bluuebunny/crossref_metadata_2025 huggingface dataset:
Destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane
Authors: Mendonca A F , Amoroso T L , Knabel S J
Published: January 2020

High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs within 15 s, whereas L. monocytogenes decreased by only 1 log in 10 min. There was a very strong correlation between the initial rate of release of material with A260 and death rate of the gram-negative pathogens (r = 0.997). At pH 12, gram-negative test cells appeared collapsed and showed evidence of lysis while gram-positive L. monocytogenes did not, when observed by scanning and transmission electron microscopy. It was concluded that destruction of gram-negative food-borne pathogens by high pH involves disruption of the cytoplasmic membrane.
Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium
Authors: Mayfield M B , Kishi K , Alic M , Gold M H
Published: January 2020

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)
Isolation, characterization, and analysis of the expression of the cbhII gene of Phanerochaete chrysosporium
Authors: Tempelaars C A , Birch P R , Sims P F , Broda P
Published: January 2020

Two cDNA sequences representing putative allelic variants of the Phanerochaete chrysosporium cbhII gene were isolated by hybridization to the Trichoderma reesei cbhII gene. Both of the equivalent genomic sequences were subsequently isolated by the inverse PCR technique. DNA sequencing showed that the cbhII open reading frame of 1,380 bp codes for a putative polypeptide of 460 amino acids which is interrupted by six introns. The domain structure found in T. reesei cbhII is conserved in the equivalent P. chrysosporium protein. The overall similarity between the two gene products is 54%, with the region of highest conservation being found in the cellulose-binding domain (65%). Unlike the cbhI gene of P. chrysosporium, cbhII does not appear to be a member of a class of closely related genes. CBHII is a new member of family B of the beta-1, 4-glucanases. Alignment of the P. chrysosporium and T. reesei CBHII protein sequences showed that all of the residues important for the formation of the extended loops of the catalytic domain and those residues that are involved in the catalytic action of the T. reesei enzyme are also present in the P. chrysosporium equivalent. The profiles of cbh gene expression in P. chrysosporium reveal that while cbhI.1 and cbhI.2 could be coregulated, cbhII can be independently controlled. The latter is so far the only cellulase gene found to be expressed when the fungus is grown on oat spelt arabinoxylan, suggesting that it may play an active role in the xylanolytic as well as the cellulolytic systems.
Anaerobic oxidation of ferrous iron by purple bacteria, a new type of phototrophic metabolism
Authors: Ehrenreich A , Widdel F
Published: January 2020

Anoxic iron-rich sediment samples that had been stored in the light showed development of brown, rusty patches. Subcultures in defined mineral media with ferrous iron (10 mmol/liter, mostly precipitated as FeCO3) yielded enrichments of anoxygenic phototrophic bacteria which used ferrous iron as the sole electron donor for photosynthesis. Two different types of purple bacteria, represented by strains L7 and SW2, were isolated which oxidized colorless ferrous iron under anoxic conditions in the light to brown ferric iron. Strain L7 had rod-shaped, nonmotile cells (1.3 by 2 to 3 microns) which frequently formed gas vesicles. In addition to ferrous iron, strain L7 used H2 + CO2, acetate, pyruvate, and glucose as substrate for phototrophic growth. Strain SW2 had small rod-shaped, nonmotile cells (0.5 by 1 to 1.5 microns). Besides ferrous iron, strain SW2 utilized H2 + CO2, monocarboxylic acids, glucose, and fructose. Neither strain utilized free sulfide; however, both strains grew on black ferrous sulfide (FeS) which was converted to ferric iron and sulfate. Strains L7 and SW2 grown photoheterotrophically without ferrous iron were purple to brownish red and yellowish brown, respectively; absorption spectra revealed peaks characteristic of bacteriochlorophyll a. The closest phototrophic relatives of strains L7 and SW2 so far examined on the basis of 16S rRNA sequences were species of the genera Chromatium (gamma subclass of proteobacteria) and Rhodobacter (alpha subclass), respectively. In mineral medium, the new isolates formed 7.6 g of cell dry mass per mol of Fe(II) oxidized, which is in good agreement with a photoautotrophic utilization of ferrous iron as electron donor for CO2 fixation. Dependence of ferrous iron oxidation on light and CO2 was also demonstrated in dense cell suspensions. In media containing both ferrous iron and an organic substrate (e.g., acetate, glucose), strain L7 utilized ferrous iron and the organic compound simultaneously; in contrast, strain SW2 started to oxidize ferrous iron only after consumption of the organic electron donor. Ferrous iron oxidation by anoxygenic phototrophs is understandable in terms of energetics. In contrast to the Fe3+/Fe2+ pair (E0 = +0.77 V) existing in acidic solutions, the relevant redox pair at pH 7 in bicarbonate-containing environments, Fe(OH)3 + HCO3-/FeCO3, has an E0' of +0.2 V. Ferrous iron at pH 7 can therefore donate electrons to the photosystem of anoxygenic phototrophs, which in purple bacteria has a midpoint potential around +0.45 V. The existence of ferrous iron-oxidizing anoxygenic phototrophs may offer an explanation for the deposition of early banded-iron formations in an assumed anoxic biosphere in Archean times.
Degradation of 1,4-dioxane by an actinomycete in pure culture
Authors: Parales R E , Adamus J E , White N , May H D
Published: January 2020

