bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus
Authors: Bezalel L , Hadar Y , Cerniglia C E
Published: January 2020

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.
Ingredient selection for plastic composite supports for L-(+)-lactic acid biofilm fermentation by Lactobacillus casei subsp. rhamnosus
Authors: Ho K L , Pometto A L , Hinz P N , Dickson J S , Demirci A
Published: January 2020

Plastic composite supports containing 50% agricultural products (oat hulls, soybean hulls, yeast extract, soybean flour, dried bovine erythrocytes, bovine albumin, and/or mineral salts) and 50% (wt/wt) polypropylene were produced by high-temperature twin-screw extrusion. The research employed two half sets of a five-factorial fractional design (2(5 - 1)) to evaluate the effects of different agricultural components on the properties of the plastic composite supports and to select the best plastic composite support formulation for lactic acid fermentation. The biofilm population was affected by the contact angle and relative hydrophobicity of the supports (r = 0.79 to 0.82). Lactic acid was produced by the suspended cells (r = 0.96) and the biofilm on the plastic composite support discs (r = 0.85). Incorporation of yeast extract into plastic composite supports enhanced growth of free and attached cells in minimal medium (P < 0.0001). The presence of soybean hulls, yeast extract, or mineral salts in plastic composite supports produced less hydrophobic supports (P < 0.0001) and enhanced cell attachment (P < 0.03). Under all conditions, suspended-cell and polypropylene disc controls gave negligible lactic acid production and cell density. Plastic composite supports containing soybean hulls, yeast extract, soybean flour, bovine albumin, and mineral salts gave the highest biofilm population (2.3 x 10(9) CFU/g of support), cell density (absorbance of 1.8 at 620 nm), and lactic acid concentration (7.6 g/liter) in minimal medium.
Nutrient leaching and end product accumulation in plastic composite supports for L-(+)-lactic Acid biofilm fermentation
Authors: Ho K G , Pometto A I , Hinz P N , Demirci A
Published: January 2020

Investigations on the leachate bioavailability, leaching rate, and lactic acid accumulation properties of plastic composite supports (PCS) were essential for large-scale or long-term lactic acid fermentation. Leachates from PCS and polypropylene discs (controls) were analyzed by the micro-Kjeldahl method; by absorbances at 260, 275, and 280 nm; and by bioassays with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The amount of leached nitrogen in a 20-ml initial soaking solution had a high correlation with the soaking solution's cell density (r = 0.87) and absorbance at 260 nm (r = 0.95). Leaching rates of various PCS were evaluated by 20 20-ml simulated repeated-batch fermentations (RBF). PCS with only yeast extract as the minor agricultural ingredient had a high leaching rate and leached out 51 to 60% of the total nitrogen during the first RBF. PCS blended with dried bovine albumin, dried bovine erythrocytes, and/or soybean flour had slowed nutrient leaching (20 to 30% of the initial leached nitrogen). Hence, they could still maintain 1 g of lactic acid per liter and measurable cell density (absorbance at 620 nm, 0.4 to 0.6) at the 20th 20-ml RBF. Lactic acid accumulation properties of PCS were evaluated by soaking the supports in a 30% lactic acid solution for 72 h at 45(deg)C. The lactic acid-soaked supports were rinsed three times and then heat treated (121(deg)C, 15 min) in 15 ml of deionized water. The results showed that lactic acid accumulation in PCS was mainly due to absorption and had no correlation with lactic acid production or biofilm formation.
Optimization of L-(+)-lactic acid production by ring and disc plastic composite supports through repeated-batch biofilm fermentation
Authors: Ho K L , Pometto A L , Hinz P N
Published: January 2020

