2 molecules present (Typas, A et al .2012 )

Robustness
 Bacterial protein networks are constantly exposed to perturbations, such as
variations in protein levels or physicochemical changes in the extracellular
environment.
 The most common source of intracellular perturbation is the inherent
stochasticity of gene transcription, or gene expression noise, which can pro-
duce large cell-to-cell variation in protein levels across a population of
genetically identical cells(Raj, A.2008).
 Such variation can occur at the level of individual genes (intrinsic noise) or
result from cell-to-cell fluctuations in common transcription factors and
general components of the transcription machinery that control the
expression of multiple genes (extrinsic noise).
 The expression noise is generally higher for genes that are expressed at a
low level, but it also depends on promoter architecture, such as the number
and strength of transcription factor binding sites, and thus can be
evolutionarily tuned independently of the mean level of gene expression.
 Although microorganisms can use noisy gene expression to their advantage
for the control of specific processes (such as sporulation) and to generate
bet-hedging strategies at the population level, such noise is usually
detrimental and needs to be buffered.
 Networks involved in signalling, metabolism and growth have thus evolved
specific mechanisms to ensure robustness of their output against stochastic
variations in protein levels, but also against genetic (mutations) or
environmental (for example, temperature) perturbations.
 In bacteria, network robustness is facilitated by the organization of genes with
common functions into operons, and by further grouping of related operons
under the same transcriptional control into regulons.

Protein – protein interaction detection methods

1. Genetic approaches - Yeast two hybrid system
2. Biochemical approaches - APMS
Affinity Purification coupled to Mass Spectrometry

Bacterial and yeast two-hybrid (B2H and Y2H) techniques:

 Commonly used in vivo methods for protein–protein interaction (PPI)

mapping.
 Both are based on protein fragment complementation, in which one fragment
of a modular protein is fused to a prey protein, and the other fragment is
fused to a bait protein.
 If the prey and bait proteins interact, the modular protein is reconstituted and
regains function, which is usually coupled to a reporter gene (such as GFP)
or growth.

Disadvantage:

 As an ex vivo system for bacteria, Y2H screens can capture only direct
pairwise interactions, including those of low stability.
 However, such an exogenous system comes with caveats, such as non-
physiological expression levels, a lack of necessary ligands or chaperones
and problems with the translocation of bacterial proteins to the yeast
nucleus, among others(Weimann, M. et al.).
 This results in low sensitivity (<0.3 in all cases reported), which means that
even in comprehensive screens
 >70% of PPIs remain undetected.
 Specificity, which was initially considered a problem118, is less problematic
nowadays, especially for genome-wide screens.
 Recently developed approaches enable the coupling of Y2H screens with
next-generation sequencing for systematic profiling of domain–domain
interactions or for mapping membrane interactomes.
B2H SYSTEM

 As B2H methods are performed in their natural environment, they detect

both direct and indirect interactions.
 The adenylyl cyclase assay is based on reconstitution of the catalytic domain
of the Bordetella pertussis toxin adenylyl cyclase and subsequent activation
of a cyclic AMP-dependent promoter; this is the most commonly used B2H
technique and is suitable for detecting interactions between membrane
proteins.( Battesti, A et .al..2012)
 Although it has never been applied at a global level for robust estimates of
its sensitivity and specificity, multisubunit protein machineries such as the
divisome have been successfully dissected using this system.
 Finally, high-throughput reverse genetics approaches can be used to infer
PPIs.
 Quantitatively measuring gene–gene (genetics) and gene–drug (chemical
genetics) interactions on a large scale is a powerful approach for linking
proteins to pathways, complexes or functional modules.
Biochemical approaches :

 In contrast to in vitro biochemical approaches, global biochemical

approaches are almost exclusively carried out in vivo.
 The most widely used method is affinity purification coupled to mass
spectrometry
 In this method, bait proteins are usually fused to a tandem AP (TAP) tag
and sequentially purified to remove prey proteins that are nonspecifically
attached(Gavin, A. C et.al.2011).
 In addition to the original TAP tag, which consists of a calmodulin-binding
peptide (CBP) and a protein A separated by a tobacco etch virus (TEV)
protease site, many variations exist today, such as the SPA tag, which was
developed for bacteria and contains three FLAG sequences instead of the
protein A.
 In AP–MS assays, interactions are probed in their natural environment, and
proteins are typically expressed at endogenous levels.
 In contrast to Y2H, AP–MS also captures co-complex associations, but fails
to detect many transient and weak PPIs. False positives can be filtered out
by different data analysis approaches all of which are geared towards
identifying promiscuously binding proteins.
  As with Y2H, false negatives are more of an inherent problem of the
methodology, in this case owing to harsh washing steps, two purification
steps, low expression of bait proteins and the use of tags that obstruct
interactions.

