bluuebunny/crossref_metadata_2025 huggingface dataset:
On-line visualization of the competitive behavior of antagonistic bacteria
Authors: H�chard Y , Jayat C , Letellier F , Julien R , Cenatiempo Y , Ratinaud M H
Published: January 2020

To study the interaction between cocultured Listeria monocytogenes and an antagonistic Leuconostoc strain producing an anti-Listeria bacteriocin, flow cytometry, a technique allowing on-line and real-time analysis, was used along with classical microbiological methods. Culture methods and flow cytometric measurements of the mixed culture over time point to a bactericidal action of the lactic acid-producing bacterial strain against L. monocytogenes cells.
Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues
Authors: Frenken L G , Egmond M R , Batenburg A M , Bos J W , Visser C , Verrips C T
Published: January 2020

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.
Evidence for Methylotrophic Symbionts in a Hydrothermal Vent Mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge
Authors: Cavanaugh Colleen M. , Wirsen Carl O. , Jannasch H. W.
Published: January 2020

Symbioses between chemolithoautotrophic bacteria and the major macrofaunal species found at hydrothermal vents have been reported for numerous sites in the Pacific Ocean. We present microscopical and enzymatic evidence that methylotrophic bacteria occur as intracellular symbionts in a new species of mytilid mussel discovered at the Mid-Atlantic Ridge hydrothermal vents. Two distinct ultrastructural types of gram-negative procaryotic symbionts were observed within gill epithelial cells by transmission electron microscopy: small coccoid or rod-shaped cells and larger coccoid cells with stacked intracytoplasmic membranes typical of methane-utilizing bacteria. Methanol dehydrogenase, an enzyme diagnostic of methylotrophs, was detected in the mytilid gills, while tests for ribulose-1,5-bisphosphate carboxylase, the enzyme diagnostic of autotrophy via the Calvin cycle, were negative. Stable carbon isotope values (δ
13
C) of mytilid tissue (−32.7 and −32.5% for gill and foot tissues, respectively) fall within the range of values reported for Pacific vent symbioses but do not preclude the use of vent-derived methane reported to be isotopically heavy relative to biogenically produced methane.
Rapid and sensitive detection of Campylobacter spp. in chicken products by using the polymerase chain reaction
Authors: Giesendorf B A , Quint W G , Henkens M H , Stegeman H , Huf F A , Niesters H G
Published: January 2020

The polymerase chain reaction (PCR) after a short enrichment culture was used to detect Campylobacter spp. in chicken products. After the 16S rRNA gene sequence of Campylobacter jejuni was determined and compared with known sequences from other enterobacteria, a primer and probe combination was selected from the region before V3 and the variable regions V3 and V5. With this primer set and probe, 426-bp fragments from C. jejuni, Campylobacter coli, and Campylobacter lari could be amplified. The detection limit of the PCR was 12.5 CFU. Chicken samples inoculated with 25 CFU of Campylobacter spp. per g were PCR positive after an 18-h enrichment, which resulted in 500 CFU/ml of culture broth. This PCR-culture assay was compared with the conventional method on naturally infected chicken products. Both methods detected the same number of positive and negative samples; however, the results of the PCR-culture assay were available within 48 h.
The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase
Authors: Fliege R , Tong S , Shibata A , Nickerson K W , Conway T
Published: January 2020

The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments.
Microbial transformation of deoxynivalenol (vomitoxin)
Authors: He P , Young L G , Forsberg C
Published: January 2020

