Nicotine vs Nicotine and Citrate

Methods

Soluble proteins

Growth, harvest and cell lysis

0.05% nicotine No nicotine

LysozymeSDSDTT

Cells in mid-exponential phase

12,000 g30 min

Protein preparation

In-gel digestion

Protein fractionationSDS-PAGE maxi

9-16% gradient gels

Peptide mixture

Identification and data analysis

Nano-liquid chromatography tandem

mass spectrometry

Protein identification by mass spectrometry

Chromosome pAO1

Data analysis: MASCOT; SCAFFOLD

Nic + - + - Nic

Proteomic analysis of nicotine response in Paenarthrobacter nicotinovorans pAO1

Marius Mihasan1,2,*, Cornelia Babii2, Devika Channaveerappa1, Roshanak Aslebagh1, Emmalyn Dupree1,Costel C. Darie1

1Biochemistry and Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY, USA2Biochemistry and Molecular Biology Laboratory, Faculty of Biology, Alexandru Ioan Cuza University, Iaşi, Romania*marius.mihasan @uaic.ro

Introduction

Figure 1. SDS-PAGE of Arthrobacter nicotinovorans proteinsgrown on citrate medium supplemented with 0.05% nicotine(left) or citrate medium without nicotine (right). Details of the66-55 kDa and 24-29 kDa areas of the same gel showing differentband patterns.

BSA

Mrk0.05% nicotine No nicotine

10 20 30 10 20 305

kDa

116

66

55

45

36

2924

20

146.5

1

2

3

4

5

6

7

8

9

1011

ResultsSDS-PAGE of proteins from the bacteria grown on nicotine-containing media showed several extra bands in the range of 60and 30 kDa. One particularity of P. nicotinovorans proteome isthe high abundance of small proteins of around 14 kDa (Fig 1).

Citrate Nicotine

Nicotine and Citrate

Nicotine vs Citrate

Citrate vs Nicotine and Citrate

Figure 2. Venn diagram illustrating overlaps between the substrate specific non redundant proteins identified by LC-MS/MS analysis of Paenarthrobacter nicotinovorans pAO1 grown of different carbon sources.

NDH 6HLNO6HDNO

KDH

DHPONH

DHPH

NBOR

MABO

AO

MAO

SsaDH

FolDPurU

PKC

NIT

Paenarthrobacter nicotinovorans is soil bacteria able use thetoxic alkaloid nicotine as carbon and energy source. This abilitywas linked to the presence of the pAO1 megaplasmid and mightoffer a unique way of exploiting nicotine containing waste forthe production of ''green'' chemicals. In the current study, weattempted to identify all the proteins expressed by P.nicotinovorans in the presence of nicotine by using shotgunproteomics.

Marius Mihasan was supported by the Romanian - U.S. Fulbright Commission

Conclusion

Deamination is preferred in the lower nicotinepathway when citrate is present. A hypotheticalpolyketide have been shown to have a nicotine-dependent expression and we hypothesize thatthe enzyme would hydrolyze the N1-C6 bondfrom the pyridine ring with the formation ofalpha-keto- glutaramate.

This work partially supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS – UEFISCDI, project number PN-III-P2-2.1-PED-2016-0177