Protein Interaction Databases

There are many softwares available to detect the protein- protein

interaction.Some of them are
1. Database of interacting proteins (DIP)
2. Molecular Interaction (MINT) database
3. Biomolecular interaction network database (BIND)
4. Biological general repository for interaction datasets (BioGRID)
5. Human Protein Interaction Database (HPID)
6. information Hyperlinked over Proteins (iHOP) database
7. Suiseki database
THE CHEMOTAXIS NETWORK

 Y2H and FRET most widely used ( but false positives CheR-CheZ and Tar-
CheY can occur)
 AP-MS INEFFICIENT
 Hub-less network - Rotary engine module-Proper assembly of structural core
assembly (Shimizu, T et .al. 2010)
 Hub-driven Hub
Export apparatus - FlhA
Chemotaxis system - CheA integrate multiple inputs and
coordinate the dynamic processes involved in protein export or
signal transduction
CELL CYCLE NETWORK

 Y2H made little contribution

 B2H & FRET have contributed much - Needed periplasmic approach
 Hierarchial manner - FtsQ and MreC acts like hub
 Downstream component feedback on dynamics
 Bacteria maintain reasonably constant growth rate and have reproducible
shape passing to progeny. This is due to ROBUSTNESS
 Extensive redundancy – Functions even when cell is not have essential
component of network when the compensatory mutations occurs in genes
 encoding them. Sharing Peptidoglycan growth and remodeling
enzymes, cell wall modifying enzyme
 Crosstalk : MreB and FtsZ ,PBP2 and PBP3
 Transient association of PBP and elongation machinery (Szwedziak, P et
al 2013)
 PBP2 move between different complexes.Contribute to peptidoglycan
synthesis at several sites .Even if PBP2 level
or halved ,E.coli maintains its growth rate
 Recently, a number of proteins controlling Z ring dynamics in response to
stress stimuli (such as DNA damage) and/or metabolic cues have been
identified
 For example, the DNA damage-induced protein SulA directly inhibits Z
ring assembly, and OpgH, an enzyme that uses UDP-glucose to produce
periplasmic glucans in E. coli, can function as a moonlighting enzyme in
its UPD-glucose-bound form to block Z ring assembly by sequestering
FtsZmonomers.
 CASE STUDY I

Introduction:

 In bacteria, the predominant family of signaling proteins is the

two-component signal transduction system.
 These signaling pathways typically consist of a sensor histidine kinase (HK)
that autophosphorylates and then transfers the phosphoryl group to a
cognate response regulator (RR) that can effect changes in cellular
physiology or behavior.
 Faithful transmission of information and avoidance of detrimental crosstalk
exquisite its specificity and how a single bacterial cell coordinates many
highly related signaling pathways and prevents crosstalk remains a major
challenge.
 Basis of specificity,molecular recognition understanding can be help in
rewiring of this specificity
Objectives

 To examine patterns of amino acid coevolution in large, multiple sequence

alignments of cognate kinase-regulator pairs.
 To find the subset of the coevolving residues is sufficient, when mutated, to
completely the switch the substrate specificity of the kinase EnvZ.

