bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway
Authors: Hur H G , Sadowsky M J , Wackett L P
Published: January 2020

The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.
Reversible Conversion of 4-Hydroxybenzoate and Phenol by
Clostridium hydroxybenzoicum
Authors: Zhang Xiaoming , Wiegel Juergen
Published: January 2020

Reversible conversion of 4-hydroxybenzoate and phenol and their analogs was observed in whole-cell suspensions and cell extracts of
Clostridium hydroxybenzoicum
grown with 4-hydroxybenzoate and 3,4-dihydroxybenzoate. Assuming that bicarbonate is the cosubstrate, the equilibrium constants calculated for the reactions 4-hydroxybenzoate
-
+ H
2
O ⇆ phenol + HCO
3
-
and 3,4-dihydroxybenzoate
-
+ H
2
O ⇆ catechol + HCO
3
-
were 11.4 (± 0.5) and 5.05 (± 0.25), respectively. In a phenol-adapted sediment slurry, 4-hydroxybenzoate and 3,4-dihydroxybenzoate were decarboxylated to phenol and to catechol, respectively, as intermediates without a lag time.
Microbial Production of Poly-β-Hydroxybutyric Acid from
d
-Xylose and Lactose by
Pseudomonas cepacia
Authors: Young Frederick K. , Kastner James R. , May Sheldon W.
Published: January 2020

Poly-β-hydroxybutyric acid (PHB) was produced from xylose and lactose by using
Pseudomonas cepacia
. Approximately 50% PHB (grams of PHB total/grams of biomass total) was produced. With a laser-based fluorescent probe, β-galactosidase activity was shown to be induced in
P. cepacia
cells grown on lactose but not in those grown on glucose or xylose.
P. cepacia
has the potential to produce biodegradable thermoplastics from hemicellulosic hydrolysates and cheese whey.
Cell culture and PCR determination of poliovirus inactivation by disinfectants
Authors: Ma J F , Straub T M , Pepper I L , Gerba C P
Published: January 2020

Inactivation of poliovirus type 1 by 1 N HCl, 1 N NaOH, 0.5 and 1.0 mg of free chlorine per liter, and UV light was compared by using cell culture and seminested PCR (30 cycles of reverse transcriptase-PCR plus 30 cycles of seminested PCR). A minimum contact time of 45 min with HCl, 3 min with NaOH, 3 and 6 min with 1.0 and 0.5 mg of free chlorine per liter, respectively, was required to render 1.64 x 10(2) PFU of poliovirus type 1 per ml undetectable by seminested PCR. In cell culture, a minimum contact time of 5 min to HCl, 30 s to NaOH, and 1 min to either chlorine concentration was required to render the viruses undetectable by the plaque assay method. No correlation was observed between results by PCR and cell culture when viruses were exposed to UV light. These data suggest that inactivated virus with intact nucleic acid sequences can be detected by PCR.
Effect of Genetically Modifying the Lactococcal Proteolytic System on Ripening and Flavor Development in Cheddar Cheese
Authors: McGarry A. , Law J. , Coffey A. , Daly C. , Fox P. F. , Fitzgerald G. F.
Published: January 2020

Three batches of six Cheddar cheeses were manufactured by using the following lactococcal strains: (i) UC317 as a control; (ii) JL3601, a proteinase-negative derivative of UC317 transformed with high-copy-number plasmid pCI3601 containing the cloned proteinase gene complex from UC317; (iii) AM312, a proteinase-negative derivative of UC317 transformed with plasmid pMG36enpr containing the neutral proteinase gene from
Bacillus subtilis
; (iv) AC322, JL3601 transformed with pMG36enpr; (v) AC311, UC317 transformed with plasmid pNZ1120, which contains the aminopeptidase N (
pepN
) gene from
Lactococcus lactis
subsp.
lactis
MG1363; and (vi) AC321, JL3601 transformed with pNZ1120. Organoleptic and chemical analyses indicated that (i) the control cheeses, which were made with UC317, were of the highest quality; (ii) cheeses made with strains harboring pCI3601 in addition to either pMG36enpr (AC322) or pNZ1120 (AC321) did not ripen in a significantly different manner than cheeses made with AM312 (containing only pMG36enpr) or AC311 (containing only pNZ1120), respectively; (iii) cheeses made with strains that overproduce
pepN
did not have improved body, texture, and flavor characteristics; and (iv) cheeses made with strains harboring the neutral proteinase from
B. subtilis
(AM312 and AC322) underwent greatly accelerated proteolysis.
Transfer in Marine Sediments of the Naturally Occurring Plasmid pRAS1 Encoding Multiple Antibiotic Resistance
Authors: Sandaa Ruth-Anne , Enger Øivind
Published: January 2020

