Nutrient-deficient Media Research Articles

Response of transgenic potato seedlings to allelopathic pressure and the effect of nutrients in the culture medium

Chinese farmers frequently use a wheat–potato cropping system. The land area planted to transgenic potatoes is increasing because transgenic potatoes have greater resistance to pests and diseases. However, little is known about the bio-compatibility of transgenic potatoes with wheat straw. The objective of this tissue culture study was to determine the allelopathic effects of wheat straw on transgenic potato seedlings. Seedlings were cultured on normal MS medium (normal treatment) and nutrient-deficient MS medium (acclimated treatment) and then transferred to MS medium, which contained wheat straw powder. Wheat straw powder inhibited potato seedling growth in both treatments. Among the parameters analyzed in this study, inhibition was greatest for plant fresh weight and least for plant height. The inhibitive effects of wheat straw were greater for seedling roots compared to shoots. Resistance to allelopathic pressure from wheat straw was greater in acclimated seedlings compared to normal seedlings. This suggested that previous pressure may have induced tolerance in the transgenic potato seedlings. Furthermore, growth inhibition of potato seedlings from the normal treatment increased as the amount of wheat straw powder in the culture medium increased. Calculations indicated that the presence of wheat straw would lead to a 55% reduction in the total biomass of normal potato seedlings compared to a 39% reduction for acclimated seedlings. Parameters such as net photosynthesis rate (Pn) and quantum yield (Y(II)) changed as the nutrient content of the culture medium increased or decreased, but the changes in the parameters were smaller for acclimated seedlings compared to normal seedlings. This suggests that nutrient status during the culture period could help transgenic potato seedlings adapt and compensate for energy loss from seedlings in defending against allelopathic pressure. In summary, the results show that previous exposure to pressures such as nutrient deficiency may increase the allelopathic pressure resistance of transgenic potato seedlings.

Localization of endophyticUndifilumfungi in locoweed seed and influence of environmental parameters on a locoweed in vitro culture system

Endophytic Undifilum oxytropis (Q. Wang, Nagao & Kakish) Pryor, Creamer, Shoemaker, McLain-Romero, & Hambleton found within toxic locoweeds ( Astragalus and Oxytropis spp.) produces the alkaloid swainsonine, which is responsible for locoism in grazing animals. We sought to determine the location of U. oxytropis within locoweed seed, develop endophyte free plants, and assess the influence of environmental stresses on locoweed and endophyte cultures. Undifilum was identified within the parenchyma layers in seeds of Astragalus lentiginosus M.E. Jones using light microscopy and polymerase chain reaction. Astragalus lentiginosus and Oxytropis sericea Nutt. seedlings produced in embryo culture without seed coats did not contain swainsonine or fungus. Plants produced from whole seed contained U. oxytropis in both foliage and root tissues. When the in-vitro cultured plants of O. sericea and U. oxytropis cultures were subjected to environmental stresses including high temperature, low and high pH media, nutrient deficient media, and polyethylene glycol (PEG) amended media to simulate water deficit, both dry mass and swainsonine levels were affected. Swainsonine levels were greatest for O. sericea and Undifilum cultures in PEG or hydrochloric acid amended media. Plants grown in PEG-amended media had significantly greater dry mass, while Undifilum grown in PEG-amended media had lower dry mass than other treatments.

Calcium-dependent Binding of HCN1 Channel Protein to Hair Cell Stereociliary Tip Link Protein Protocadherin 15 CD3

The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform expressed in trout saccular hair cells, was found by yeast two-hybrid protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin 15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair cell stereocilia, consistent with gradated distribution expected for tip link sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries of rat cochlear inner and outer hair cells, and HCN1 protein was immunolocalized to cochlear hair cell stereocilia. As predicted by the trout hair cell model, the amino terminus of rat organ of Corti HCN1 was found by yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15 CD3, a tip link protein implicated in mechanosensory transduction. Specific binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down assays and surface plasmon resonance analysis, both predicting dependence on Ca(2+). In the presence of calcium chelators, binding between HCN1 and protocadherin 15 CD3 was characterized by a K(D) = 2.39 x 10(-7) m. Ca(2+) at 26.5-68.0 microm promoted binding, with K(D) = 5.26 x 10(-8) m (at 61 microm Ca(2+)). Binding by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179 (GenBank accession number XP_238200), with homology to the comparable region in trout hair cell protocadherin 15a-like protein, as necessary for binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to underlie HCN1 channel formation, was also found to be Ca(2+)-dependent, although the binding was skewed toward a lower effective maximum [Ca(2+)] than for the HCN1 interaction with protocadherin 15 CD3. Competition may therefore exist in vivo between the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3 favored between 26.5 and 68 microm Ca(2+). Taken together, the evidence supports a role for HCN1 in mechanosensory transduction of inner ear hair cells.

