bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and field application of a quantitative method for examining natural assemblages of protists with oligonucleotide probes
Authors: Lim E L , Caron D A , Delong E F
Published: January 2020

A fluorescent in situ hybridization method that uses rRNA-targeted oligonucleotide probes for counting protists in cultures and environmental water samples is described. Filtration, hybridization, and enumeration of fixed cells with biotinylated eukaryote-specific probes and fluorescein isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, Mass.). Counts of various species of cultured protists by this probe hybridization method were not significantly different from counts obtained by the 4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. However, counts of total nanoplankton (TNAN) based on probe hybridizations in several field samples and in samples collected from a mesocosm experiment were frequently higher than TNAN counts obtained by staining with DAPI or AO. On the basis of these results, 25 to 70% of the TNAN determined with probes were not detectable by DAPI or AO staining. The underestimation of TNAN abundances in samples stained with DAPI or AO was attributed to the existence of small nanoplanktonic cells which could be detected with probes but not DAPI or AO and the difficulty associated with distinguishing DAPI- or AO-stained protists attached to or embedded in aggregates. We conclude from samples examined in this study that enumeration of TNAN with oligonucleotide probes provides estimates of natural TNAN abundances that are at least as high as (and in some cases higher than) counts obtained with commonly employed fluorochrome stains. The quantitative in situ hybridization method we have described here enables the direct enumeration of free-living protists in water samples with oligonucleotide probes. When combined with species-specific probes, this method will enable quantitative studies of the abundance and distribution of specific protistan taxa.
Sporulation-promoting ability of Clostridium perfringens culture fluids
Authors: Shih N J , Labbé R G
Published: January 2020

The culture supernatant fluids (CSFs) of 12 strains of Clostridium perfringens types A, B, C, and D stimulated sporulation of test strains NCTC 8238 and NCTC 8449 of this organism. The sporulation-promoting ability was present in vegetative and sporulating CSFs of both enterotoxin-positive (Ent+) and Ent- strains. The sporulation factor possessed a molecular weight between 1,000 and 5,000 and was heat and acid stable. This study suggests a potential role for Ent- strains in food-borne disease outbreaks caused by Ent+ strains of C. perfringens type A.
Cometabolic transformation and cleavage of nitrodiphenylamines by three newly isolated sulfate-reducing bacterial strains
Authors: Drzyzga O , Schmidt A , Blotevogel K
Published: January 2020

Three sulfate-reducing bacterial strains (Desulfovibrio sp. strain SHV, Desulfococcus sp. strain WHC, and Desulfomicrobium sp. strain WHB) with the capacity to cometabolize 2-nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were newly isolated. Before breaking down the diphenylamine structure, these strains cometabolically reduce the nitrodiphenylamines to the corresponding aminodiphenylamines during anaerobic oxidation of the growth substrate lactate (Desulfovibrio strain SHV and Desulfomicrobium strain WHC) or benzoate (Desulfococcus strain WHB), leading to the formation of aniline and a smaller quantity of methylaniline. These compounds were not further metabolized by the sulfate reducers. The anaerobic metabolism of aminodiphenylamines also led to the formation of heterocyclic condensation products such as phenazine and acridine derivatives, provided that they contained an amino group in the ortho position of the diphenylamine (e.g., 2-aminodiphenylamine or 2,4-diaminodiphenylamine). In addition, low levels of indole and benzothiazole derivatives were identified, but these also were not further metabolized by the three sulfate-reducing strains.
Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal
Authors: Hill R T , Straube W L , Palmisano A C , Gibson S L , Colwell R R
Published: January 2020

Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site. C. perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca. 48 km) and 50 nautical miles (ca. 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site. C. perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site. At stations where C. perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm. In some cases, C. perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation. We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site.
A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate
Authors: Coventry M J , Gordon J B , Alexander M , Hickey M W , Wan J
Published: January 2020

Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification.
Stable Carbon Isotope Evidence for Atmospheric CO(inf2) Uptake by Cyanobacterial Surface Scums in a Eutrophic Lake
Authors: Gu B , Alexander V
Published: January 2020