An actinomycete capable of sustained aerobic growth on 1,4-dioxane was isolated from a dioxane-contaminated sludge samples. The actinomycete, CB1190, grows on 1,4-dioxane as the sole carbon and energy source with a generation time of approximately 30 h. CB1190 degrades 1,4-dioxane at a rate of 0.33 mg of dioxane min-1 mg of protein-1 and mineralizes 59.5% of the dioxane to CO2. CB1190 also grows with other cyclic and linear ethers as the sole carbon and energy sources, including 1,3-dioxane, 2-methyl-1,3-dioxolane, tetrahydrofuran, tetrahydropyran, diethyl ether, and butyl methyl ether. CB1190 is capable of aerobic autotrophic growth on H2 and CO2.
Demethylation of dimethylsulfoniopropionate to 3-mercaptopropionate by an aerobic marine bacterium
Authors: Visscher P T , Taylor B F
Published: January 2020

A bacterium, strain BIS-6, that grew aerobically on dimethylsulfoniopropionate (DMSP) was isolated from an intertidal mud sample. Strain BIS-6 quantitatively demethylated DMSP and 3-methiolpropionate to 3-mercaptopropionate. Strain BIS-6 was a versatile methylotroph growing on the osmolytes DMSP and glycine betaine and their methylated degradation products (dimethyl glycine, sarcosine, methylamines, and dimethyl sulfide.
Serological and Ecological Characteristics of a Nodule-Dominant Serotype from an Indigenous Soil Population of
Rhizobium leguminosarum
bv. trifolii
Authors: Leung Kamtin , Yap Kathryn , Dashti Narjes , Bottomley Peter J.
Published: January 2020

Although at least 13 antigenically distinct serotypes of
Rhizobium leguminosarum
bv. trifolii exist in an Abiqua silty clay loam soil, one serotype, AS6, occupies ≥50% of the root nodules formed on field-grown subclover and between 33 and 78% of the nodules formed on five annual clover species grown in the same soil under laboratory conditions. The dominance of subclover nodules by serotype AS6 was reproducible over a 4-year sampling period and throughout the entire 200- by 100-m pasture examined. Serotype AS6 was composed of three antigenically distinct subtypes (AS6-a, AS6-b, and AS6-c). Each subtype contributed about one-third of the AS6 isolates recovered from nodules of field-grown subclover plants and maintained similar population densities in nonrhizosphere and rhizosphere soil. Rhizobia with the AS6 antigenic signature accounted for from 20 to 100% of the soil populations of
R. leguminosarum
in arable and pasture soils under legumes throughout the state of Oregon. Over a 12-month period, the population densities of the serotype AS6 complex and three minor nodule-occupying serotypes (AG4, AP17, and AS21) were measured in the rhizospheres of field-grown subclover and orchard grass and in nonrhizosphere Abiqua soil. Regardless of season or serotype, the orchard grass rhizosphere effect was minimal, with the ratio between rhizosphere and nonrhizosphere serotype population densities ranging between 2.5 (midsummer) and 10.5 (spring). In contrast, the magnitude of the subclover rhizosphere effect varied seasonally and among serotypes. Between October and December the ratios for all serotypes were similar (12.5 to 25.5). However, in the spring (April and May), the magnitude of the rhizosphere effect varied among the indigenous serotypes (ratios, 10.5 to 442) and for minor nodule-occupying serotypes AS21 (ratio, 442) and AP17 (ratio, 47) was as great as, or even greater than, the magnitude of the rhizosphere effect observed with the AS6 complex (ratio, 65.5).
Mineral Precipitation by Epilithic Biofilms in the Speed River, Ontario, Canada
Authors: Konhauser K. O. , Schultze-Lam S. , Ferris F. G. , Fyfe W. S. , Longstaffe F. J. , Beveridge T. J.
Published: January 2020

