Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production.The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5 mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast, the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50 mM for thiolutin (133 mg/g DCW), 1.25 mM for senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/g DCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

Effect of amino acids containing sulfur on dithiolopyrrolone antibiotic productions by Saccharothrix algeriensis NRRL B-24137

AIMS: To study the effect of sulfur-containing amino acids (L-cysteine, L-cystine, L-methionine and DL-ethionine) on the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137. METHODS AND RESULTS: The production levels of dithiolopyrrolones were investigated by using high performance liquid chromatography in a chemically semi-synthetic medium. The production of the studied antibiotics depends upon the nature, concentration and the time of addition of these sources in the culture medium. Both cysteine and cystine favoured the specific productions of dithiolopyrrolones; iso-butyryl-pyrrothine (ISP) by cysteine, however butanoyl-pyrrothine, senecioyl-pyrrothine and tigloyl-pyrrothine by cystine, when added initially to the culture medium. The maximum specific productions of dithiolopyrrolones were observed in the presence of 5 mmol l(-1) cystine for thiolutin, 5 mmol l(-1) cysteine for ISP, and 10 mmol l(-1) cystine for others studied dithiolopyrrolones as shown in Fig. 3. The production of these antibiotics was decreased when the concentrations of cysteine and cystine were in excess. All dithiolopyrrolone specific productions were strongly inhibited by addition of methionine and ethionine, without inhibition of mycelial growth. CONCLUSIONS: Among all studied amino acids, cystine and cysteine can be used as supplements for improvement the production of dithiolopyrrolone antibiotics by S. algeriensis NRRL B-24137. SIGNIFICANCE AND IMPACT OF THE STUDY: Dithiolopyrrolone antibiotics have many important applications for employing them as medicaments, particularly in the treatment of human and animal cancers. In the present work, the influence of containing-sulfur amino acids on dithiolopyrrolone antibiotic productions was studied. The obtained results can be employed for the optimization of the culture medium for the dithiolopyrrolone productions in higher quantities.     

Expression of pyrrothine N-acyltransferase activities in Saccharothrix algeriensis NRRL B-24137: new insights into dithiolopyrrolone antibiotic biosynthetic pathway

Aims:  The hypothetical dithiolopyrrolone biosynthetic pathway includes a final step of pyrrothine nucleus acylation. The presence of an enzymatic activity catalysing this reaction was investigated in Saccharothrix algeriensis NRRL B-24137. To understand the effect exerted by organic acids on the level of dithiolopyrrolone production, their influence on enzymatic expression was studied.
Methods and Results:  The transfer of acetyl-CoA or benzoyl-CoA on pyrrothine was assayed in the cell-free extract of Sa. algeriensis NRRL B-24137. This study reports the presence of an enzymatic activity catalysing this reaction that was identified as either pyrrothine N -acetyltransferase or N -benzoyltransferase. The stimulation of benzoyl-pyrrothine (BEP) production by addition of benzoic acid at 1·25 mmol l⁻¹ into the culture medium was demonstrated, and results showed that under the same conditions of growth, pyrrothine N -benzoyltransferase specific activity was doubled.
Conclusions:  This study shows that BEP production is enhanced in the presence of benzoic acid partly because of an induction of pyrrothine N -benzoyltransferase.
Significance and Impact of the Study:  The antitumor and antibiotic properties of dithiolopyrrolones are related to their variable acyl groups. New insights into regulation of biosynthetic pathway, especially the step of pyrrothine acylation, could lead after further studies to yield improvement and to selective production of dithiolopyrrolones with new biological activities.     

The final acylation step in aromatic dithiolopyrrolone biosyntheses: Identification and characterization of the first bacterium N-benzoyltransferase from Saccharothrix algeriensis NRRL B-24137

The last step in the biosynthesis of dithiolopyrrolone antibiotics was thought to involve the transfer of acyl group from acyl-CoA to pyrrothine/holothin core. In Saccharothrix algeriensis NRRL B-24137, two acyltransferases, an acetyltransferase and a benzoyltransferase were proposed to catalyze this step. We have previously identified, in Sa. algeriensis genome, two open read frames, actA and actB patiently encoded these enzymes. This study focuses primarily on the characterization of the protein encoded by actA. After cloning and expressing of actA in Escherichia coli BL21, the recombinant protein encoded by actA was purified. Selectivity of ActA for pyrrothine/holothin as substrate and different acyl-CoA as co-substrate was evaluated using two acyls-groups, linear and aromatic. The enzyme was shown to prefer aromatic groups over linear groups as donor group; further neither product nor transfer was observed for linear groups. Therefore ActA has been determined to be a pyrrothine/holothin N-benzoyltransferase which can either pyrrothine (K_m of 72 μM) or holothin (K_m of 129.5 μM) as substrates and benzoyl-CoA (K_m of 348.65 and 395.28 μM) as co-substrates for pyrrothine and holothin, respectively. The optimum pH and temperature has been shown to be 8, 40 °C, respectively. ActA is the first enzyme characterized as N-benzoyltransferase in bacteria.     

