bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms for soil moisture effects on activity of nitrifying bacteria
Authors: Stark J M , Firestone M K
Published: January 2020

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.
The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis
Authors: Drainas C , Vartholomatos G , Panopoulos N J
Published: January 2020

The expression of the ice nucleation gene inaZ from Pseudomonas syringae in Zymomonas mobilis strains under the control of three different promoters was investigated to establish the utility of the gene as a reporter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterless version of the inaZ gene was placed under the control of three different promoters: P(infpdc) (pyruvate decarboxylase), a homologous strong promoter from Z. mobilis; P(infbla) ((beta)-lactamase) of plasmid pBR325; and P(infhrpR), the promoter of hrpR, a regulatory gene from P. syringae pv. phaseolicola. The apparent strengths of all three promoters, measured by quantifying the ice nucleation activity at -9 deg C, were lower in Z. mobilis than in Escherichia coli. The levels of ice nucleation activity expressed under the P(infpdc) promoter were significantly higher than those obtained with the two heterologous promoters in Z. mobilis. Plasmid pCG4521 (RK2 replicon) gave much lower levels of ice nucleation activity when propagated in strain uvs-51, a plasmid instability mutant of Z. mobilis, compared with the wild-type strain. The ice nucleation activity in Z. mobilis cultures showed unusual partitioning in that the culture supernatants obtained after low-speed centrifugation contained the majority of ice nuclei. Analysis of the ice nucleation spectra revealed that the cell pellets contained both "warm" and "cold" nuclei, while the culture supernatant contained primarily cold nuclei, suggesting that the cold nucleus activity may be extracellular. However, all nucleation activity was retained by 0.22-(mu)m-pore-size filters.
Competitive metabolism of naphthalene, methylnaphthalenes, and fluorene by phenanthrene-degrading pseudomonads
Authors: Stringfellow W T , Aitken M D
Published: January 2020

Polynuclear aromatic hydrocarbons (PAHs) typically exist as complex mixtures in contaminated soils, yet little is known about the biodegradation of PAHs in mixtures. We have isolated two physiologically diverse bacteria, Pseudomonas stutzeri P-16 and P. saccharophila P-15, from a creosote-contaminated soil by enrichment on phenanthrene as the sole carbon source and studied their ability to metabolize several other two- and three-ring PAHs. Naphthalene, 1-methylnaphthalene, and 2-methylnaphthalene served as growth substrates for both organisms, while fluorene was only cometabolized. We also studied the effects of these compounds on initial rates of phenanthrene uptake in binary mixtures. Lineweaver-Burk analysis of kinetic measurements was used to demonstrate competitive inhibition of phenanthrene uptake by all four compounds, suggesting that multiple PAHs are being transformed by a common enzyme pathway in whole cells. Estimates of the inhibition coefficient, Ki, are reported for each compound. The occurrence of competitive metabolic processes in physiologically diverse organisms suggests that competitive metabolism may be a common phenomenon among PAH-degrading organisms.
Active site analysis and stabilization of sarcosine oxidase by the substitution of cysteine residues
Authors: Nishiya Y , Zuihara S , Imanaka T
Published: January 2020

Two cysteine residues (C-265 and C-318) in the putative hydrophilic regions of sarcosine oxidase were substituted by using site-directed mutagenesis. Since the mutant with the C-to-S mutation at position 318 (C318S) lost the enzyme activity, C-318 (conserved among sarcosine oxidases) is most likely a part of the active site. C265S, C265A, C265D, and C265R showed nearly the same enzymatic properties as those of the wild type. However, they were much more stable than the wild type in the presence of inhibitors that modified the thiol group. Moreover, they were extremely stable throughout the cultivation of the recombinant strains or even in cell extracts.
Growth and survival of Escherichia coli O157:H7 under acidic conditions
Authors: Conner D E , Kotrola J S
Published: January 2020