Microbial inocula from rumen fluid, soil, and contents of the large intestines of chickens (CLIC) and of swine (SLIC) were tested for their ability to transform deoxynivalenol (vomitoxin) in vitro. Microorganisms in (CLIC) completely transformed pure vomitoxin, and this activity was retained through six serial subcultures. No alteration of the toxin by incubation with SLIC was detected, whereas 35% of the vomitoxin was metabolized in the original culture of rumen fluid and 50% was metabolized by the soil sample, though metabolism was decreased in subsequent subcultures of either sample. A single metabolite was isolated and identified as deepoxy vomitoxin. The increase in concentration of deepoxy vomitoxin in the culture medium corresponded with the decrease in vomitoxin concentration. The vomitoxin transformation rate was not affected by either the ratio of CLIC to vomitoxin (5 to 0.2 g of CLIC per mg of vomitoxin) or the initial concentration of vomitoxin (14 to 1,400 ppm) in the medium. Biotransformation of vomitoxin was completely inhibited when the pH in the medium was lowered to 5.20. Sodium azide at a 0.1% (wt/vol) concentration in the medium blocked the transformation of vomitoxin, suggesting that the deepoxidation of vomitoxin is an energy-dependent process. About 50% of the vomitoxin in moldy corn in culture medium was transformed by microorganisms from CLIC. The vomitoxin transformation rate in moldy corn was not affected when the concentration of CLIC changed from 0.2 to 0.8 g/ml of medium. Vomitoxin in the moldy corn was not transformed when CLIC were added to corn without culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of hydrogen peroxide in loss of culturability mediated by visible light in Escherichia coli in a freshwater ecosystem
Authors: Arana I , Muela A , Iriberri J , Egea L , Barcina I
Published: January 2020

A study was made of the mechanisms by which visible light produces cell dormancy in Escherichia coli, resulting in loss of culturability. Visible light may act directly on the cells or generate photoproducts with a negative effect on the cells. In nonilluminated microcosms the addition of increasing concentrations of hydrogen peroxide, one of the photoproducts formed in natural aquatic systems, gave rise to the formation of nonculturable cells and injured culturable cells, and this negative effect depended on the concentration of peroxide. On the other hand, in illuminated microcosms the addition of compounds which eliminate hydrogen peroxide (i.e., catalase, sodium pyruvate, and thioglycolate) had a protective effect on the E. coli cells, as the CFU counts on minimal medium and on recuperation medium were significantly higher (P < 0.05) than those detected in the absence of these compounds. Furthermore, when hydrogen peroxide was eliminated, the CFU counts on recuperation medium did not fall significantly, indicating that nonculturable cells did not form. These results rule out the direct effect of visible light on the cells and show that hydrogen peroxide, generated photochemically, may be the cause of the loss of culturability of E. coli in illuminated systems.
Heterotrophic bacteria in an air-handling system
Authors: Hugenholtz P , Fuerst J A
Published: January 2020

Heterotrophic bacteria from structural surfaces, drain pan water, and the airstream of a well-maintained air-handling system with no reported building-related illness were enumerated. Visually the system appeared clean, but large populations of bacteria were found on the fin surface of the supply-side cooling coils (10(5) to 10(6) CFU cm-2), in drain pan water (10(5) to 10(7) CFU ml-1), and in the sump water of the evaporative condenser (10(5) CFU ml-1). Representative bacterial colony types recovered from heterotrophic plate count cultures on R2A medium were identified to the genus level. Budding bacteria belonging to the genus Blastobacter dominated the supply surface of the coil fins, the drain pan water, and the postcoil air. These data and independent scanning electron microscopy indicated that a resident population of predominantly Blastobacter bacteria was present as a biofilm on the supply-side cooling coil fins.
Determination of virulence of different strains of Listeria monocytogenes and Listeria innocua by oral inoculation of pregnant mice
Authors: Lammerding A M , Glass K A , Gendron-Fitzpatrick A , Doyle M P
Published: January 2020

A pregnant mouse model was developed to follow the course of infection after peroral inoculation with six different strains of Listeria monocytogenes and one strain of Listeria innocua. Tissues were sampled and analyzed by microbiologic and histologic methods for 5 days postinoculation. In gnotobiotic pregnant BALB/c mice, L. monocytogenes Scott A (SA), serotype 4b, colonized the gastrointestinal tract, translocated to the livers and spleens of mice by day 1 postinoculation, and multiplied in these tissues until day 4. Infection of the placental tissues occurred by days 3 and 4 and was followed by infection of the fetuses. Little damage of colonic and cecal tissues was evident by histologic examination. Livers and spleens showed a cellular immune response; a similar immune response was not detected in the placentas or fetuses. A rough variant of L. monocytogenes SA which was as virulent as the parent strain in mice when injected intraperitoneally was less virulent perorally and did not consistently infect the fetuses. L. monocytogenes ATCC 19113, serotype 3a, did not colonize the gastrointestinal tract, nor was it isolated from any internal tissue. L. monocytogenes strains of serotypes 1/2a and 1/2b behaved like the SA strain in this mouse model. L. innocua colonized the gastrointestinal tract and translocated to the livers and spleens but did not survive in these organs and rapidly disappeared without infecting placental and fetal tissues. In comparison with gnotobiotic mice, conventional pregnant mice inoculated with L. monocytogenes strains showed less consistent infection. These results suggest that the gnotobiotic pregnant mouse is a useful model for detecting differences in virulence relating to colonization, invasiveness, and uteroplacental infection which cannot be detected by intraperitoneal inoculation of mice.
Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths
Authors: Ninet B , Bannerman E , Bille J
Published: January 2020

