th  - 1988 - 51st ENC Conference

I NATURAL ABUNDANCE 13 C and 14 N NMR (IF BACTERIAL OSIIOLYIES IN VlYO. B.A.
THU 9 • 3 0 .I Lewis,eS.C.Cayley, S. Padmanabhan, and II.T. Record, dr. Dept. of Chemistry,
University of Wisconsin, Madison Wl 53706.
Bacteria such as E. Coil and_5, lyphimurium are capable ol growir 0 under conditions of moderately
high osmotic stress, up to about 0.7 molar salt. To adapt to such hi~h-osmolarity enviror~ments, the b~~l.erial
cell accumulates potassium ions and also synthesizes or accurnulal.es one or more small orgonic molecules.
These include the anion glutamate and the neutral or zwitterionic molec..ules prol ine, glycine betaine (I,I,I,I,H-
trimethyl glycine) and/or trehalase, a glucose dimer. Because these small molecules are accumulated to
intracellular concentrations on the order of 0.5 molal, they are re~Jily observed in dense cell slurries by
natural abundance 13 C NMR on our Bruker AM360 with a I 0 turn br-c,~lband probe. I '-1N NI'IR is al~ u.~ful to
observe glycine betaine, which h~ a relatively narrow 14 N spectrum due to the symmetric environment of
the nitrogen and its lack of exchangeable protons.
We are able to measure the relative and absolute amounts of the various or~nic osmolytes ~..~:umulaled
by the bacteria in vivo under a variety of environmental conditions. In minimal medium with 0.5 M l'laCI,
trehalose and glutamate are the on!y small organic molecules pre~nt in high amounts. If I mM proline is added
to the medium, it is accumulated to nearly 0.4 M intracellular ly, with some diminution of the trehsl~-e., and
glutamate levels. Glycine betaine, however, also supplied at I raM, is accumulated to about 0.5 M, and
trehalose is completely eliminated.
Under these, high salt conditions, significant amounts of rf power are absorbed by the sample,
particularly at the high frequencies of 90 IiHz for 13 C and 360 for i H. Thus for- the 13 C e×periments we
employ gated proton decoupling to minimize sample heating. In ~.klition, the pulse lengths must be calibrated
for each sample, and internal standards must be used for quantitative measurement.
64