Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824.

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the beta-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures.

The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacillus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods. All the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 degrees C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC 11454 which had been grown at 4 and 10 degrees C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.     

Bacteriocin-mediated inhibition of Clostridium botulinum spores by lactic acid bacteria at refrigeration and abuse temperatures.

The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacillus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 degrees C was investigated by modified deferred and simultaneous antagonism methods. All the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 degrees C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC 43200 and 43201 were bacteriocinogenic at 4, 10, and 15 degrees C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC 11454 which had been grown at 4 and 10 degrees C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards.     

Development and Characterization of a Gene Expression Reporter System for Clostridium acetobutylicum ATCC 824

A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene. In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively. Detection of β-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional β-galactosidase in C. acetobutylicum. In addition, time course studies revealed differences in the β-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters. The stability of the β-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis. The data indicated that the β-galactosidase produced by the lacZ gene from T. thermosulfurogenes EM1 was stable in the exponential phase of growth. In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of β-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.     

Acetone-butanol fermentation of xylose and sugar mixtures

Summary Three strains ofCl. acetobutylicum and one ofCl. butyricum have been tested for their ability to ferment xylose to butanol. ATCC 824 and NRRL 527 produced 0.28 g solvents/g xylose, while ATCC 8260 and NRRL 594 produced much butyric acid. In 2-stage fermentations in which ATCC 8260 or NRRL 594 acted upon xylose for 12 to 20 h, followed by NRRL 527 for a total of 3 days, yields of solvent were better, 0.32 g/g xylose. Upon fermenting a mixture of sugars simulating sulphite waste liquor 0.36 g solvents/g sugar were obtained. Sugar consumption in both cases was about 96%.     

Isolation and properties of Bacillus brevis mutants unable to produce tyrocidine.

Mutants of Bacillus brevis ATCC 10068 were isolated which produced less than 1/100 of the amount of tyrocidine produced by the parent strain. These mutants produced spores at the same frequency and which were as resistant to heating at 80 degrees C for up to 3 h as were those produced by the parent strain. A partially purified tyrocidine synthetase from strain ATCC 10068 catalyzed [32P]PPi-ATP exchange reactions dependent on added tyrocidine-constituent amino acids. These activities were separated into three groups (I, II, and III) by fractionation on an Ultrogel AcA34 column. Each group was similar to one of the three components (heavy, intermediate, and light, respectively) found previously for strain ATCC 8185 except that glutamate-dependent activity was not detected in the group I activities and some amino acyl-tRNA synthetase activities were associated with the group III activities. Some of the mutants were shown to have defective tyrocidine synthetase enzymes. Mutant BH30 was defective in two of the group II amino acid-dependent [32P]PPi-ATP exchange reactions, mutant BH16 was defective in one of the group I and one of the group II reactions, and mutant BH34 had alterations to activities in all of the groups. It is unlikely that any of these mutants could synthesise tyrocidine. We conclude that tyrocidine is not involved in either the sporulation process or the resistance of spores of B. brevis ATCC 10068 to heating at 80 degrees C for up to 3 h.     

Biochemical and chemical studies on strains designated Prevotella intermedia and proposal of a new pigmented species, Prevotella nigrescens sp. nov.

A total of 31 strains of Prevotella intermedia were subjected to DNA-DNA hybridization and were characterized by performing physiological tests and by performing a multilocus enzyme analysis, using malate dehydrogenase and glutamate dehydrogenase. All of the strains assigned to P. intermedia fermented glucose and sucrose, hydrolyzed starch but not esculin, and produced indole, acetic, isobutyric, isovaleric, and succinic acids as metabolic end products. The results of DNA reassociation experiments performed with the reference probe permitted separation of the strains into two well-defined homology groups. In addition, strains with DNAs that hybridized with DNA from strain ATCC 25611T (T = type strain) had high levels of peptidase activity and cleaved lipid substrates (4-methylumbelliferyl laurate and 4-methylumbellifelyl elaidate). Multilocus enzyme electrophoresis revealed two electromorphic profiles, one characteristic of strain ATCC 25611T and the other characteristic of strain ATCC 33563T. We propose that a new species, Prevotella nigrescens, should be created for the genetically distinct group of strains that hybridized with strain ATCC 33563T. Strain ATCC 33563 is designated the type strain of P. nigrescens.     