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.
Species-specific oligonucleotide probes for five Bifidobacterium species detected in human intestinal microflora
Authors: Yamamoto T , Morotomi M , Tanaka R
Published: January 2020

Portions of the 16S rRNA from closely related species of the genus Bifidobacterium that are found in the human intestinal microflora were sequenced in order to design species-specific oligonucleotide probes. Five oligonucleotide probes ranging from 16 to 19 bases in length and complementary to 16S rRNA sequences from Bifidobacterium adolescentis, B. bifidum, B. breve, B. infantis, and B. longum were synthesized. With crude high-molecular-weight RNA preparations as targets, these probes showed the desired species specificity, even down to a 1-nucleotide difference. For the practical evaluation of these probes, their specificity and sensitivity were tested against seven strains of the same species and 54 strains of heterologous bacteria with fixed whole cells as targets. The probes for B. adolescentis, B. breve, and B. longum showed efficient and specific hybridization. Although the probes for B. bifidum and B. infantis cross-reacted with a few bacterial strains not isolated from humans, these probes showed species specificity for human intestinal bacteria. These 16S rRNA probes should prove valuable for the identification and detection of human intestinal Bifidobacterium species.
Cell surface redox potential as a mechanism of defense against photosensitizers in fungi
Authors: Sollod C C , Jenns A E , Daub M E
Published: January 2020

The phytotoxin cercosporin, a singlet oxygen-generating photosensitizer, is toxic to plants, mice, and many fungi, yet the fungi that produce it, Cercospora spp., are resistant. We hypothesize that resistance to cercosporin may result from a reducing environment at the cell surface. Twenty tetrazolium dyes differing in redox potential were used as indicators of cell surface redox potential of seven fungal species differing in resistance to cercosporin. Resistant fungi were able to reduce significantly more dyes than were sensitive fungi. A correlation between dye reduction and cercosporin resistance was also observed when resistance levels of Cercospora species were manipulated by growth on different media. The addition of the reducing agents ascorbate, cysteine, and reduced glutathione (GSH) to growth media decreased cercosporin toxicity for sensitive fungi. None of these agents directly reduced cercosporin at the concentrations at which they protected fungi. Spectral and thin-layer chromatographic analyses of cercosporin solutions containing the different reducing agents indicated that GSH, but not cysteine or ascorbate, reacted with cercosporin. Resistant and sensitive fungi did not differ in endogenous levels of cysteine, GSH, or total thiols. On the basis of data from this and other studies, this report presents a model which proposes that cercosporin resistance results from the production of reducing power at the surfaces of resistant cells, leading to transient reduction and detoxification of the cercosporin molecule.
DNA relatedness among strains of the sweet potato pathogen Streptomyces ipomoea (Person and Martin 1940) Waksman and Henrici 1948
Authors: Labeda D P , Lyons A J
Published: January 2020

DNA relatedness among 28 putative strains of Streptomyces ipomoea from geographically diverse locations and the type strain, NRRL B-12321, was determined spectrophotometrically. The data confirm that these 28 strains are not closely related genetically to the plant-pathogenic species Streptomyces scabies (39% DNA relatedness) or Streptomyces acidiscabies (17% DNA relatedness) or any other major blue-spored Streptomyces species (less than 30% DNA relatedness). Of the 29 strains examined, 4 could be clearly distinguished from S. ipomoea on the basis of morphological criteria, i.e., they had gray rather than blue spores and produced melanin pigment, and their low DNA relatedness to authentic S. ipomoea strains confirmed their original misidentification. The remaining 25 S. ipomoea strains exhibited high DNA relatedness among themselves (76 to 100% homology), even though they had been isolated in different locations throughout the United States and Japan. The avirulent type strain, NRRL B-12321, exhibited slightly lower DNA relatedness with the virulent strains of S. ipomoea (85% average DNA relatedness) than was observed among the virulent strains (average of 96% DNA relatedness).
Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms
Authors: Amann R I , Stromley J , Devereux R , Key R , Stahl D A
Published: January 2020