Results:

Specificity of phosphotransfer residues in dhp domain of histidine kinase

 Histidine kinase and Response Reulator

 EnvZ - OmpR ,CC1181 - CC1182 , RstB - RstA
 Fusion of DHp domains of CC1181 (from Caulobacter crescentus) and the
CA domain of E. coli EnvZ have a response regulator of CC1182
 CC1181 of DHp domain-EnvZ of CA domain - CC1182
 Fusion of DHp domains of RstB (from E. coli) to the CA domain of E. coli
EnvZ have a response regulator of RstA
 RstB of DHp domain-EnvZ of CA domain - RstA

This indicates phosphotransfer residues in DHp domain of histidine kinase

Analysis of subdomain chimeric histidine kinase

 Covariation analysis-7 residues within DHp domain

 Subdomain chimera is made .
 EnvZ – OmpR. RstB - RstA ,Chim 1 - Rst A not to OmpR
 They found that within 7 residues within DHp domain is responsible for
specificity
Rewiring histidine kinases in vitro by mutating
specificity-determining residues
 Mutating only the residues would be able to change the substrate
selectivity of EnvZ
 Three residue sufficient to change the substrate preference of EnvZ
to that of RstB
Experimental procedures

Computational Analyses:

 Putative, cognate two-component proteins were identified in sequenced

bacterial genomes by selecting adjacent genes predicted to encode a histidine
kinase (HK) and a response regulator (RR), by using custom PERL scripts.
 For histidine kinases, the sensor and transmembrane domains were eliminated,
and, for response regulators, the output domains were removed.
 This procedure retained the dimerization and histidine phosphotransfer (DHp)
domain as well as the catalytic and ATP-binding (CA) domain of the histidine
kinases and the receiver domains (RDs) of the response regulators.
 For each cognate HK-RR pair, the DHp, CA, and RD domains were
concatenated into a single sequence and aligned with PCMA with some manual
adjustment.

Cloning and Protein Purification

Phosphorylation and Phosphotransfer Assays

 In vitro analyses of phosphorylation and phosphotransfer were performed as

previously described.
 Kinase and regulator were present at 2.5 mM each. Reactions were incubated
at room temperature, and products were then separated by 10% SDS-PAGE,
exposed to a phosphor screen, and quantified by using a Typhoon 9400
Scanner (GE Healthcare)
 For analysis of phosphotransfer kinetics, autophosphorylated kinases were
purified by ultrafiltration before incubation with response regulators.
 Initial rates were determined by measuring the rate of phosphorylation of a
kinase’s cognate substrate between 0 and 10 s and of noncognate substrates
between 0 and 300 s.

In Vivo Analysis of Specificity Mutations

 pEnvZ contains full-length envZ under control of the lac promoter.

 Plasmids expressing three of the chimeras, p(MI+loop2), p(MI+loop3), and
p(MI+loop5), were constructed by replacing the EnvZ DHp domain in pEnvZ
 with the corresponding chimeric sequences by using the restriction sites NdeI
and RsrII.
 Overnight cultures were diluted 1:1000 into fresh media and grown at 37C
with aeration to an OD600 of 0.1–0.2.
 GFP fluorescence of AFS161 cultures was measured with a
spectrofluorometer).
 YFP fluorescence of AFS237 cultures was measured by fluorescence
microscopy and was normalized by CFP fluorescence.
 For each culture, the average YFP:CFP fluorescence ratio was computed for
150 cells.
Importance

 Cell wall synthesis machinery depends on a host of enzymes,most notably

the Penicillin Binding Proteins(PBP)
 In that PBP2 is thought to be the major enzyme responsible for
transpeptidase activity moving in diffusive motion
 MreB is an bacterial actin homologue involved in elongation machinery
which moves in circumferential motion
 The PBP2 and MreB interaction was found to be transient interaction.
 They found it is not stable interaction as in multiprotein complex as E.coli
maintains its growth rate even when cellular pool of PBP2 is halved.
 Transient interaction heps in maintaining its robustness

Objectives

 To develop model of PBP2 interaction with MreB2

 To determine whether growth rate is maintained during depletion of PBP2
making a network more robust to fluctuations in its limiting components
 To confirm the transient interaction of PBP2 and MreB2
 To determine the efficient strategy for spatiotemporal coordination of
protein activities
Materials and methods

Strain Construction

 Plasmids were constructed using enzymatic assembly methods .

