bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli
Authors: Wright R M , Yablonsky M D , Shalita Z P , Goyal A K , Eveleigh D E
Published: January 2020

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.
Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae
Authors: Siragusa G R
Published: January 2020

A bovine intestinal bacterial isolate, identified as Enterococcus hirae, was found to produce a bacteriocin (designated hiraecin S) inhibitory to Listeria monocytogenes and other Listeria spp. Identification to species level was determined by comprehensive biochemical and morphological tests which were verified by DNA-DNA homology assays. The antimicrobial agent was inactivated by pronase and papain and was insensitive to catalase. The antimicrobial activity was not due to hydrogen peroxide or acid formation, nor was lysozyme or muramidase activity observed in cell-free bacteriocin preparations. Inhibition of selected gram-negative bacteria was not observed. Other enterococci were sensitive to the bacteriocin, and except for Listeria spp., no other gram-positive bacteria tested were inhibited.
Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1
Authors: Chaudhuri K , Bhadra R K , Das J
Published: January 2020

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.
The anaerobic degradation of 3-chloro-4-hydroxybenzoate in freshwater sediment proceeds via either chlorophenol or hydroxybenzoate to phenol and subsequently to benzoate
Authors: Zhang X , Wiegel J
Published: January 2020

To study the anaerobic degradation of the chimera 3-chloro-4-hydroxybenzoate (3-Cl,4-OHB), anaerobic freshwater sediment samples from the vicinity of Athens, Ga., were adapted for the transformation of 4-hydroxybenzoate (4-OHB), 3-chlorobenzoate (3-CB), 2-chlorophenol (2-CP), and 2,4-dichlorophenol (2,4-DCP). In nonadapted samples, both 4-OHB (product of aryl dechlorination) and 2-CP (product of aryl decarboxylation) were observed as intermediates in the transformation of 3-Cl,4-OHB to phenol. The accumulated phenol was subsequently transformed to benzoate, an intermediate in the conversion to methane and CO2. In 4-OHB-adapted samples (i.e., samples adapted for aryl decarboxylation), 2-CP was the first intermediate which was subsequently dechlorinated to phenol. In 3-CB-adapted samples (i.e., samples adapted for meta-chlorobenzoate dehalogenation), 3-Cl,4-OHB was stoichiometrically dechlorinated to 4-OHB. In 2-CP-adapted samples (i.e., samples adapted for ortho-chlorophenol dehalogenation), 4-OHB was the first major intermediate. Furthermore, 3-CB was not dechlorinated in 2-CP-adapted sediment samples, suggesting the possibility that different 3-Cl,4-OHB dechlorinating systems were induced in the 2-CP- and 3-CB-adapted sediments. Adaptation of sediment samples for dechlorination of 2,4-DCP did not lead to adaptation for dechlorination of 3-Cl,4-OHB. However, 3-Cl,4-OHB was dechlorinated to 4-OHB in our stable, sediment-free 2,4-DCP-dechlorinating enrichment, isolated previously from the same environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Assessment of [
3
H]Thymidine Incorporation into DNA as a Method To Determine Bacterial Productivity in Stream Bed Sediments
Authors: Kaplan Louis A. , Bott Thomas L. , Bielicki John K.
Published: January 2020

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25�C, but hydrolysis at 120�C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 μM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.
Adaptation of model genetically engineered microorganisms to lake water: growth rate enhancements and plasmid loss
Authors: Sobecky P A , Schell M A , Moran M A , Hodson R E
Published: January 2020