In vitro study of bacterial degradation of ethyl glucuronide and ethyl sulphate

Recent studies show that ethyl glucuronide (EtG) can be decomposed by bacteria; whilst so far no degradation of ethyl sulphate (EtS) has been observed. In the present study, in vitro experiments with bacterial colonies were performed. Bacteria (Escherichia coli, Klebsiella pneumoniae, Clostridium sordellii) were isolated from autopsy material (liver, heart blood, urine, ascites, pericardial fluid, pleural fluid) tested for beta-glucuronidase activity, and three bacterial strains were added to nutrient-deficient medium containing EtG and/or EtS and incubated at 36 +/- 1 degrees C. Samples were taken after various intervals up to 11 days, and EtG and EtS were determined by electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). EtG was degraded by E. coli and C. sordellii--complete degradation occurred in the range of 3-4 days--and these bacteria exhibited beta-glucuronidase activity. EtS was not affected within 11 days of incubation.

Cellulose as an Extracellular Matrix Component Present in Enterobacter sakazakii Biofilms

Cellulose was identified and characterized as an extracellular matrix component present in the biofilm of an Enterobacter sakazakii clinical isolate grown in nutrient-deficient (M9) medium. Using a bacterial artificial cloning approach in Escherichia coli and subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. In addition to the genes already described for cellulose production, two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass coverslips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.

Role of the GGDEF protein family in Salmonella cellulose biosynthesis and biofilm formation.

Salmonella enterica serovar Typhimurium is capable of producing cellulose as the main exopolysaccharide compound of the biofilm matrix. It has been shown for Gluconacetobacter xylinum that cellulose biosynthesis is allosterically regulated by bis-(3',5') cyclic diguanylic acid, whose synthesis/degradation depends on diguanylate cyclase/phosphodiesterase enzymatic activities. A protein domain, named GGDEF, is present in all diguanylate cyclase/phosphodiesterase enzymes that have been studied to date. In this study, we analysed the molecular mechanisms responsible for the failure of Salmonella typhimurium strain SL1344 to form biofilms under different environmental conditions. Using a complementation assay, we were able to identify two genes, which can restore the biofilm defect of SL1344 when expressed from the plasmid pBR328. Based on the observation that one of the genes, STM1987, contains a GGDEF domain, and the other, mlrA, indirectly controls the expression of another GGDEF protein, AdrA, we proceeded on a mutational analysis of the additional GG[DE]EF motif containing proteins of S. typhimurium. Our results demonstrated that MlrA, and thus AdrA, is required for cellulose production and biofilm formation in LB complex medium whereas STM1987 (GGDEF domain containing protein A, gcpA) is critical for biofilm formation in the nutrient-deficient medium, ATM. Insertional inactivation of the other six members of the GGDEF family (gcpB-G) showed that only deletion of yciR (gcpE) affected cellulose production and biofilm formation. However, when provided on plasmid pBR328, most of the members of the GGDEF family showed a strong dominant phenotype able to bypass the need for AdrA and GcpA respectively. Altogether, these results indicate that most GGDEF proteins of S. typhimurium are functionally related, probably by controlling the levels of the same final product (cyclic di-GMP), which include among its regulatory targets the cellulose production and biofilm formation of S. typhimurium.

Culture Studies of Alexandrium affine (Dinophyceae), a Non-Toxic Cyst Forming Dinoflagellate from Bahía Concepción, Gulf of California

Alexandrium affine (Inoue et Fukuyo) Balech, isolated from Bahia ConcepciOn (Gulf of California), was studied to determine the effect of environmental factors on cyst germination and vegetative growth. Alexandrium affine was homothallic and isogamous, and formed cysts in nutrient-deficient (N- or P-limiting) medium. The maturation period of newly formed cysts varied between two weeks and three months, depending on the storage temperature, with colder temperatures prolonging the process. The rate of germination increased with increasing temperature, and was not significantly influenced by light. Germination experiments suggest a broad temperature window for A. affine cysts, ranging from 5 to 25 °C. The optimal vegetative growth rates were 0.25 to 0.34 day - 1 at 20-30 °C. No vegetative growth was observed below 15 °C or above 35 °C. With HPLC toxin analyses, we confirm that this species does not produce saxitoxins. These data on the dormancy, excystment, and growth characteristics seem to be regulated by the environmental constraints of this subtropical bay.