The lack of identity between Bacillus stearothermophilus and Bacillus thermoglucosidasius in serological patterns of exo-oligo-1,6-glucosidase and exo-α-1,4-glucosidase

Summary Among 16 Bacillus stearothermophilus strains, 11 strains (ATCC 7953, ATCC 10149, ATCC 12976, ATCC 12978, ATCC 12980, ATCC 15951, ATCC 21365, IAM 11001, IAM 11004, IAM 11062 and IFO 12550) produced a protein reactable on double immuno-diffusion with the antiserum against Bacillus thermoglucosidasius KP 1006 (DSM 2542) exo-oligo-1,6-glucosidase (dextrin 6-glucanohydrolase, EC.3.2.1.10). However, these antigens in part shared their antigenic determinants. In addition to an exo-oligo-1,6-glucosidase, 6 B. thermoglucosidasius strains [KP 1006, KP 1012, KP 1013, KP 1014, KP 1019 and KP 1022 (DSM 2543)] formed a protein cross-reacted with the antiserum against B. stearothermophilus ATCC 12016 exo--1,4-glucosidase (-d-glucoside glucohydrolase, EC.3.2.1.20). These two antigens showed, however, a partial coincidence in their antigenic determinant groups. Of 16 B. stearothermophilus strains, 3 strains (ATCC 8005, ATCC 12016 and ATCC 15952) produced a protein immunologically compatible with the -1,4-glucosidase, while 4 strains (ATCC 12979, ATCC 12980, ATCC 15951 and IAM 11001) made the other protein which showed certain differences partly from this enzyme in its antigenic groups. No protein precipitated with the anti--1,4-glucosidase occurred in the remaining 9 B. stearothermophilus strains (ATCC 7953, ATCC 10149, ATCC 12976, ATCC 12977, ATCC 12978, ATCC 21365, IAM 11004, IAM 11062 and IFO 12550). These data indicate no serological identity between two thermophilic Bacillus species in their glucosidase patterns.     

A newly isolated Streptomyces sp. CS392 producing three antimicrobial compounds

With the aim of isolating new microbes capable of producing strong antimicrobial substances, strain CS392 was screened from 700 soil isolates preserved in our laboratory. The strain was related to genus Streptomyces based on various characteristics. Three highly active antimicrobial compounds, C1, C2 and C3, produced by the strain were purified by solvent extraction followed by silica gel column chromatography. These compounds were highly active against various Gram-positive resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococcus (VRE). Among three, C3 was the most active against MRSA and VRSA with minimal inhibitory concentration (MIC) of 2 μg/ml while C2 and C3 had MIC values of 4 μg/ml for the strains. In case of Bacillus subtilis ATCC6633, C1 and C3 were more effective with MIC values of 0.5 μg/ml than C2 with MIC of 2 μg/ml. Those antibiotics were variably active (MIC of 4-32 μg/ml) against Micrococcus luteus ATCC 9341, Enterococcus faecalis ATCC 29212, Mycobacterium smegmatis ATCC 9341 and VRE.     

Heat resistance of Desulfotomaculum nigrificans spores in soy protein infant formula preparations.

The heat resistance of Desulfotomaculum nigrificans spores was determined in soy protein infant formula preparations. Methods of sporulation were developed and evaluated. D. nigrificans spores of highest heat resistance were produced in a 40% infusion of spent mushroom compost. Fraction-negative D121 degrees C-values obtained in modified soy formula were 25.8 min for spores of ATCC 7946 produced at 55 degrees C and 54.4 min for an isolate designated RGI 1, which was sporulated at 66 degrees C. From the fraction-negative D-values, z-values were obtained of 6.7 degrees C for ATCC 7946 and 9.5 degrees C for RGI 1. Survivor-curve D121 degrees C-values were 5.6 min for ATCC 7946 and 2.7 min for RGI 1 sporulated at 55 degrees C and heated in modified soy formula. Corresponding D121 degrees C-values in Butterfield phosphate buffer (pH 7.2) were 3.3 min (ATCC 7946) and 1.1 min (RGI 1). The z-values generated from survivor-curve D-values were similar to those obtained by using fraction-negative procedures. In all instances the inactivation kinetics appeared to be linear. The isolate designated RGI 1, when sporulated at 66 degrees C and heated in a modified infant soy formula, exhibited an extraordinary heat resistance far in excess of previous reports.     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