When a genetically engineered microorganism (GEM) is released into a natural ecosystem, its survival, and hence its potential environmental impact, depends on its genetic stability and potential for growth under highly oligotrophic conditions. In this study, we compared plasmid stability and potential for growth on low concentrations of organic nutrients of strains of Pseudomonas putida serving as model GEMs. Plasmid-free and plasmid-bearing (NAH7) prototrophic isogenic strains and two amino-acid auxotrophs, all containing antibiotic resistance markers, were held physically separate from but in chemical contact with lake water containing the natural bacterium-sized microbial populations. Cells were reisolated at intervals over a 2-month period to determine the percent retaining the plasmid and the specific growth rate on various media. Plasmid stability in lake water was strongly strain specific; the NAH7 plasmid was stably maintained by the prototrophic strain for the duration of the test but was lost within 24 h by both of the auxotrophs. Specific growth rates of reisolates, compared with those of the corresponding non-lake water-exposed strains (i.e., parental strains), were not different when measured in rich medium (Luria-Bertani broth). However, specific growth rates were 42, 55, and 63% higher in reisolates of auxotrophs and the plasmid-free prototroph, respectively, when measured in 10-fold-diluted medium after exposure of 15 days or longer to lake water. Moreover, lake water-exposed strains grew actively when reintroduced into sterile lake water (28- to 33-fold increase in numbers over 7 days), while the corresponding unadapted parental strains exhibited no growth over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)
Introduction of a de novo bioremediation ability, aryl reductive dechlorination, into anaerobic granular sludge by inoculation of sludge with Desulfomonile tiedjei
Authors: Ahring B K , Christiansen N , Mathrani I , Hendriksen H V , Macario A J , Conway de Macario E
Published: January 2020

Methanogenic upflow anaerobic granular-sludge blanket (UASB) reactors treat wastewaters at a high rate while simultaneously producing a useful product, methane; however, recalcitrant environmental pollutants may not be degraded. To impart 3-chlorobenzoate (3-CB)-dechlorinating ability to UASB reactors, we inoculated granular sludge in UASB reactors with either a pure culture of Desulfomonile tiedjei (a 3-CB-dechlorinating anaerobe) or a three-member consortium consisting of D. tiejei, a benzoate degrader, and an H2-utilizing methanogen. No degradation occurred in an uninoculated control reactor which was started with the same granular sludge, but inoculated reactors and granules from the inoculated UASB systems rapidly transformed 3-CB (54 mumol/day/g of granule biomass). After several months at a hydraulic retention time of 0.5 day, much shorter than the generation time of D. tiedjei, the reactors still dechlorinated 3-CB. This indicated that the bacteria were immobilized in the reactor granules, and by using an antibody probe for D. tiedjei, we demonstrated that this microorganism had colonized the sludge granules. These results represent the first addition of a pure culture or a defined microbial mixture to a viable waste treatment process to introduce a specific de novo degradative pathway into a granular-sludge consortium.
Mechanisms and Rates of Decay of Marine Viruses in Seawater
Authors: Suttle Curtis A. , Chen Feng
Published: January 2020

Loss rates and loss processes for viruses in coastal seawater from the Gulf of Mexico were estimated with three different marine bacteriophages. Decay rates in the absence of sunlight ranged from 0.009 to 0.028 h
-1
, with different viruses decaying at different rates. In part, decay was attributed to adsorption by heat-labile particles, since viruses did not decay or decayed very slowly in seawater filtered through a 0.2-μm-pore-size filter (0.2-μm-filtered seawater) and in autoclaved or ultracentrifuged seawater but continued to decay in cyanide-treated seawater. Cyanide did cause decay rates to decrease, however, indicating that biological processes were also involved. The observations that decay rates were often greatly reduced in 0.8- or 1.0-μm-filtered seawater, whereas bacterial numbers were not, suggested that most bacteria were not responsible for the decay. Decay rates were also reduced in 3-μm-filtered or cycloheximide-treated seawater but not in 8-μm-filtered seawater, implying that flagellates consumed viruses. Viruses added to flagellate cultures decayed at 0.15 h
-1
, corresponding to 3.3 viruses ingested flagellate
-1
h
-1
. Infectivity was very sensitive to solar radiation and, in full sunlight, decay rates were 0.4 to 0.8 h
-1
. Even when UV-B radiation was blocked, rates were as high as 0.17 h
-1
. Calculations suggest that in clear oceanic waters exposed to full sunlight, most of the virus decay, averaged over a depth of 200 m, would be attributable to solar radiation. When decay rates were averaged over 24 h for a 10-m coastal water column, loss rates of infectivity attributable to sunlight were similar to those resulting from all other processes combined. Consequently, there should be a strong diel signal in the concentration of infectious viruses. In addition, since sunlight destroys infectivity more quickly than virus particles, a large proportion of the viruses in seawater is probably not infective.

