The Bacterial Spore Proteome; Identifying Targets for

Spore Germination and Outgrowth Inhibition.

Stanley Brul, Wishwash Abhyankar, Rachna Pandey, Johan van Beilen,

Norbert Vischer, Erik Manders, Alex Ter Beek, Leo de Koning and Chris de

Koster

Van Leeuwenhoek Centre for Advanced Microscopy,

Mass Spectrometry of Biomacromolecules &

Molecular Biology & Microbial Food Safety

Swammerdam Institute for Life Sciences

University of Amsterdam

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Genomes & proteomes are accessible (see Mol. Biol. of the Cell 2015)

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Spore physiology & proteomics

Study of the effect of growth conditions on spore outer layers

Study of the effect of sporulation temperature on the spore coat proteome and stress resistance

GeLC-MS/MS study of spore IM germination receptors & associated targets

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Study of the effect of growth conditions on spore outer layers and stress resistance

B. subtilis PY79

Kamphorst et al., 2016; Manuscript under preperation

Bacillus subtilis studies (genomes available)

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Spores cultured on solid agar medium possess higher thermal resistance than spores cultured in a liquid medium.

How does the sporulation environment affect spore resistance?

Comparison of the outer layer protein levels of spores cultures on solid and liquid medium using metabolic 15N labeling.

Study aims

Kamphorst et al., 2016; Manuscript under preperation

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14N spores made on solid 2xSG agar medium plates.

15N spores made in 15NH4Cl supplied MOPS buffered liquid medium.

Sporulation allowed for 120 hrs. (5 days).

Sporulation was induced by glucose starvation and cell crowding.

Mixing based on OD600.

Kamphorst et al., 2016; Manuscript under preperation

Relative quantification approach

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TS agar plate

TSB liquid medium

14N 2xSG liquid

medium

15N MOPS liquid

medium

15N MOPS liquid

medium

15N MOPS liquid medium

14N 2xSG Agar plates

5 day incubation at 37°C

Growth at 37°C till OD600 ~ 0.3-0.4

Growth at 37°C till OD600 ~ 0.3-0.4

Growth at 37°C till OD600 ~ 0.3-0.4

Growth at 37°C till max. dilution reaches

OD600 ~ 0.3-0.4

Growth at 37°C till

OD600 ~ 0.3-0.4

5 day incubation at 37°C

Mixing 14N & 15N spores (1:1 mixing based on OD600)

14N 2xSG liquid

medium

Spore coat isolation & SDS extraction

Reduction, Alkylation & Trypsin digestion

LC-FT-ICR-MS/MS analysis &

14N/15N ratio calculations

Observations of spore crops

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14N 15N

14N spores 15N spores

Kamphorst et al., 2016; Manuscript under preperation

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Up regulated in solid medium

Up regulated in liquid medium

14N 15N

Kamphorst et al., 2016; Manuscript under preperation

Outer Coat & Crust Inner Coat

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Electron micrographs of 14N & 15N spores

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14N (solid medium) 15N (liquid medium)

Thickness of Outer coat layer varies significantly in the two spore populations!

Inner coat layers possibly more cross-linked Kamphorst et al., 2016; Manuscript under preperation

Spore morphology

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Spores cultured in the liquid medium have significant thicker outer

coat than spores cultured on the solid medium.

Spores cultured in the liquid medium have a significant different coat

protein composition compared to the spores cultured on solid medium.

The large variation over the replica’s in the 14N/15N peptide ratios for

certain proteins suggest variations in cross-linking between these

coat proteins.

Kamphorst et al., 2016; Manuscript under preperation

Summary

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Possible cross-link targets in the coat

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Cross-linking of coat

proteins?

30% of the coat

proteins are resistent to

extraction

Inner coat Outer coat

Pe

ptid

ogly

ca

n

Cortex S

po

IVA

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Abhyankar et al., 2015. Food Microbiology

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Spore physiology & Proteomics

Study of the effect of sporulation temperature on the spore coat proteome

B. weihenstephanensis WSBC10204

Psychrotolerant spore former (genome was unavailable)

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The newest genome of B. weihenstephanensis

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12°C 30°C

Spore harvest

Spore coat isolation & protein extraction

Tryptic Soy Broth liquid medium

Minimal Sporulation

medium

Overnight, 30°C

10 days 5 days

Reduction, Alkylation & Trypsin digestion

LC-FT-ICR-MS/MS analysis

Method

Stelder et al., 2016; Manuscript under review

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Temperature dependence of identified proteins

17 3 3

Proteins with unknown function

27 10 8

Proteins with known spore association 18 9 14

Non-spore associated proteins

30°C 12°C

Stelder et al., 2016; Manuscript under review

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B. Weihenstephanensis data indicate arginase as 12ºC target

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12 ºC

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Spores physiology & Proteomics

GeLC-MS/MS study of spore IM germination receptors & associated targets

B. subtilis PY79

Zheng et al., 2016

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Zheng et al., 2016

Method

Proteomic analysis of the purified

B. subtilis spore inner membrane

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IM protein identification

Zheng et al., 2016

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Some specific proteins identified:

Enzymes involved in Coenzyme A synthesis viz. PanB, PanC, PanE, CoaBC, CoaE and IlvD.

CoA reported to be di-sulfide cross-linked to proteins in B. megaterium spores Setlow & Setlow, 1977

Role of CoA as a modulator of metabolism in germinating spores is worth studying.

SpeA & SpeE involved in biosynthesis of Spermidine (polyamine) were identified.