Epilithic microbial communities, ubiquitously found in biofilms on submerged granite, limestone, and sandstone, as well as on the concrete support pillars of bridges, were examined in the Speed River, Ontario, Canada. Transmission electron microscopy showed that attached bacteria (on all substrata) were highly mineralized, ranging from Fe-rich capsular material to fine-grained (<1 μm) authigenic (primary) mineral precipitates. The authigenic grains exhibited a wide range of morphologies, from amorphous gel-like phases to crystalline structures. Energy-dispersive X-ray spectroscopy indicated that the most abundant mineral associated with epilithic bacteria was a complex (Fe, Al) silicate of variable composition. The gel-like phases were similar in composition to a chamositic clay, whereas the crystalline structures were more siliceous and had compositions between those of glauconite and kaolinite. The consistent formation of (Fe, Al) silicates by all bacterial populations, regardless of substratum lithology, implies that biomineralization was a surface process associated with the anionic nature of the cell wall. The adsorption of dissolved constituents from the aqueous environment contributed significantly to the mineral formation process. In this regard, it appears that epilithic microbial biofilms dominate the reactivity of the rock-water interface and may determine the type of minerals formed, which will ultimately become part of the riverbed sediment. Because rivers typically contain high concentrations of dissolved iron, silicon, and aluminum, these findings provide a unique insight into biogeochemical activities that are potentially widespread in natural waters.
Bacterial succession in necrotic tissue of agria cactus (Stenocereus gummosus)
Authors: Foster J L , Fogleman J C
Published: January 2020

The bacterial communities associated with the development of necroses in injured agria cactus tissue were examined in the field by using both human-induced injuries and cactus tissue inoculated with cactophilic bacteria. Whole-cell bacterial fatty acids were used to determine when and where each of 23 detected species occurred. This information was then used to describe successional patterns of bacterial colonization. Although the number of bacterial species in human-induced injuries reached a maximum on day 16, the Shannon-Weaver diversity index increased to a plateau, which reflects a stable bacterial community. The potential of the bacterial community to macerate the injured cactus tissue was also examined, and the results indicate that the bacteria initially colonizing the newly injured cactus tissue were more likely to contain pectinolytic, proteolytic, and lipolytic enzymes than were the bacteria entering the injuries once tissue maceration had already begun. The cactophilic fruit fly Drosophila mojavensis has been previously shown to carry bacteria to newly injured cactus tissue. In these studies, exclusion of these insects did not significantly affect bacterial succession or community structure. This supports our contention that bacteria colonize injured tissue primarily by passive transport, e.g., on wind-blown particles.
Staphylococcin 1580 is identical to the lantibiotic epidermin: implications for the nature of bacteriocins from gram-positive bacteria
Authors: Sahl H G
Published: January 2020

Staphylococcin 1580 was purified to homogeneity from culture supernatants of Staphylococcus epidermidis 1580 by means of adsorption to XAD 2, cation exchange chromatography, and high-performance liquid chromatography on reversed-phase C18. The purified active substance migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent M(r) of approximately 2,000. Amino acid analysis, mass determination (2,165 Da) and N-terminal sequencing (Ile-Ala-Xaa-Lys-Phe-Ile-Xaa-Xaa-Pro-Gly-Xaa-Ala-Lys-block) demonstrated that staphylococcin 1580 is identical to epidermin, a lanthionine-containing antibiotic peptide (lantibiotic).
Lignin-Degrading Enzymes of the Commercial Button Mushroom,
Agaricus bisporus
Authors: Bonnen Alice M. , Anton Lori H. , Orth Ann B.
Published: January 2020

