May 25, 1989 - LINDA TOMBRAS SMITH'* AND GARY M. SMITH'. Plant Growth .... processor) revealed a positive Cotton effect at 212 nm and a negative effect ...
JOURNAL OF BACTERIOLOGY, Sept. 1989, p. 4714-4717
Vol. 171, No. 9
0021-9193/89/094714-04$02.00/0 Copyright © 1989, American Society for Microbiology
An Osmoregulated Dipeptide in Stressed Rhizobium meliloti LINDA TOMBRAS SMITH'* AND GARY M. SMITH' Plant Growth Laboratorv1 and DeparBtment of Food Science anid Technology, Universit Davis, California 95616
of
California,
Received 15 March 1989/Accepted 25 May 1989
One common mechanism of cellular adaptation to osmotic stress is the accumulation of organic solutes in the cytosol. We have used natural-abundance 13C nuclear magnetic resonance to identify all organic solutes that accumulate to significant levels in Rhizobium meliloti. Our studies led to the discovery of a new dipeptide, N-acetylglutaminylglutamine amide (NAGGN), which is accumulated during osmotic stress. Only rarely have peptides been shown to function in bacteria, and furthermore, this is the first example of a peptide playing a role in osmoregulation. Evidence for the biological role of NAGGN in osmotic-stress protection is presented.
Osmotic stress is a problem with which all forms of life must deal. Adaption to osmotic stress, termed osmoregulation, allows cells to tolerate adverse conditions such as
bial strains were maintained on solid mannitol-salts-yeast extract (3), and cultures were grown in malate-Casamino
Acids (MCAA) medium (19) for NMR experiments. Glucose minimal medium (8) was used for experiments with Pseuldomonas fluores(cens. Sample preparation and NMR spectroscopy. Cultures (1 liter) in MCAA medium, plus additions where indicated, were harvested at late log phase, and the protein contents were determined (14). The pelleted cells were extracted three times with 7% perchloric acid (total volume, 10 ml) and neutralized with KOH, and the resulting KCl04 was removed by centrifugation. The neutralized extract was passed through a 0.5-ml column of Chelex-100 and lyophilized, and the lyophilizate was dissolved in 15% D20. Overall yields were consistently 70 to 80% as determined by the addition of a known amount of amino acid (e.g., alanine or glycine) to a number of harvested cultures. '3C NMR spectra were obtained at 90.5 MHz by using a General Electric NT-360 spectrometer with a probe temperature of 25°C. A single pulse, producing a 70° nutation angle, was used for data acquisition. Broad-band proton decoupling was employed during signal digitization. The decoupler power was switched to a lower level between acquisition and the subsequent pulse to maintain the nuclear Overhauser effect but prevent undue sample heating. A pulse repetition interval of 3 s was found not to cause significant suppression of resonances of aliphatic carbons. A 20-KHz spectral window was employed in the quadrature phase detection mode, with 8,192 time-domain points. The line broadening applied during processing varied between 5 and 12 Hz. 13C NMR chemical shifts, reported relative to that of tetramethylsilane, were obtained by assuming that the chemical shift of internal dioxane in 15% D20 is 67.80 ppm. The concentration of osmolytes was quantitated by comparing peak heights with those of standard amino acids added to the prepared sample and also with values obtained by amino acid analysis. All experiments were run in duplicate, generally with no more than 10% error between samples. Synthesis of NAGGN. Synthetic N-acetyl-L-glutaminylL-glutamine amide was prepared from L-glutamyl-L-glutamate (Sigma Chemical Co.) by acetylation with acetic anhydride (pH 11). Excess acetic acid was removed by repeated rotoevaporation, and the product was amidated by reaction with NH4CI and a threefold excess of the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma) (pH 5; reaction time, 2 h). Because NAGGN is uncharged, it was purified from contaminants by passage over an AG501-8X
drought or high salinity. A common mechanism of osmoregulation is the accumulation of inorganic or organic solutes or both in the cytosol to restore turgor in plants and microbes or to control cell volume in animals (13, 22). The osmotically active organic solutes (osmolytes) fall into three general classes (22): polyols (sugars, sugar alcohols, glycerol), amino acids and amino acid derivatives (glutamate, proline, betaines, y-aminobutyric acid, taurine), and urea and methylamines (trimethylamine-N-oxide). Examples of these osmolytes can be found in microbes, plants, and animals. Even in mammals, osmolytes are observed: the osmotically stressed accumulation of glycine betaine and taurine in kidney (6) and heart tissues (21), respectively, has been reported. Although osmolytes are usually identified and quantitated by chemical means, natural-abundance 13C nuclear magnetic resonance (NMR) spectroscopy has been particularly useful in the study of osmoregulation because all classes of organic compounds can be detected by this method. For example, although it was known for some time that Escherichia coli accumulates glutamate (16), Larsen et al. (12) recently found that the major osmolyte is actually trehalose. Also, the cyanobacterium Synechococcus sp., which was thought to accumulate only inorganic ions, quite unexpectedly was found to contain a high concentration of glucosylglycerol (4). Our investigation is concerned with osmoregulation in Rhizobium meliloti, the root nodule symbiont of alfalfa. This bacterial species accumulates glutamate (5) and possibly K+ (23) when osmotically stressed, but no general investigation of osmoregulation in this species has been carried out. In this report we describe the use of natural-abundance '3C NMR spectroscopy to identify all major organic osmolytes accumulated by R. meliloti. This approach led to the identification of a new osmoregulated compound, N-acetylglutaminylglutamine amide (NAGGN). Preliminary results of this work have been presented elsewhere (L. T. Smith and G. M. Smith, J. Cell Biol. 107:629, 1988). MATERIALS AND METHODS
Strains and media. R. meliloti 102F34 was used in most experiments. Other strains used are listed in Table 2. Rhizo*
Corresponding author. 4714
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B
200
180
160
140
120
100
80
60
40
20
0
PPM
FIG. 1. (A) Natural-abundance 13C NMR spectrum of a cell extract of R. meliloti 102F34 grown in 0.5 M NaCl. Details for sample preparation and NMR parameters are given in Materials and Methods. Resonances at 182.5, 177.0, 56.0, 34.7, and 28.9 ppm arise from glutamate. (B) Natural-abundance 13C NMR spectrum of synthetic NAGGN.
