Case Study: Sporulation (c. 2010) Sporulation Initiation ...durand/Phylogenetics/2017/Lectures/Phylogeneti… · Recall: Specificity residues control signal fidelity 54 Antibiotic

10/9/2017

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Phylogenetics of the Sporulation Initiation Pathway

Philip Davidson, Ph.D.

Carnegie Mellon University

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Subject: Evolution of Bacterial Signaling Pathways

Antibiotic

Salt

Glucose

Membrane Repair

Antibiotic Resistance

Metabolism

Histidine Kinase (HK)

Response Regulator (RR)

… … HK RR

Cognate Pair

Case Study: Sporulation Initiation (Spo0) Pathway

in Firmicutes Bacteria

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Bacillus subtilis

Spo0F

Sporulation Kinases

Spo0B Spo0A

Endospores

Identified by Burbulys & Hoch in 1991

Sporulation (c. 2010)

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Spo0F

Sporulation Kinase

Spo0B Spo0A

Phosphorelay

Bacillus subtilis

Paenibacillus polymyxa

Bacillus anthacis

Clostridia

Bacilli

?

Sporulation has different interaction architectures in each class (c. 2015)

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Spo0F

Sporulation Kinase

Spo0B Spo0A

Phosphorelay

Bacillus subtilis


Bacillus anthacis

Clostridia

Bacilli

Sporulation Kinase

Spo0A

Direct phosphorylation

Clostridium acetobutylicum

Clostridiodes difficile

Lachnoclostridium thermocellum

Same phenotype, Same master regulator,

Different interaction pattern


Phosphorelay

Phylogenetic Question

Homology of Spo0A and Phenotype suggest that there is a common ancestor

How do two different pathways evolve from the same common ancestor?

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How do two different pathways evolve from the same common ancestor?

• To investigate this problem we need…

– A Firmicutes species tree

– Distribution of sporulation initiation pathway architectures

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How to construct a species tree

• Step 0 – Choose a set of genomes

• Step 1 – Choose data (Sequence acquisition)

• Step 2 – Multiple Sequence Alignment

• Step 3 – Choose substitution model

• Step 4 – Build your tree

• Step 5 – Analyze your tree

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Step 0 - Genomes

• Broad distribution (84 Firmicutes)

– Both Clostridia and Bacilli species sporulate

– Spore-formers and non-spore-formers?

• Outgroup species for rooting (1 Fusobacteria)

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Step 1 – Common types of data for species trees

• Necessary (ideal) characteristics:

– One unit per genome

– No horizontal gene transfer

• Common Types

– 16S ribosomal RNA

– Ribosomal proteins

– All singleton or “core” proteins (closely related)

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16s rRNA vs. Protein data – What’s the big deal?

• 16s rRNA

– Only 4 values (Low information content)

– Insubstantial biochemical selection

• Protein Data (Singleton, Core, Ribosomal)

– 20 values!

– Biochemical selection

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Species Tree

• Step 1 - Data – 50 ribosomal proteins

• Step 2, 3, and 4 - Tree construction – MAFFT/GUIDANCE

– Trim gapped col.

– RAxML

• Step 5 - Bootstrapped – 100 times

• n/a is black

• 0 is red

• 50 is yellow

• 100 is green

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Facultative anaerobes

Obligate anaerobes

Bacilli

Clostridia

Firmicutes

We can now ask our phylogenetic question in terms of the species tree!

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No spores

No spores

Firmicutes

Spore-former

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Spo0F

Sporulation Kinases

Spo0B Spo0A

Firmicutes

All sporulators must have a Spo0 pathway

Sporulation Kinases Spo0A

Architecture is poorly understood in many genomes


Phosphorelay

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Spo0F

Sporulation Kinases

Spo0B Spo0A

What was the ancestral pathway?


How did the pathway change?

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Spo0F

Sporulation Kinases

Spo0B Spo0A

Direct phosphorylation of Spo0A in ancestral pathway,

phosphorelay emerged in Bacilli


Standing Hypothesis: Ancestral direct phosphorylation

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Evidence: - Clostridium are anaerobic

like ancestor - Simpler pathway

Spo0F

Sporulation Kinases

Spo0B Spo0A


Standing Hypothesis: Ancestral direct phosphorylation

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Spo0F

Sporulation Kinases

Spo0B Spo0A


Standing hypothesis is testable!

