8/8/2019 Poster Edinburgh 2009

1/1

New structural and functional defects in polyphosphate deficient bacteria: A cellular andproteomic study

Cristian Varela 1, Cecilia Mauriaca 1, Alberto Paradela 2, Juan P Albar 2, Carlos A Jerez 1, Francisco P Chvez 11 Laboratory of Molecular Microbiology and Biotechnology & Millennium Institute of Cell Dynamics and Biotechnology (ICDB). Faculty of Sciences, University of Chile

2 Proteomic Service, National Center of Biotechnology. CSIC. Madrid, Espaa

RESULTS

ABSTRACTInorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2) and degraded by exopolyphosphatase (PPX). Bacterialcells with polyP deficiencies by knocking out the ppk1 gene were affected in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence among others. The cause of this pleiotropy is not entirely understood.In bacteria, polyP depletions have been also achieved by the overexpression of exopolyphosphates. By using this approach in the polyP-accumulating bacteria Pseudomonas sp. B4, we mimicked some pleitropic defects found in Pseudomonasaeruginosa PAO1 ppk1 . Why polyphosphate deficiency in bacteria causes multiplestructural and functional defects can be clarify by using OMICS and mathematical modelling approachs. We used comparative proteomics to elucidate the cellular adjustments that occurred during polyP deficiency in this bacterium and suggested that during polyP deficiency energy metabolism and particularly nucleoside triphosphate(NTP) formation were affected and that bacterial cells overcame this problem by increasing the flux of energy-generating metabolic pathways s uch as tricarboxilic acid (TCA) cycle, B-oxidation and oxidative phosphorylation and by reducing energy-consuming ones such as active transporters and aminoacid biosynthesis. Furthermore,our results suggested that during polyP deficiency a general stress response also took place.

2D-PAGE of polyP-deficient and control Pseudomonassp . B4 cell. Panel shows theseparation of protein in the pI range 5-8 and 4.7-5.9. Numbers with arrows indicate the spotnumber used for MS/MS analyses (Tables).

Summary of protein spots identified whose expr ession increases during polyP eficiency

Summary of protein spots identified whose expression decreases during polyP deficiency

A.-Planktonic cultures from exponential phase. B.-Planktonic cultures fromstationary phase. C.-Colonies grown on LB agar plates.

F at ty A ci d s G lu co s e

Pyruvate

Glyc o ly sis

P y ruvateDeh y drogenase

Normal polyP Acetyl-CoA

Citric AcidCycleNADH

FADH 2 ATPGTP

E l e c tron transfer &Oxidative phospor yl ation

F-oxidation

polyPPPKs

PiPPX

F at ty Ac id s G lu co s e

Pyruvate

Glyc oly sis

P y ruvateDeh y drogenase

LowpolyP Acetyl-CoA

Citric AcidCycle

NADHFADH 2

ATPGTP

E l ec tron transfer &Oxidative phospor yl ation

F-oxidation

polyP

PPKs

Pi

PPX

PROPOSED MODEL

TABLES

PolyP ATP PPK

Polyphosphatekinase

PPX Pi

Exopolyphosphatase

polyP(-)

Control

250 nm