Agaricus bisporus
, grown under standard composting conditions, was evaluated for its ability to produce lignin-degrading peroxidases, which have been shown to have an integral role in lignin degradation by wood-rotting fungi. The activity of manganese peroxidase was monitored throughout the production cycle of the fungus, from the time of colonization of the compost through the development of fruit bodies. Characterization of the enzyme was done with a crude compost extract. Manganese peroxidase was found to have a pI of 3.5 and a pH optimum of 5.4 to 5.5, with maximal activity during the initial stages of fruiting (pin stage). The activity declined considerably with fruit body maturation (first break). This apparent developmentally regulated pattern parallels that observed for laccase activity and for degradation of radiolabeled lignin and synthetic lignins by
A. bisporus
. Lignin peroxidase activity was not detected in the compost extracts. The correlation between the activities of manganese peroxidase and laccase and the degradation of lignin in
A. bisporus
suggests significant roles for these two enzymes in lignin degradation by this fungus.
Seasonal and spatial population dynamics of the nitrogen-efficient guild in a desert bajada grassland
Authors: Herman R P , Provencio K , Torrez R , Seager G M
Published: January 2020

This study examined the temporal and spatial variation in the populations of bacteria from a Chihuahuan desert bajada grassland that could grow on nearly nitrogen-free medium, a nitrogen-efficient guild (NEG). A Curtis similarity index of 0.876 and nearly identical diversity and equability indexes (H' = 1.22, J = 0.46 and H' = 1.27, J = 0.48 for the crown edge and interplant samples, respectively) indicated that there was no qualitative difference between the NEG assemblages isolated from samples taken at Bouteloua eriopoda plant crowns and in nonvegetated areas 45 cm from crowns. The difference in NEG populations between the crown and interplant samples was only 2-fold, despite a 9.5-fold difference in root biomass between the sites. These differences, while consistent, were statistically significant at only 50% of the sampling times. There was over an order of magnitude difference in NEG numbers in root-associated soil and in bulk soil from the crown or intercrown sites. The typical trend for temporal variation in NEG numbers was that they increased in the spring, fluctuated dramatically over the summer, and declined at the summer's end. The pattern of soil moisture change was the only abiotic variable which showed the same fluctuation pattern as NEG numbers.
Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry
Authors: Breeuwer P , Drocourt J L , Rombouts F M , Abee T
Published: January 2020

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.
Identification of capsule-forming Bacillus anthracis spores with the PCR and a novel dual-probe hybridization format
Authors: Reif T C , Johns M , Pillai S D , Carl M
Published: January 2020

Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis. The virulent strains of B. anthracis are encapsulated and toxigenic. In this paper we describe the development of a PCR technique for identifying spores of B. anthracis. Two 20-mer oligonucleotide primers specific for the capB region of 60-MDa plasmid pXO2 were used for amplification. The amplification products were detected by using biotin- and fluorescein-labeled probes in a novel dual-probe hybridization format. Using the combination of PCR amplification and dual-probe hybridization, we detected two copies of the bacterial genome. Because the PCR assay could detect a minimum of 100 unprocessed spores per PCR mixture, we attempted to facilitate the release of DNA by comparing the effect of limited spore germination with the effect of mechanical spore disruption prior to PCR amplification. The two methods were equally effective and allowed us to identify single spores of B. anthracis in PCR mixtures.
Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar
Authors: Beecher D J , Wong A C
Published: January 2020

Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections. Evidence is accumulating that hemolysin BL is a major B. cereus virulence factor. We describe two methods for detection of hemolysin BL in crude samples and on primary culture media. In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains. In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood. Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B. cereus isolates and in 46 of 136 (34%) presumptive B. cereus isolates from soil. All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL. The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.
An Outbreak of
Aeromonas hydrophila
Infection in Turtles (
Pseudemis scripta
)
Authors: Pasquale Vincenzo , Baloda Suraj B. , Dumontet Stefano , Krovacek Karel
Published: January 2020

An outbreak of
Aeromonas hydrophila
infection with a high rate of mortality (95%) in turtles (
Pseudemis scripta
) in Italy is reported. Pure cultures of the pathogen were isolated from liver, lung, kidney, and heart specimens of the turtles. The
A. hydrophila
isolate was resistant to amoxicillin, ampicillin, cephalothin, and trimethoprim-sulfamethoxazole but was sensitive to a number of other antibiotics tested. The study indicates that pet turtles can act as reservoirs of this pathogen and may play an important role in the etiology of
Aeromonas
-associated human infections.
Role of mycelium and extracellular protein in the biodegradation of 2,4,6-trichlorophenol by Phanerochaete chrysosporium
Authors: Armenante P M , Pal N , Lewandowski G
Published: January 2020