The effect of pH reduction with acetic (pH 5.2), citric (pH 4.0), lactic (pH 4.7), malic (pH 4.0), mandelic (pH 5.0), or tartaric (pH 4.1) acid on growth and survival of Escherichia coli O157:H7 in tryptic soy broth with 0.6% yeast extract held at 25, 10, or 4 degrees C for 56 days was determined. Triplicate flasks were prepared for each acid treatment at each temperature. At 25 degrees C, populations increased 2 to 4 log10 CFU/ml in all treatments except that with mandelic acid, whereas no growth occurred at 10 or 4 degrees C in any treatments except the control. However, at all sampling times, higher (P < 0.05) populations were recovered from treatments held at 4 degrees C than from those held at 10 degrees C. At 10 degrees C, E. coli O157:H7 was inactivated at higher rates in citric, malic, and mandelic acid treatments than in the other treatments. At the pH values tested, the presence of the organic acids enhanced survival of the pathogen at 4 degrees C compared with the unacidified control. E. coli O157:H7 has the ability to survive in acidic conditions (pH, > or = 4.0) for up to 56 days, but survival is affected by type of acidulant and temperature.
Detection of Escherichia coli O157:H7 in meat by an enzyme-linked immunosorbent assay, EHEC-Tek
Authors: Johnson R P , Durham R J , Johnson S T , MacDonald L A , Jeffrey S R , Butman B T
Published: January 2020

Investigation of the specificity of an enzyme-linked immunosorbent assay (ELISA) for detection of Escherichia coli O157:H7 in raw meats (EHEC-Tek; Organon Teknika Corp.) revealed that the target antigens of the detection reagent, monoclonal antibody 4E8C12, were present in numerous serotypes of E. coli and that their ELISA reactivity was influenced by bile salts, acriflavine, and heat. The specificity of the ELISA was improved by a modified test protocol incorporating immunocapture.
Unusual Growth Phase and Oxygen Tension Regulation of Oxidative Stress Protection Enzymes, Catalase and Superoxide Dismutase, in the Phytopathogen Xanthomonas oryzae pv. oryzae
Authors: Chamnongpol S , Mongkolsuk S , Vattanaviboon P , Fuangthong M
Published: January 2020

The enzymes catalase and superoxide dismutase play major roles in protecting phytopathogenic bacteria from oxidative stress. In Xanthomonas species, these enzymes are regulated by both growth phase and oxygen tension. The highest enzyme levels were detected within 1 h of growth. Continued growth resulted in a decline of both enzyme activities. High oxygen tension was an inducing signal for both enzyme activities. An 80,000-Da monofunctional catalase and a manganese superoxide dismutase were the major forms of the enzymes detected at different stages of growth. The unusual regulatory patterns are common among several Xanthomonas strains tested and may be advantageous to Xanthomonas species during the initial stage of plant-microorganism interactions.
Effects of electron donor and acceptor conditions on reductive dehalogenation of tetrachloromethane by Shewanella putrefaciens 200
Authors: Picardal F , Arnold R G , Huey B B
Published: January 2020

Shewanella putrefaciens 200 is a nonfermentative bacterium that is capable of dehalogenating tetrachloromethane to chloroform and other, unidentified products under anaerobic conditions. Since S. putrefaciens 200 can respire anaerobically by using a variety of terminal electron acceptors, including NO3-, NO2-, and Fe(III), it provides a unique opportunity to study the competitive effects of different electron acceptors on dehalogenation in a single organism. The results of batch studies showed that dehalogenation of CT by S. putrefaciens 200 was inhibited by O2, 10 mM NO3-, and 3 mM NO2-, but not by 15 mM Fe(III), 15 mM fumarate, or 15 mM trimethylamine oxide. Using measured O2, Fe(III), NO2-, and NO3- reduction rates, we developed a speculative model of electron transport to explain inhibition patterns on the basis of (i) the kinetics of electron transfer at branch points in the electron transport chain, and (ii) possible direct inhibition by nitrogen oxides. In additional experiments in which we used 20 mM lactate, 20 mM glucose, 20 mM glycerol, 20 mM pyruvate, or 20 mM formate as the electron donor, dehalogenation rates were independent of the electron donor used. The results of other experiments suggested that sufficient quantities of endogenous substrates were present to support transformation of tetrachloromethane even in the absence of an exogenous electron donor. Our results should be significant for evaluating (i) the bioremediation potential at sites contaminated with both halogenated organic compounds and nitrogen oxides, and (ii) the bioremediation potential of iron-reducing bacteria at contaminated locations containing significant amounts of iron-bearing minerals.
Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil
Authors: Wright D A , Killham K , Glover L A , Prosser J I
Published: January 2020

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.
Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins
Authors: Popham D L , Sengupta S , Setlow P
Published: January 2020

Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores. Spores lacking the two major alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha-beta- spores) have the same core water content as do wild-type spores, but alpha-beta- dacB spores had more core water than did dacB spores. The resistance of alpha-beta-, alpha-beta- dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide. These data (i) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of alpha/beta-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (ii) suggest that binding of alpha/beta-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (iii) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (iv) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by alpha/beta-type SASP.
Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts
Authors: Powers E M
Published: January 2020

A simple and rapid (< 60 s) nonstaining technique with 3% potassium hydroxide to determine Gram reactions was tested with 495 food-borne and waterborne bacteria and yeasts. In KOH, suspensions of gram-negative bacteria become viscous and string out. Gram-positive bacteria are not affected. There was 100% correlation between the KOH string test results and gram-positive and gram-negative strains.
Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes
Authors: Jelén H H , Mirocha C J , Wasowicz E , Kamiński E
Published: January 2020

Twenty-five strains of Fusarium sambucinum grown on wheat kernels were examined for trichothecene production and the synthesis of volatile sesquiterpenes. The volatiles were purged with air and collected on Tenax traps. Adsorbed compounds were eluted from the traps and injected into a gas chromatograph coupled with a mass spectrometer. Ten strains isolated from potato tubers produced high amounts of diacetoxyscirpenol and its derivatives. These strains were characterized by the production of high amounts of diverse sesquiterpenes. In 10 cultures, 19 compounds were detected, of which 6 were predominant and composed as much as 82% of the volatile sesquiterpene fraction (e.g., beta-farnesene, beta-chamigrene, beta-bisabolene, alpha-farnesene, trichodiene, and an unidentified compound). Fifteen strains isolated from various sources that did not produce trichothecenes produced much less volatile sesquiterpenes, with less chemical diversity. No more than six compounds were present in cultures. Two of these compounds were present in the toxigenic strains isolated from potatoes (beta-farnesene and acoradiene), but four were unique to the strains not producing trichothecenes (longifolene, isocaryophyllene, delta-elemene, and an unidentified one). The pattern of volatile sesquiterpenes was characteristic and distinctive for both toxic and nontoxic strains.
Survival and Plant Growth Promotion of Detergent-Adapted Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7NSK2
Authors: Devliegher W , Arif M , Verstraete W
Published: January 2020

Four detergents were tested as selective C sources for the plant growth-promoting rhizobacteria Pseudomonas aeruginosa 7NSK2 and Pseudomonas fluorescens ANP15. CO-720 (Igepal CO-720) or DOS (dioctyl sulfosuccinate), applied at 0.2% to the soil, increased the number of detergent-adapted, inoculated strains by almost 1.5 log units after 25 days, accounting for virtually the entire increase in total bacteria. The same dose of Tween 80 or N-laurylsarcosine, on the other hand, increased the indigenous populations by almost 2.5 log units, with only minor increases in the number of detergent-adapted inoculated strains. When CO-720 or DOS was initially supplied, the number of detergent-adapted 7NSK2 organisms was about 2 log units higher after 3 months of incubation than for the detergent-unadapted strain. This better survival resulted in a significantly higher root colonization of maize in a pot experiment with soil inoculation, with a significantly (P <= 0.05) higher shoot dry weight (18 to 33%). In a first field experiment with rhizosphere inoculation of 1-month-old maize plants, no effects on the height of two maize cultivars could be observed 1 month after inoculation. In a second field experiment, leaf and stem dry weights of yellow mustard and grass dry weight were increased in the treatments with seed and soil inoculation of the detergent-adapted 7NSK2 in combination with CO-720 application by, respectively, 7 to 8%, 19 to 23%, and 20 to 31%, although only the increases in grass dry weight were statistically significant at P <= 0.1. To some extent, 7NSK2 and DOS application also positively affected the mineral content of yellow mustard.
Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium
Authors: Dietrich D , Hickey W J , Lamar R
Published: January 2020

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.
Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases
Authors: Reid J R , Coolbear T , Moore C H , Harding D R , Pritchard G G
Published: January 2020

Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two regions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to PI- and PIII-type lactococcal proteinases. In each series, one oligopeptide had an identical sequence to that of the kappa-casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His<-->Glu. Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of kappa-casein resulted in reduced cleavage of susceptible bonds by the PI-type proteinase and increased cleavage of susceptible bonds by the PIII-type proteinase. In the case of the oligopeptide corresponding to residues 153 to 169 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the PI-type enzyme). Substitution of Glu by His in this oligopeptide, even in the P7 position, resulted in increased cleavage of the bond by the PI-type proteinase and reduced cleavage by the PIII-type proteinase. C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolic engineering of Lactococcus lactis: influence of the overproduction of alpha-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions
Authors: Platteeuw C , Hugenholtz J , Starrenburg M , van Alen-Boerrigter I , de Vos W M
Published: January 2020

