Conclusion - Alexandru Ioan Cuza University
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Nicotine vs Nicotine and Citrate Methods Soluble proteins Growth, harvest and cell lysis 0.05% nicotine No nicotine Lysozyme SDS DTT Cells in mid-exponential phase 12,000 g 30 min Protein preparation In-gel digestion Protein fractionation SDS-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 Mihasan 1,2,* , Cornelia Babii 2 , Devika Channaveerappa 1 , Roshanak Aslebagh 1 , Emmalyn Dupree 1 ,Costel C. Darie 1 1 Biochemistry and Proteomics Group, Department of Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY, USA 2 Biochemistry 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 proteins grown on citrate medium supplemented with 0.05% nicotine (left) or citrate medium without nicotine (right). Details of the 66-55 kDa and 24-29 kDa areas of the same gel showing different band patterns. BSA Mrk 0.05% nicotine No nicotine 10 20 30 10 20 30 5 kDa 116 66 55 45 36 29 24 20 14 6.5 1 2 3 4 5 6 7 8 9 10 11 Results SDS-PAGE of proteins from the bacteria grown on nicotine- containing media showed several extra bands in the range of 60 and 30 kDa. One particularity of P. nicotinovorans proteome is the 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 6HLNO 6HDNO KDH DHPONH DHPH NBOR MABO AO MAO SsaDH FolD PurU PKC NIT Paenarthrobacter nicotinovorans is soil bacteria able use the toxic alkaloid nicotine as carbon and energy source. This ability was linked to the presence of the pAO1 megaplasmid and might offer a unique way of exploiting nicotine containing waste for the production of ''green'' chemicals. In the current study, we attempted to identify all the proteins expressed by P. nicotinovorans in the presence of nicotine by using shotgun proteomics. Marius Mihasan was supported by the Romanian - U.S. Fulbright Commission Conclusion Deamination is preferred in the lower nicotine pathway when citrate is present. A hypothetical polyketide have been shown to have a nicotine- dependent expression and we hypothesize that the enzyme would hydrolyze the N1-C6 bond from the pyridine ring with the formation of alpha-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
Transcript of Conclusion - Alexandru Ioan Cuza University
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