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.
Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringiensis subsp. kenyae
Authors: Masson L , Moar W J , van Frankenhuyzen K , Bossé M , Brousseau R
Published: January 2020

A protoxin gene, localized to a high-molecular-weight plasmid from Bacillus thuringiensis subsp. kenyae, was cloned on a 19-kb BamHI DNA fragment into Escherichia coli. Characterization of the gene revealed it to be a member of the CryIE toxin subclass which has been reported to be as toxic as the CryIC subclass to larvae from Spodoptera exigua in assays with crude E. coli extracts. To directly test the purified recombinant gene product, the gene was subcloned as a 4.8-kb fragment into an expression vector resulting in the overexpression of a 134-kDa protein in the form of phase-bright inclusions in E. coli. Treatment of solubilized inclusion bodies with either trypsin or gut juice from the silkworm Bombyx mori resulted in the appearance of a protease-resistant 65-kDa protein. In force-feeding bioassays, the purified activated protein was highly toxic to larvae of B. mori but not to larvae of Choristoneura fumiferana. In diet bioassays with larvae from S. exigua, the purified protoxin was nontoxic. However, prior activation of the protoxin by tryptic digestion resulted in the appearance of some toxic activity. These results demonstrate that this new subclass of protein toxin may not be useful for the control of Spodoptera species as previously reported. Hierarchical clustering of the nine known lepidopteran-specific CryI toxin subclasses through multiple sequence alignment suggests that the toxins fall into four possible subgroups or clusters.
Filtration sizes of human immunodeficiency virus type 1 and surrogate viruses used to test barrier materials
Authors: Lytle C D , Tondreau S C , Truscott W , Budacz A P , Kuester R K , Venegas L , Schmukler R E , Cyr W H
Published: January 2020

Filters with well-defined holes were used to determine the effective diameters in buffer of human immunodeficiency virus type 1, herpes simplex virus type 1, and four bacteriophages (phi X174, T7, PRD1, and phi 6), which may serve as surrogate viruses for testing barrier materials. Bacteriophages phi 6 and PRD1 most closely model human immunodeficiency virus type 1 in filtration size.
Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction
Authors: Tsai Y L , Olson B H
Published: January 2020

Polymerase chain reaction was used to amplify the low copy number of two 16S ribosomal gene fragments from soil and sediment extracts. Total DNA for polymerase chain reaction was extracted from 1 g of seeded or unseeded samples by a rapid freeze-and-thaw method. Amplified DNA fragments can be detected in DNA fractions isolated from seeded soil containing less than 3 Escherichia coli cells and from seeded sediments containing less than 10 cells. This research demonstrated that coupling polymerase chain reaction to direct DNA extraction improves sensitivity by 1 and 2 orders of magnitude for sediments and soils, respectively. This technique could become a powerful tool for genetic ecology studies.
Strain selection in carbon-limited chemostats affects reproducibility of Thermoanaerobacter ethanolicus fermentations
Authors: Lacis L S , Lawford H G
Published: January 2020

We found that the reproducibility of chemostat trials can be improved by using chemostat-adapted strains. Our experimental findings are consistent with adaptation that involves an improvement in culture fitness and an alteration of the fermentation genotype.
Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp
Authors: Wernars K , Heuvelman K , Notermans S , Domann E , Leimeister-Wächter M , Chakraborty T
Published: January 2020

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.
Isolation and characterization of cesium-accumulating bacteria
Authors: Tomioka N , Uchiyama H , Yagi O
Published: January 2020