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Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants
Authors: Laitinen S , Nevalainen A , Kotimaa M , Liesivuori J , Martikainen P J
Published: January 2020

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.
Production of Eicosapentaenoic Acid (20:5 n-3) by
Vibrio pelagius
Isolated from Turbot (
Scophthalmus maximus
(L.)) Larvae
Authors: Ring� Einar , Sinclair Pauline D. , Birkbeck Harry , Barbour Andrew
Published: January 2020

Fourteen bacterial strains isolated from turbot,
Scophthalmus maximus
(L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species,
Vibrio pelagius,
contained a high proportion of EPA in cellular lipid. This finding was confirmed by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4�C.
Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene
Authors: Gustafson C E , Thomas C J , Trust T J
Published: January 2020

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.
The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme
Authors: Gibbs M D , Saul D J , Lüthi E , Bergquist P L
Published: January 2020

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.
Monoclonal antibodies that detect live salmonellae
Authors: Torensma R , Visser M J , Aarsman C J , Poppelier M J , van Beurden R , Fluit A C , Verhoef J
Published: January 2020

Nine immunoglobulin G and nine immunoglobulin M murine monoclonal antibody-producing hybridomas reactive with live Salmonella bacteria were obtained from several fusions of immune spleen cells and Sp2/0 myeloma cells. The antibodies were selected by the magnetic immunoluminescence assay. The monoclonal antibodies were reactive with serogroups A, B, C1, C2, D, E, and K and Salmonella choleraesuis subsp. diarizonae. Each monoclonal antibody proved to be reactive with a distinct serotype. Clinical isolates belonging to these Salmonella serogroups could be detected. Reactivity with non-Salmonella bacteria proved to be minor.
Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain
Authors: Fenton A M , Stephens P M , Crowley J , O'Callaghan M , O'Gara F
Published: January 2020

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.
Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae
Authors: Avery S V , Tobin J M
Published: January 2020

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.
Physiological Events in
Clostridium acetobutylicum
during the Shift from Acidogenesis to Solventogenesis in Continuous Culture and Presentation of a Model for Shift Induction
Authors: Grupe Helga , Gottschalk Gerhard
Published: January 2020

The pH of continuous cultures of
Clostridium acetobutylicum
growing at pH 5.6 was allowed to decrease to 4.3 after acid production and thereby to shift the cultures from acetate and butyrate to acetone and butanol formation. Several parameters were determined during the shift. An increase in the intracellular acid concentration to 440 mM was recorded. An excess of undissociated butyric acid but not of acetic acid just before the shift to solventogenesis was followed by a decline in acid production and subsequently by the uptake of acids. The intracellular ATP concentration reached a minimum before the onset of solventogenesis; this presumably reflects the ATP-consuming proton extrusion connected with the increase in the ΔpH from 0.7 to 1.4 units. The pool of NADH plus NADPH exhibited a drastic increase until solventogenesis was induced. The changes in the ATP and ADP and NADH plus NADPH pools during these pH shift experiments were the beginning of a stable metabolic oscillation which could also be recorded as an oscillation of the culture redox potential under steady-state solventogenic conditions. Similar changes were observed when the shift was induced by the addition of butyrate and acetate (50 mM each) to the continuous culture. However, when methyl viologen was added, important differences were found: ATP levels did not reach a minimum, acetoacetate decarboxylase activity could not be measured, and butanol but not acetone was produced. A model for the shift is proposed; it assumes the generation of two signals, one by the changed ATP and ADP levels and the other by the increased NAD(P)H level.
Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species
Authors: Donovan W P , Rupar M J , Slaney A C , Malvar T , Gawron-Burke M C , Johnson T B
Published: January 2020

Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.
Effect of sodium chloride on the intracellular solute pools of Listeria monocytogenes
Authors: Patchett R A , Kelly A F , Kroll R G
Published: January 2020