Two molecular forms of penicillin amidase synthesized by Escherichia coli

The Swatek's method was further simplified for the assay of penicillin amidase activity. The absorbance of colour obtained during determination of 6-aminopenicillanic acid was dependent on concentration of 4-dimethylaminobenzaldehyde and on temperature. Antiodies induced in rabbits with one molecular form of penicillin amidase from E. coli PCM 271 (PA-1 or PA-2) did not cross-react with the other amidase form. No differences in substrate specificity on inactivation with SDS and in alkaline medium between the two amidase forms were observed. Concentrated urea inactivated PA-2 irreversibly and PA-1 reversibly. N-Bromosuccinimide inactivated almost completely only PA-1. Two E. coli PCM 271 strain variants were separated by microbial selection. Each of them produced only one amidase form. Also two amidase forms were found in cells of E. coli ATCC 11105, whereas E. coli ATCC 9636 and ATCC 9637 synthesize only PA-1.     

Preparation and regeneration of bacteriophage PL-1 enzyme-induced Lactobacillus casei protoplasts.

Lactobacillus casei ATCC 27092 protoplasts were obtained by treatment with a bacteriolytic enzyme, which was produced in the PL-1 phage lysates of this bacterium, at 37 degrees C for 1.5 h in 50 mM Tris hydrochloride buffer (pH 7.2) containing 20% sucrose and 10 mM MgSO4. The protoplasts lacked the cell wall layer but retained the ability to transport L-[3H]glutamine. The frequency of regeneration was about 1%, whereas about 99% of the cells were osmotically sensitive.     

Mycophenolic Acid Production by Drug-resistant and Methionine or Glutamic-Acid Requiring Mutants of Pénicillium brevicompactum

Penicillium brevicompactum ATCC 16024 produced 1.7 g/1 of mycophenolic acid (MPA) in the culture medium. Various drug-resistant mutants, showing resistance to such as polyene antibiotics, chemotherapeutic agents, redox indicator and surfactants, were derived from the fungus. Most of the mutants produced 2.0 ~2.5 g/1 of MPA. A clofibrate and dodecyltrimethylammonium chloride double resistant mutant, No. 4–23–11, produced 4.7 g/1 of MPA. A monofluoroacetic acid resistant strain, No. 5–1, derived from No. 4–23–11 produced 5.3 g/1 of MPA.

A methionine auxotroph, M-l, derived from ATCC 16024, produced 4.0 g/1 of MPA. A glutamate auxotroph, G-42, derived from strain No. 4–23–11 produced 5.8 g/1 of MPA. G-42 grew on l-aspartate instead of l-glutamate, and showed one-third the pyruvate carboxylase activity of the parent. Another glutamate auxotroph, G-78, did not produce MPA but accumulated 1.5 g/1 of acetate in the culture medium, and showed one-fifth the citrate synthase activity of the parent strain.     

Metabolism of naphthalene by pseudomonads: salicylaldehyde as the first possible inducer in the metabolic pathway.

Pseudomonas ATCC 17483 produced enzymes for naphthalene metabolism when growing in a medium containing succinate and naphthalene. Mutants for naphthalene metabolism produced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were able to produce these enzymes only when the metabolic pathway was intact as far as salicylaldehyde, which was therefore identified as the first possible inducer.     

Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora

Methane fermentation of Japanese cedar wood was carried out after pretreatment with four strains of white rot fungi, Ceriporiopsis subvermispora ATCC 90467, CZ-3, CBS 347.63 and Pleurocybella porrigens K-2855. These fungi were cultivated on wood chip media with and without wheat bran for 4-8 weeks. The pretreated wood chip was fermented anaerobically with sludge from a sewage treatment plant. Pretreatments with C. subvermispora ATCC 90467, CZ-3 and CBS 347.63 in the presence of wheat bran for 8 weeks decreased 74-76% of beta-O-4 aryl ether linkages in the lignin to accelerate production of methane. After fungal treatments with C. subvermispora ATCC 90467 and subsequent 30-days methane fermentation, the methane yield reached 35 and 25% of the theoretical yield based on the holocellulose contents of the decayed and original wood, respectively. In contrast, treatment with the three strains of C. subvermispora without wheat bran cleaved 15-26% of the linkage and produced 6-9% of methane. There were no significant accelerating effects in wood chips treated with P. porrigens which has a lower ability to decompose the lignin. Thus, it was found that C. subvermispora, with a high ability to decompose aryl ether bonds of lignin, promoted methane fermentation of softwood in the presence of wheat bran.     

Xylitol production by Candida species grown on a grass hydrolysate

Xylitol was produced by selected species of the yeast Candida after growth on a medium containing a hydrolysate of the North American perennial prairie grass big bluestem. The grass was hydrolysed by a combination of dilute acid and enzymatic treatments. After growth on the medium for 120 h at 30 °C, Candida tropicalis ATCC 750 produced a 1.4-fold higher level of xylitol than did C. tropicalis ATCC 20215 while biomass production by C. tropicalis ATCC 750 was 1.7-fold higher than Candida guilliermondii ATCC 20216. The xylitol yields observed for C. tropicalis ATCC 750, Candida mogii ATCC 18364 and C. guilliermondii ATCC 20216 were at least 1.4-fold higher than the yield observed for C. tropicalis ATCC 20215 after growth for 120 h at 30 °C.     

Citric acid production by Aspergillus niger on wet corn distillers grains

AIMS: To determine which citric acid-producing strain of Aspergillus niger utilized wet corn distillers grains most effectively to produce citric acid. METHODS AND RESULTS: Citric acid and biomass production by the fungal strains were analysed on the untreated grains or autoclaved grains using an enzyme assay and a gravimetric method respectively. Fungal citric acid production on the grains was found to occur on the untreated or autoclaved grains. The highest citric acid level on the grains was produced by A. niger ATCC 9142. The autoclaved grains supported less citric acid production by the majority of strains screened. Biomass production by the fungal strains on the untreated or autoclaved grains was quite similar. The highest citric acid yields for A. niger ATCC 9142, ATCC 10577, ATCC 11414, ATCC 12846 and ATCC 26550 were found on the untreated grains. Treatment of the grains had little effect on citric acid yields based on reducing sugars consumed by A. niger ATCC 9029 and ATCC 201122. CONCLUSIONS: It is feasible for citric acid-producing strains of A. niger to excrete citric acid on wet corn distillers grains whether the grains are treated or untreated. The most effective citric acid-producing strain of A. niger was ATCC 9142. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that the ethanol processing co-product wet corn distillers grains could be utilized as a substrate for the commercial production of citric acid by A. niger without treatment of the grains.     

Selection of Streptomyces ambofaciens mutants that produce large quantities of spiramycin and determination of optimal conditions for spiramycin production

The aim of this work was to develop a strategy to isolate a morphologically stable mutant of Streptomyces ambofaciens ATCC 15154 which produced high titers of spiramycin. The rationale was to grow a nitrosoguanidine-mutated population for many generations under nonselective conditions followed by two cycles of protoplast formation and regeneration. A total of 2,400 surviving colonies were then screened for spiramycin production and subsequently checked for stability. From this experiment, strain 6-37 was isolated that produced 181 mg of spiramycin per liter and only one morphological type. The parent strain (ATCC 15154) produced 107 mg of spiramycin per liter and four morphological types. Strain 6-37 was then mutated with nitrosoguanidine, and 14,000 colonies were screened for spiramycin production. From this experiment, five strains were isolated that produced titers ranging from 187 to 373 mg of spiramycin per liter. Subsequent media and time studies with these strains resulted in a fermentation that produced 1,728 mg of spiramycin per liter.