The carbon isotope composition of photosynthetic organisms reflects the (delta)(sup13)C of their inorganic carbon sources. The large difference between (delta)(sup13)C in cyanobacterial surface scums (-25 to -18(permil)) and subsurface blooms (-34 to -30(permil)) in a subarctic lake indicates direct atmospheric CO(inf2) fixation by the scums.
Detection of infectious astroviruses in water
Authors: Pinto R M , Abad F X , Gajardo R , Bosch A
Published: January 2020

A method based on the infection of CaCo-2 cells and molecular hybridization with a specific cDNA probe has been developed for the detection of infectious astroviruses in environmental samples. By this procedure wild-type astroviruses have been detected in water from an area where a concurrent gastroenteritis outbreak was reported.
Microbial degradation of hydrochlorofluorocarbons (CHCl2F and CHCl2CF3) in soils and sediments
Authors: Oremland R S , Lonergan D J , Culbertson C W , Lovley D R
Published: January 2020

The ability of microorganisms to degrade trace levels of the hydrochlorofluorocarbons HCFC-21 and HCFC-123 was investigated. Methanotroph-linked oxidation of HCFC-21 was observed in aerobic soils, and anaerobic degradation of HCFC-21 occurred in freshwater and salt marsh sediments. Microbial degradation of HCFC-123 was observed in anoxic freshwater and salt marsh sediments, and the recovery of 1,1,1-trifluoro-2-chloroethane indicated the involvement of reductive dechlorination. No degradation of HCFC-123 was observed in aerobic soils. In some experiments, HCFCs were degraded at low (parts per billion) concentrations, raising the possibility that bacteria in nature remove HCFCs from the atmosphere.
Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains
Authors: Gouka R J , Punt P J , Hessing J G , van den Hondel C A
Published: January 2020

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.
Formation of Fatty Acid-degrading, anaerobic granules by defined species
Authors: Wu W , Jain M K , Zeikus J G
Published: January 2020

An endospore-forming, butyrate-degrading bacterium (strain BH) was grown on butyrate in monoxenic coculture with a methanogen. The culture formed dense aggregates when Methanobacterium formicicum was the methanogenic partner, but the culture was turbid when Methanospirillum hungatei was the partner. In contrast, a propionate-degrading, lemon-shaped bacterium (strain PT) did not form aggregates with Methanobacterium formicicum unless an acetate-degrading Methanosaeta sp. was also included in the culture. Fatty acid-degrading methanogenic granules were formed in a laboratory-scale upflow reactor at 35(deg)C fed with a medium containing a mixture of acetate, propionate, and butyrate by using defined cultures of Methanobacterium formicicum T1N, Methanosaeta sp. strain M7, Methanosarcina mazei T18, propionate-degrading strain PT, and butyrate-degrading strain BH. The maximum substrate conversion rates of these granules for acetate, propionate, and butyrate were 43, 9, and 17 mmol/g (dry weight)/day, respectively. The average size of the granules was about 1 mm. Electron microscopic observation of the granules revealed that the cells of Methanobacterium formicicum, Methanosaeta sp., butyrate-degrading, and propionate-degrading bacteria were dispersed in the granules. Methanosarcina mazei existed inside the granules as aggregates of its own cells, which were associated with the bulk of the granules. The interaction of different species in aggregate formation and granule formation is discussed in relation to polymer formation of the cell surface.
Induction of the d-Amino Acid Oxidase from Trigonopsis variabilis
Authors: Horner R , Wagner F , Fischer L
Published: January 2020

Induction of the d-amino acid oxidase (EC. 1.4.3.3) from the yeast Trigonopsis variabilis was investigated by using a minimal medium containing glucose as the carbon and energy source, (NH(inf4))(inf2)SO(inf4) as the nitrogen source, and various d- and dl-amino acid derivatives as inducers. The best new inducers found were N-carbamoyl-d-alanine, N-acetyl-d-tryptophan, and N-chloroacetyl-d-(alpha)-aminobutyric acid; when the induction effects of these compounds were compared with the effects of d-alanine as the nitrogen source and inducer, the resulting activities of d-amino acid oxidase per gram of dried yeast were 4.2, 2.1, and 1.5 times higher, respectively. The optimum concentration of the best inducer, N-carbamoyl-d-alanine, was 5 mM. This inducer could also be used in its racemic form. The induction was pH dependent. After cultivation of the yeast in a 50-liter bioreactor, d-amino acid oxidase activity of about 3,850 (mu)kat (231,000 U) was obtained. In addition, production of the d-amino acid oxidase was found to be significantly dependent on the metal salt composition of the medium. Addition of zinc ions was required to obtain high d-amino acid oxidase levels in the cells. The optimum concentration of ZnSO(inf4) was about 140 (mu)M.
Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development
Authors: Suárez A M , Rodríguez J M , Hernández P E , Azcona-Olivera J I
Published: January 2020