The als gene for alpha-acetolactate synthase of Lactococcus lactis MG1363 was cloned on a multicopy plasmid under the control of the inducible L. lactis lacA promoter. More than a hundredfold overproduction of alpha-acetolactate synthase was obtained in L. lactis under inducing conditions as compared with that of the host strain, which contained a single chromosomal copy of the als gene. The effect of alpha-acetolactate synthase overproduction on the formation of end products in various L. lactis strains was studied under different fermentation conditions. Under aerobic conditions and with an initial pH of 6.0, overexpression of the als gene resulted in significant acetoin production that amounted to more than one-third of the pyruvate converted. However, the effect of the alpha-acetolactate synthase overproduction was even more pronounced in the lactate dehydrogenase-deficient strain L. lactis NZ2700. Anaerobic cultivation of this strain resulted in a doubling of the butanediol formation of up to 40% of the converted pyruvate. When cultivated aerobically at an initial pH of 6.8, overexpression of the als gene in L. lactis NZ2700 resulted in the conversion of more than 60% of the pyruvate into acetoin, while no butanediol was formed. Moreover, at an initial pH of 6.0, similar amounts of acetoin were obtained, but in addition approximately 20% of the pyruvate was converted into butanediol. These metabolic engineering studies indicate that more than 80% of the lactose can be converted via the activity of the overproduced alpha-acetolactate synthase in L. lactis.
Distribution of Aeromonas species in the intestinal tracts of river fish
Authors: Sugita H , Tanaka K , Yoshinami M , Deguchi Y
Published: January 2020

Aeromonas isolates were obtained from fish intestines, water, and sediments from an urban river and identified by the DNA-DNA microplate hybridization method. The isolates were Aeromonas veronii (22%), Aeromonas caviae (18%), Aeromonas hydrophila (13%), Aeromonas sobria (8%), Aeromonas jandaei (7%), and other Aeromonas spp. (33%). Aeromonas species occurred at high densities with high incidences, regardless of season. The results strongly suggest that aeromonads are indigenous in fish intestines, water, and sediments of rivers and have the potential to be predominant in aquatic environments.
Rapid and accurate identification of Escherichia coli K-12 strains
Authors: Kuhnert P , Nicolet J , Frey J
Published: January 2020

A specific PCR for the identification of K-12 strains, based on the genetic structure of the O-antigen gene cluster (rfb) of Escherichia coli K-12, is described. The assay clearly differentiates E. coli K-12-derived strains from other E. coli strains used in the laboratory or isolated from human and animal clinical specimens, from food, or from environmental samples. Moreover, lineages of K-12 strains can be distinguished with a second PCR based on the same gene cluster. The method presents a useful tool in identifying K-12 for monitoring strains which are used as biologically safe vehicles in biotechnological research, development, and production processes.
Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases
Authors: Rinas U , Hellmuth K , Kang R , Seeger A , Schlieker H
Published: January 2020

Endogenous and exogenous accumulation of nucleobases was observed when Escherichia coli entered the stationary phase. The onset of the stationary phase was accompanied by excretion of uracil and xanthine. Except for uracil and xanthine, other nucleobases (except for minor amounts of hypoxanthine), nucleosides, and nucleotides (except for cyclic AMP) were not detected in significant amounts in the culture medium. In addition to exogenous accumulation of nucleobases, stationary-phase cells increased the endogenous concentrations of free nucleobases. In contrast to extracellular nucleobases, hypoxanthine was the dominating intracellular nucleobase and xanthine was present only in minor concentrations inside the cells. Excretion of nucleobases was always connected to declining growth rates. It was observed in response to entry into the stationary phase independent of the initial cause of the cessation of cell growth (e.g., starvation for essential nutrients). In addition, transient accumulation of exogenous nucleobases was observed during perturbations of balanced growth conditions such as energy source downshifts. The nucleobases uracil and xanthine are the final breakdown products of pyrimidine (uracil and cytosine) and purine (adenine and guanine) bases, respectively. Hypoxanthine is the primary degradation product of adenine, which is further oxidized to xanthine. The endogenous and exogenous accumulation of these nucleobases in response to entry into the stationary phase is attributed to degradation of rRNA.
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1 genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase
Authors: Walfridsson M , Hallborn J , Penttil� M , Ker�nen S , Hahn-H�gerdal B
Published: January 2020