What is the present-day architecture?

Homology identification for the Spo0 Pathway

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Antibiotic

Salt

Glucose

Membrane Repair


Metabolism

HK RR

Cognate Pairs

B. Subtilis Spo0 Pathway

Easy to find signal transduction proteins, hard to tell them apart

• Domain-based homology identification

• Spo0A – Conserved output

domain

• Spo0 Kinases – Not adjacent to any RR

– PAS in sensor region

Spo0Fs are difficult to identify

Problem – Lack of output domain – “stand-alone receiver”

– Stand-alone receivers not unique to this pathway

Spo0A

Spo0F

Spo0B


Solution – Identifiable by conservation of sequence features

– Identifiable by conservation of genome neighborhood

Spo0A

Spo0F

Spo0B

Bacillus subtilis

Spo0F is encoded in a conserved neighborhood

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Bacillus subtilis

Bacillus anthracis

Bacillus amyloliquefaciens

Paenibacillus mucilaginosus

Clostridia acetobutylicum

Desulfotomaculum acetoxidans

Desulfosporosinus meridiei

Thermoanaerobacter. thermosaccharolyticum

Legend

– Transaldolase

– CTP synthase

– Fructose bisphosphate aldolase

• Look for neighboring genes to find Spo0F ( )

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Spo0F results


Problem – Lack of output domain – “stand-alone receiver”

– Stand-alone receivers not unique to this pathway

Spo0A

Spo0F

Spo0B


Solution – Identifiable by conservation of sequence features

– Identifiable by conservation of genome neighborhood

Spo0A

Spo0F

Spo0B

Bacillus subtilis

BLAST with B. subtilis Spo0B

Results are weakly supported

Most are called “Signal transduction Histidine kinase, regulating citrate/malate metabolism”

BLAST performed 10/14/13, including Firmicutes, excluding Bacilli family species

Spo0B Interaction Domain Conservation

• Weak conservation across whole protein

• Significant conservation in interaction domain

Spo0B is encoded in a conserved neighborhood

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• Legend

– rProtein L21p

– rProtein L27

– ObgE-like GTPase

Bacillus subtilis

Bacillus lichenformis


Bacillus amyloliquefaciens

Thermoanaerobact. thermosaccharolyticum


Desulfosporosinus meridiei

Clostridia acetobutylicum

• Examine genome neighborhoods for Spo0B( )

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Spo0B results

Spo0F

Sporulation Kinases

Spo0B Spo0A


Standing hypothesis predictions are not consistent with observed

distribution


Spo0F

Sporulation Kinases

Spo0B Spo0A


Presence of phosphorelay proteins suggests function, but….

Are these actually phosphorelays? Test a clostridial phosphorelay

D. acetoxidans

Experimental Verification of Phosphotransfer

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P P

P P

P

P P

P

P P

P

P P

P

P P

A bacterium

P

P P

P P

P KP

KP Spo0F Spo0B

Spo0A

0F

Phosphotransfer Profiling Experimental Design for phosphorelays

K1 Hot ATP Spo0F Spo0B

Separate on PAGE gel

Expose on film

1 Spo0A

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Spo0A

Spo0A

15

K1

Spo0B

Spo0F

K1

Dtox_1918 ATP [γ-32P] Spo0F Spo0B

Experimental Results

18 21 24 27 30

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Spo0F

Dtox_1918

Spo0A

Spo0B

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Is each component necessary?

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10 min

Dtox_1918 Spo0F

Spo0A

0A

K1

0F

K1

10 min

Dtox_1918

Spo0B Spo0A

0A

K1

0B

K1

10 min

Dtox_1918

Spo0A

0A

K1 K1

No direct transfer from

Kinase to Spo0A

Transfer to Spo0A not observed

without Spo0B

Transfer to Spo0A not observed

without Spo0F

Transfer to Spo0A requires all phosphorelay components.