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.
Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei
Authors: Margolles-Clark E , Hayes C K , Harman G E , Penttilä M
Published: January 2020

The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.
Reduction and Acetylation of 2,4-Dinitrotoluene by a Pseudomonas aeruginosa Strain
Authors: Noguera D R , Freedman D L
Published: January 2020

Aerobic and anoxic biotransformation of 2,4-dinitrotoluene (DNT) was examined by using a Pseudomonas aeruginosa strain isolated from a plant treating propellant manufacturing wastewater. DNT biotransformation in the presence and absence of oxygen was mostly reductive and was representative of the type of cometabolic transformations that occur when a high concentration of an easily degradable carbon source is present. P. aeruginosa reduced both nitro groups on DNT, with the formation of mainly 4-amino-2-nitrotoluene and 2-amino-4-nitrotoluene and small quantities of 2,4-diaminotoluene. Acetylation of the arylamines was a significant reaction. 4-Acetamide-2-nitrotoluene and the novel compounds 2-acetamide-4-nitrotoluene, 4-acetamide-2-aminotoluene, and 2,4-diacetamidetoluene were identified as DNT metabolites. The biotransformation of 2,4-diaminotoluene to 4-acetamide-2-aminotoluene was 24 times faster than abiotic transformation. 2-Nitrotoluene and 4-nitrotoluene were also reduced to their corresponding toluidines and then acetylated. However, the yield of 4-acetamidetoluene was much higher than that of 2-acetamidetoluene, demonstrating that acetylation at the position para to the methyl group was favored.
Bacterial Growth on Distant Naphthalene Diffusing through Water, Air, and Water-Saturated and Nonsaturated Porous Media
Authors: Harms H
Published: January 2020

The influence of substrate diffusion on bacterial growth was investigated. Crystalline naphthalene was supplied as the substrate at various distances in the range of centimeters from naphthalene-degrading organisms separated from the substrate by agar-solidified mineral medium. Within 2 weeks, the cells grew to final numbers which were negatively correlated with the distance from the substrate. A mathematical model that combined (i) Monod growth kinetics extended by a term for culture maintenance and (ii) substrate diffusion could explain the observed growth curves. The model could also predict growth on naphthalene that was separated from the bacteria by air. In addition, the bacteria were grown on distant naphthalene that had to diffuse to the cells through water-saturated and unsaturated porous media. The growth of the bacteria could be used to calculate the effective diffusivity of naphthalene in the three-phase system. Diffusion of naphthalene in the pore space containing 80% air was roughly 1 order of magnitude faster than in medium containing only 20% air because of the high Henry's law coefficient of naphthalene. It is proposed that the effective diffusivities of the substrates and the spatial distribution of substrates and bacteria are the main determinants of final cell numbers and, consequently, final degradation rates.
Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp
Authors: Matheson V G , Forney L J , Suwa Y , Nakatsu C H , Sexstone A J , Holben W E
Published: January 2020

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.
Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains
Authors: Goupil N , Corthier G , Ehrlich S D , Renault P
Published: January 2020

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.
Recruitment and expression of toluene/trichloroethylene biodegradation genes in bacteria native to deep-subsurface sediments
Authors: Romine M F , Brockman F J
Published: January 2020

Four plasmids, each encoding a combination of either an Escherichia coli or Pseudomonas putida promoter and either toluene dioxygenase or toluene monooxygenase, were electroporated into five bacterial strains isolated from sediments found at depths of 91 to 295 m. Four of these engineered bacterial strains demonstrated both toluene and trichloroethylene degradation activities.
Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB)
Authors: Sylvestre M , Hurtubise Y , Barriault D , Bergeron J , Ahmad D
Published: January 2020