The biodegradation of 2,4,6-trichlorophenol (2,4,6-TCP) by Phanerochaete chrysosporium was studied in batch systems. In experiments with mycelial suspension, the degradation of 2,4,6-TCP was found to occur in the absence of ligninase. Chloride ion was recovered in nearly stoichiometric amounts at the end of the process. The microorganism did not retain its degradation ability for more than 6 days under substrate-deficient conditions. Neither the mycelium nor the extracellular protein alone could degrade 2,4,6-TCP; both were required for complete degradation to occur. In experiments in which 2,4,6-TCP was exposed to the culture supernatant separated from its mycelium, negligible degradation was obtained and no chloride ion was recovered. No degradation was observed even when the supernatant was supplemented with hydrogen peroxide as a possible cosubstrate. In experiments performed with washed mycelium separated from its supernatant, no degradation took place until the mycelium released additional extracellular protein 5 to 6 h into the incubation. Additions of washed mycelium separated from its supernatant to active cultures also produced an increase in the rate of degradation in correspondence with the protein release. The protein release was independent of the presence of 2,4,6-TCP. The addition of cycloheximide to inhibit the synthesis of de novo proteins completely suppressed the release of protein by the mycelium and resulted in no 2,4,6-TCP degradation. Additions of culture supernatants containing a high concentration of extracellular protein to active cultures produced an increase in the rate of 2,4,6-TCP degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
13
C Nuclear Magnetic Resonance Studies of Citrate and Glucose Cometabolism by
Lactococcus lactis
Authors: Ramos Ana , Jordan Kieran N. , Cogan Timothy M. , Santos Helena
Published: January 2020

13
C nuclear magnetic resonance (
13
C-NMR) was used to investigate the metabolism of citrate plus glucose and pyruvate plus glucose by nongrowing cells of
Lactococcus lactis
subsp.
lactis
19B under anaerobic conditions. The metabolism of citrate plus glucose during growth was also monitored directly by in vivo NMR. Although pyruvate is a common intermediate metabolite in the metabolic pathways of both citrate and glucose, the origin of the carbon atoms in the fermentation products was determined by using selectively labeled substrates, e.g., [2,4-
13
C]citrate, [3-
13
C]pyruvate, and [2-
13
C]glucose. The presence of an additional substrate caused a considerable stimulation in the rates of substrate utilization, and the pattern of end products was changed. Acetate plus acetoin and butanediol represented more than 80% (molar basis) of the end products of the metabolism of citrate (or pyruvate) alone, but when glucose was also added, 80% of the citrate (or pyruvate) was converted to lactate. This result can be explained by the activation of lactate dehydrogenase by fructose 1,6-bisphosphate, an intermediate in glucose metabolism. The effect of different concentrations of glucose on the metabolism of citrate by dilute cell suspensions was also probed by using analytical methods other than NMR. Pyruvate dehydrogenase (but not pyruvate formate-lyase) was active in the conversion of pyruvate to acetyl coenzyme A. α-Acetolactate was detected as an intermediate metabolite of citrate or pyruvate metabolism, and the labeling pattern of the end products agrees with the α-acetolactate pathway. It was demonstrated that the contribution of the acetyl coenzyme A pathway for the synthesis of diacetyl, should it exist, is lower than 10%. Evidence for the presence of internal carbon reserves in
L. lactis
is presented.
Improved Method for Recovery of mRNA from Aquatic Samples and Its Application to Detection of
mer
Expression
Authors: Jeffrey Wade H. , Nazaret Sylvie , Von Haven Robin
Published: January 2020