Visualization of grapevine root colonization by the Saharan soil isolate Saccharothrix algeriensis NRRL B-24137 using DOPE-FISH microscopy

Background and aim

There is currently a gap of knowledge regarding whether some beneficial bacteria isolated from desert soils can colonize epi- and endophytically plants of temperate regions. In this study, the early steps of the colonization process of one of these bacteria, Saccharothrix algeriensis NRRL B-24137, was studied on grapevine roots to determine if this beneficial strain can colonize a non-natural host plant. An improved method of fluorescence in situ hybridization (FISH), the double labeling of oligonucleotide probes (DOPE)-FISH technique was used to visualize the colonization behavior of such bacteria as well as to determine if the method could be used to track microbes on and inside plants.

Methods

A probe specific to Saccharothrix spp. was firstly designed. Visualization of the colonization behavior of S. algeriensis NRRL B-24137 on and inside roots of grapevine plants was then carried out with DOPE-FISH microscopy.

Results

The results showed that 10 days after inoculation, the strain could colonize the root hair zone, root elongation zone, as well as root emergence sites by establishing different forms of bacterial structures as revealed by the DOPE-FISH technique. Further observations showed that the strain could be also endophytic inside the endorhiza of grapevine plants.

Conclusions

Taking into account the natural niches of this beneficial strain, this study exemplifies that, in spite of its isolation from desert soil, the strain can establish populations as well as subpopulations on and inside grapevine plants and that the DOPE-FISH tool can allow to detect it.     

Nutritional requirements for the production of dithiolopyrrolone antibiotics by Saccharothrix algeriensis NRRL B-24137

The amino acid and humic acid requirements of Saccharothrix algeriensis NRRL B-24137 for growth and production of the dithiolopyrrolone antibiotics were studied in a semi-synthetic medium (SSM). Nature and concentration of amino acids and humic acid strongly influenced the growth and dithiolopyrrolone specific production.

The highest value of thiolutin (acetyl-pyrrothine) specific production was obtained in the presence of 1 g/l humic acid (336 mg/g DCW), and in the presence of 5 mM l-cystine (309 mg/g DCW) as compared to 19 mg/g DCW obtained with the control. Furthermore, thiolutin production was increased about six-fold, four-fold and three-fold in the presence of l-proline, l-glutamic acid and dl-histidine, respectively. In contrast, the production of thiolutin was reduced by addition of other amino acids such as l-glutamine, dl-ethionine, l-methionine and l-arginine. The highest value of isobutyryl-pyrrothine production was obtained in the presence of 2,6-diaminopimelic acid and l-lysine (7.8 and 1.0 mg/g DCW, respectively). However, the highest value of butanoyl-pyrrothine production was obtained in the presence of humic acid (6.6 mg/g DCW), followed by l-cysteine and l-proline (3.6 and 3.2 mg/g DCW, respectively). In addition, the maximum specific production of senecioyl-pyrrothine (29 mg/g DCW) and tigloyl-pyrrothine (21 mg/g DCW) was obtained in the presence of humic acid. We found that, except for isobutyryl-pyrrothine, production of all dithiolopyrrolones was favoured by addition of l-proline. The maximum specific production was obtained with l-proline at concentrations of 2.50 mM for thiolutin (133 mg/g DCW), 1.25 mM for senecioyl-pyrrothine, tigloyl-pyrrothine and butanoyl-pyrrothine production (29, 23 and 3.9 mg/g DCW, respectively). Production of all dithiolopyrrolones strongly decreased as the l-methionine or dl-ethionine concentration was increased in the culture medium.     

The Saharan isolate Saccharothrix algeriensis NRRL B-24137 induces systemic resistance in Arabidopsis thaliana seedlings against Botrytis cinerea

Background and aim

Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea.

Methods

The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3, AtrhbohF-3, and ups1-1 were also screened to determine other parts of the mechanisms involved.

Results

The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved.

Conclusions

The isolate NRRL B-24137, after colonizing the root systems of A. thaliana, induces an ISR against B. cinerea, which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.     

Yield enhancement strategy of dithiolopyrrolone from Saccharothrix algeriensis by aliphatic alcohols supplementation

The production of dithiolopyrrolones by Saccharothrix algeriensis was investigated after supplementing the culture medium with ethanol and/or 1-butanol. Optimal conditions for the addition of ethanol to the culture medium provided a maximal dithiolopyrrolone titer of about 200 mg⋅L⁻¹ after 5 days of culture, roughly corresponding to a 600%-increase. Using NAD(P)H oxidase inhibitor (diphenyleneiodonium) or reactive oxygen species scavenger (para-aminobenzoic acid), we suppose that ethanol promotes the formation of reactive oxygen species in Saccharothrix algeriensis, which, in turn, could induce biomass decline and dithiolopyrrolone overproduction. However, the underlying mechanisms remain to be elucidated. These results may be helpful for the control of dithiolopyrrolone yields from Saccharothrix algeriensis cultures.     

