Listeria monocytogenes is a gram-positive food-borne pathogen that is notably resistant to osmotic stress and can grow at refrigerator temperatures. These two characteristics make it an insidious threat to public health. Like several other organisms, L. monocytogenes accumulates glycine betaine, a ubiquitous and effective osmolyte, intracellularly when grown under osmotic stress. However, it also accumulates glycine betaine when grown under chill stress at refrigerator temperatures. Exogenously added glycine betaine enhances the growth rate of stressed but not unstressed cells, i.e., it confers both osmotolerance and cryotolerance. Both salt-stimulated and cold-stimulated accumulation of glycine betaine occur by transport from the medium rather than by biosynthesis. Direct measurement of glycine betaine uptake shows that cells transport betaine 200-fold faster at high salt concentration (4% NaCl) than without added salt and 15-fold faster at 7 than at 30 degrees C. The kinetics of glycine betaine transport suggest that the two transport systems are indistinguishable in terms of affinity for betaine and may be the same. Hyperosmotic shock and cold shock experiments suggest the transport system(s) to be constitutive; activation was not blocked by chloramphenicol. A cold-activated transport system is a novel observation and has intriguing implications concerning the physical state of the cell membrane at low temperature.

The full-length cDNA sequence (P93622_VITVI) of polyphenol oxidase (PPO) cDNA from grape Vitis vinifera L., cv Grenache, was found to encode a translated protein of 607 amino acids with an expected molecular weight of ca. 67 kDa and a predicted pI of 6.83. The translated amino acid sequence was 99%, identical to that of a white grape berry PPO (1) (5 out of 607 amino acid potential sequence differences). The protein was purified from Grenache grape berries by using traditional methods, and it was crystallized with ammonium acetate by the hanging-drop vapor diffusion method. The crystals were orthorhombic, space group C222(1). The structure was obtained at 2.2 A resolution using synchrotron radiation using the 39 kDa isozyme of sweet potato PPO (PDB code: 1BT1 ) as a phase donor. The basic symmetry of the cell parameters (a, b, and c and alpha, beta, and gamma) as well as in the number of asymmetric units in the unit cell of the crystals of PPO, differed between the two proteins. The structures of the two enzymes are quite similar in overall fold, the location of the helix bundles at the core, and the active site in which three histidines bind each of the two catalytic copper ions, and one of the histidines is engaged in a thioether linkage with a cysteine residue. The possibility that the formation of the Cys-His thioether linkage constitutes the activation step is proposed. No evidence of phosphorylation or glycoslyation was found in the electron density map. The mass of the crystallized protein appears to be only 38.4 kDa, and the processing that occurs in the grape berry that leads to this smaller size is discussed.

We have investigated the spectral response of front-surface-illuminated GaN and AlGaN/GaN p-i-n ultraviolet photodetectors prepared by reactive molecular beam epitaxy on sapphire substrates. GaN homojunction p-i-n photodiodes exhibited a peaked response near the band edge. This enhanced response was absent in the AlGaN/GaN heterojunction p-i-n detectors. We analyzed the effect of p-layer thickness of the GaN p-i-n diodes on the magnitude of the peak photoresponse. The AlGaN/GaN photodiodes had a maximum zero-bias responsivity of 0.12 A/W at 364 nm, which decreased by more than 3 orders of magnitude for wavelengths longer than 390 nm. A reverse bias of −10 V raised the responsivity to 0.15 A/W without any significant increase in noise. The root-mean-square noise current in a 1 Hz bandwidth is ∼1.0 pA, corresponding to a noise-equivalent-power of ∼8.3 pW. We measured extremely fast decay times of 12 ns for the AlGaN/GaN and 29 ns for the GaN photodiodes.

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 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 (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 cen...

The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.The animal and human pathogen Listeria monocytogenes is the causative agent of listeriosis, a food-borne disease primarily affecting immunocompromised individuals with a 20 to 30% mortality rate (28, 36). L. monocytogenes has long been recognized as an organism capable of proliferating at refrigeration temperatures (46). During the last decade, several aspects of the L. monocytogenes physiology have been identified that are linked to the ability of this otherwise mesophilic pathogen to adapt to environments of low temperature, including the expression of cold shock proteins, the retailoring of the membrane lipid composition, and the accumulation of compatible solutes.Cold-stress (cold-shock and cold acclimation) proteins whose synthesis is increased after temperature downshifts have been isolated, but for the most part their identities and functions remain uncertain (6,16,32,47). Analogous proteins have been studied for Escherichia coli and Bacillus subtilis and have been postulated to function as RNA chaperons, transcription antiterminators, or transcription activators (9,13,14,19). A recent study of gene expression in response to growth of L. monocytogenes at 10°C showed that the pathogen's acclimation involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and...