Previously described methods for extraction of mRNA from environmental samples may preclude detecting transcripts from genes that were present in low abundance in aquatic bacterial communities. By combining a boiling sodium dodecyl sulfate-diethylpyrocarbonate lysis step with acid-guanidinium extraction, we improved recovery of target mRNA from both pure cultures and environmental samples. The most significant advantage of the new protocol is that it is easily adapted to yield high recovery of mRNA from 142-mm-diameter flat filters and high-capacity cartridge filters. The lysis and extraction procedures are more rapid than previously described methods, and many samples can be handled at once. RNA extracts have been shown to be free of contaminating DNA. The lysis procedure does not damage target mRNA sequences, and mRNA can be detected from fewer than 10
6
bacterial cells. We used the new method to examine transcripts of genes responsible for detoxification of mercurial compounds. Induction of
merA
(specifying mercuric reductase) transcripts in stationary-phase
Pseudomonas aeruginosa
containing Tn
501
occurred within 60 s of HgCl
2
addition and was proportional to the amount of Hg(II) added. The new technique also allowed the detection of
merA
transcripts from the microbial community of a mercury-contaminated pond (Reality Lake, Oak Ridge, Tenn.). Significant differences in
merA
transcript abundance were observed between different locations associated with the lake. The results indicate that the new method is simple and rapid and can be applied to the study of
mer
gene expression of aquatic communities in their natural habitats.
Displacement of Enterococcus faecalis from hydrophobic and hydrophilic substrata by Lactobacillus and Streptococcus spp. as studied in a parallel plate flow chamber
Authors: Millsap K , Reid G , van der Mei H C , Busscher H J
Published: January 2020

The displacement of Enterococcus faecalis 1131 from hydrophobic and hydrophilic substrata by isolates of Lactobacillus casei 36 and Streptococcus hyointestinalis KM1 was studied in a parallel plate flow chamber. The experiments were conducted with either 10 mM potassium phosphate buffer or human urine as the suspending fluid, and adhesion and displacement were measured by real-time in situ image analysis. The results showed that E. faecalis 1131 was displaced by lactobacilli (31%) and streptococci (74%) from fluorinated ethylene propylene in buffer and that displacement by lactobacilli was even more effective on a glass substratum in urine (54%). The passage of an air-liquid interface significantly impacted on adhesion, especially when the surface had been challenged with lactobacilli (up to 100% displacement) or streptococci (up to 94% displacement). These results showed that the parallel plate flow system with real-time in situ image analysis was effective for studying bacterial adhesion and that uropathogenic enterococci can be displaced by indigenous bacteria.
Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses
Authors: Chen K H , Credi R , Loi N , Maixner M , Chen T A
Published: January 2020

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)
Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1
Authors: Tatum F M , Briggs R E , Halling S M
Published: January 2020

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.
Inhibitory Effects of Turf Pesticides on
Bacillus popilliae
and the Prevalence of Milky Disease
Authors: Dingman Douglas W.
Published: January 2020

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide
Bacillus popilliae,
the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-
o
-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-
o
tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of
B. popilliae,
and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.
Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar
Authors: Brosch R , Chen J , Luchansky J B
Published: January 2020

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.
Isolation of a Bacterial Culture That Degrades Methyl
t
-Butyl Ether
Authors: Salanitro J. P. , Diaz L. A. , Williams M. P. , Wisniewski H. L.
Published: January 2020

We have isolated a mixed bacterial culture (BC-1) which is capable of degrading the gasoline oxygenate methyl
t
-butyl ether (MTBE). BC-1 was developed from seed microorganisms present in a chemical plant biotreater sludge. This enrichment culture has been maintained in continuous culture treating high concentrations of MTBE (120 to 200 mg/liter) as the sole carbon source in a simple feed containing NH
4
+
, PO
4
3-
, Mg
2+
, and Ca
2+
nutrients. The unit had a stable MTBE removal rate when maintained with a long cell retention time (ca. 80 to 90 days); however, when operated at a ≤50-day cell waste rate, loss of MTBE-degrading activity was observed. The following three noteworthy experimental data show that MTBE is biodegraded extensively by BC-1: (i) the continuous (oxygen-sparged) culture was able to sustain a population of autotrophic ammonia-oxidizing bacteria which could nitrify influent NH
4
+
concentrations at high rates and obtain CO
2
(sole carbon source for growth) from the metabolism of the alkyl ether, (ii) BC-1 metabolized radiolabeled either (
14
CH
3
O-MTBE) to
14
CO
2
(40%) and
14
C-labeled cells (40%), and (iii) cell suspensions of the culture were capable of degrading (substrate depletion experiments) MTBE to
t
-butyl alcohol, a primary metabolite of MTBE. BC-1 is a mixed culture containing several bacterial species and is the first culture of its kind which can completely degrade an alkyl ether.
Roles of bacterial attachment and spontaneous partitioning in the biodegradation of naphthalene initially present in nonaqueous-phase liquids
Authors: Ortega-Calvo J J , Alexander M
Published: January 2020