The results of microcosm experiments performed with the fish-pathogenic bacterium
Aeromonas salmonicida
acting as a donor showed that promiscuous plasmid pRAS1, which encodes tetracycline resistance, is transferred at a high frequency in marine sediments even in the absence of a selective factor. The presence of oxytetracycline resulted in an increase in the transfer frequency compared with that of a microcosm to which no selective factor was added. Transfer frequencies of 3.4 × 10
-1
transconjugant per recipient and 3.6 transconjugants per donor cell were obtained in a microcosm to which oxytetracycline had been added. Hybridization with a DNA probe specific for plasmid pRAS1 revealed that 45.8% of the oxytetracycline-resistant isolates obtained from a microcosm with no selective pressure carried the plasmid, while 86.8% of the isolates obtained from a microcosm to which oxytetracycline had been added carried the plasmid. Phenotypic characterization of the transconjugants revealed that the plasmid had been transferred to a variety of different biotypes in both microcosms. The diversity among the transconjugants isolated from the microcosm to which oxytetracycline had been added was substantially lower than the diversity among the transconjugants isolated from the microcosm to which no selective agent had been added.
Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts
Authors: Corbin D R , Greenplate J T , Wong E Y , Purcell J P
Published: January 2020

We cloned and sequenced structural gene choM, which encodes an insecticidally active cholesterol oxidase in Streptomyces sp. strain A19249. The primary translation product was predicted to be a 547-amino-acid protein whose first 43 amino acids constitute a secretory signal peptide. Expression of the gene with the signal sequence in Escherichia coli resulted in production of a protein that had enzymatic and insecticidal properties which were indistinguishable from those of the cholesterol oxidase secreted by Streptomyces sp. strain A19249. Expression of the gene with or without the signal sequence in tobacco protoplasts resulted in production of an enzymatically active cholesterol oxidase.
Mimosine, a Toxin Present in Leguminous Trees (
Leucaena
spp.), Induces a Mimosine-Degrading Enzyme Activity in Some
Rhizobium
Strains
Authors: Soedarjo Muchdar , Hemscheidt Thomas K. , Borthakur Dulal
Published: January 2020

Thirty-seven
Rhizobium
isolates obtained from the nodules of leguminous trees (
Leucaena
spp.) were selected on the basis of their ability to catabolize mimosine, a toxin found in large quantities in the seeds, foliage, and roots of plants of the genera
Leucaena
and
Mimosa
. A new medium containing mimosine as the sole source of carbon and nitrogen was used for selection. The enzymes of the mimosine catabolic pathway were inducible and were present in the soluble fraction of the cell extract of induced cells. On the basis of a comparison of the growth rates of
Rhizobium
strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.
Degradation of Raw Starch by a Wild Amylolytic Strain of
Lactobacillus plantarum
Authors: Giraud Eric , Champailler Alain , Raimbault Maurice
Published: January 2020

Lactobacillus plantarum
A6, isolated from fermented cassava, can break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high α-amylase activity (60 U/ml). Synthesis of the enzyme occurred during the stationary phase and resulted in full hydrolysis of the cassava starch granules. This gave 41 g of lactic acid from 45 g of raw starch after 3 days of fermentation. Enzymatic attack was evident under scanning electron microscopy in the rougher appearance of the surface of starch granules and in the presence of large cavities in some of them. In contrast, when the pH was not regulated, only a small amount of α-amylase activity was produced (2 U/ml) and no decrease in the starch content of the medium was observed. However, under scanning electron microscopy, some granules displayed a rougher surface, which might have been the result of weak enzymatic attack.
Direct measurement of chlorine penetration into biofilms during disinfection
Authors: De Beer D , Srinivasan R , Stewart P S
Published: January 2020