D. acetoxidans Spo0 pathway is a phosphorelay!

Spo0F

Sporulation Kinases

Spo0B Spo0A


Are these actually phosphorelays? 1. D. acetoxidans verified as phosphorelay

2. Are other predicted Spo0F and Spo0B proteins functional?

Similar specificity to experimentally verified Spo0 architectures suggests similar patterns of interaction

Kinase Spo0F Spo0B Spo0A

Bacilli

Clostridia

Encode Spo0F and Spo0B

Spo0F

Sporulation Kinases

Spo0B Spo0A

NELLEYD

QLGNSL

QGILEVD

TAGFQL

TGGFQL

TAGFQL

TSGFQI

TVGFQL

B. subtilis

Spo0F

Sporulation Kinases

Spo0B Spo0A


Experimental and bioinformatic analyses support Clostridial

phosphorelays

Spo0F

Sporulation Kinases

Spo0B Spo0A



How did the pathway change?

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Bird’s eye view of the Firmicutes

Paenibacillaceae

Erysipelatrichaceae

Bacillaceae

Lactobacillaceae

Sporomusaceae

Veillonellaceae

Peptococcaceae

Thermoanaerobacterales

Clostridium sensu stricto

Ruminoccaceae

Lachnospiraceae

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Spo0 Architecture Distribution

• Patchy distribution in both classes

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Evolutionary history of Spo0 Architecture

• What is the architecture of the common ancestral pathway?

• In which lineage(s) did the phosphorelay arise?

• In which lineages did remodeling occur?

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What was the architecture of the ancestral pathway?

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Ancestral direct phosphorylation Ancestral phosphorelay

Multiple emergence of phosphorelay?

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Spo0F

Sporulation Kinases

Spo0B Spo0A

Spo0F

Sporulation Kinases

Spo0B Spo0A

B. subtilis

D. acetoxidans

Test by conservation of

interaction


Bacillus subtilis

Bs0F/Dt0F

Bs0B

Bs0A

Bsub_KinA

Dt0A

Bs0B - tag

Dt0B Dtox_1918

Bs0F Dt0F Bs0B Dt0F

Dt0B Bs0F Bs0F

Dt0A Dt0B Bs0F

Bs0A Bs0B

Bs0F

Dt0A Bs0B Dt0A Dt0A

Bs0F Dtox1918 BsKinA

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• Each Desulfotomaculum Spo0 component is able to recapitulate the function of its counterpart

• Unlikely to arise coincidentally

Preservation of specificity What was the architecture of the ancestral pathway?

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Ancestral direct phosphorylation Ancestral phosphorelay

Conservation of interaction suggests a shared origin for

the phosphorelay.

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Alternative Single Origin Hypothesis

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Transfers into each lineage, regardless of origin

Later origin of phosphorelay, with transfer

Multiple transfers to obtain each component

B. Subtilis Spo0 Pathway

Bacillus subtilis

Each transfer must insert into same neighborhood

Spo0F

Spo0B

Transfer of the phosphorelay is unlikely

Hypothesis: Ancestral phosphorelay, with multiple instances of pathway remodeling

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Multiple remodeling events from phosphorelay to direct

phosphorylation

Spo0F

Sporulation Kinases

Spo0B Spo0A Sporulation Kinases

Spo0A

Ancestral phosphorelay

Requires gain of interaction between kinase and Spo0A

What were the changes that allowed this to happen?

• Multiple remodeling events – Requires gain of

interaction between kinase and Spo0A

• What were the changes that allowed this to happen?

Specificity residues maintain signal fidelity and prevent crosstalk

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Antibiotic

Salt

Glucose

Membrane Repair


Metabolism

Interaction of HK and RR

TRLAEM?

DRLLRYN!

VRYREM?

TRLGAL? DEVLAYP >:( DEVLAYP!

DELLELN!

HK

RR

Small changes in specificity residues are tolerated

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Antibiotic

Salt

Glucose

Membrane Repair


Metabolism

DRLLRYN!

TRLAEM?