Four customized bioreactors, three with plastic composite supports (PCS) and one with suspended cells (control), were operated as repeated-batch fermentors for 66 days at pH 5 and 37 degrees C. The working volume of each customized reactor was 600 ml, and each reactor's medium was changed every 2 to 5 days for 17 batches. The performance of PCS bioreactors in long-term biofilm repeated-batch fermentation was compared with that of suspended-cell bioreactors in this research. PCS could stimulate biofilm formation, supply nutrients to attached and free suspended cells, and reduce medium channelling for lactic acid production. Compared with conventional repeated-batch fermentation, PCS bioreactors shortened the lag time by threefold (control, 11 h; PCS, 3.5 h) and sixfold (control, 9 h; PCS, 1.5 h) at yeast extract concentrations of 0.4 and 0.8% (wt/vol), respectively. They also increased the lactic acid productivity of Lactobacillus casei subsp. rhamnosus (ATCC 11443) by 40 to 70% and shortened the total fermentation time by 28 to 61% at all yeast extract concentrations. The fastest productivity of the PCS bioreactors (4.26 g/liter/h) was at a starting glucose concentration of 10% (wt/vol), whereas that of the control (2.78 g/liter/h) was at 8% (wt/vol). PCS biofilm lactic acid fermentation can drastically improve the fermentation rate with reduced complex-nutrient addition.
Anaerobic and aerobic degradation of pyridine by a newly isolated denitrifying bacterium
Authors: Rhee S K , Lee G M , Yoon J H , Park Y H , Bae H S , Lee S T
Published: January 2020

New denitrifying bacteria that could degrade pyridine under both aerobic and anaerobic conditions were isolated from industrial wastewater. The successful enrichment and isolation of these strains required selenite as a trace element. These isolates appeared to be closely related to Azoarcus species according to the results of 16S rRNA sequence analysis. An isolated strain, pF6, metabolized pyridine through the same pathway under both aerobic and anaerobic conditions. Since pyridine induced NAD-linked glutarate-dialdehyde dehydrogenase and isocitratase activities, it is likely that the mechanism of pyridine degradation in strain pF6 involves N-C-2 ring cleavage. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, and nitrous oxide as electron acceptors. In a batch culture with 6 mM nitrate, degradation of pyridine and denitrification were not sensitively affected by the redox potential, which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated during denitrification significantly inhibited cell growth and pyridine degradation. Growth yield on pyridine decreased slightly under denitrifying conditions from that under aerobic conditions. Furthermore, when the pyridine concentration used was above 12 mM, the specific growth rate under denitrifying conditions was higher than that under aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages over strictly aerobic bacteria in field applications.
Intraspecific Differentiation of Vibrio vulnificus Biotypes by Amplified Fragment Length Polymorphism and Ribotyping
Authors: Arias C R , Verdonck L , Swings J , Garay E , Aznar R
Published: January 2020

The intraspecific genomic relatedness of 80 Vibrio vulnificus isolates, 44 of biotype 1 and 36 of biotype 2, from different geographic origins and sources was evaluated by ribotyping and AFLP (amplified fragment length polymorphism) fingerprinting. Ribopatterns of DNAs digested with KpnI and hybridized with an oligonucleotide complementary to a highly conserved sequence in the 23S rRNA gene revealed up to 19 ribotypes in the species, which were different for the two biotypes. Sixteen different ribotypes were found within biotype 1 strains from clinical and environmental sources, and only three, recovered mainly from diseased eels, were found within biotype 2. Within this biotype, 96% of the strains showed the same ribopattern. The closest similarity was shown by the strains coming from the same eel farm, irrespectively of biotype. AFLP fingerprints obtained by selective PCR amplification of HindIII-TaqI double-restricted DNA fragments exhibited a strain-specific pattern which allowed the finest differentiation of subgroups within the eel-pathogenic isolates sharing the same ribopattern. Both techniques revealed good genetic markers for intraspecific differentiation of V. vulnificus. Ribotyping clearly separated the eel-pathogenic strains from the clinical and environmental isolates, whereas AFLP enabled the monitoring of individual strains and therefore constitutes one of the most discriminative tools for epidemiological and ecological studies.
Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid Tolerance Response in Listeria monocytogenes LO28
Authors: O'driscoll B , Gahan C , Hill C
Published: January 2020

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.
Changes in Cellular States of the Marine Bacterium Deleya aquamarina under Starvation Conditions
Authors: Joux F , Lebaron P , Troussellier M
Published: January 2020