Spermidine produced in germinating spores of B. megaterium. What is its role there?

Taken from Hobley et al. 2012; established by Setlow, 1974)

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HTrC & YpeB are found in the inner membrane and may be targets to interfere

with the basis of germination triggering SleB and thus cortex and coat lysis (Zheng et al. 2016, Journal Proteome Res.; see also Bernhards et al. J. Bact. 2015; Meany et al. Anaerobe 2015 )

SleB

CwlJ

(hydrolases)

Germinants

Coat

CORTEX ? ?

Ca2+ DPA

HtcR YpeB

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Spores in AmBiC buffer

Bead beating + Urea+ DTT

Addition of Acetonitrile Alkylation with Iodoacetamide

LysC

Trypsin

ZIC-HILIC peptide pre-fractionation

FT-ICR-MS/MS

Double digestion of proteins

Disrupted spores

Swarge et al., Manuscript under preparation

Recent developments in the studies of sporulation

and germination processes have brought forward

the need of comprehensive time lapse spore

proteome analyses.

In order to enable monitoring of protein levels

during sporulation and germination we have

developed a ‘one pot’ spore processing method for

mass spectrometric analysis of proteins from all

spore layers. The method is applicable to Bacillus

subtilis, B. cereus and Clostridium difficile.

‘One pot’ sample processing method

for time resolved spore proteomics

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Further developments 1:

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Nowadays using 'omics' tools molecular mechanistic data can be gathered to answer questions pertaining to predictive molecular modelling of microbial behavior.

Cooperativity of SpoVA channel gating introduces spore memory for germination stimuli

New:

Single spore & cell analysis using Spore-Tracker Papers

Accurate single cell germination and outgrowth data is needed for building accurate models (use of reporter proteins)!

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Further developments 2:

Pandey et al., 2013, 2015 & Manuscript under review

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Spore-tracker allows single spore data analysis (Pandey et al. 2013, 2015)

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3 mM Potassium Sorbate Control

Observation of germination and outgrowth

Heterogenity of wt and sorbic acid stressed

B.subtilis spores

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pHi measurements in single bacteria with improved (I)pHluorin possible

Van Beilen & Brul (2013)

Three “pH probes” using three promoters at the

amyE locus to drive IpHluorin:

PptsG → vegetative cell specific (growth on

glucose)

PspoIIA → mothercell specific

PsspE → forespore specific

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PptsG-IpHluorin PspoIIA-IpHluorin PsspE-IpHluorin

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Lineage tracing allows identification of single cell heterogeneity in microcolonies under acid stress

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Phase contrast 390 nm 470 nm

Conclusions

Live-imaging of intracellular pH using IpHluorin in

Bacillus subtilis is possible in vegetative cells (and now

germinating / outgrowing spores).

Heterogeneity in stress response to potassium acetate

and sorbate (food preservatives) exists at the

microcolony level.

Lineage tracing shows similar heterogeneity in stress

response in individual cells of micro-colonies upon

(acetate) acid stress.

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Cooperativity of SpoVA channel gating introduces spore memory for germination stimuli

New:

Accurate single cell germination and outgrowth data is needed for building accurate models (use of reporter proteins)!

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Further developments 2:

Nowadays using 'omics' tools molecular mechanistic data can be gathered to answer questions pertaining to predictive molecular modelling of microbial behavior.

Quantitative proteomics for Mathematical modelling to describe and pinpoint the molecular details of the germination DPA release target

Ultimately we aim at quantitative prediction of the spore population germination time distribution and germination efficiency.

Recently, it was found that spores exhibit memory for germination.

Spores given a short germinant pulse respond more readily to a second brief exposure.

The experiments provide excellent transient kinetic data for mathematical modelling of the germinosome mode of action.

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Wang et al. MBio. 2015.

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Germinosome of a B. subtilis spore

Coats

Cortex

(PG)

Core

(CaDPA)

Outer

membrane

Inner

membrane

GerP

germinosome SpoVA

channel

CLEs

Water Nutrient

gerninant

Spore germination triggered by nutrient

Signal transduction pathway

outcome

SpoVAD: ~6,500

molecules/spore.

Germinosome: GerAA and GerAC

each at ~1,100, and GerBA, GerBC and

GerKA each at~700. GerD, ~3,500

and……?

Stewart and Setlow 2013 & Wang et al. 2015;

New candidates from Zheng et al. 2016.

Germinants

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Molecular model for SpoVA channel opening that exhibits memory

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germinant inactive receptor active receptor

closed channel

activated closed channel

open channel

k1

k-1

k2

k3

k4

k5 k5

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De Koster et al., Manuscript in preparation

Germinant concentration and opening of SpoVA channels

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Time (min) Time (min)

4.0 – 2.7 mM 2.6– 1.8 mM

1.7– 1.0 mM

Two germinant pulses @ 2 < t < 2.2 min @ 20 < t < 20.2 min

Interestingly, also a time lag period from germinant stimulus to opening is predicted.

Cooperativity of SpoVA channel gating introduces spore memory for germination stimuli

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De Koster et al., Manuscript in preparation

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Proteomics approaches aid the understanding of spore physiology.

Proteomics methods can allow for comprehensive time resolved

spore proteome analyses.

Proteomics methods lay the basis for quantitative cellular

physiology i.e. the germination kinetics of bacterial spores.

Overall Summary

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Acknowledgements

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Mass Spectrometry of

BioMacromolecules Molecular Biology and Microbial Food

Safety

Universiteit van Amsterdam

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