RRantibiotics Signal Strength

Recall: Specificity residues control signal fidelity

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Antibiotic

Salt

Glucose

Membrane Repair


Metabolism

RRantibiotics

HKsalt

RRsalt

HKantibiotics HKglucose

RRglucose

Signal fidelity and crosstalk within a cell

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Antibiotic

Salt

Glucose

Membrane Repair


Metabolism

RRantibiotics

HKsalt

RRsalt

HKantibiotics HKglucose

RRglucose

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Specificity spectra of the phosphorelay

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Spo0F

KP Spo0B

Spo0A

Spo0F

Phosphorelay Kinase

Spo0A

Spo0B

Remodeling was mediated by changes in…

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Spo0F

KP Spo0B

Spo0AP

KP

KD

KD

Spo0AD

Spo0AP

Spo0AD

Spo0A

KP KD

Sporulation Kinases

KD

Spo0AD Spo0AD

KD

Spo0F

KP

Spo0AP

KP

Spo0AP

Spo0B KD

KP Spo0B

Test remodeling by cross-species interactions

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Spo0F

Sporulation Kinase

Spo0B Spo0A

Spo0F

Sporulation Kinase

Spo0B Spo0A

B. subtilis

D. acetoxidans

Sporulation Kinase Spo0A

C. acetobutylicum

Cross-species experiment results

– Bacillar phosphorelay (B. subtilis)

– Clostridial phosphorelay (D. acetoxidans)

– Direct phosphorylation (C. acetobutylicum)

Spo0F

Bacillus subtilis


Clostridium acetobutylicum

Spo0F Spo0A

Spo0A

Spo0A

Remodeling was mediated through changes in sporulation kinases

Remodeling was mediated by changes in…

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Spo0F

KP Spo0B

Spo0AP

KP

KD

KD

Spo0AD

Spo0AP

Spo0AD

Spo0A

KP KD

Sporulation Kinases

KD

Spo0AD Spo0AD

KD

Spo0F

KP

Spo0AP

KP

Spo0AP

Spo0B KD

KP Spo0B

Overview

• Background: Bacterial signaling pathways

• Sporulation Initiation (Spo0) Pathway: a case study

The evolutionary history of the Spo0 pathway

– Ancestral phosphorelay

– Multiple remodeling events resulted in transition for phosphorelay to direct phosphorylation

– Remodeling was mediated through changes to sporulation kinases, not Spo0A

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Overview



• The evolutionary history of the Spo0 pathway

– Ancestral Spo0 Pathway was a Phosphorelay

– Remodeling to a direct phosphorylation pathway was mediated through changes in Spo Kinases.

Discussion

– How could this remodeling happen?

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Remodeling through changes in phosphorelay kinase specificity

Spo0F

Cell1

KP 0B

Spo0A

Cell1 Phosphorelay

KP

Cell1 Direct phosphorylation

Remodeling through acquisition of a kinase specific for Spo0A

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Spo0F

Cell1

Cell2

K1

K2

0B

RR2

Spo0A

Spo0F1

Cell1

Cell2

K1

K2

Loss of K1/0F/0B

Acquisition of K2

Spo0A1

K2

Spo0A1

Cell1 Spo0A2 Spo0F2

0B

Cell1

Cell2

Overview



• The evolutionary history of the Spo0 pathway

– Ancestral Spo0 Pathway was a Phosphorelay

– Remodeling to a direct phosphorylation pathway was mediated through changes in Spo Kinases.

Discussion

– How could this remodeling happen?

– Why does it happen so often?

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Constrained nature of phosphorelay

• Spectrum of Spo0F must overlap with Spo0A

• Few mutations may be required to change targets

Spo0F

TSGFQL

Spo0A

TSGFET

Acquisition of new signals for sporulation

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• Sporulation linked to survival

• New kinase may target Spo0A directly

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Recovery from loss of sporulation

• Recovery from loss of sporulation

– Loss of Spo0F or Spo0B = loss of sporulation

– Direct phosphorylaton may recover sporulation

Spo0F

KP

Spo0A

KD Spo0B

Summary

• Identification of Spo0 proteins

• Ancestral phosphorelay hypothesis

– Direct phosphorylation architecture is a derived state

– Remodeling occurs through changes in kinases not Spo0A

• Molecular recognition phosphorelay is flexible, permitting expansion

Documents

Case Study: Sporulation (c. 2010) Sporulation Initiation ...durand/Phylogenetics/2017/Lectures/Phylogeneti… · Recall: Specificity residues control signal fidelity 54 Antibiotic