The concentrations of intracellular solutes in Listeria monocytogenes were examined in cells grown at various concentrations of NaCl. At 5% NaCl, cells contained elevated concentrations of potassium and glycine betaine compared with concentrations in cells grown without NaCl. At 7.5% NaCl, cells contained increased concentrations of K+, glycine betaine, glycine, alanine, and proline. Only glycine betaine, choline, or glycine promoted growth on a solidified defined medium containing 4% NaCl; there was no growth at higher concentrations of NaCl in the defined medium.
Presence of human immunodeficiency virus nucleic acids in wastewater and their detection by polymerase chain reaction
Authors: Ansari S A , Farrah S R , Chaudhry G R
Published: January 2020

The human immunodeficiency virus type 1 (HIV-1) released by infected individuals or present in human and hospital wastes can potentially cause contamination problems. The presence of HIV-1 was investigated in 16 environmental samples, including raw wastewater, sludge, final effluent, soil, and pond water, collected from different locations. A method was developed to extract total nucleic acids in intact form directly from the raw samples or from the viral concentrates of the raw samples. The isolated nucleic acids were analyzed for the presence of HIV-1 by using in vitro amplification of the target sequences by the polymerase chain reaction (PCR) method. HIV-1-specific proviral DNA and viral RNA were detected in the extracted nucleic acids obtained from three wastewater samples by this method. The specificity of the PCR-amplified products was determined by Southern blot hybridization with an HIV-1-specific oligonucleotide probe, SK19. The isolated nucleic acids from wastewater samples were also screened for the presence of poliovirus type 1, representing a commonly found enteric virus, and simian immunodeficiency virus, representing, presumably, rare viruses. While poliovirus type 1 viral RNA was found in all of the wastewater samples, none of the samples yielded a simian immunodeficiency virus-specific product. No PCR-amplified product was yielded when wastewater samples were directly used for the detection of HIV-1 and poliovirus type 1. The wastewater constituents appeared to be inhibitory to the enzymes reverse transcriptase and DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)
Development of a Method for Detection of Lactic Acid Bacteria Producing Exclusively the
l
-(+)- Isomer of Lactic Acid
Authors: Jehanno D. , Thuault D. , Bourgeois C. M.
Published: January 2020

A method was developed for the detection and isolation, within a population of lactic acid bacteria, of strains producing exclusively the
l
-(+)- isomer of lactic acid; the visual detection of colonies of these particular strains can be carried out directly on agar plates (50 to 70 colonies per plate). The method is based on an enzymatic stereospecific reaction involving
d
-(−)-lactate dehydrogenase and linked to a staining reaction; the diffusion area of the
d
-(−)- isomer stains red around the
d
-(−)- and the
dl
-lactic acid-producing colonies, while the colonies producing exclusively
l
-(+)-lactic acid are detected by the absence of the colored halo. The intensity of staining was increased when cellulose powder and Tween 20 were added to the agar medium.
Direct extraction of bacterial plasmids from food for polymerase chain reaction amplification
Authors: Andersen M R , Omiecinski C J
Published: January 2020

In this report we describe a simple and rapid technique using DNA affinity columns that permits direct extraction of bacterial plasmids from a variety of foods for polymerase chain reaction amplification. The procedure was used to detect virulent enteroinvasive Escherichia coli in several artificially seeded matrices, including seafoods, greens, dairy products, enrichment media, and water. Polymerase inhibitors present in both foods and enrichment media were removed efficiently.
Applicability of an immunoblot technique combined with a semiautomated electrophoresis system for detection of staphylococcal enterotoxins in food extracts
Authors: Orden J A , Goyache J , Hernández J , Doménech A , Suárez G , Gómez-Lucía E
Published: January 2020

We studied the usefulness of an immunoblot technique for the detection of staphylococcal enterotoxins (SEs) in strains and food extracts. Food samples (milk, yogurt, hot dog sausage, cheese, and mayonnaise) were artificially contaminated with SEA through SEE. Protein A did not interfere with the results; it appeared on electrophoresis gels as bands with molecular weights higher than those of the SEs. Other food proteins were not revealed by the technique. The immunoblot technique proved to be fast, specific, and sensitive for the detection of SEs in foods.
Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway
Authors: Sondossi M , Sylvestre M , Ahmad D
Published: January 2020

