IN VIVO 1HNMR STUDY OF DIFFUSIONAL WATER

1
IN VIVO 1H-NMR STUDY OF DIFFUSIONAL WATER
PERMEABILITY OF BACTERIAL CELLS
T. Danevcic (1), A. Sepe (2), G. Lahajnar (2), D.
Stopar (1)
(1) University of Ljubljana, Biotechnical
faculty, Department of Food Technology,
Laboratory of Microbiology, Vecna pot 111, 1000
Ljubljana, Slovenia (2) Joef Stefan Institute,
Jamova 39, 1000 Ljubljana, Slovenia  
Water is the universal biological solvent. Since
the rate of water transport across the cell
membrane controls many physiological processes in
the cell it is important to develop noninvasive
methods for the study of membrane permeability to
water. In this study, in vivo NMR spectroscopy
was used to measure membrane water diffusional
permeability, Pd, of the Gram-negative bacteria
Escherichia coli K12 in cell suspension. In
particular, the water proton NMR transverse
relaxation curves of cell suspensions
paramagnetically doped with MnCl2 were measured,
using the standard Carr-Purcell-Meilboom-Gill
(CPMG) pulse method.
E.coli cells were grown in a triptone-yeast
extract broth, harvested in a late exponential
phase by centrifugation, resuspended in a
Tris-HCl buffer at pH 7.4, put in a NMR tube,
and measured on a Tecmag Apollo 100 MHz NMR
spectrometer. A two site-exchange model of water
exchange between diamagnetic cell interior
(compartment B) and the paramagnetic
extracellular solution (compartment A) was
assumed. The experimental NMR transverse
magnetization decay curves (black squares) were
fitted to a double exponential decay superimposed
on a constant baseline (compartment C). That is,
the following equation was applied to analyze the
data
where T2a and T2b are the relaxation times
observed in the presence of water exchange
between the compartments A and B.
Napis k ordinati te slike se naj glasi relative
change in Pd
Water permeation across E.coli cell membranes was
studied upon addition of either NaCl, ethanol or
HgCl2. All solute concentrations were normalized
to 1.0 which indicates final concentrations of
200mM NaCl, 5mM HgCl2, or of 10 (V/V) ethanol,
respectively. Water permeability across the
nontreated bacterial membrane was estimated to be
3.5 0.2 10-3 cm/s. Pd is found to increase
when either ethanol or NaCl is added. On the
other hand, upon addition of HgCl2 Pd is found
to decrease.
The total cell volume (B compartment) was found
to decrease slowly with time, when cells were in
LB growth medium or Tris-HCl buffer, pH7.4. The
decrease, however, was more pronounced upon
addition of ethanol, NaCl or HgCl2. The results
indicate that NMR conditions (i.e., high cell
density, less oxygen) are not optimal for growth
of the bacterial cells. In addition, solvents
can dramatically reduce cell viability. For
example, almost all bacterial cells (99.5) were
severly disturbed by addition of 5 mM HgCl2 after
15 min of incubation.

• CONCLUSIONS
• 1H-NMR pulse technique appears to be a powerful
  method for in vivo monitoring of the bacterial
  membrane diffusional water permeability
• it enables real time monitoring of bacterial cell
  aggregation and dying process
• diffusional water permeability (relative to that
  of control cells) is found to increase upon
  addition of NaCl or ethanol, and to decrease upon
  addition of HgCl2.

The extent of cell aggregation (propotional to
the magnitude of compartment C) was found to
decrease during the experiment. This can be
observed because the water that escapes from the
C compartment is no longer prevented from
paramagnetic interactions with Mn ions. This
escape can be the consequence of (i)
solubilization of the bacterial cells, (ii)
breaking tight junctions between the bacterial
cells, however, not breaking the aggregates,
and/or (iii) dying of the bacterial cells during
the experiment.