Cesium-accumulating bacteria, strains CS98 and CS402, were isolated from soil by a radioactive autoradiographic method using 137Cs. These strains displayed the rod-coccus growth cycle and contained mesodiaminopimelic acid, mycolic acids, and tuberculostearic acids. The major menaquinone of CS98 was MK-8(H2). On the basis of these characteristics, strain CS98 was identified as Rhodococcus erythropolis and strain CS402 was classified in the genus Rhodococcus. The maximum values of cesium removal efficiencies in the liquid culture containing 10 mumol of cesium per liter for strains CS98 and CS402 were 90 and 47%, respectively. The maximum cesium contents in strains CS98 and CS402 were 52.0 and 18.8 mumol/g (dry weight) of cells, respectively. Maximum values of cesium concentration factors for strains CS98 and CS402 were 3.5 x 10(4) and 3.6 x 10(3), respectively.
Monitoring of Yersinia enterocolitica in murine and bovine feces on the basis of the chromosomally integrated luxAB marker gene
Authors: Kaniga K , Sory M P , Delor I , Saegerman C , Limet J N , Cornelis G R
Published: January 2020

We previously integrated the luxAB gene into the Yersinia enterocolitica chromosome. In this article, we assessed, by luminometry, the survival of the engineered strain KNG1024 in the digestive tracts of mice and cows. In situ detection and a count of the released strain were performed on feces from orally inoculated BALB/c mice for 24 days. This method is a rapid and reliable system for long-term monitoring of genetically engineered bacteria. In cow feces, the count of Y. enterocolitica ranged from 210 to 6,000 CFU/g of feces. This very low count was not detectable by direct luminometry.
Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria
Authors: Greer L E , Robinson J A , Shelton D R
Published: January 2020

Seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria, including Pseudomonas, Alcaligenes, and Bordetella spp., were compared on the basis of growth kinetics. Estimates of maximum growth rate (mu max, k1) and half-saturation growth constant (Ks, k3) were obtained by fitting substrate depletion curves to a four-parameter version of the integrated Monod equation. Estimates of Ks ranged from 2.2 micrograms/ml (10 microM) to 33.8 micrograms/ml (154 microM), and estimates of mu max ranged from 0.20 h-1 (Td = 3.5 h) to 0.32 h-1 (Td = 2.2 h). Estimates of mu max, but not Ks, were affected by changes in initial inoculum density. Maximum growth rates (mu max) were also estimated from turbidity measurements. They ranged from 0.10 h-1 (Td = 6.9 h) to 1.0 h-1 (Td = 0.7 h). There was no correlation between estimates of mu max derived from substrate depletion curves and those derived from turbidity measurements (P = 0.20).
Natamycin as a Fungicide in Agar Media
Authors: Pedersen Jens Christian
Published: January 2020

Fungal inhibition in four commonly used agar media was improved by substituting natamycin (pimaricin) for cycloheximide. The recovery of bacteria was not affected by natamycin, whereas fungal contamination from a variety of samples was significantly suppressed. Furthermore, natamycin lacks the occupational health hazards of cycloheximide. Medium-dependent natamycin degradation occurred during the preparation and refrigerated storage of agar plates, but the addition of natamycin at 21.6 μg/ml resulted in effective residual activity.
Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene
Authors: Théberge M , Lacaze P , Shareck F , Morosoli R , Kluepfel D
Published: January 2020

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.
Analysis of two chondroitin sulfate utilization mutants of Bacteroides thetaiotaomicron that differ in their abilities to compete with the wild type in the gastrointestinal tracts of germfree mice
Authors: Hwa V , Salyers A A
Published: January 2020

Previously, we isolated two mutants of Bacteroides thetaiotaomicron that were unable to grow on the mucopolysaccharide chondroitin sulfate (CS). One of these mutants (46-1) was outcompeted by the wild type in the intestinal tracts of germfree mice, whereas the other mutant (46-4) competed equally with the wild type. In the present article, we report a detailed characterization of these two mutants. Assays of enzymes in the CS utilization pathway revealed that 46-1 did not express one of these enzymes, chondro-6-sulfatase. The absence of chondro-6-sulfatase activity in extracts from 46-1 allowed us to detect a previously unknown activity of another enzyme in the CS breakdown pathway, beta-glucuronidase. In addition to hydrolyzing its normal substrate (an unsulfated disaccharide), beta-glucuronidase also hydrolyzed the 6-sulfated disaccharide subunit of CS. Two-dimensional gel analysis of polypeptides produced by 46-1 showed that several proteins other than the 6-sulfatase were either missing or expressed aberrantly. Thus, 46-1 could be a regulatory mutant. Mutant 46-4 was unable to grow on CS, hyaluronic acid, or disaccharides of CS. Thus, expression of the CS pathway enzymes could not be induced. Nonetheless, the growth pattern of 46-4 and some other findings indicate that the structural genes for these enzymes were still intact. The most likely target of mutant 46-4 is a regulatory locus that is required for expression of CS utilization genes. A surprising characteristic of 46-1 was its inability to grow on heparin, a mucopolysaccharide which is structurally similar to CS but is utilized by a different pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
Fungal metabolism and detoxification of fluoranthene
Authors: Pothuluri J V , Heflich R H , Fu P P , Cerniglia C E
Published: January 2020