Bacterial conversion of biphenyl (BP) and chlorobiphenyls (CBPs) to benzoates and chlorobenzoates (CBAs) proceeds by introduction of molecular oxygen at the 2,3 position, followed by a 1,2-meta cleavage of the molecule. Complete mineralization of CBPs requires the presence of two sets of genes, one for the transformation fo CBPs into CBAs and a second for the degradation of CBAs. It has been shown previously that removal of the CBAs produced from the degradation of CBPs is essential for efficient degradation of CBPs. In this study we confirmed that CBAs inhibit BP and CBP transformation in Pseudomonas testosteroni B-356. Among the three monochlorobenzoates tested, 3-chlorobenzoate was the most effective inhibitor. Furthermore, we found that in strain B-356, CBA transformation is controlled by BP-induced oxygenases that are not present in benzoate-grown cells. We found that this BP-linked CBA transformation pathway transformed CBAs produced from CBPs into several metabolites, including chlorocatechols and corresponding muconic semialdehydes. These metabolites inhibited the 2,3-dihydroxybiphenyl 1,2-dioxygenase, while CBAs by themselves had no effect on this enzyme. Therefore, on the basis of this and other observations, it appears that when CBAs produced from CBPs accumulate in the growth medium, they are converted into unproductive metabolites that reduce the flux of the BP and CBP degradation pathway. The practical implications of these interactions on the microbial degradation of polychlorinated BPs are also discussed.
Metabolites formed during anaerobic transformation of toluene and o-xylene and their proposed relationship to the initial steps of toluene mineralization
Authors: Evans P J , Ling W , Goldschmidt B , Ritter E R , Young L Y
Published: January 2020

Strain T1 is a facultative bacterium that is capable of anaerobic toluene degradation under denitrifying conditions. While 80% of the carbon from toluene is either oxidized to carbon dioxide or assimilated into cellular carbon, a significant portion of the remainder is transformed into two dead-end metabolites. These metabolites were produced simultaneous to the mineralization of toluene and were identified as benzylsuccinic acid and benzylfumaric acid. Identification was based on comparison of mass spectra of the methyl esters of the metabolites and authentic compounds that were chemically synthesized. Strain T1 is also capable of o-xylene transformation during growth on toluene. o-Xylene does not serve as a source of carbon and is not mineralized. Rather, it is transformed to analogous dead-end metabolites, (2-methylbenzyl)-succinic acid and (2-methylbenzyl)-fumaric acid. o-Xylene transformation also occurred during growth on succinic acid, which suggests that attack of the methyl group by succinyl-coenzyme A is a key reaction in this transformation. We reason that the main pathway for toluene oxidation to carbon dioxide involves a mechanism similar to that for the formation of the metabolites and involves an attack of the methyl group of toluene by acetyl-coenzyme A.
Isolation and partial characterization of an 87-kilodalton beta-1,3-glucanase from Bacillus circulans IAM1165
Authors: Aono R , Sato M , Yamamoto M , Horikoshi K
Published: January 2020

Bacillus circulans IAM1165 produces at least two extracellular beta-1,3-glucanases that lyse fungal cell walls. One of these extracellular enzymes was purified to homogeneity. The molecular mass was 87 kDa, and the pI was 4.3. The optimum temperature of the enzyme reaction was 70 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate. The pH range of the enzyme was broad (pH 4.5 to 9.0), and the optimum pH was 6.5. The enzyme is an endo beta-1,3-glucanase and has a random cleavage pattern.
Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum
Authors: Olsson J C , Westerdahl A , Conway P L , Kjelleberg S
Published: January 2020