Transient chlorine concentration profiles were measured in biofilms during disinfection by use of a microelectrode developed for this investigation. The electrode had a tip diameter of ca. 10 microm and was sensitive to chlorine in the micromolar range. The biofilms contained Pseudomonas aeruginosa and Klebsiella pneumoniae. Chlorine concentrations measured in biofilms were typically only 20% or less of the concentration in the bulk liquid. Complete equilibration with the bulk liquid did not occur during the incubation time of 1 to 2 h. The penetration depth of chlorine into the biofilm and rate of penetration varied depending on the measurement location, reflecting heterogeneity in the distribution of biomass and in local hydrodynamics. The shape of the chlorine profiles, the long equilibration times, and the dependence on the bulk chlorine concentration showed that the penetration was a function of simultaneous reaction and diffusion of chlorine in the biofilm matrix. Frozen cross sections of biofilms, stained with a redox dye and a DNA stain, showed that the area of chlorine penetration overlapped with nonrespiring zones near the biofilm-bulk fluid interface. These data indicate that the limited penetration of chlorine into the biofilm matrix is likely to be an important factor influencing the reduced efficacy of this biocide against biofilms as compared with its action against planktonic cells.
Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes
Authors: Buchrieser C , Weagant S D , Kaspar C W
Published: January 2020

Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.
Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer
Authors: Mills D A , Choi C K , Dunny G M , McKay L L
Published: January 2020

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.
Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides
Authors: Sundin G W , Demezas D H , Bender C L
Published: January 2020

We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.
Uniparental Mitochondrial Transmission in the Cultivated Button Mushroom,
Agaricus bisporus
Authors: Jin Tianru , Horgen Paul A.
Published: January 2020

A uniparental mitochondrial (mt) transmission pattern has been previously observed in laboratory matings of the cultivated mushroom
Agaricus bisporus
on petri dishes. In this study, four sets of specific matings were further examined by taking mycelial plugs from the confluent zone of mated homokaryons and inoculating these plugs into rye grain for laboratory fruiting and for fruiting under industrial conditions. Examination of the mt genotype of each individual fruit body for mt-specific restriction fragment length polymorphisms further confirmed that the mt genome was inherited uniparentally. The vegetative radial growth and the fruiting activity of two pairs of intraspecific heterokaryons, each pair carrying the same combination of nuclear genomes but different mt genotypes, were compared. Our results suggested that the mt genotype did not appreciably affect radial growth or fruiting activity. The failure to recover both heterokaryons, each carrying either parental mt genotype in any given cross, therefore clearly indicated that in matings of
A. bisporus
, the mt genome from one of the parental homokaryons is either selectively excluded in the newly formed heterokaryon or selectively eliminated in the immediate heterokaryotic mitotic progeny of the newly formed heterokaryon.
Factors involved in adherence of lactobacilli to human Caco-2 cells
Authors: Greene J D , Klaenhammer T R
Published: January 2020

A quantitative assay performed with bacterial cells labelled with [3H]thymidine was used to investigate factors involved in the adherence of human isolates Lactobacillus acidophilus BG2FO4 and NCFM/N2 and Lactobacillus gasseri ADH to human Caco-2 intestinal cells. For all three strains, adherence was concentration dependent, greater at acidic pH values, and significantly greater than adherence of a control dairy isolate, Lactobacillus delbrueckii subsp. bulgaricus 1489. Adherence of L. acidophilus BG2FO4 and NCFM/N2 was decreased by protease treatment of the bacterial cells, whereas adherence of L. gasseri ADH either was not affected or was enhanced by protease treatment. Putative surface layer proteins were identified on L. acidophilus BG2FO4 and NCFM/N2 cells but were not involved in adherence. Periodate oxidation of bacterial cell surface carbohydrates significantly reduced adherence of L. gasseri ADH, moderately reduced adherence of L. acidophilus BG2FO4, and had no effect on adherence of L. acidophilus NCFM/N2. These results indicate that Lactobacillus species adhere to human intestinal cells via mechanisms which involve different combinations of carbohydrate and protein factors on the bacterial cell surface. The involvement of a secreted bridging protein, which has been proposed as the primary mediator of adherence of L. acidophilus BG2FO4 in spent culture supernatant (M.-H. Coconnier, T. R. Klaenhammer, S. Kernéis, M.-F. Bernet, and A. L. Servin, Appl. Environ. Microbiol. 58:2034-2039, 1992), was not confirmed in this study. Rather, a pH effect on Caco-2 cells contributed significantly to the adherence of this strain in spent culture supernatant.(ABSTRACT TRUNCATED AT 250 WORDS)
Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic
Bacillus
sp
Authors: Chauthaiwale Jyoti , Rao Mala
Published: January 2020