Five metabolites produced by Cunninghamella elegans from fluoranthene (FA) in biotransformation studies were investigated for mutagenic activity towards Salmonella typhimurium TA100 and TA104. Whereas FA displayed positive, dose-related mutagenic responses in both tester strains in the presence of a rat liver homogenate fraction, 3-FA-beta-glucopyranoside, 3-(8-hydroxy-FA)-beta-glucopyranoside, FA trans-2,3-dihydrodiol, and 8-hydroxy-FA trans-2,3-dihydrodiol were negative. 9-Hydroxy-FA trans-2,3-dihydrodiol showed a weak positive response in S. typhimurium TA100. Mutagenicity assays performed with samples extracted at 24-h intervals during incubation of C. elegans with FA for 120 h showed that mutagenic activity decreased with time. Comparative studies with rat liver microsomes indicated that FA trans-2,3-dihydrodiol, the previously identified proximal mutagenic metabolite of FA, was the major metabolite. The circular dichroism spectrum of the rat liver microsomal FA trans-2,3-dihydrodiol indicated that it was optically active. In contrast, the circular dichroism spectrum of the fungal FA trans-2,3-dihydrodiol showed no optical activity. These results indicate that C. elegans has the potential to detoxify FA and that the stereochemistry of its trans-2,3-dihydrodiol metabolite reduces its mutagenic potential.
Fumonisin B1 production by Fusarium species other than F. moniliforme in section Liseola and by some related species
Authors: Nelson P E , Plattner R D , Shackelford D D , Desjardins A E
Published: January 2020

Strains of Fusarium proliferatum, F. subglutinans, F. anthophilum, F. annulatum, F. succisae, F. beomiforme, F. dlamini, F. napiforme, and F. nygamai from a variety of substrates and geographic areas were tested for the production of fumonisin B1 in culture. None of the cultures of F. subglutinans (0 of 23), F. annulatum (0 of 1), F. succisae (0 of 2), or F. beomiforme (0 of 15) produced fumonisin B1 in culture. Strains of F. proliferatum (19 of 31; 61%) produced fumonisin B1 in amounts ranging from 155 to 2,936 ppm, strains of F. anthophilum (3 of 17; 18%) produced fumonisin B1 in amounts ranging from 58 to 613 ppm, strains of F. dlamini (5 of 9; 56%) produced fumonisin B1 in amounts ranging from 42 to 82 ppm, strains of F. napiforme (5 of 33; 15%) produced fumonisin B1 in amounts ranging from 16 to 479 ppm, and strains of F. nygamai (10 of 27; 37%) produced fumonisin B1 in amounts ranging from 17 to 7,162 ppm. Of the species tested, F. proliferatum is the most important producer of fumonisin B1 because of its association with corn and animal mycotoxicoses such as porcine pulmonary edema. F. napiforme and F. nygamai also may be important because of their association with the food grains millet and sorghum. At present, F. anthophilum and F. dlamini are of minor importance because they are not associated with corn or other major food grains and have only a limited geographic range. This is the first report of the production of fumonisins by F. anthophilum, F. dlamini, and F. napiforme.
Characterization of culturability, protistan grazing, and death of enteric bacteria in aquatic ecosystems
Authors: González J M , Iriberri J , Egea L , Barcina I
Published: January 2020