mixed bed resin (Bio-Rad Laboratories, Inc.). The molecular mass (315) was confirmed by fast-atom bombardment mass spectrometry on a ZAB-HS-2F mass spectrometer (VG Analytical). Analysis of the compound by circular dichroism (J-500C spectropolarimeter [Jasco] with a DP-501N data processor) revealed a positive Cotton effect at 212 nm and a negative effect at 228 nm. Mass spectral analysis. Structural characterization of the dipeptide isolated from R. meliloti was conducted by generating a collision-activated dissociation-mass-analyzed ion kinetic energy daughter ion spectrum of the (M + H)+ ion, which was obtained by using fast-atom bombardment ionization. Helium was used as the collision gas in the second field-free region of the mass spectrometer at a pressure of 10-6 mb (1 bar = -100 kPa) (measured at the diffusion pump stack). High-pressure liquid chromatography analysis. The purity of synthetic and naturally isolated NAGGN was ascertained by high-pressure liquid chromatography analysis on a Hewlett-Packard HP 1090 liquid chromatograph with a diode array detector using a Supelco C18 DB microbore column (25 cm by 2 mm) with 5-pum packing. The mobile phase was water, and the retention time of both synthetic and natural NAGGN was 5.3 min, at a flow rate of 0.3 ml/min. RESULTS Identification of NAGGN. A 13C NMR spectrum of an acid extract of stressed R. meliloti 102F34 was analyzed (Fig.
1A). As expected, the spectrum shows that glutamate (182.5, 177.0, 56.0, 34.7, and 28.9 ppm) was the major osmolyte. However, a number of additional, unidentified resonances were also present. The occurrence of these resonances in the same ratio in several samples suggested that all of the unidentified resonances arose from the same compound. To identify this compound, several NMR experiments were conducted, including a heteronuclear 1H-13C shift correlation map (15) and a J-resolved two-dimensional proton spectrum (1) which were obtained at a proton frequency of 360 MHz (NT-360 spectrometer, General Electric Co.). Also, a homonuclear COSY (2) spectrum was obtained at 500 MHz (NM-500 spectrometer; General Electric). The chemical shifts and spin-coupling patterns derived from these experiments indicated that the compound is closely related to glutamine or a similar derivative of glutamate. Also, the presence of two resonances of equal intensity at 54.6 and 54.3 ppm, which resembled the resonances of two ox carbons, suggested that the compound was a dipeptide. Furthermore, the resonance at 23 ppm is characteristic of N-acetyl groups, suggesting an amino-blocked peptide. These interpretations are in accord with ninhydrin analyses in which more ninhydrin-positive material is obtained from acid-hydrolyzed extracts than from untreated extracts. To aid in analysis, a purified (deionized) sample was subjected to mass spectrometry. The spectrum yielded a molecular ion (M + H)+ mass of 316 and major fragments with masses of 299, 244, 171, 146, and 129. The mass and
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SMITH AND SMITH
J. BACTERIOL.
TABLE
TABLE 1. Osmolytes accumulated by R. toeliloti 102F34 grown under osmotic stress" Addition to MCAA medium
0.5 M NaCl 0.5 M KCI 0.45 M K2SO4 0.7 M Sucrose 0.4 M NaCi'
Accumulated osmolyte (nmol/mg of protein) Glutamate NAGGN Trehalose
550 450 560 210 400
360 330 340 72 250
UD T T UD 50
" Cultures were grown and samples were prepared as described in Materials and Methods. The concentration of osmolyte was quantitated by 13C NMR using 100 ,umol of alanine as a standard and by amino acid analyses. UD. Undetectable (