 Expression plasmids were constructed using a low-copy plasmid with a
pSC101 origin (pRM102), and coding sequences for the relevant genes
were amplified from E. coli MG1655 with the appropriate homology
regions for assembly.
 Gene deletions and fluorescent protein fusions were introduced into the
chromosome using allelic exchange methods with suicide plasmids (30).
The desired sequences for integration were amplified by PCR and cloned
into pDS132.
 MFDpir cells were transformed with the resulting plasmids and used for
conjugative transfer into the recipient strain. Resulting merodiploids were
selected on lysogeny broth (LB) plates supplemented with chloramphenicol.
Strains that had lost the integrated plasmid (andsacB gene) by homologous
recombination were selected on LB plates containing 5% (wt/vol) sucrose.
 All chromosomal modifications were confirmed by amplification and
sequencing of the targeted region.
Microscopy
 Single-particle tracking was performed on a TIRF microscope built with a
Ti-E Eclipse stand.
 The objectives used were either an Apo TIRF 100× (N.A. 1.49) or a Plan
Apo Lambda 100× DM, depending on whether phase-contrast images were
acquired concurrently.
 Shuttering of the laser illumination was controlled by an acoustooptic
tunable filter before the fiber coupler.
 Images were acquired with an iXon3+ 887 EMCCD camera, and
synchronization between components was achieved using μManager with a
microcontroller
Single-Particle Imaging

 Cells expressing fluorescently labeled proteins were grown to saturation

overnight in the rich medium EZ-RDM with 0.2% glucose and then diluted
1:100 in fresh medium and incubated with shaking at 37 °C for 2 h.
 Cells were spotted onto 1% agarose pads with EZ-RDM plus 0.2% glucose
and covered with argon plasma-cleaned coverslips.
 For drift correction, To measure MreB speeds, we used an imaging
sequence consisting of a 50-ms exposure with an activation laser (405 nm at
∼0.05 kW/cm2) followed by capture of 10 images with an imaging laser
(561 nm at ∼0.5 kW/cm2) every 2.5 s, which was repeated for a total of
500–1,000 cycles.
 To capture the more rapid PBP2 dynamics, we simultaneously exposed the
cells to both activation and imaging lasers (405 nm at ∼0.05 kW/cm2 and
561 nm at ∼1 kW/cm2) for 10-ms exposures every 45.3 ms (except for the
truncation mutant, which required a shorter imaging interval.
Colocalization of PBP2 and MreB

Estimate of the no of PBP2 Molecules required for growth

 Previous studies have estimated that there are ∼100 PBP2 molecules per E.
coli cell
 To estimate whether each PBP2 could participate in five cross-linking
events per second, we assume that cell wall material is synthesized in a
spatially uniform fashion across the cylindrical portion of the cell, which has
an average length of 3 µm and an average surface area of ∼9 µm2.
 Thus, each of the 500 new cross-links corresponds to a region with area of
∼0.02 µm2.
 Assuming that the transpeptidation reaction is fast relative to the speed of
PBP2 motion across this area, a PBP2 molecule has to achieve an MSD of at
least 0.1 µm2within 1 s to visit all five cross-linking sites. Our measured
diffusion constant of ∼0.06 µm2/s corresponds to an MSD of 0.24 µm2 in 1 s
based on the relationship MSD(t) = 4Dt for 2D diffusion, and is therefore
sufficient for coverage of cross-linking across the entire cell surface during
cell elongation.
Single-cell & colony growth Imaging

Results:

 Difference in the mobilities of MreB and PBP2 excluded the possibility that
they function together in a long-lived synthesis complex
 Weak, significant, correlation suggesting that PBP2 spends at most a small
fraction of its time interacting with MreB
 Model for PBP2 interaction with MreB.
 o PBP2 moves rapidly within the inner membrane (IM), forming transient
interactions with sites of peptidoglycan (PG) synthesis through its
transpeptidase (TP) domain, either with the peptidoglycan elongation
machinery (PGEM), or with the wall itself.
o PBP2 also contains transmembrane (TM) and nonpenicillin binding
(nPB) domains, which may mediate interactions with other
morphogenetic proteins.
 The Transpeptidase domain but not catalytic activity influences PBP2
motion
 Growth rate is maintained during depletion of PBP2
o Growth rate is unaffected by substantial PBP2 depletion. Time-lapse
microscopy of E. coli TKL141cells growing during PBP2 depletionis
specified in minutes.
o Even though cells lose their rod-shaped morphology, they continue to
grow at a rate comparable to wild-type cells until two to three divisions
have taken place.
o Growth rate during PBP2 depletion is quantitatively unaffected over
more than two doubling times.
 Mecillinam treatment reduces growth rate and MREb speed in a dose-
dependent manner.

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