The mineralization by an Arthrobacter sp. of naphthalene initially dissolved in di(2-ethylhexyl)phthalate exhibited a slow phase followed by a rapid phase. Triton X-100, which inhibited cell attachment, prevented the onset of the second phase. Triton X-100 increased the extent of mineralization of naphthalene initially present in 2,2,4,4,6,8,8-heptamethylnonane. Cells attached to the interface mineralized the aromatic hydrocarbon at a rate four times higher than the rate of partitioning in the absence of microorganisms, but this microbial activity was markedly reduced by Triton X-100. We suggest that utilization of naphthalene originally present in nonaqueous-phase liquids may involve a partitioning-limited initial stage carried out by bacteria freely suspended in the aqueous phase and a subsequent, more rapid stage effected by bacteria present directly at the nonaqueous-liquid-water interface.
Design and performance of a trickling air biofilter for chlorobenzene and o-dichlorobenzene vapors
Authors: Oh Y S , Bartha R
Published: January 2020

From contaminated industrial sludge, two stable multistrain microbial enrichments (consortia) that were capable of rapidly utilizing chlorobenzene and o-dichlorobenzene, respectively, were obtained. These consortia were characterized as to their species composition, tolerance range, and activity maxima in order to establish and maintain the required operational parameters during their use in biofilters for the removal of chlorobenzene contaminants from air. The consortia were immobilized on a porous perlite support packed into filter columns. Metered airstreams containing the contaminant vapors were partially humidified and passed through these columns. The vapor concentrations prior to and after biofiltration were measured by gas chromatography. Liquid was circulated concurrently with the air, and the device was operated in the trickling air biofilter mode. The experimental arrangement allowed the independent variation of liquid flow, airflow, and solvent vapor concentrations. Bench-scale trickling air biofilters removed monochlorobenzene, o-dichlorobenzene, and their mixtures at rates of up to 300 g of solvent vapor h(-1) m(-3) filter volume. High liquid recirculation rates and automated pH control were critical for stable filtration performance. When the accumulating NaCl was periodically diluted, the trickling air biofilters continued to remove chlorobenzenes for several months with no loss of activity. The demonstrated high performance and stability of the described trickling air biofilters favor their use in industrial-scale air pollution control.
Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene
Authors: Duetz W A , de Jong C , Williams P A , van Andel J G
Published: January 2020

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.
Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish
Authors: Magariños B , Romalde J L , Lemos M L , Barja J L , Toranzo A E
Published: January 2020

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes
Authors: Burggraf S , Mayer T , Amann R , Schadhauser S , Woese C R , Stetter K O
Published: January 2020

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.
Indigenous Arbuscular Mycorrhizal Fungi Associated with
Acacia albida
Del. in Different Areas of Senegal
Authors: Diop Tahir A. , Gueye Mamadou , Dreyfus Bernard L. , Plenchette Christian , Strullu Desire G.
Published: January 2020

The influences of seasons, plant age, and physicochemical properties of the soil on surface and deep biological arbuscular mycorrhizal fungus parameters associated with
Acacia albida
were assessed in different areas of Senegal. More indigenous arbuscular mycorrhizal propagules were found in the localities of the Sudano-Guinean zone (Djinaki and Kabrousse) than in those of the Sahelian zone (Louga and Diokoul), and species belonging to the genera
Glomus, Gigaspora, Acaulospora
, and
Sclerocystis
prevailed. The numbers of total and viable spores increased more during the rainy season than during the dry season (about 108% more total spores and 262% more viable spores). Similarly, both total and viable spores were more prevalent around young
Acacia
trees than old trees. However, the intensities of root colonization did not differ in each ecoclimatic zone.
Nutritional requirements for growth of Helicobacter pylori
Authors: Nedenskov P
Published: January 2020

A chemically defined medium consisting of a buffered mineral base supplemented with amino acids, a purine, and thiamine supported growth of 23 clinical isolates and the type strain of Helicobacter pylori. The growth of four strains was inhibited by the presence of certain amino acids. All but one strain required alanine for growth. The amino acids leucine, valine, phenylalanine, methionine, arginine, and histidine were generally required. Isoleucine either was required or stimulated growth. Strains could be differentiated into groups on the basis of a requirement for one or more of the amino acids cysteine, serine, and proline. Only one strain however, demonstrated a requirement for all three of these amino acids.