In this study, we have used different fluorescent dyes and techniques to characterize the heterogeneity and changes of the physiological states encountered by the marine bacterium Deleya aquamarina during a 92-day starvation survival experiment at 20 and 5(deg)C. Changes of physiological states were investigated on a single-cell basis by flow cytometry and epifluorescence microscopy in conjunction with fluorescent dyes specific for various cellular functions and constituents. Heterogeneities within populations with regard to functions (respiration, substrate responsiveness, enzymatic activity, and cytoplasmic membrane permeability), constituent (DNA), and cell volume (light scatter) were compared to the evolution of viable plate counts (CFU). At 20(deg)C, CFU changes were divided into three stages corresponding to stability up to day 13 followed by a rapid drop between days 13 and 42 and then by stabilization at a level of 10 to 20% during the remaining survival period. Most of the cellular fractions showing a metabolic activity were close to the evolution of the culturable cells, suggesting the absence of viable but nonculturable cells. On the other hand, cells with selective cytoplasmic membrane permeability but without any metabolic activity were observed, and this stage was followed by DNA alteration occurring at different rates after the loss of membrane cytoplasmic permeability. We observed a greater maintenance of culturability, physiological functions, DNA, and cellular volume at the lower temperature. These results have different ecological implications from both methodological and conceptual viewpoints.
The human Lactobacillus acidophilus strain LA1 secretes a nonbacteriocin antibacterial substance(s) active in vitro and in vivo
Authors: Bernet-Camard M F , Liévin V , Brassart D , Neeser J R , Servin A L , Hudault S
Published: January 2020

The adhering human Lactobacillus acidophilus strain LA1 inhibits the cell association and cell invasion of enteropathogens in cultured human intestinal Caco-2 cells (M. F. Bernet, D. Brassard, J. R. Neeser, and A. L. Servin, Gut 35:483-489, 1994). Here, we demonstrate that strain LA1 developed its antibacterial activity in conventional or germ-free mouse models orally infected by Salmonella typhimurium. We present evidence that the spent culture supernatant of strain LA1 (LA1-SCS) contained antibacterial components active against S. typhimurium infecting the cultured human intestinal Caco-2 cells. The LA1-SCS antibacterial activity was observed in vitro against a wide range of gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, S. typhimurium, Shigella flexneri, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae. By contrast, no activity was observed against species of the normal gut flora, such as lactobacilli and bifidobacteria. The LA1-SCS antibacterial activity was insensitive to proteases and independent of lactic acid production.
Glucose metabolism in the yeast Schwanniomyces castellii: role of phosphorylation site I and an alternative respiratory pathway
Authors: Zimmer E , Blanchard S , Boze H , Moulin G , Galzy P
Published: January 2020

Glucose metabolism in a Crabtree-negative yeast, Schwanniomyces castellii, and a cytochrome b-deficient mutant of this strain was investigated in chemostat culture. The wild-type and mutant strains exhibited the same behavior. Oxidative metabolism was observed when the substrate uptake rate (qS) was low. Fermentative metabolites were excreted when the qS value was higher than 0.40 g.g-1.h-1, indicating the occurrence of a respirofermentative metabolism; however, the respiratory quotient (RQ) remained near 1. When fermentation occurred, the cytochrome pathway was repressed but not the salicylhydroxamic acid (SHAM)-sensitive pathway. The presence of an alternative SHAM-sensitive respiratory pathway and the presence of phosphorylation site I in all metabolic conditions explained the RQ value of 1 and accounted for high biomass yields in oxidative metabolism conditions (0.62 g.g-1 for the wild-type strain and 0.31 g.g-1 for the cytochrome b-deficient mutant strain).
Biochemical and molecular characterization of the insecticidal fragment of CryV
Authors: Sekar V , Held B , Tippett J , Amirhusin B , Robeff P , Wang K , Wilson H M
Published: January 2020