Saccharomyces cerevisiae was metabolically engineered for xylose utilization. The Pichia stipitis CBS 6054 genes XYL1 and XYL2 encoding xylose reductase and xylitol dehydrogenase were cloned into S. cerevisiae. The gene products catalyze the two initial steps in xylose utilization which S. cerevisiae lacks. In order to increase the flux through the pentose phosphate pathway, the S. cerevisiae TKL1 and TAL1 genes encoding transketolase and transaldolase were overexpressed. A XYL1- and XYL2-containing S. cerevisiae strain overexpressing TAL1 (S104-TAL) showed considerably enhanced growth on xylose compared with a strain containing only XYL1 and XYL2. Overexpression of only TKL1 did not influence growth. The results indicate that the transaldolase level in S. cerevisiae is insufficient for the efficient utilization of pentose phosphate pathway metabolites. Mixtures of xylose and glucose were simultaneously consumed with the recombinant strain S104-TAL. The rate of xylose consumption was higher in the presence of glucose. Xylose was used for growth and xylitol formation, but not for ethanol production. Decreased oxygenation resulted in impaired growth and increased xylitol formation. Fermentation with strain S103-TAL, having a xylose reductase/xylitol dehydrogenase ratio of 0.5:30 compared with 4.2:5.8 for S104-TAL, did not prevent xylitol formation.
Bioconversion of 2,4-diamino-6-nitrotoluene to a novel metabolite under anoxic and aerobic conditions
Authors: Gilcrease P C , Murphy V G
Published: January 2020

Under nitrate-reducing, nongrowth conditions, a Pseudomonas fluorescens species reduced 2,4,6-trinitrotoluene to aminodinitrotoluenes, which were then further reduced to diaminonitrotoluenes. 2,4-Diamino-6-nitrotoluene (2,4-DANT) was further transformed to a novel metabolite, 4-N-acetylamino-2-amino-6-nitrotoluene (4-N-AcANT), while 2,6-diamino-4-nitrotoluene (2,6-DANT) was persistent. Efforts to further degrade 2,4-DANT and 2,6-DANT under aerobic, nitrogen-limited conditions were unsuccessful; 2,6-DANT remained persistent, and 2,4-DANT was again transformed to the 4-N-AcANT compound.
Comparison of biomass dry weights and radial growth rates of fungal colonies on media solidified with different gelling compounds
Authors: Reeslev M , Kjoller A
Published: January 2020

Penicillium commune, Aureobasidium pullulans, and Paecilomyces farinosus were grown on two different media solidified with agar, Pluronic F-127, Carrageenan X-4910, or Carrageenan X-4910 overlaid with cellophane. Growth on Carrageenan X-4910 was generally the same as that on agar, as was the visual appearance of the colonies, e.g., the pigmentation. The Carrageenan X-4910 gels had a melting point, depending on the medium, of 41 to 46(deg)C, and the dry weights of the colonies were readily determined at 60(deg)C. To determine the dry weights of the colonies grown on agar plates, the gels were boiled for 10 min to melt the agar. Comparison of these two procedures showed that the boiling procedure resulted in a 22% reduction of the biomass dry weight. Cellophane membranes did not affect the radial growth rate profoundly. The biomass density was almost halved for P. commune and P. farinosus grown with membranes, whereas the presence of the membrane did not affect the biomass density of A. pullulans. The biomass densities of the colonies grown on Pluronic F-127 were significantly reduced, while in most cases, the radial growth rates of colonies grown on Pluronic F-127 were significantly higher than those obtained on agar or Carrageenan X-4910. Furthermore, the morphology of the leading hyphae was altered, and the hyphal growth unit length was more than twice that obtained on agar and Carrageenan X-4910. Carrageenan X-4910 is a valuable gelling compound for the study of the growth of fungi, as the biomass dry weight is readily determined and growth is similar to that obtained on agar gels.
Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp
Authors: Morais P B , Martins M B , Klaczko L B , Mendon�a-Hagler L C , Hagler A N
Published: January 2020

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.
Differential inhibition by allylsulfide of nitrification and methane oxidation in freshwater sediment
Authors: Roy R , Knowles R
Published: January 2020