Glucose and nutrient concentrations affect the expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes.

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in Listeria monocytogenes. Here, the expression of the 104-kDa Listeria adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K(2)HPO(4) reduced this effect. L. monocytogenes cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while L. monocytogenes cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative L. monocytogenes strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.

Zinc acclimation and its effect on the zinc tolerance of Raphidocelis subcapitata and Chlorella vulgaris in laboratory experiments

The effect of zinc acclimation of Raphidocelis subcapitata (syn. Selenastrum capricornutum) and Chlorella vulgaris on their sensitivity towards this metal was examined in a series of laboratory experiments. These two commonly used algal species were acclimated to 65 μg Zn/l and changes in zinc tolerance were monitored using standard growth inhibition tests. The chemically defined ISO medium was used as a control culture medium. Both species demonstrated a maximum increase in zinc tolerance of a factor of 3 after 100 days of acclimation. Shifts in the shape of the concentration–response curve due to acclimation were observed for R. subcapitata. Compared to non-acclimated algae, acclimated R. subcapitata exhibited higher growth rates in all zinc treatments as well as in the controls. This suggests that the use of ISO-medium results in sub-optimal growth due to zinc deficiency. These effects could not be demonstrated for C. vulgaris. The zinc tolerance of both species decreased significantly one week after returning the acclimated algae to control (ISO) medium. 72hEC50 values based on growth rate were two to four times higher than those calculated using biomass measurements. Algal toxicity test results, particularly if used for metal risk assessments, must not be conducted using nutrient deficient media.

Akinete Formation in Tribonema bombycinum Derbes et Solier (Xanthophyceae) in Relation to Freezing Tolerance

(Xanthophyceae), was examined. T. bombycinum shifted from vegetative cells to akinetes with starving by a prolonged batch culture, by culture with a diluted medium, or by culture with a single nutrient-deficient medium. In addition, akinetes developed by desiccation, but cold treatment at 4 C did not facilitate akinete formation. During starving, the vegetative cells, which had a large central vacuole in the protoplasm and thin cell walls, finally changed to akinetes, which had many small vacuoles and oil droplets in the protoplasm and thick cell walls. During akinete formation by starving, the freezing tolerance (LT50) increased gradually from −3 C in vegetative cells to far below −30 C in akinetes. When vegetative cells were subjected to equilibrium freezing, their size shrank greatly and aparticulate domains accompanied by fracture-jump lesions developed in the plasma membranes. Akinetes subjected to equilibrium freezing showed little shrinkage, and freezing-induced ultrastructural changes did not occur in the plasma membranes. The morphological changes in the process of akinete formation and the responses to equilibrium freezing resembled those of cold-acclimated terrestrial plants.

Cutaneous Zygomycosis Due toSaksenaea Vasiformis: Case Report and Literature Review

Cutaneous Zygomycosis Due to<i>Saksenaea Vasiformis</i>: Case Report and Literature Review

Metabolic response of the halotolerant green algaDunaliella bardawil to nitrogen: Phosphorus ratios in batch culture

The effect of N/P ratios and salinity on the growth as well as some metabolic activities by the halotolerant green algaDunaliella bardawil were followed. In nutrient-deficient media (N0 or P0) growth was retarded and cells accumulated large amounts of carotene, glycerol and saccharides while the contents of protein and amino acids were significantly decreased. In contrast, in nutrient-enriched media (N5 and/or P5), the growth and protein content in addition to the oxygen evolution were significantly increased at the expense of glycerol and saccharide content.

9α-Hydroxylation of 4-androstene-3,17-dione by resting Rhodococcus sp. cells

The effect of nitrogen and carbon nutrition, growth and biomass storage on the effectiveness of microbial 9α-hydroxylation of 4-androstene-3,17-dione was studied. The transformation was carried out by resting Rhodococcus sp. cells in nutrient deficient transformation medium at different pHs as well as in the presence of several solvents of the steroid compounds. A conversion ratio of ca 0·75 was observed with stationary phase cells grown on medium containing glucose and non-dehydrated casein hydrolysate. A strong influence of the nitrogen source on the effectiveness of the process was noticed. The presence of different alcohols in the reaction medium was found to increase the substrate conversion ratio to ca 0·85. A decrease in the transformation ability of stored cells was a logarithmic function of the storage time.