Two C-terminal deletion constructs were made to study the effect of such deletions on the biological activity of the CryV protein of Bacillus thuringiensis subsp. kurstaki. The results of feeding on neonatal larvae of Ostrinia nubilalis (European corn borer [ECB]) indicated that the 50% lethal dose of the full-length CryV protein was 3.34 micrograms/g of diet (95% fiducial limits, 2.53 to 4.32 micrograms/g of diet). Removal of 71 amino acids (aa) from the C terminus had little effect on toxicity, whereas deletion of 184 aa abolished the insecticidal activity of the CryV protein completely. Truncations of the full-length CryV protein were also generated with trypsin and the midgut protease of ECB. The proteolytically treated products were characterized by determining their N-terminal amino acid sequences. The CryV protein was found to be cleaved by both proteases through a two-step process. Initially an intermediary form was generated which contained aa 45 of full-length CryV as its N-terminal end. The C-terminal end of this peptide was not experimentally determined. However, analysis of the deduced amino acid sequence of CryV indicated that the C-terminal end of the intermediary form is likely either aa 655 or 659. Further N-terminal processing of the intermediary form resulted in a protease-resistant core form. The core included aa 156 to aa 655 or 659. While the intermediary form retained 100% of the ECB larval toxicity, the core form exhibited only approximately 22% of the toxicity of the full-length protein.
Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle
Authors: Elskens M T , Penninckx M J
Published: January 2020

A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH. Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG). Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase. These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast. Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide. Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1. Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast. The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity. Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.
Complete reductive dechlorination of 1,2-dichloropropane by anaerobic bacteria
Authors: Loffler F E , Champine J E , Ritalahti K M , Sprague S J , Tiedje J M
Published: January 2020

The transformation of 1,2-dichloropropane (1,2-D) was observed in anaerobic microcosms and enrichment cultures derived from Red Cedar Creek sediment. 1-Chloropropane (1-CP) and 2-CP were detected after an incubation period of 4 weeks. After 4 months the initial amount of 1,2-D was stoichiometrically converted to propene, which was not further transformed. Dechlorination of 1,2-D was not inhibited by 2-bromoethanesulfonate. Sequential 5% (vol/vol) transfers from active microcosms yielded a sediment-free, nonmethanogenic culture, which completely dechlorinated 1,2-D to propene at a rate of 5 nmol min(sup-1) mg of protein(sup-1). No intermediate formation of 1-CP or 2-CP was detected in the sediment-free enrichment culture. A variety of electron donors, including hydrogen, supported reductive dechlorination of 1,2-D. The highest dechlorination rates were observed between 20(deg) and 25(deg)C. In the presence of 1,2-D, the hydrogen threshold concentration was below 1 ppm by volume (ppmv). In addition to 1,2-D, the enrichment culture transformed 1,1-D, 2-bromo-1-CP, tetrachloroethene, 1,1,2,2-tetrachloroethane, and 1,2-dichloroethane to less halogenated compounds. These findings extend our knowledge of the reductive dechlorination process and show that halogenated propanes can be completely dechlorinated by anaerobic bacteria.
Conservation of plasmid-encoded dibenzothiophene desulfurization genes in several rhodococci
Authors: Denis-Larose C , Labbé D , Bergeron H , Jones A M , Greer C W , al-Hawari J , Grossman M J , Sankey B M , Lau P C
Published: January 2020

The cloned sulfur oxidation (desulfurization) genes (sox) for dibenzothiophene (DBT) from the prototype Rhodococcus sp. strain IGTS8 were used in Southern hybridization and PCR experiments to establish the DNA relatedness in six new rhodococcal isolates which are capable of utilizing DBT as a sole sulfur source for growth. The ability of these strains to desulfurize appears to be an exclusive property of a 4-kb gene locus on a large plasmid of ca. 150 kb in IGTS8 and ca. 100 kb in the other strains. Besides a difference in plasmid profile, IGTS8 is distinguishable from the other strains in at least the copy number of the insertion sequence IS1166, which is associated with the sox genes.
Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster
Authors: Chary V K , de la Fuente J L , Liras P , Martin J F
Published: January 2020

Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.
Reduction of Selenium Oxyanions by Enterobacter cloacae SLD1a-1: Isolation and Growth of the Bacterium and Its Expulsion of Selenium Particles
Authors: Losi M E , Frankenberger W T
Published: January 2020