An alkaliphilic, thermophilic
Bacillus
sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg
2+
, Co
2+
, and Mn
2+
were required for maximum activity of the enzyme. The

K
m

values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while
K
cat
values were 2.3 × 10
2
s
-1
and 0.5 × 10
2
s
-1
, respectively.
Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation
Authors: Brussow H , Fremont M , Bruttin A , Sidoti J , Constable A , Fryder V
Published: January 2020

In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation. Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed. The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups. Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences. Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges. All but one cheese phage isolate showed serological cross-reactivity with yogurt phages. A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S. thermophilus phages from our collection. With the sequence information from an unusually conserved S. thermophilus phage DNA element (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes. PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml).
Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase
Authors: Trimbur D E , Gutshall K R , Prema P , Brenchley J E
Published: January 2020

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.
Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp
Authors: Morikawa M , Izawa Y , Rashid N , Hoaki T , Imanaka T
Published: January 2020

A hyperthermophilic archaeon strain, KOD1, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100 degrees C, and the optimal temperature was 95 degrees C. The anaerobic strain was an S0-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110 degrees with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90 degrees C for 2 h, and the half-life of enzymatic activity at 100 degrees C was 60 min. The proteolytic activity was significantly inhibited by p-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.
Analysis of Expression of a Phenazine Biosynthesis Locus of
Pseudomonas aureofaciens
PGS12 on Seeds with a Mutant Carrying a Phenazine Biosynthesis Locus-Ice Nucleation Reporter Gene Fusion
Authors: Georgakopoulos Dimitrios G. , Hendson Mavis , Panopoulos Nickolas J. , Schroth Milton N.
Published: January 2020

A derivative of
Pseudomonas aureofaciens
PGS12 expressing a promoterless ice nucleation gene under the control of a phenazine biosynthesis locus was used to study the expression of a phenazine antibiotic locus (Phz) during bacterial seed colonization. Seeds of various plants were inoculated with wild-type PGS12 and a PGS12 ice nucleation-active
phz:inaZ
marker exchange derivative and planted in soil, and the expression of the reporter gene was monitored at different intervals for 48 h during seed germination.
phz
gene expression was first detected 12 h after planting, and the expression increased during the next 36-h period. Significant differences in expression of bacterial populations on different seeds were measured at 48 h. The highest expression level was recorded for wheat seeds (one ice nucleus per 4,000 cells), and the lowest expression level was recorded for cotton seeds (one ice nucleus per 12,000,000 cells). These values indicate that a small proportion of bacteria in a seed population expressed phenazine biosynthesis. Reporter gene expression levels and populations on individual seeds in a sample were lognormally distributed. There was greater variability in reporter gene expression than in population size among individual seeds in a sample. Expression on sugar beet and radish seeds was not affected by different inoculum levels or soil matric potentials of -10 and -40 J/kg; only small differences in expression on wheat and sugar beet seeds were detected when the seeds were planted in various soils. It is suggested that the nutrient level in seed exudates is the primary reason for the differences observed among seeds. The lognormal distribution of phenazine expression on seeds and the timing and difference in expression of phenazine biosynthesis on seeds have implications for the potential efficacy of biocontrol microorganisms against plant pathogens.
Nitrogen Assimilation: Optimization of
15
N-Labelled Azoverdin Production by
Azomonas macrocytogenes
ATCC 12334
Authors: Dobišová Magda , Abdallah Mohamed A. , Kyslík Pavel
Published: January 2020

The effects of various nitrogen sources and of the cultivation time on the biosynthesis of
15
N-labelled azoverdin were studied in batch cultures of
Azomonas macrocytogenes
ATCC 12334. Growth for 48 h in the presence of ammonium sulfate (0.2 g/liter) resulted in 95% labelling of azoverdin with
15
N. A growth-related synthesis of azoverdin, under both nitrogen-fixing and ammonium-assimilating conditions, was observed.
Isolation of three hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading species of the family Enterobacteriaceae from nitramine explosive-contaminated soil
Authors: Kitts C L , Cunningham D P , Unkefer P J
Published: January 2020