THE ROLE OF THE SIGMA FACTOR σs (KatF) IN BACTERIAL GLOBAL REGULATION

The protein encoded by katF (also known as nur, appR, csi-2, abrD, and rpoS in various alleles) has been biochemically confirmed to be an alternate sigma transcription factor and renamed sigma S. Its synthesis is controlled transcriptionally and posttranscriptionally by as yet undefined mechanisms that are active well into stationary phase. sigma S controls a regulon of 30 or more genes expressed in response to starvation and during the transition to stationary phase. Proteins in the regulon, many of which have not been characterized, enhance long-term survival in nutrient-deficient medium and have a diverse group of functions including protection against DNA damage, the determination of morphological changes, the mediation of virulence, osmoprotection, and thermotolerance. Differential expression of subfamilies of genes within the regulon is effected by supplementary regulatory factors, working both individually and in combination to modulate activity of different sigma S-dependent promoters.

Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

The effects of nutrients and temperature on biomass, growth, lipid production, and fatty acid composition ofCyclotella cryptica Reimann, Lewin, and Guillard

Batch cultures ofCyclotella cryptica Reimann, Lewin, and Guillard were grown at two different nutrient (nitrogen/silica) levels and at two different temperatures (20 and 30°C). Biomass and cell yields decreased with decreased levels of both the nutrients (nitrogen and silica) at 20 and 30°C; whereas lipids (total, neutral, and polar) increased with decreased levels of nutrients at these two temperatures. Changes in fatty acid composition were also noted; the diatoms produced increased amounts of fatty acids C14:0 and C16:0 when grown in nutrient-deficient medium. The influence of nutrient stress and temperature is discussed.

Changes in Photosynthesis/Respiration Ratio and Levels of Few Carbohydrates in Leaves of Nutrient Depleted Barley and Pea

Changes in carbon (C) partitioning in response to an induced phosphorus (P) or nitrogen (N) deficiency were studied in mature leaves of barley ( Hordeum vulgare L. cv. Agneta) and pea ( Pisum sativum L. cv. Marma). Optimally growing, 7-day-old pea plants or 15-day-oldbarley plants were transferred to Nor P-free but otherwise complete media for a period of 14–20 days. The responses of the two species to both types of nutrient deprivation can be divided into two phases: an early sucrose and starch accumulating phase, lasting for 8–10 days, and a late phase during which these carbohydrate levels decreased. During the first phase, the rate of the dark respiration decreased sharply whereas the CO 2 assimilation rate decreased only slightly. An increase in the photosynthesis/respiration ratio had already become apparent after 2 days of growth on the nutrient deficient media. In the later phase, the starch and sucrose pools decreased to levels even lower than the initial ones. Simultaneously the relative decrease in CO 2 assimilation rate surpassed the decrease in dark respiration rate, i.e. the photosynthesis/respiration ratio began to decrease, eventually falling below the initial value measured on day o. In both species, accumulations of starch and soluble sugars in both mature leaves and sink tissues (roots) were substantially higher in N-deprived plants than in P-deprived ones.

Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.

Adhesion of Mycoplasma pneumoniae to Sulfated Glycolipids and Inhibition by Dextran Sulfate

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).

Growth Response of Putrefactive Anaerobe 3679 to Combinations of Potassium Sorbate and Some Common Curing Ingredients (Sucrose, Salt, and Nitrite), and to Noninhibitory Levels of Sorbic Acid

ABSTRACT The effects of some common curing ingredients (sucrose, sodium chloride, and nitrite) on sorbate-induced inhibition were tested in putrefactive anaerobe (PA) 3679, a proteolytic species of Clostridium. A noninhibitory concentration (1%) of sucrose was not synergistic with the inhibitory action of potassium sorbate (30 mM; pH 6). The effect of sodium chloride depended upon the concentration used; a combination of 3% NaCl and sorbate (30 mM; pH 6) was more inhibitory than either compound alone but 1% NaCl reduced the inhibitory action of sorbate (30 mM; pH 6). Combining a noninhibitory concentration of nitrite (100 pM) and sorbate (30 mM; pH 6) was bactericidal to PA 3679. Nitrite raised the intracellular pH (pHi) and increased the protonmotive force (PMF) of untreated and sorbate-treated cells. Growth of PA 3679 was stimulated by the addition of a noninhibitory level of undissociated sorbic acid (5.5 mM; pH 7) or 0.1% glucose (5.5 mM) to a nutrient-deficient medium (0.25% trypticase [BBL]) but growth initiated more rapidly when sorbic acid was added than when the medium was supplemented with glucose. Results of this study are discussed in relation to the possible mechanisms of preservative action and suggest that certain recent changes in methods of food processing and packaging may increase the possibility of growth of clostridial organisms, including Clostridium botulinum.