A facultative bacterium capable of removing the selenium (Se) oxyanions selenate (SeO(inf4)(sup2-)) and selenite (SeO(inf3)(sup2-)) from solution culture in flasks open to the atmosphere was isolated and studied with the goal of assessing its potential for use in bioremediation of seleniferous agricultural drainage water. Elemental Se (Se(sup0)) was confirmed as a product of the reaction. The organism, identified as Enterobacter cloacae and designated strain SLD1a-1 (ATCC 700258), removed from 61.5 to 94.5% of added SeO(inf4)(sup2-) (the primary species present in agricultural drainage water) at concentrations from 13 to 1,266 (mu)M. Equimolar amounts of nitrate (NO(inf3)(sup-)), which interferes with SeO(inf4)(sup2-) reduction in some organisms, did not influence the reaction in growth experiments but had a slight inhibitory effect in a washed-cell suspension. Washed-cell suspension experiments also showed that (i) SeO(inf3)(sup2-) is a transitory intermediate in reduction of SeO(inf4)(sup2-), being produced and rapidly reduced concomitantly; (ii) NO(inf3)(sup-) is also reduced concomitantly and at a much higher rate than SeO(inf4)(sup2-); and (iii) although enzymatic, reduction of either oxyanion does not appear to be an inducible process. Transmission electron microscopy revealed that precipitate particles are <0.1 (mu)m in diameter, and these particles were observed free in the medium. Evidence indicates that SLD1a-1 uses SeO(inf4)(sup2-) as an alternate electron acceptor and that the reaction occurs via a membrane-associated reductase(s) followed by rapid expulsion of the Se particles.
Identification and modification of biphenyl dioxygenase sequences that determine the specificity of polychlorinated biphenyl degradation
Authors: Mondello F J , Turcich M P , Lobos J H , Erickson B D
Published: January 2020

The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria. The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners. Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners. BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity. The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV. Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707. Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners. However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative. These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase.
Evidence for paralytic shellfish poisons in the freshwater cyanobacterium Lyngbya wollei (Farlow ex Gomont) comb. nov
Authors: Carmichael W W , Evans W R , Yin Q Q , Bell P , Moczydlowski E
Published: January 2020

Lyngbya wollei (Farlow ex Gomont) comb. nov., a perennial mat-forming filamentous cyanobacterium prevalent in lakes and reservoirs of the southeastern United States, was found to produce a potent, acutely lethal neurotoxin when tested in the mouse bioassay. Signs of poisoning were similar to those of paralytic shellfish poisoning. As part of the Tennessee Valley Authority master plan for Guntersville Reservoir, the mat-forming filamentous cyanobacterium L. wollei, a species that had recently invaded from other areas of the southern United States, was studied to determine if it could produce any of the known cyanotoxins. Of the 91 field samples collected at 10 locations at Guntersville Reservoir, Ala., on the Tennessee River, over a 3-year period, 72.5% were toxic. The minimum 100% lethal doses of the toxic samples ranged from 150 to 1,500 mg kg of lyophilized L. wollei cells-1, with the majority of samples being toxic at 500 mg kg-1. Samples bioassayed for paralytic shellfish toxins by the Association of Official Analytical Chemists method exhibited saxitoxin equivalents ranging from 0 to 58 micrograms g (dry weight)-1. Characteristics of the neurotoxic compound(s), such as the lack of adsorption by C18 solid-phase extraction columns, the short retention times on C18 high-performance liquid chromatography (HPLC) columns, the interaction of the neurotoxins with saxiphilin (a soluble saxitoxin-binding protein), and external blockage of voltage-sensitive sodium channels, led to our discovery that this neurotoxin(s) is related to the saxitoxins, the compounds responsible for paralytic shellfish poisonings. The major saxitoxin compounds thus far identified by comparison of HPLC fluorescence retention times are decarbamoyl gonyautoxins 2 and 3. There was no evidence of paralytic shellfish poison C toxins being produced by L. wollei. Fifty field samples were placed in unialgal culture and grown under defined culture conditions. Toxicity and signs of poisoning for these laboratory-grown strains of L. wollei were similar to those of the field collection samples.
Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae
Authors: Berka R M , Schneider P , Golightly E J , Brown S H , Madden M , Brown K M , Halkier T , Mondorf K , Xu F
Published: January 2020

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.
Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities
Authors: Jaspers E , Overmann J
Published: January 2020

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.
Partitioning of Symbiotic Bacteria between Generations of an Insect: a Quantitative Study of a Buchnera sp. in the Pea Aphid (Acyrthosiphon pisum) Reared at Different Temperatures
Authors: Humphreys N J , Douglas A E
Published: January 2020