在培养基中加入抗生素防止万年青茎段培养污染的研究

以玛丽安万年青茎段为试验材料,将浓度为50mg·L1的青霉素、氯霉素、利福平、庆大霉素、先锋霉素6号、链霉素和卡拉霉素7种抗生素分别经过过滤灭菌后加入到MS基本培养基,从中筛选出防止污染效果较好的两种抗生素,分别按50、100、150mg·L1的浓度加入到MS培养基中培养,以筛选出最适合浓度;然后再筛选出利福平和氯霉素的最佳浓度组合。结果表明:7种抗生素中以利福平和氯霉素防止污染效果较好,未污染率分别为68.42%和58.33%,成活率分别为65.79%和52.78%,利福平和氯霉素的浓度均为150mg·L1时防止污染的效果最佳;25mg·L1利福平+50mg·L1氯霉素和50mg·L1利福平+50mg·L1氯霉素组合,未污染率分别达到78.46%、85.18%,极显著高于50mg·L1利福平单独处理的效果。     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Optimization of lactic acid production in SSF by Lactobacillus amylovorus NRRL B-4542 using Taguchi methodology

Lactic acid production parameter optimization using Lactobacillus amylovorus NRRL B-4542 was performed using the design of experiments (DOE) available in the form of an orthogonal array and a software for automatic design and analysis of the experiments, both based on Taguchi protocol. Optimal levels of physical parameters and key media components namely temperature, pH, inoculum size, moisture, yeast extract, MgSO4 . 7H20, Tween 80, and corn steep liquor (CSL) were determined. Among the physical parameters, temperature contributed higher influence, and among media components, yeast extract, MgSO4 . 7H20, and Tween 80 played important roles in the conversion of starch to lactic acid. The expected yield of lactic acid under these optimal conditions was 95.80% and the actual yield at optimum conditions was 93.50%.     

Development of a continuous system for the degradation of a cyanuric acid by adsorbed Pseudomonas sp. NRRL B-12228

Cyanuric acid in high concentrations (15.5 mm) was degraded completely by Pseudomonas sp. NRRL B-12228 independently of glucose concentration. In the batch fermentations there was a relation between the glucose concentration, on the one hand, and the liberation of ammonia or production of protein, on the other. The greater the supply of carbon, the more biomass was produced, and fewer NH _inf4 ^sup+ ions were released. Continuous fermentations using adsorbed cells could be performed to degrade cyanuric acid. In spite of different glucose feeding there was only a negligible difference in residues of s-triazine. In a one-step continuous system with dilution rates between 0.021 h^–1 and 0.035 h^–1, even a ratio of 0.65 between glucose and cyanuric acid was not sufficient to degrade the cyanuric acid supplied (320–540 mol l^–1 h^–1) completely. When a continuous two-step system was applied with dilution rates between 0.035 h^–1 and 0.056 h^–1, the consumption of carbon source could be minimized while s-triazine degradation up to 860 mol l^–1 h^–1 was complete. In this way the ratio between glucose and cyanuric acid could be increased to 0.25 (molar C:N ratio = 0.33:1). Thereby the process was made considerably more economic.     

培养基中添加海藻糖对大球盖菇、斑玉蕈菌丝生长的影响

【背景】大球盖菇和斑玉蕈是食药兼用且具有开发潜力的珍稀食用菌,培养基对菌丝生长及子实体发育具有重要作用,优化培养基显得尤为重要。【目的】筛选出最适合培养大球盖菇、斑玉蕈的新型培养基。【方法】使用添加海藻糖的新型培养基,对不同培养基培养的大球盖菇及斑玉蕈菌株的菌丝生长状况、生长速度和生物量及纤维素酶、漆酶活性进行测定与分析。【结果】相较于PDA培养基,添加海藻糖的培养基能够提高菌丝生长速度、增加生物量,海藻糖添加的比例对纤维素酶和漆酶的影响较大,对大球盖菇及斑玉蕈菌丝的生长产生显著的促进作用,但不会改变其蛋白质的组成。【结论】大球盖菇最适合选用PTA-5培养基,斑玉蕈的最佳培养基是PDTA培养基。     

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2 h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.     

Effect of exogeneous methyl oleate on the time course of some parameters of Streptomyces hygroscopicus NRRL B-1865 culture

Addition of methyl oleate to a Streptomyces hygroscopicus NRRL B-1865 culture modified the metabolic properties of this strain. This addition decreased the pH of the medium, increased the valine uptake of the cells and reduced their consumption of glucose until the beginning of antibiotic biosynthesis, which was delayed. At the same time, an increase in growth (× 1.8) and a marked improvement in antibiotic production (× 20) could be observed. The use of labelled methyl oleate showed that methyl oleate was not a precursor of antibiotics produced by S. hygroscopicus NRRL B-1865. It is suggested that methyl oleate addition may cause some alteration in membrane permeability, inducing an increase in H⁺ extrusion and stimulating the accumulation of branched amino acids, known to be direct precursors of polyether antibiotics. Correspondence to: L. David     

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid

The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.