Of more than 400 bacteria isolated from turbot (Scophthalmus maximus), 89 have previously been shown to inhibit the in vitro growth of the fish pathogen Vibrio anguillarum. The aim of the present study was to investigate the potential of seven of these strains, as well as of intestinal isolates (four strains) from a closely related fish, dab (Limanda limanda), for colonizing farmed turbot as a means of protecting the host from infection by V. anguillarum. In addition, the inhibitory effect of these strains on the pathogen was further studied. Colonization potential was measured by the capacity of the strains to adhere to and grow in turbot intestinal mucus. These parameters were also used to investigate the potential of V. anguillarum to amplify in the turbot intestinal tract. Because of the observed rapid growth of V. anguillarum in intestinal mucus, it can be proposed that the intestinal tract is a site for V. anguillarum multiplication. Strains isolated from the intestine showed greater capacity for adhesion to and growth in fish intestinal mucus than did the pathogen and the skin mucus isolates. All of the isolates released metabolites into the culture medium that had inhibitory effects against V. anguillarum. The results are discussed with emphasis on administering bacteria of host origin to farmed turbot in order to control V. anguillarum-induced disease.
Cloning, sequencing, and expression in Escherichia coli of lcnB, a third bacteriocin determinant from the lactococcal bacteriocin plasmid p9B4-6
Authors: van Belkum M J , Kok J , Venema G
Published: January 2020

On the bacteriocin plasmid p9B4-6 of Lactococcus lactis subsp. cremoris 9B4, a third bacteriocin determinant was identified. The genes encoding bacteriocin production and immunity resided on a 1.2-kb CelII-ScaI fragment and were located adjacent to one of two previously identified bacteriocin operons (M. J. van Belkum, B. J. Hayema, R. E. Jeeninga, J. Kok, and G. Venema, Appl. Environ. Microbiol. 57:492-498, 1991). The fragment was sequenced and analyzed by deletion and mutation analyses. The bacteriocin determinant consisted of two genes which were transcribed as an operon. The first gene (lcnB), containing 68 codons, was involved in bacteriocin activity. The second gene (lciB) contained 91 codons and was responsible for immunity. The specificity of this novel bacteriocin, designated lactococcin B, was different from that of the other two bacteriocins specified by p9B4-6. Part of the nucleotide sequence of the lactococcin B operon was similar to a nucleotide sequence also found in the two other bacteriocin operons of p9B4-6. This conserved region encompassed a nucleotide sequence upstream of the bacteriocin gene and the 5' part of the gene. When the lactococcin B operon was expressed in Escherichia coli by using a T7 RNA polymerase-specific promoter, antagonistic activity could be detected.
Effects of Temperature on Two Psychrophilic Ecotypes of a Heterotrophic Nanoflagellate,
Paraphysomonas imperforata
Authors: Choi Joon W. , Peters Francesc
Published: January 2020

Two different psychrophilic types of the heterotrophic nanoflagellate
Paraphysomonas imperforata
were isolated from Newfoundland coastal waters and the Arctic Ocean. When fed bacteria without food limitation, both isolates were able to grow at temperatures from -1.8 to 20°C, with maximum growth rates of 3.28 day
-1
at 15°C and 2.28 day
-1
at 12.3°C for the Newfoundland and the Arctic isolates, respectively. Ingestion rates increased with temperature from 14 to 62 bacteria flagellate
-1
h
-1
for the Newfoundland isolate and from 30 to 99 bacteria flagellate
-1
h
-1
for the Arctic isolate. While temperature did not affect cell yields (number of protozoa produced divided by number of bacteria consumed), it affected flagellate sizes. This differential effect of temperature on cell yield and cell size resulted in a changing gross growth efficiency (GGE) in terms of biovolume; colder temperatures favored higher GGEs. The comparison of
Q
10
values for growth rates and ingestion rates between the isolates shows that the Arctic isolate is better adapted to extremely cold temperature than the Newfoundland isolate. At seawater-freezing temperature (-1.8°C), the estimated maximum growth rates and maximum ingestion rates are 0.81 day
-1
and 30 bacteria flagellate
-1
h
-1
for the Arctic isolate and 0.54 day
-1
and 12 bacteria flagellate
-1
h
-1
for the Newfoundland isolate. Our findings about psychrophilic nanoflagellates fit the general characteristics of cold-water-dwelling organisms: reduced physiological rates and higher GGEs at lower temperatures. Because of the large and persistent differences between the isolates, we conclude that they are ecotypes adapted to specific environmental conditions.
Formation of Novel Polysaccharides by
Bradyrhizobium japonicum
Bacteroids in Soybean Nodules
Authors: Streeter John G. , Salminen Seppo O. , Whitmoyer Robert E. , Carlson Russell W.
Published: January 2020