Nonstained bacteria (NSB), rhodamine-stained bacteria (RSB), and fluorescence-labeled bacteria (FLB) were prepared from two enteric bacterial species, Escherichia coli and Enterococcus faecalis. Counts of CFU of NSB and RSB and total numbers of RSB and FLB were monitored over time, both in the presence and in the absence of natural microbiota. In the presence of natural microbiota, no differences were observed between CFU counts of NSB and RSB, but RSB total numbers were 1 to 4 orders of magnitude higher than CFU numbers. Therefore, the use of standard bacteriological media causes an important underestimation of the total number of enteric bacteria. In the absence of natural microbiota, the total numbers of NSB, RSB, and FLB remained constant over time. These results showed that RSB are a reliable indicator of the decay in both the total number and the CFU of enteric bacteria in natural water samples. By using RSB, enteric bacteria were classified as culturable cells, nonculturable cells (or somnicells), and dead cells in the presence of natural microbiota. In the presence of natural microbiota, differences between RSB and FLB direct counts were detected for E. coli, but not for E. faecalis. These differences were explained by size-selective grazing. Thus, protistan grazing was found to be the main cause of the decrease in total numbers of enteric bacteria in our experiments.
Purification and Characterization of an α-
l
-Arabinofuranosidase from
Butyrivibrio fibrisolvens
GS113
Authors: Hespell Robert B. , O'Bryan Patricia J.
Published: January 2020

An α-
l
-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of
Butyrivibrio fibrisolvens
GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45°C and was stable to 55°C. The
K

m

and
V
max
for
p
-nitrophenyl-α-
l
-arabinofuranoside were 0.7 mM and 109 μmol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-α-
l
-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since
B. fibrisolvens
GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.
Redox Cycling of Iron Supports Growth and Magnetite Synthesis by
Aquaspirillum magnetotacticum
Authors: Guerin William F. , Blakemore Richard P.
Published: January 2020

Under anaerobic conditions and in the absence of alternative electron acceptors, growth of the magnetic bacterium
Aquaspirillum magnetotacticum
MSI was iron concentration dependent. Weak chelation of the iron (with quinate, oxalate, or 2,3-dihydroxybenzoate) enhanced growth, whereas strong chelation (with EDTA, citrate, or nitrilotriacetic acid) retarded the growth of strain MSI relative to that of controls lacking chelators. Growth was proportional to the percentage of unchelated iron in medium containing EDTA in various molar ratios to iron. Addition of the respiratory inhibitors antimycin A (5 μM), NaCN (10 mM), and NaN
3
(10 mM) inhibited growth with Fe(III) or NO
3
-
as the terminal electron acceptor. Growth with O
2
and NO
3
-
was inhibited by 2-heptyl-4-hydroxyquinolone-
N
-oxide (HOQNO) but not with 2 mM Fe(III). Under strongly reducing conditions, strain MS1 survived but grew poorly and became irreversibly nonmagnetic. Growth and iron reduction in anaerobic cultures were stimulated by the provision of small amounts of O
2
or H
2
O
2
. Slow infusion of air to cultures which had reduced virtually all of the Fe(III) in the medium (2 mM) supported a high rate of iron reoxidation (relative to killed controls) and growth in proportion to the amount of iron reoxidized. Oxygen consumption by iron-reducing cultures was predominantly biological, since NaCN and HOQNO both inhibited consumption. Inhibition of oxygen consumption (and iron reoxidation) by the addition of ferrozine and the inhibition of iron oxidation (and oxygen consumption) by the addition of HOQNO suggest that iron oxidation by strain MS1 is an aerobic respiratory process, perhaps tied to energy conservation. Iron oxidation was also necessary for magnetite synthesis, since in microaerobic denitrifying cultures, sequestration of reduced iron by ferrozine present in 10-fold molar excess to the available iron resulted in loss of magnetism and a severe drop in the average magnetosome number of the cells.
Anaerobic dechlorination of polychlorobiphenyls (Aroclor 1242) by pasteurized and ethanol-treated microorganisms from sediments
Authors: Ye D , Quensen J F , Tiedje J M , Boyd S A
Published: January 2020