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.
Chloroform mineralization by toluene-oxidizing bacteria
Authors: McClay K , Fox B G , Steffan R J
Published: January 2020

Seven toluene-oxidizing bacterial strains (Pseudomonas mendocina KR1, Burkholderia cepacia G4, Pseudomonas putida F1, Pseudomonas pickettii PKO1, and Pseudomonas sp. strains ENVPC5, ENVBF1, and ENV113) were tested for their ability to degrade chloroform (CF). The greatest rate of CF oxidation was achieved with strain ENVBF1 (1.9 nmol/min/mg of cell protein). CF also was oxidized by P. mendocina KR1 (0.48 nmol/min/mg of cell protein), strain ENVPC5 (0.49 nmol/min/mg of cell protein), and Escherichia coli DH510B(pRS202), which contained cloned toluene 4-monooxygenase genes from P. mendocina KR1 (0.16 nmol/min/mg of cell protein). Degradation of [14C]CF and ion analysis of culture extracts revealed that CF was mineralized to CO2 (approximately 30 to 57% of the total products), soluble metabolites (approximately 15%), a total carbon fraction irreversibly bound to particulate cellular constituents (approximately 30%), and chloride ions (approximately 75% of the expected yield). CF oxidation by each strain was inhibited in the presence of trichloroethylene, and acetylene significantly inhibited trichloroethylene oxidation by P. mendocina KR1. Differences in the abilities of the CF-oxidizing strains to degrade other halogenated compounds were also identified. CF was not degraded by B. cepacia G4, P. putida F1, P. pickettii PKO1, Pseudomonas sp. strain ENV113, or P. mendocina KRMT, which contains a tmo mutation.
Survival of Escherichia coli O157:H7 in broth and processed salami as influenced by pH, water activity, and temperature and suitability of media for its recovery
Authors: Clavero M R , Beuchat L R
Published: January 2020

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.
Effects of phenol feeding pattern on microbial community structure and cometabolism of trichloroethylene
Authors: Shih C , Davey M E , Zhou J , Tiedje J M , Criddle C S
Published: January 2020

Cometabolism of trichloroethylene (TCE) by phenol-fed enrichments was evaluated in four reactors with distinct phenol feeding patterns. The reactors were inoculated from the same source, operated at the same average dilution rate, and received the same mass of phenol over time. Only the timing of phenol addition differed. Reactor C received phenol continuously; reactor SC5 received phenol semicontinuously--alternating between 5 h of feed and 3 h without feed; reactor SC2 alternated between 2 h of feed and 6 h without feed; and reactor P received a single pulse every 24 h. The structure of the enrichments and their capacity for TCE transformation were analyzed. In long-term operation, reactors C and SC5 were dominated by fungi, had higher levels of predators, were more susceptible to biomass fluctuations, and exhibited reduced capacity for TCE transformation. Reactors P and SC2 were characterized by lower levels of fungi, higher bacterial biomass, higher concentrations of TCE-degrading organisms, and higher rates of TCE transformation. After 200 days of operation, rates of TCE transformation increased 10-fold in reactor P, resulting in TCE transformation rates that were 20 to 100 times higher than the rates of the other reactor communities. The cause of this shift is unknown. Isolates capable of the highest rates of TCE transformation were obtained from reactor P. We conclude that cometabolic activity depends upon microbial community structure and that the community structure can be manipulated by altering the growth substrate feeding pattern.
Inhibition of sulfate respiration by 1,8-dihydroxyanthraquinone and other anthraquinone derivatives
Authors: Cooling F I , Maloney C L , Nagel E , Tabinowski J , Odom J M
Published: January 2020