Certain strains of
Bradyrhizobium japonicum
form a previously unknown polysaccharide in the root nodules of soybean plants (
Glycine max
(L.) Merr.). The polysaccharide accumulates inside of the symbiosome membrane—the plant-derived membrane enclosing the bacteroids. In older nodules (60 days after planting), the polysaccharide occupies most of the symbiosome volume and symbiosomes become enlarged so that there is little host cytoplasm in infected cells. The two different groups of
B. japonicum
which produce different types of polysaccharide in culture produce polysaccharides of similar composition in nodules. Polysaccharide formed by group I strains (e.g., USDA 5 and USDA 123) is composed of rhamnose, galactose, and 2-
O
-methylglucuronic acid, while polysaccharide formed by group II strains (e.g., USDA 31 and USDA 39) is composed of rhamnose and 4-
O
-methylglucuronic acid. That the polysaccharide is a bacterial product is indicated by its composition plus the fact that polysaccharide formation is independent of host genotype but is dependent on the bacterial genotype. Polysaccharide formation in nodules is common among strains in serogroups 123, 127, 129, and 31, with 27 of 39 strains (69%) testing positive. Polysaccharide formation in nodules is uncommon among other
B. japonicum
serogroups, with only 1 strain in 18 (6%) testing positive.
Total Degradation of EDTA by Mixed Cultures and a Bacterial Isolate
Authors: Nörtemann Bernd
Published: January 2020

A bacterial mixed culture, which was obtained from sewage by a special enrichment procedure, utilized EDTA as the sole source of carbon and nitrogen for growth. High concentrations of mineral salts, particularly CaCl
2
, or the use of a mineral base without nitrogen protected the cells from inactivation after transfer into fresh medium containing 200-mg/liter (0.67 mM) EDTA. The chemical speciation did not influence the biodegradability of EDTA. However, when resting cells of the mixed culture were incubated with EDTA in the presence of an equivalent molar amount of FeCl
3
, the reaction came to a halt before the complete consumption of the substrate. A gram-negative isolate from the mixed population, BNC1, also metabolized EDTA in monoculture. Growth of the pure culture was promoted by biotin or folic acid but was always accompanied by the accumulation of unidentified metabolites and was slow (μ
max
, 0.024 h
-1
) compared with that of the original community (μ
max
, 0.036 h
-1
).
Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7
Authors: Daya-Mishne N , Shabtai Y
Published: January 2020

Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.
Light-dependent degradation of nitrophenols by the phototrophic bacterium Rhodobacter capsulatus E1F1
Authors: Blasco R , Castillo F
Published: January 2020

Rhodobacter capsulatus E1F1, a phototrophic purple nonsulfur bacterium capable of photoassimilating nitrate or nitrite, grew phototrophically in the presence of mono- and dinitrophenols with acetate as a carbon source, the highest growth levels being obtained under microaerobic conditions. Utilization of 2,4-dinitrophenol was strictly light dependent, was inhibited by O2 and by ammonium, and took place with the simultaneous and stoichiometric production of 2-amino-4-nitrophenol, which accumulated in the medium and was poorly used for further growth in anaerobiosis. Metabolism of mononitrophenols was also light dependent but was activated by O2 and by ammonium. Metabolism of nitrophenols seemed to depend on inducible systems which were repressed in nitrogen-starved cells. Induction of the in vivo 2,4-dinitrophenol reducing system was strongly inhibited by chloramphenicol.
IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis
Authors: Romero D A , Klaenhammer T R
Published: January 2020

The lactococcal insertion sequence IS946 was used to construct suicide vectors for insertion of heterologous DNA into chromosomal and plasmid sequences of Lactococcus lactis subsp. lactis. Electroporation of L. lactis strains, including the recombination-deficient strain MMS362, with the suicide vector pTRK145 yielded 10(1) to 10(3) transformants per micrograms of DNA. pTRK145 insertions occurred primarily in the chromosome, with one insertion detected in a resident plasmid. Vector-specific probes identified junction fragments that varied among transformants, indicating random insertions of pTRK145.
Detection of Pediococcus spp. in brewing yeast by a rapid immunoassay
Authors: Whiting M , Crichlow M , Ingledew W M , Ziola B
Published: January 2020