Three species of the family Enterobacteriaceae that biochemically reduced hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were isolated from nitramine explosive-contaminated soil. Two isolates, identified as Morganella morganii and Providencia rettgeri, completely transformed both RDX and the nitroso-RDX reduction intermediates. The third isolate, identified as Citrobacter freundii, partially transformed RDX and generated high concentrations of nitroso-RDX intermediates. All three isolates produced 14CO2 from labeled RDX under O2-depleted culture conditions. While all three isolates transformed HMX, only M. morganii transformed HMX in the presence of RDX.
Immunological detection of Salmonella paratyphi A in raw prawns
Authors: Korbsrisate S , Sarasombath S , Janyapoon K , Ekpo P , Pongsunk S
Published: January 2020

A slot blot enzyme-linked immunosorbent assay, using monoclonal antibodies specific only for Salmonella paratyphi A, to detect S. paratyphi A contamination in raw prawns has been established. When artificially contaminated prawn samples were tested. S. paratyphi A contamination could be identified correctly within 20 h. No false positives from samples artificially contaminated by other microorganisms were obtained. The sensitivity was such that as few as 1 S. paratyphi A organism per g of raw prawn could be detected. Therefore, the assay constituted a promising test for the rapid and specific detection of S. paratyphi A in prawns.
Cloning of organic solvent tolerance gene ostA that determines n-hexane tolerance level in Escherichia coli
Authors: Aono R , Negishi T , Nakajima H
Published: January 2020

A variety of genes are involved in determining the level of organic solvent tolerance of Escherichia coli K-12. Gene ostA is one of the genes contributing to the level of organic solvent tolerance. This gene was cloned from an n-hexane-tolerant strain of E. coli, JA300. A JA300-based n-hexane-sensitive strain, OST4251, was converted to the n-hexane-tolerant phenotype by transformation with DNA containing the ostA gene derived from JA300. Thus, the cloned ostA gene complemented the n-hexane-sensitive phenotype of OST4251.
Cross-Resistance to
Bacillus thuringiensis
Toxin CryIF in the Diamondback Moth (
Plutella xylostella
)
Authors: Tabashnik Bruce E. , Finson Naomi , Johnson Marshall W. , Heckel David G.
Published: January 2020

Selection with
Bacillus thuringiensis
subsp.
kurstaki,
which contains CryIA and CryII toxins, caused a >200-fold cross-resistance to CryIF toxin from
B. thuringiensis
subsp.
aizawai
in the diamondback moth,
Plutella xylostella.
CryIE was not toxic, but CryIB was highly toxic to both selected and unselected larvae. The results show that extremely high levels of cross-resistance can be conferred across classes of CryI toxins of
B. thuringiensis.
Purification and Characterization of Two Epoxide Hydrolases from
Corynebacterium
sp. Strain N-1074
Authors: Nakamura Tetsuji , Nagasawa Toru , Yu Fujio , Watanabe Ichiro , Yamada Hideaki
Published: January 2020

Enzymes II
a
and II
b
, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from
Corynebacterium
sp. strain N-1074, which catalyzes the formation of (
R
)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II
a
for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II
b
. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.
Tyrosinase Reaction and Subsequent Chitosan Adsorption for Selective Removal of a Contaminant from a Fermentation Recycle Stream
Authors: Payne Gregory F. , Sun Wei-Qiang
Published: January 2020

In the industrial production of penicillin V, the phenoxyacetate precursor is added to the fermentor to direct biosynthesis. When used for producing semisynthetic penicillins, the penicillin V is often hydrolyzed to 6-aminopenicillanic acid with the regeneration of the phenoxyacetate precursor. To reduce raw-material as well as waste-disposal costs, it is desirable to recycle the phenoxyacetate precursor. Unfortunately, the recycle stream is generally contaminated by the
p
-hydroxylated derivative of this precursor. We examined a two-step approach to eliminate this contaminant. In the first step the tyrosinase enzyme was used to selectively convert the
p
-hydroxyphenoxyacetate contaminant to a reactive intermediate—presumably its quinone. In the second step, the tyrosinase-generated reactive intermediate was allowed to react with and strongly bind to chitosan. In contrast, the phenoxyacetate precursor was neither oxidized by tyrosinase nor bound to chitosan. When concentrated phenoxyacetate solutions were tested, the combination of tyrosinase and chitosan effectively converted low levels of the
p
-hydroxyphenoxyacetate contaminant and removed its products from solution, while the concentration of the phenoxyacetate precursor was unaffected.
Effect of microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores
Authors: Welt B A , Tong C H , Rossen J L , Lund D B
Published: January 2020

Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating.
Influence of the Gas-Water Interface on Transport of Microorganisms through Unsaturated Porous Media
Authors: Wan Jiamin , Wilson John L. , Kieft Thomas L.
Published: January 2020

In this article, a new mechanism influencing the transport of microorganisms through unsaturated porous media is examined, and a new method for directly visualizing bacterial behavior within a porous medium under controlled chemical and flow conditions is introduced. Resting cells of hydrophilic and relatively hydrophobic bacterial strains isolated from groundwater were used as model microorganisms. The degree of hydrophobicity was determined by contact-angle measurements. Glass micromodels allowed the direct observation of bacterial behavior on a pore scale, and three types of sand columns with different gas saturations provided quantitative measurements of the observed phenomena on a porous medium scale. The reproducibility of each break-through curve was established in three to five repeated experiments. The data collected from the column experiments can be explained by phenomena directly observed in the micromodel experiments. The retention rate of bacteria is proportional to the gas saturation in porous media because of the preferential sorption of bacteria onto the gas-water interface over the solid-water interface. The degree of sorption is controlled mainly by cell surface hydrophobicity under the simulated groundwater conditions because of hydrophobic forces between the organisms and the interfaces. The sorption onto the gas-water interface is essentially irreversible because of capillary forces. This preferential and irreversible sorption at the gas-water interface strongly influences the movement and spatial distribution of microorganisms.
Engineering and production of streptokinase in a Bacillus subtilis expression-secretion system
Authors: Wong S L , Ye R , Nathoo S
Published: January 2020

Streptokinase is one of the major blood-clot-dissolving agents used in many medical treatments. With the cloned streptokinase gene (skc) available, production of the secreted streptokinase from various Bacillus subtilis strains was studied. The use of the six-extracellular-protease-deficient strain, WB600, greatly improved the production yield of the secreted streptokinase. A modified skc which has the original skc promoter and signal sequence replaced with the B. subtilis levansucrase promoter and signal sequence was also constructed. B. subtilis carrying either the wild-type or the modified skc produces streptokinase at a comparable level. Even with WB600 as the expression host, a C-terminally-processed streptokinase was also observed. Through region-specific combinatorial mutagenesis around the C-terminal processing sites, streptokinase derivatives resistant to C-terminal degradation were engineered. One of the derivatives showed a 2.5-fold increase in specific activity and would potentially be a better thrombolytic agent.
Genetic and Biochemical Characterization of
Nectria haematococca
Strains with Adhesive and Adhesion-Reduced Macroconidia
Authors: Epstein L. , Kwon Y. H. , Almond D. E. , Schached L. M. , Jones M. J.
Published: January 2020

A previous study reported the isolation of two mutants (LE1 and LE2) of the plant pathogenic fungus
Nectria haematococca
(anamorph,
Fusarium solani
f. sp.
cucurbitae
) with macroconidia with reduced ability to adhere (Att
-
) to zucchini fruits and polystyrene. The adhesion-reduced-phenotype in LE1 and LE2 macroconidia is temperature sensitive and dependent on the concentration of nutrients. Classical genetic analysis of progeny derived from LE1 identified a mutation in a genetic locus, named
Att1
. The 90-kDa glycoprotein and macroconidial tip mucilage which were previously associated with the development of adhesion competence in Att
+
macroconidia are specifically associated with macroconidia; neither is produced on microconidia, which are relatively nonadherent. However, macroconidia of both Att
+
and Att
-
strains produce the 90-kDa glycoprotein and the macroconidial tip mucilage.
Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis
Authors: Ma K , Robb F T , Adams M W
Published: January 2020

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)
Two Genomic Regions Involved in Catechol Siderophore Production by
Erwinia carotovora
Authors: Bull Carolee T. , Ishimaru Carol A. , Loper Joyce E.
Published: January 2020