Derivatives of 9,10-anthracenedione, or anthraquinone, were shown to inhibit respiratory sulfate reduction by pure cultures of sulfate-reducing bacteria, as well as by crude enrichment cultures. Structure-activity studies showed that an increasing degree of substitution of the anthraquinone nucleus resulted in increasing 50% inhibition (I(inf50)) values for sulfate respiration. Addition of charged ring substituents also resulted in an increase in the I(inf50) concentration. Experiments carried out with 1,8-dihydroxyanthraquinone demonstrated inhibition of hydrogen-dependent sulfate respiration but not hydrogen-dependent sulfite or thiosulfate respiration. Addition of pyruvate resulted in stimulation of sulfate-dependent hydrogen oxidation in the presence of the anthraquinone. These observations, together with a direct demonstration of uncoupling in French press vesicle preparations, suggest that the underlying mechanism of inhibition is uncoupling of ATP synthesis from electron transfer reactions. The low I(inf50) values for inhibition (0.5 to 10 (mu)M) and the relatively low general toxicity of anthraquinones suggest that these compounds may be useful for inhibition of sulfide generation in situations which are incompatible with the use of broadly toxic biocides.
Production of kanosamine by Bacillus cereus UW85
Authors: Milner J L , Silo-Suh L , Lee J C , He H , Clardy J , Handelsman J
Published: January 2020

Bacillus cereus UW85 produces two antibiotics that contribute to its ability to suppress certain plant diseases (L. Silo-Suh, B. Lethbridge, S. J. Raffel, H. He, J. Clardy, and J. Handelsman, Appl. Environ. Microbiol. 60:2023-2030, 1994). To enhance the understanding of disease suppression by UW85, we determined the chemical structure, regulation, and the target range of one of the antibiotics. The antibiotic was identified as 3-amino-3-deoxy-D-glucose, also known as kanosamine. Kanosamine was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested. Maximum accumulation of kanosamine in B. cereus UW85 culture supernatants coincided with sporulation. Kanosamine accumulation was enhanced by the addition of ferric iron and suppressed by addition of phosphate to rich medium. Kanosamine accumulation was also enhanced more than 300% by the addition of alfalfa seedling exudate to minimal medium.
Comparison of Quasielastic Light Scattering and Laser Diffractometry as Nondestructive Probes into the Structure of Bacillus sphaericus Spores Produced at Different Temperatures
Authors: Molina-Garcia A D , de Pieri L A , Ludlow I , Waites W M , de la Torre J G , Harding S E
Published: January 2020

Volume 62, no. 5, p. 1702, column 2, equation 3: the equation should read as follows. g(sup1)((tau)) = [g(sup2)((tau)) - 1](sup1/2) = exp[-K(sup2)(D(inf1) cos(sup2)(alpha) + D(inf2) sin(sup2)(alpha))(tau)] (3) [This corrects the article on p. 1699 in vol. 62.].
Characterization of thermophilic consortia from two souring oil reservoirs
Authors: Mueller R F , Nielsen P H
Published: January 2020

The microbial consortia from produced water at two different oil fields in Alaska (Kuparuk) and the North Sea (Ninian) were investigated for sulfate-reducing and methanogenic activity over a range of temperatures and for a variety of substrates. The consortia were sampled on site, and samples were either incubated on site at 60(deg)C with various substrates or frozen for later incubation and analyses. Temperature influenced the rates of sulfate reduction, hydrogen sulfide production, and substrate oxidation, as well as the cell morphology. The highest rates of sulfate reduction and substrate oxidation were found between 50 and 60(deg)C. Formate and n-butyrate were the most favorable electron donors at any tested temperature. Acetate was utilized at 35(deg)C but not at 50 or 70(deg)C and was produced at 60(deg)C. This indicates that the high levels of acetate found in produced water from souring oil formations are due mainly to an incomplete oxidation of volatile fatty acids to acetate. The cell size distribution of the microbial consortium indicated a nonuniform microbial composition in the original sample from the Kuparuk field. At different temperatures, different microbial morphologies and physiologies were observed. Methane-producing activity at thermophilic temperatures (60(deg)C) was found only for the Kuparuk consortium when hydrogen and carbon dioxide were present. No methane production from acetate was observed. Suppression of methanogenic activity in the presence of sulfate indicated a competition with sulfate-reducing bacteria for hydrogen.
Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism
Authors: Lee D H , Zo Y G , Kim S J
Published: January 2020

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.
Responses of methanotrophic activity in soils and cultures to water stress
Authors: Schnell S , King G M
Published: January 2020