Addition of nitrapyrin, allylthiourea, C(inf2)H(inf2), and CH(inf3)F to freshwater sediment slurries inhibited CH(inf4) oxidation and nitrification to similar extents. Dicyandiamide and allylsulfide were less inhibitory for CH(inf4) oxidation than for nitrification. Allylsulfide was the most potent inhibitor of nitrification, and the estimated 50% inhibitory concentrations for this process and CH(inf4) oxidation were 0.2 and 121 (mu)M, respectively. At a concentration of 2 (mu)M allylsulfide, growth and CH(inf4) oxidation activity of Methylosinus trichosporium OB3b were not inhibited. Allylsulfide at 200 (mu)M inhibited the growth of M. trichosporium by approximately 50% but did not inhibit CH(inf4) oxidation activity. Nitrite production by cells of M. trichosporium was not significantly affected by allylsulfide, except at a concentration of 2 mM, when growth and CH(inf4) oxidation were also inhibited by about 50%. Methane monooxygenase activity present in soluble fractions of M. trichosporium was not inhibited significantly by allylsulfide at either 200 (mu)M or 2 mM. These results suggest that the partial inhibition of CH(inf4) oxidation in sediment slurries by high allylsulfide concentrations may be caused by an inhibition of the growth of methanotrophs rather than an inhibition of methane monooxygenase activity specifically. We conclude that allylsulfide is a promising tool for the study of interactions of methanotrophs and nitrifiers in N cycling and CH(inf4) turnover in natural systems.
Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40
Authors: Garvey P , Fitzgerald G F , Hill C
Published: January 2020

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.
Role of Glycine Betaine and Related Osmolytes in Osmotic Stress Adaptation in Yersinia enterocolitica ATCC 9610
Authors: Park S , Smith L T , Smith G M
Published: January 2020

Yersinia enterocolitica is a gram-negative, food-borne pathogen that can grow in 5% NaCl and at refrigerator temperatures. In this report, the compatible solutes (osmolytes) which accumulate intracellularly and confer the observed osmotic tolerance to this pathogen were identified. In minimal medium, glutamate was the only detectable osmolyte that accumulated in osmotically stressed cells. However, when the growth medium was supplemented with glycine betaine, dimethylglycine, or carnitine, the respective osmolyte accumulated intracellularly to high levels and the growth rates of the osmotically stressed cultures improved from 2.4- to 3.5-fold. Chill stress also stimulated the intracellular accumulation of glycine betaine, but the growth rate was only slightly improved by this osmolyte. Both osmotic upshock and temperature downshock stimulated the rate of uptake of [(sup14)C]glycine betaine by more than 30-fold, consistent with other data indicating that the osmolytes are accumulated from the growth medium via transport.
Detection of small round structured viruses in shellfish by reverse transcription-PCR
Authors: Lees D N , Henshilwood K , Green J , Gallimore C I , Brown D W
Published: January 2020

We describe the application of a previously developed sample extraction procedure to the detection of small round structured viruses (SRSVs) in shellfish. Initial seeding experiments showed that PCR inhibitor removal and virus recoveries were comparable to those in previous studies with poliovirus. Shellfish from a range of sewage-contaminated sites were then tested for the presence of SRSVs by using broadly reactive PCR primers followed by Southern blotting with internal probe sites. Positive results were obtained from 5 of 31 field samples tested. Four of these positive samples were from highly polluted sites. PCR product sequence analysis confirmed their identity as SRSV and showed sequence diversity compared with virus controls, suggesting that the results were not a consequence of PCR cross-contamination. Finally, shellfish associated with four separate outbreaks of viral gastroenteritis were tested by PCR and Southern blot for the presence of SRSVs. All outbreak samples tested gave positive results. As far as we are aware, this is the first demonstration of the detection in environmentally contaminated shellfish of the SRSVs responsible for human gastroenteritis. This development may help contribute to the further development of public health controls for molluscan shellfish.
Studies of Streptomyces reticuli cel-1 (cellulase) gene expression in Streptomyces strains, Escherichia coli, and Bacillus subtilis
Authors: Walter S , Schrempf H
Published: January 2020

Various streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Strepotmyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.
Species limits in Rhizobium populations that nodulate the common bean (Phaseolus vulgaris)
Authors: Eardly B D , Wang F S , Whittam T S , Selander R K
Published: January 2020