Two regions involved in catechol biosynthesis (
cbs
) of
Erwinia carotovora
W3C105 were cloned by functional complementation of
Escherichia coli
mutants that were deficient in the biosynthesis of the catechol siderophore enterobactin (
ent
). A 4.3-kb region of genomic DNA of
E. carotovora
complemented the
entB402
mutation of
E. coli.
A second genomic region of 12.8 kb complemented
entD, entC147, entE405
, and
entA403
mutations of
E. coli.
Although functions encoded by catechol biosynthesis genes (
cbsA, cbsB, cbsC, cbsD
, and
cbsE
) of
E. carotovora
were interchangeable with those encoded by corresponding enterobactin biosynthesis genes (
entA, entB, entC, entD
, and
entE
), only
cbsE
hybridized to its functional counterpart (
entE
) in
E. coli.
The
cbsEA
region of
E. carotovora
W3C105 hybridized to genomic DNA of 21 diverse strains of
E. carotovora
but did not hybridize to that of a chrysobactin-producing strain of
Erwinia chrysanthemi.
Strains of
E. carotovora
fell into nine groups on the basis of sizes of restriction fragments that hybridized to the
cbsEA
region, indicating that catechol biosynthesis genes were highly polymorphic among strains of
E. carotovora.
Monoclonal antibody-based enzyme-linked immunosorbent assay of Fusarium T-2 and zearalenone toxins in cereals
Authors: Barna-Vetró I , Gyöngyösi A , Solti L
Published: January 2020

Direct, competitive enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies have been developed for quantitative determination of trichothecene T-2 toxin (T-2), and zearalenone (F-2) from different cereals: Among the several extraction solvents tried, 89% acetonitrile with additives was chosen. The extracts were then used without cleanup in the ELISA. With appropriate dilution of the samples (1:25 or 1:50), the matrix effects caused by lipid and/or protein content of the samples can be diminished to the extent that the assay is no longer impaired. The mean recoveries from cereals infected with 100 to 2,000 ng of T-2 and 50 to 500 ng of F-2 per g were 85 and 91%, respectively. The measuring range of the T-2 test is 100 to 2,000 ng/g, and that of the F-2 test is 25 to 400 ng/g. The mean within-assay and interassay coefficients of variation of standard curves are both less than 10%. According to recovery results with artificially infected cereals, our tests proved to be suitable for rapid screening of food and feed samples for the presence of T-2 and F-2 toxins.
Utilization of Glucose and Xylose in Ruminal Strains of
Butyrivibrio fibrisolvens
Authors: Marounek M. , Kopečný J.
Published: January 2020

The dual-substrate utilization pattern in cultures of five ruminal strains of
Butyrivibrio fibrisolvens
growing on glucose and xylose was investigated. Strains ATCC 19171 and 86 utilized glucose and xylose simultaneously. Other strains exhibited diauxic growth. Strains X1 and CE 51 exhibited classical diauxic growth in which glucose was utilized during the first phase. Strain X2D62 displayed atypical diauxic growth in which slow utilization of xylose was followed by rapid utilization of glucose after the xylose depletion. The ATP-dependent phosphorylation of glucose was found in all strains tested. The phosphoenolpyruvate-dependent phosphorylation of glucose was detected only in
B. fibrisolvens
CE 51.
Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane
Authors: Abee T , Klaenhammer T R , Letellier L
Published: January 2020

The bacteriocin lactacin F is bactericidal against Lactobacillus delbrueckii, Lactobacillus helveticus, and Enterococcus faecalis. Activity against L. delbrueckii was recently shown to be dependent on two peptides, LafA and LafX, which are encoded within the lactacin F operon (T. R. Klaenhammer, FEMS Microbiol. Rev. 12:39-87, 1993). It has been proposed that two peptides form an active lactacin F complex. In this study, the action of lactacin F against E. faecalis ATCC 19443 and the effects of various environmental parameters were investigated in detail. Addition of lactacin F induced the loss of K+ from cells of L. delbrueckii, Lactobacillus johnsonii 88-4, and E. faecalis, while the lactacin F producer L. johnsonii VPI 11088 was not affected by the bacteriocin. Lactacin F caused an immediate loss of cellular K+, depolarization of the cytoplasmic membrane, and hydrolysis of internal ATP in E. faecalis. Lactacin F induced loss of K+ in 3,3',4',5-tetrachlorosalicylanilide-treated cells, indicating that pores are formed in the absence of a proton motive force. ATP hydrolysis was not due to dissipation of the proton motive force but was most likely caused by efflux of inorganic phosphate, resulting in a shift of the ATP hydrolysis equilibrium. Action of lactacin F was optimal at acidic pH values and was reduced in the presence of di- and trivalent cations. The lanthanide gadolinium (Gd3+) prevented action of lactacin F completely at a concentration of 0.2 mM. Lactacin F-induced loss of cell K+ was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)