Diffusive gas transport at high water contents and physiological water stress at low water contents limited atmospheric methane consumption rates during experimental manipulations of soil water content and water potential. Maximum rates of atmospheric methane consumption occurred at a soil water content of 25% (grams per gram [dry weight]) and a water potential of about -0.2 MPa. In contrast, uptake rates were highest at a water content of 38% and a water potential of -0.03 MPa when methane was initially present at 200 ppm. Uptake rates of atmospheric and elevated methane decreased when water potentials were reduced by adding either ionic or nonionic solutes to soils with a fixed water content. Uptake rates during these manipulations were lower when sodium chloride or potassium chloride was used to adjust water potential rather than sucrose. The response of methane consumption by soils to water potential was somewhat less pronounced than the response of methanotrophic cultures (e.g., Methylosinus trichosporium OB3b, Methylomonas rubra [= M. methanica], an isolate from a freshwater peat, and an isolate from an intertidal marine mudflat). However, unlike soils, methanotrophic cultures exhibited a stronger adverse response to nonionic solutes than to sodium chloride.
Biodegradation of organic wastes containing surfactants in a biomass recycle reactor
Authors: Konopka A , Zakharova T , Oliver L , Camp D , Turco R F
Published: January 2020

The microbial biodegradation of simulated graywater, containing 21.5 mg of linear alkylbenzene sulfonate liter-1, was investigated with a continuous-flow bioreactor with 100% biomass recycle. Low concentrations of organic matter in the ultrafiltration eluate were achieved by hydraulic residence times as short as 1.6 h and for periods of up to 74 days at a hydraulic residence time of 6 h. Upon a shift from the chemostat to the biomass recycle mode, the increase in biomass with time approximated a linear rather than an exponential function. Biomass densities as high as 6.8 g of cell protein liter-1 were reached; this was 50-fold higher than the steady-state biomass level in chemostats fed the same medium. We assessed physiological changes in the microbial community after a switch from the chemostat to the biomass recycle mode. Over 150 h, there was a two- to fourfold decrease in the respiratory potential of the microbes. After this decrease, respiratory potentials were relatively constant up to 74 days of operation. A decline in reactivity was also indicated by increasing lag periods before growth in response to organic nutrient inputs and by a decrease in the proportion of cells able to reduce tetrazolium dye. However, the bioreactor system was still capable of rapidly metabolizing inputs of organic matter, because of the very high biomass concentrations. It appears that < 10% of the organic carbon inputs accumulate as biomass.
Optical fiber-based in situ spectroscopy of pigmented single colonies
Authors: Wiggli M , Ghosh R , Bachofen R
Published: January 2020

We have adapted a commercially available fiber-optic spectroradiometer with diode array detection to record reflection and absorption spectra from single, 1-mm-diameter bacterial colonies. A careful assessment of the performance of the spectroradiometer for this application is reported. In a model study employing colonies from various phototrophic bacteria, we show that the reflectance spectra are reliable within the range of 450 to 820 nm, whereas the transmission spectra yield accurate peak intensities and absorption maxima from 400 to 900 nm. For screening of populations of about 10(sup4) colonies, fiber-optic transmission spectroscopy provides an attractive and inexpensive alternative to present techniques based on charge-coupled device imaging technology.
Improved cultivation systems for isolation of the colorado potato beetle spiroplasma
Authors: Konai M , Hackett K J , Williamson D L , Lipa J J , Pollack J D , Gasparich G E , Clark E A , Vacek D C , Whitcomb R F
Published: January 2020

In North America, the Colorado potato beetle, Leptinotarsa decemlineata, is often infected with the host-specific, gut-inhabiting Colorado potato beetle spiroplasma (CPBS). CPBS is apparently a commensal, but it may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivating the organism, however, has hindered the development of a suitable transformation system. In this study, we eliminated the need for coculturing CPBS with insect cells. CPBS was reliably isolated with the BBL Anaerobic GasPak Jar system (low redox, enhanced CO(inf2)), which was easier to use and less expensive than insect cell coculture methods. A further advantage is a reduction in contaminating insect cell components. Use of anaerobiosis should facilitate early-passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. The use of low-pH (6.0 to 6.5) media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. These formulations permitted the first cultivation of CPBS on solid media, an accomplishment that will simplify the selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.
Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains
Authors: Sadowsky M J , Kinkel L L , Bowers J H , Schottel J L
Published: January 2020