A polychlorobiphenyl (PCB)-dechlorinating inoculum eluted from upper Hudson River sediments was treated with either heat or ethanol or both. The treated cultures retained the ability to dechlorinate PCBs (Aroclor 1242) under strictly anaerobic conditions. The dechlorination activity was maintained in serial cultures inoculated with transfers of 1% inoculum when the transferred inoculum was treated each time in the same manner. No methane production was detected in any treated culture, although dechlorination of PCBs in the untreated cultures was always accompanied by methane production. All treated cultures preferentially removed meta chlorines, yielding a dechlorination pattern characterized by accumulation of certain ortho- and para-subsituted congeners such as 2-4-chlorobiphenyl (2-4-CB), 2,4-2-CB, and 2,4-4-CB. In contrast, the untreated cultures showed more extensive dechlorination activities, which almost completely removed both meta and para chlorines from Aroclor 1242. These results suggest that microorganisms responsible for the dechlorination of PCBs in the upper Hudson River sediments can be grouped into two populations according to their responses to the heat and ethanol treatments. Microorganisms surviving the heat and ethanol treatments preferentially remove meta chlorines, while microorganisms lost from the enrichment mainly contribute to the para dechlorination activity. These results indicate that anaerobic sporeformers are at least one of the physiological groups responsible for the reductive dechlorination of PCBs. The selection of a dechlorinating population by such treatments may be an important step in isolation of PCB-dechlorinating microorganisms.
Use of Hydrogen Peroxide Treatment and Crystal Violet Agar Plates for Selective Recovery of Bacteriophages from Natural Environments
Authors: Asghari Abdolkarim , Farrah Samuel R. , Bitton Gabriel
Published: January 2020

Hydrogen peroxide inactivated bacteriophages and bacteria at different rates. A concentration of 0.1% hydrogen peroxide reduced the numbers of several bacteria by an average of 94% but caused an average of 25% inactivation in the numbers of bacteriophages tested. Treating natural samples with hydrogen peroxide selectively reduced the indigenous bacterial flora and permitted better visualization of plaques of lawns of
Escherichia coli
C-3000. In some cases indigenous gram-positive bacteria were relatively resistant to hydrogen peroxide, but their growth could be limited by incorporation of crystal violet into the bottom agar used for plaque assays. The use of hydrogen peroxide treatment and crystal violet-containing plates permitted recovery of more phages from natural samples than did other procedures, such as chloroform pretreatment or the use of selective plating agar such as EC medium.
Aerobic vinyl chloride metabolism in Mycobacterium aurum L1
Authors: Hartmans S , De Bont J A
Published: January 2020

Mycobacterium aurum L1, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain L1 is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain L1, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain L1 and were also identified as Mycobacterium aurum.
Complete oxidation of linear alkylbenzene sulfonate by bacterial communities selected from coastal seawater
Authors: Sigoillot J C , Nguyen M H
Published: January 2020

Anionic surfactants, especially alkylbenzene sulfonates, are discharged into marine areas in great quantities. Because of their poor biodegradability, linear alkylbenzene sulfonates accumulate in seawater and sediments. Bacterial communities that can degrade surfactants were selected from coastal seawater contaminated by urban sewage. All the isolated strains consisted of gram-negative, strictly aerobic rods or helical bacteria. Some of these, though isolated from coastal seawater, did not need sodium for growth and appeared to be related to the genera Alcaligenes and Pseudomonas. Complete surfactant biodegradation was achieved by three important steps: terminal oxidation of the alkyl chain, desulfonation, and aromatic-ring cleavage. Only a few strains were able to carry out the first two steps. The aromatic ring was then cleaved by other strains that possess very specific enzymatic activities. Finally, a number of strains grew on short acids that were end-of-metabolism products of the others.
Conditions for protoplasting, regenerating, and transforming the calicheamicin producer, Micromonospora echinospora
Authors: Love S F , Maiese W M , Rothstein D M
Published: January 2020

We report methods to generate protoplasts, to regenerate mycelia, and to transform Micromonospora echinospora. This actinomycete produces the unusual antitumor antibiotics, the calicheamicins. These protocols may be applied to other actinomycetes that have been difficult to transform. These methods also may facilitate the cloning of calicheamicin biosynthetic genes by genetic complementation.
Use of a genetically engineered Escherichia coli strain to produce 1,2-dihydroxy-4'-chlorobiphenyl
Authors: Khan A A , Walia S K
Published: January 2020

Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.
Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C
Authors: Garcia-Alvarado J S , Labbé R G , Rodriguez M A
Published: January 2020

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).
Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp
Authors: Stoffels G , Nissen-Meyer J , Gudmundsdottir A , Sletten K , Holo H , Nes I F
Published: January 2020

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.