The population of symbiotic Buchnera bacteria in parthenogenetic females of the pea aphid Acyrthosiphon pisum was determined by quantitative hybridization of a DNA probe (groESL) to aphid homogenates. The aphids bore 1 x 10(sup7) to 2 x 10(sup7) bacterial cells per mg (fresh weight). In teneral aphids (i.e., aphids that had moulted to adulthood but that had not initiated reproduction), >75% of the bacteria were in the embryos, and the density of bacteria in the embryos was consistently greater than that in the maternal tissues. The bacterial density in teneral aphids increased from 1.3 x 10(sup7) to 2.0 x 10(sup7) cells mg (fresh weight) of aphids(sup-1) with temperature between 15 and 25(deg)C. This variation could be attributed to a temperature-dependent increase in both the density of bacteria in the embryos and embryo content of the aphids.
Analysis of fecal populations of bifidobacteria and lactobacilli and investigation of the immunological responses of their human hosts to the predominant strains
Authors: Kimura K , McCartney A L , McConnell M A , Tannock G W
Published: January 2020

The bifidobacterial and lactobacillus populations of fecal samples collected from 10 human subjects were studied. The numbers of bifidobacteria were similar in the fecal samples of all of the subjects, but lactobacillus numbers varied, even between samples collected from the same individual. Analysis of the composition of the bacterial populations by ribotyping and pulsed-field gel electrophoresis to differentiate between strains showed that, at least for the numerically predominant strains, each subject harbored a unique collection of bifidobacteria and lactobacilli. Predominant bifidobacterial and lactobacillus strains detected in the feces of each subject were used in immunological assays (lymphocyte transformation, serum antibody titers) to determine the influence of the bacteria on the immune system of their host. Immunoglobulin G antibodies reactive with lactobacilli were detected at high concentrations; antibodies reactive with bifidobacteria were present at lower concentrations. The antibodies appeared to be genus specific rather than strain specific. The results of the study emphasized the complexity of the relationship that exists between the intestinal microflora and the human host.
Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess
Authors: Flikweert M T , van Dijken J P , Pronk J T
Published: January 2020

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.
New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917
Authors: Framson P E , Nittayajarn A , Merry J , Youngman P , Rubens C E
Published: January 2020

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.
Microbial Utilization of Free and Clay-Bound Insecticidal Toxins from Bacillus thuringiensis and Their Retention of Insecticidal Activity after Incubation with Microbes
Authors: Koskella J , Stotzky G
Published: January 2020

The insecticidal toxins produced by Bacillus thuringiensis subspp. kurstaki and tenebrionis were resistant when bound on clays, but not when free, to utilization by pure and mixed cultures of microbes as sources of carbon and carbon plus nitrogen, and their availability as a nitrogen source was reduced. The bound toxins retained insecticidal activity both before and after exposure to microbes or pronase. The insecticidal activity of the toxins persisted for 40 days (the longest time evaluated) in nonsterile soil continuously maintained at the -33-kPa water tension and room temperature, alternately air dried and rewetted to the -33-kPa water tension, or alternately frozen and thawed, although alternate drying and wetting reduced the activity.
Vertical Patterns of Nitrogen Transformations during Infiltration in Two Wetland Soils
Authors: Davidsson T E , Stepanauskas R , Leonardson L
Published: January 2020

The (sup15)N isotope dilution and pairing methods were applied to investigate the vertical distribution of nitrogen transformations during infiltration in one peaty soil and one sandy soil. Water containing (sup15)N-nitrate (99.9%; 200 (mu)M) as the only nitrogen fraction was infiltrated through cores containing homogenized soil, with lengths varying from 5.5 to 38 cm. Oxygen and nitrogen dynamics were investigated by measuring inflowing and outflowing water. The experimental design allowed determinations of vertical profiles of aerobic respiration, nitrification, and coupled and uncoupled denitrification and ammonification. In the sandy soil, all oxygen was consumed in the upper 14 cm and nitrate was subsequently consumed and removed, up to a maximum of 70% in the longest core (28 cm). In the peaty soil, oxygen was consumed in the upper 7.5 cm and all nitrate was denitrified in the top 20 cm. In both soils, nitrogen removal by denitrification was counteracted by the release of ammonium and dissolved organic nitrogen. In the sandy soil, net nitrogen removal occurred in cores of 14 cm and longer; in the longest core, 40% was removed. In the peaty soil, release was equal to removal in the top 14 cm but release exceeded removal in the deeper layers, leading to a 100% increase of total nitrogen in the effluent water from the longest core (38 cm).
Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction
Authors: Kudlich M , Keck A , Klein J , Stolz A
Published: January 2020