Evolutionary genetic relationships among 146 bean-nodulating Rhizobium strains, including 94 field isolates from three localities in Colombia and 36 strains from Mexico, were examined by multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis of a PCR-amplified 260-bp segment of the 16S rRNA gene. Seventy-five electrophoretic types (ETs), corresponding to multilocus enzyme genotypes, were identified, including a genotypically diverse group of 18 ETs in Colombia that is strongly differentiated from the ETs of R. etli, which occur in Mexico, Colombia, and Brazil. Most strains of the distinctive Colombian ETs carried the same 16S rRNA allele as did strains of R. etli, but, surprisingly, 17 isolates of two of these ETs had the allele that is characteristic of R. leguminosarum, and strains of two other divergent groups of ETs were also polymorphic for the two alleles. No fully satisfactory explanation for the occurrence of the R. leguminosarum 16S rRNA allele in three distantly related groups of strains is available, but horizontal transfer and recombination of the gene, in whole or in part, would seem to be more plausible than convergence in nucleotide sequence.
Possible regulatory role for nonaromatic carbon sources in styrene degradation by Pseudomonas putida CA-3
Authors: O'Connor K , Buckley C M , Hartmans S , Dobson A D
Published: January 2020

Styrene metabolism in styrene-degrading Pseudomonas putida CA-3 cells has been shown to proceed via styrene oxide, phenylacetaldehyde, and phenylacetic acid. The initial step in styrene degradation by strain CA-3 is oxygen-dependent epoxidation of styrene to styrene oxide, which is subsequently isomerized to phenylacetaldehyde. Phenylacetaldehyde is then oxidized to phenylacetic acid. Styrene, styrene oxide, and phenylacetaldehyde induce the enzymes involved in the degradation of styrene to phenylacetic acid by P. putida CA-3. Phenylacetic acid-induced cells do not oxidize styrene or styrene oxide. Thus, styrene degradation by P. putida CA-3 can be subdivided further into an upper pathway which consists of styrene, styrene oxide, and phenylacetaldehyde and a lower pathway which begins with phenylacetic acid. Studies of the repression of styrene degradation by P. putida CA-3 show that glucose has no effect on the activity of styrene-degrading enzymes. However, both glutamate and citrate repress styrene degradation and phenylacetic acid degradation, showing a common control mechanism on upper pathway and lower pathway intermediates.
Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli
Authors: Rüdiger A , Jorgensen P L , Antranikian G
Published: January 2020

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.
Characterization of the endosymbiont of a deep-sea bivalve, Calyptogena soyoae
Authors: Kim Y W , Yasuda M , Yamagishi A , Oshima T , Ohta S
Published: January 2020

We have purified DNA from gill tissue of a marine bivalve, Calyptogena soyoae, collected from the deep-sea cold seep communities in Sagami Bay, Japan. An rRNA gene was amplified, cloned, and sequenced. In situ hybridization revealed that the sequence is that of a bacterial endosymbiont within the gill of C. soyoae.
Effect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for neonatal BALB/c mice
Authors: Fayer R
Published: January 2020

Oocysts of Cryptosporidium parvum suspended in 5.25, 2.63, or 1.31% aqueous sodium hypochlorite (Clorox laundry bleach) for 10, 30, 60, or 120 min at 21 degrees C were administered by gastric intubation to neonatal BALB/c mice. Microscopic examination of intestinal tissue sections revealed developmental stages of C. parvum in all of the mice.
Regeneration of phosphorus and nitrogen by four species of heterotrophic nanoflagellates feeding on three nutritional States of a single bacterial strain
Authors: Eccleston-Parry J D , Leadbeater B
Published: January 2020

Three physiological states of a single bacterial strain, namely, balanced, phosphorus-rich, and nitrogen-rich bacteria, were obtained by culturing a bacterial strain in chemostats under three different nutrient regimens. Each was shown to be distinctly different in elemental composition with respect to C/N/P ratio. These bacteria were fed to four species of heterotrophic nanoflagellates in batch culture grazing experiments, and the percent regeneration efficiencies of bacterium-bound nitrogen and phosphorus by the flagellates were compared. All flagellate species regenerated comparable amounts of nitrogen, which was thought to be due to their similar internal C/N ratios. There was, however, interspecies variation with regard to phosphorus regeneration: the two faster-growing species (Paraphysomonas imperforata and Bodo designis) released significantly more phosphorus than the two slower-growing species (Stephanoeca diplocostata and Jakoba libera). The observed differences were thought to have been influenced by a combination of life cycle strategies and internal C/P ratios.