PCR DNA fingerprinting using repetitive intergenic DNA sequences (rep-PCR) was investigated as a means of differentiating between closely related strains of Streptomyces which were, in some cases, indistinguishable by other classification methods. Our results demonstrated that the majority of strains had unique rep-PCR DNA fingerprints and established that the technique could be a very useful tool in rapidly determining strain identity.
Differential media for quantitative recovery of waterborne Aeromonas hydrophila
Authors: Handfield M , Simard P , Letarte R
Published: January 2020

Because of the ubiquity of Aeromonas spp., their prevalence in drinking water, and the increasing number of reports on Aeromonas sp.-related infections, a standard method for routine and quantitative recovery had to be defined. On the basis of a survey of 10 media for recovery analysis and subsequent differentiation assays in mixed cultures, we conclude that ampicillin-dextrin agar performed the best for the recovery of Aeromonas spp. in drinking water and the differentiation by simple criteria of that genus from other common waterborne bacteria.
Cross-Polarized Magic-Angle Spinning (sup13)C Nuclear Magnetic Resonance Spectroscopic Characterization of Soil Organic Matter Relative to Culturable Bacterial Species Composition and Sustained Biological Control of Pythium Root Rot
Authors: Boehm M J , Wu T , Stone A G , Kraakman B , Iannotti D A , Wilson G E , Madden L V , Hoitink H
Published: January 2020

We report the use of a model system that examines the dynamics of biological energy availability in organic matter in a sphagnum peat potting mix critical to sustenance of microorganism-mediated biological control of pythium root rot, a soilborne plant disease caused by Pythium ultimum. The concentration of readily degradable carbohydrate in the peat, mostly present as cellulose, was characterized by cross-polarized magic-angle spinning (sup13)C nuclear magnetic resonance spectroscopy. A decrease in the carbohydrate concentration in the mix was observed during the initial 10 weeks after potting as the rate of hydrolysis of fluorescein diacetate declined below a critical threshold level required for biological control of pythium root rot. Throughout this period, total microbial biomass and activity, based on rates of [(sup14)C]acetate incorporation into phospholipids, did not change but shifts in culturable bacterial species composition occurred. Species capable of inducing biocontrol were succeeded by pleomorphic gram-positive genera and putative oligotrophs not or less effective in control. We conclude that sustained efficacy of naturally occurring biocontrol agents was limited by energy availability to this microflora within the organic matter contained in the potting mix. We propose that this critical role of organic matter may be a key factor explaining the variability in efficacy typically encountered in the control of pythium root rot with biocontrol agents.
Nonanoic Acid, a Fungal Self-Inhibitor, Prevents Germination of Rhizopus oligosporus Sporangiospores by Dissipation of the pH Gradient
Authors: Breeuwer P , De Reu J C , Drocourt J , Rombouts F M , Abee T
Published: January 2020

Germination of Rhizopus oligosporus sporangiospores is characterized by swelling of the spores and subsequent emergence of germ tubes. Changes in spore morphology and alterations in intracellular pH (pH(infin)) of the sporangiospores were assessed during the germination process by flow cytometry. Sporangiospores were stained with carboxyfluorescein by incubation with carboxyfluorescein diacetate. The nonfluorescent carboxyfluorescein diacetate is passively transported into intact cells and subsequently cleaved by esterases, which results in intracellular accumulation of the fluorescent carboxyfluorescein. Given that the fluorescence of carboxyfluorescein is pH dependent, the pH(infin) of the individual spores could be assessed simultaneously with spore size. For R. oligosporus, swelling of the sporangiospores was accompanied by an increase of pH(infin). In the presence of nonanoic acid, a self-inhibitor produced by various fungi, increase of the pH(infin) was prevented and swelling was inhibited at concentrations of less than 1 mM. Octanoic acid and decanoic acid were equally effective. Acetic acid also inhibited germination but at much higher concentrations (>8 mM). The mechanism of action of these fatty acids is most likely dissipation of the pH gradient. A model is proposed in which the pH(infin) plays a crucial role in the germination of R. oligosporus sporangiospores.