The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown.
Flow cytometric analysis of peripheral blood lymphocyte immunophenotypes in persons infected with Treponema pallidum
Authors: Pope V , Larsen S A , Rice R J , Goforth S N , Parham C E , Fears M B
Published: January 2020

To characterize the human immune response to syphilis, we determined the effect of infection with Treponema pallidum on the percentage of the various lymphocyte subpopulations in the peripheral blood of infected and uninfected persons. Monoclonal antibodies labeled with either fluorescein isothiocyanate or phycoerythrin were used to perform dual color analysis on a FACScan with the following markers: CD3 for total T cells, CD4 for T helper cells, CD8 for T suppressor cells, CD19 for B cells, and CD16 plus CD56 for natural killer cells. Lymphocyte immunophenotype results were analyzed by the stage of untreated syphilis and by gender. Although they were within the ranges of the normal distribution of immunophenotypes, the percentages of CD4+ cells were significantly lower (P < 0.001) and those of CD8+ cells were higher (P = 0.03) in patients with syphilis than in the uninfected population. For infected versus uninfected subjects, both women and men, the differences in the mean percentages of CD3+ and CD4+ cells were significant (P < or = 0.05). Significant differences were noted between the sexes in secondary syphilis only in the mean percentages of cells positive for CD3, CD4, CD8, and CD16 plus CD56. Gender had no effect on lymphocyte subpopulations in subjects with primary or latent syphilis. In the control population, significant differences due to gender were observed in the percentages of cells positive for CD3, CD4, and CD16 plus CD56.
A confocal microscopy study of anticytoskeletal antibody activity in patients with connective tissue disease
Authors: French P W , Penny R , Yang J L
Published: January 2020

The significance of the presence of antibodies to cytoskeleton proteins in patients with connective tissue diseases is not clear, as there is a high level of these antibodies in healthy controls. In an attempt to improve the visualization of the immunofluorescence binding pattern of autoantibodies to cytoskeletal structures in cultured fibroblasts, we have used confocal microscopy. Of the 256 serum samples tested, 155 (61%) WERE reactive with cytoplasmic structures. These reactive samples could be divided into seven patterns of binding, as determined by double-blind examination of single-section confocal images. While confirming the results of previous immunofluorescence studies which have shown that autoantibodies that bind to filamentous structures in the cytoplasm of cultured cells are common in patients with connective tissue diseases, we were able to identify three patterns of cytoskeletal binding which may be useful as an adjunct to other tests for the diagnosis of some connective tissue diseases, in particular systemic sclerosis (scleroderma) and rheumatoid arthritis/Sjogren's syndrome. None of the seven patterns was exclusive to a particular disease. We conclude that confocal microscopy may be of limited use as an adjunct to other serological assays in the diagnosis of some forms of connective tissue disease.
Role of human natural killer cells in health and disease
Authors: Whiteside T L , Herberman R B
Published: January 2020

Natural killer (NK) cells, the CD3- CD56+ CD16+ subset of peripheral blood lymphocytes, have long been known to be involved in non-major histocompatibility complex-restricted natural immunity to virally infected and malignant target cells. The association of abnormalities in NK cell numbers or functions with a broad spectrum of human diseases has been more clearly defined in recent years as a result of the improved knowledge of NK cell physiology and advances in monitoring of NK cell functions in health and disease. The ability to reliably measure changes in NK activity and/or numbers during the course of disease or response to treatment has focused attention on the role of the NK cell in disease pathogenesis. The improved understanding of NK cell deficiency in disease has opened a way for therapies specifically designed to improve NK cell function. The therapeutic use of biologic response modifiers capable of augmenting NK cell activity in vivo and of adoptive transfer of highly enriched, activated autologous NK cells in diseases such as cancer and AIDS is being evaluated. The importance of NK cells in health and the consequences of NK cell deficiency or excess are likely to be more extensively monitored in the future.