Identification of Intracellular Metabolites in Shewanella oneidensis MR-1 upon Salt Stress Sandra Villa2, Dominique Joyner1,3, Chris Petzold2, Edward E. Baidoo1,3, Peter Benke1,3, Francesco Pingitore1,3 Aindrila Mukhopadhyay1,3, Terry Hazen1,3, Julie

1
Identification of Intracellular Metabolites in
Shewanella oneidensis MR-1 upon Salt Stress
Sandra Villa2, Dominique Joyner1,3, Chris
Petzold2, Edward E. Baidoo1,3, Peter Benke1,3,
Francesco Pingitore1,3Aindrila Mukhopadhyay1,3,
Terry Hazen1,3, Julie A. Leary4, Jay D.
Keasling1,2,31Virtual Institute for Microbial
Stress and Survival (VIMSS), 2University of
California Berkeley 3Lawrence Berkeley National
Laboratory, 4University of California Davis
Abstract
Results
The goals of this project are to develop and
utilize methods to quantify and identify
metabolites in Shewanella oneidensis MR-1 and to
study how metabolism is influenced by
perturbations at the physiological level. S.
oneidensis is able to reduce various organic and
inorganic substrates1, including toxic metal
oxides commonly found in a number of DOE sites.
The metabolic versatility of MR-1 led it to be a
potential candidate for bioremediation of metal
contaminants. To further our understanding upon
physiological perturbations, metabolic tools are
needed to comprehensively identify and quantify
metabolites under defined conditions. To this
end, a capillary-electrophoresis quadrupole mass
spectrometry method was applied for the targeted
identification of intracellular metabolites.
Further, a non-targeted study of S. oneidensis
metabolome, after salt stress, using direct
infusion FTMS will be presented.
Partial List of Metabolites Identified via direct
infusion FTMS analysis ? Total number of ions
assigned a unique empirical formula 56.
Common osmoprotectants
Experimental Design
Procedure for Identification of Metabolites

Glycine Betaine
Glutamate
Control 0.1M NaCl
Mass Spectrum

Empirical formula assigned using Metacyc
database
C1
t1 290mins
148.0606
Intens.
Intens.
8
x10
8
x10
1.0
1.0
C5H10NO4 148.0605 error -0.7 ppm
Proline
0.8
0.8
0.7
0.6
0.6
0.4
0.4
t 0
0.2
0.2
0.0
0.0
C0
147.00
148.05
149.00
m/z
V1
0.4
Growth (OD)
Stress 700mM additional NaCl
Assign putative metabolite structure (s)
Metacyc DB (2,200)
e.g.

• Table 1. Partial list of metabolites identified
  from the data collected in the positive ion mode
  only. The identifications for the salt stress
  related compounds were verified using CID
  fragmentation studies. () Further studies are
  needed to delineate structural isomers. (--)
  The ion was only observed in one sample.

Time (hours)
Glutamic Acid
O-Acetyl-serine
Verify using analytical standards
Figure 1. Shown is a general schematic of S.
oneidensis growth under control and salt stressed
conditions. The cells were grown in modified M1
media (40mM lactate, no vitamins).
Towards the Targeted Analysis of the Central
Metabolic Pathways
Components
glyoxylate
pyruvate
fumarate
succinate
oxoloacetate
malate
2-oxo-glutarate
citrate/isocitrate
acetyl-CoA
succinyl CoA
Extraction Procedure
Scripts for the Rapid Identification of
Metabolites
Oxoloacetate

• The manual assignment of metabolite structures
  to a single measured ion accurate mass is a e
• time consuming process without a curated database
  and scripts to aid in the assignment of
• structures to masses. Three scripts were written
  to aid in the identification of metabolites.
• Accurate Mass Measurements
• - Identification of metabolite based on the mass
  difference of the measured accurate mass to the
  theoretical exact mass of
  the database.
• Data Base
• - Metacyc DB contains 2,200 compounds, more
  than 240 organisms
• - adducts searched
• - Positive mode protonated, sodiated, and
  di-sodiated
• - Negative mode deprotonated
• Mass Comparison (control vs. stressed)
• - Two data files are compared, two ions are
  considered the same based on a set error and only
  ions whose mass error of the measured
  accurate mass to the theoretical exact mass of
  the DB are within a certain
  range are displayed.
• Intensity Comparison (control vs. stressed)
• - Two data files are compared, two data files
  are considered the same based on a set error and
  only displays whose mass error
  is within a certain range and intensity changed
  by a certain amount

All steps performed at cold temperatures
Citrate/ Isocitrate
Pyruvic Acid
Glyoxylate
Bruker Apex 7T FTMS
Conclusions and Future Work
Identification of metabolites only using accurate
mass measurements can be complicated, specially
if using large databases such as Metacyc where
several structural isomers can be present for a
single theoretical exact mass. The use of smaller
databases with known metabolites found in S.
oneidensis can aid in delineating structural
isomers. Further, the use of a high resolution
separation technique such as capillary
electrophoresis can provide an extra parameter to
help in the identification of metabolites.
Biologically, S. oneidensis under the present
conditions responds similarly to the current salt
stress model2. The use of a better quantitative
technique (such as CE-MS) and a more targeted
approach towards the central metabolic pathways
and intermediates leading to the osmoprotectants
of interest may lead to a more complete picture
of the metabolic response of S. oneidensis under
salt stress.
Figure 3. Shown is the schematic of the FTMS. Its
high mass accuracies (lt2ppm) enables the
assignment of elemental formulas to measured
ions. From elemental formulas, metabolite
structures can be assigned.
References and Acknowledgements

• Nealson et al. (2002) Antonie van Leewenhoek 81
  215-222.
• Storz, G. Hengge-Aronis, R. Bacterial Stress
  Responces. United States, ASM Press, 2000. pp.
  79-97.