A membrane immunofluorescent-antibody test was developed to detect diacetyl-producing Pediococcus contaminants in brewery pitching yeast (yeast [Saccharomyces cerevisiae] slurry collected for reinoculation). Centrifugations at 11 and 5,100 x g separate yeast cells from bacteria and concentrate the bacteria, respectively. Pelleted bacteria resuspended and trapped on a black membrane filter are reacted with monoclonal antibodies specific for cell surface antigens and then with fluorescein-conjugated indicator antibodies. Whether pitching yeast is contaminated with pediococci at 0.001% is determined in less than 4 h. The sensitivity of the assay is 2 orders of magnitude below the Pediococcus detection limit of direct microscopy.
Fluorimetric Detection of a
Bacillus stearothermophilus
Spore-Bound Enzyme, α-
d
-Glucosidase, for Rapid Indication of Flash Sterilization Failure
Authors: Vesley Donald , Langholz Ann C. , Rohlfing Stephen R. , Foltz William E.
Published: January 2020

A biological indicator based on fluorimetric detection within 60 min of a
Bacillus stearothermophilus
spore-bound enzyme, α-
d
-glucosidase, has been developed. Results indicate that the enzyme survived slightly longer than spores observed after 24 h of incubation. The new system shows promise for evaluating flash sterilization cycles within 60 min compared with conventional 24-h systems.
Comparison of assimilable organic carbon and UV-oxidizable carbon for evaluation of ultrapure-water systems
Authors: Governal R A , Yahya M T , Gerba C P , Shadman F
Published: January 2020

Bacterial growth potential was measured in an ultrapure-water pilot plant by modified assimilable organic carbon (AOC) and UV-oxidizable carbon tests. An ion-exchange unit increased UV-oxidizable carbon, yet did not significantly (P greater than or equal to 0.05) alter AOC values. UV radiation decreased UV-oxidizable carbon and increased AOC.
Preparation of Encapsulated Microbial Cells for Environmental Applications
Authors: Stormo Keith E. , Crawford Ronald L.
Published: January 2020

An improved method for the encapsulation of bacteria into microspheres of alginate, agarose, or polyurethane is described. Cell suspensions were passed through a low-pressure nozzle into an aqueous phase where matrix polymerization or gelation yielded beads 2 to 50 μm in diameter. Trials with a chlorophenol-degrading
Flavobacterium
species showed that cells entrapped by these procedures were as catabolically active as free cells. These types of beads should have numerous applications in the fields of environmental science and engineering.
Effect of Yeast Extract and Vitamin B
12
on Ethanol Production from Cellulose by
Clostridium thermocellum
I-1-B
Authors: Sato Kanji , Goto Shingo , Yonemura Sotaro , Sekine Kenji , Okuma Emiko , Takagi Yoshio , Hon-Nami Koyu , Saiki Takashi
Published: January 2020

Addition to media of yeast extract, a vitamin mixture containing vitamin B
12
, biotin, pyridoxamine, and
p
-aminobenzoic acid, or vitamin B
12
alone enhanced formation of ethanol but decreased lactate production in the fermentation of cellulose by
Clostridium thermocellum
I-1-B. A similar effect was not observed with
C. thermocellum
ATCC 27405 and JW20.
Antifungal activity of n-tributyltin acetate against some common yam rot fungi
Authors: Olurinola P F , Ehinmidu J O , Bonire J J
Published: January 2020

The antifungal activity of n-tributyltin acetate (TBTA) was examined in relation to combating yam rot disease. TBTA exhibited a significant effect in vitro and in vivo on four yam rot fungal isolates tested. However, the in vitro toxicity of TBTA was drastically reduced when 2.5% Tween 80 was the solvent instead of 25% acetone, as indicated by the MICs of 156.0 and 5.0 micrograms/ml, respectively.