bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138
Authors: Tamaru Y , Araki T , Amagoi H , Mori H , Morishita T
Published: January 2020

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.
Bacillus subtilis Vegetative Catalase Is an Extracellular Enzyme
Authors: Naclerio G , Baccigalupi L , Caruso C , De Felice M , Ricca E
Published: January 2020

Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault.
Population sizes and growth pressure responses of intestinal microfloras of deep-sea fish retrieved from the abyssal zone
Authors: Yano Y , Nakayama A , Yoshida K
Published: January 2020

The intestinal floras of seven deep-sea fish retrieved at depths of from 3,200 to 5,900 m were examined for population sizes and growth responses to pressure. Large populations of culturable bacteria, ranging from 1.1 x 10(sup6) to 3.6 x 10(sup8) cells per ml of contents, were detected when samples were incubated at conditions characteristic of those of the deep sea. Culturable cell counts at in situ pressures were greater than those at atmospheric pressure in all samples. Most of the strains isolated by the spread-plating method at atmospheric pressure later proved barophilic. Barophilic bacteria were the predominant inhabitants of the abyssal fish intestines.
Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8
Authors: Piddington C S , Kovacevich B R , Rambosek J
Published: January 2020

Dibenzothiophene (DBT), a model compound for sulfur-containing organic molecules found in fossil fuels, can be desulfurized to 2-hydroxybiphenyl (2-HBP) by Rhodococcus sp. strain IGTS8. Complementation of a desulfurization (dsz) mutant provided the genes from Rhodococcus sp. strain IGTS8 responsible for desulfurization. A 6.7-kb TaqI fragment cloned in Escherichia coli-Rhodococcus shuttle vector pRR-6 was found to both complement this mutation and confer desulfurization to Rhodococcus fascians, which normally is not able to desulfurize DBT. Expression of this fragment in E. coli also conferred the ability to desulfurize DBT. A molecular analysis of the cloned fragment revealed a single operon containing three open reading frames involved in the conversion of DBT to 2-HBP. The three genes were designated dszA, dszB, and dszC. Neither the nucleotide sequences nor the deduced amino acid sequences of the enzymes exhibited significant similarity to sequences obtained from the GenBank, EMBL, and Swiss-Prot databases, indicating that these enzymes are novel enzymes. Subclone analyses revealed that the gene product of dszC converts DBT directly to DBT-sulfone and that the gene products of dszA and dszB act in concert to convert DBT-sulfone to 2-HBP.
Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk
Authors: Teraguchi S , Shin K , Ozawa K , Nakamura S , Fukuwatari Y , Tsuyuki S , Namihira H , Shimamura S
Published: January 2020

When milk-fed mice were orally inoculated with Clostridium ramosum C1, this strain proliferated in the gut and became the dominant component of the fecal microflora. In this experimental model, bovine lactoferrin (bLF) administered with milk suppressed the proliferation of this strain in vivo and decreased the numbers of C. ramosum and other bacteria in the feces. This bacteriostatic effect of bLF was dependent on the concentration of bLF, the duration of feeding, and the administered dose of C. ramosum C1. Compared with bovine serum albumin, ovalbumin, bovine whey protein isolate, or bovine casein, only bLF showed this specific activity. A similar effect of bLF was observed after oral inoculation with C. ramosum JCM 1298, C. paraputrificum VPI 6372, or C. perfringens ATCC 13124. A hydrolysate prepared by digestion of bLF with porcine pepsin showed the same inhibitory effect on proliferation of C. ramosum in vivo as occurred with undigested bLF. These results indicate that ingested bLF can exert a bacteriostatic effect against clostridia in the gut even after it has been digested to some extent.
Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes
Authors: Bidochka M J , Walsh S R , Ramos M E , Leger R J , Silver J C , Roberts D W
Published: January 2020

The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers.
Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter
Authors: Aagnes T H , Sørmo W , Mathiesen S D
Published: January 2020

In free-living (FL) reindeer eating a natural mixed winter diet dominated by lichens, captive (CF) reindeer fed pure lichens ad libitum, and CF reindeer subsequently starved for 1 day (CS1 reindeer) or 4 days (CS4 reindeer), the dominant rumen anaerobic bacteria were characterized, their population densities were estimated, and ruminal pH and volatile fatty acid concentrations were determined. In the FL reindeer, the total median viable anaerobic bacterial population ranged from 18 x 10(8) to 35 x 10(8) cells per ml of rumen fluid (n = 4), compared with 26 x 10(8) to 34 x 10(8) and 0.09 x 10(8) to 0.1 x 10(8) cells per ml of rumen fluid in CF reindeer (n = 2) and CS4 reindeer (n = 2), respectively. The median bacterial population adhering to the rumen solids ranged from 260 x 10(8) to 450 x 10(8), 21 x 10(8) to 38 x 10(8), and 0.5 x 10(8) cells per g (wet weight) of rumen solids in FL, CF, and CS4 reindeer, respectively. Although there were variations in the rumen bacterial composition among the FL reindeer (n = 4), strains of Bacteroides, Fibrobacter, Streptococcus, and Clostridium dominated in the rumen fluid. Streptococcus spp. and Clostridium spp. were the dominant bacteria in the CF reindeer (n = 2), while in the CS4 reindeer (n = 2) the dominant bacteria were Fusobacterium spp., members of the family Enterobacteriaceae, and Eubacterium spp. Transmission electron micrographs of lichen particles from the rumen of one FL reindeer, one CF reindeer, and one CS4 reindeer show bacteria resembling Bacteroides spp. adhering to the lichen particles, evidently digesting the lichen hyphae from the inside.(ABSTRACT TRUNCATED AT 250 WORDS)
Dot Blot Enzyme-Linked Immunosorbent Assay for Monitoring the Fate of Insecticidal Toxins from Bacillus thuringiensis in Soil
Authors: Tapp H , Stotzky G
Published: January 2020

The release of transgenic plants and microorganisms expressing truncated genes from Bacillus thuringiensis that code for active insecticidal toxins rather than for the inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clay minerals and other soil particles. To monitor the fate of these toxins in soil, a dot blot enzyme-linked immunosorbent assay (ELISA) that detects free and particle-bound toxins from B. thuringiensis subsp. kurstaki and subsp. tenebrionis was developed. The lower limit of detection of the toxins, either free or adsorbed or bound on the clay minerals montmorillonite (M) or kaolinite (K) or on the clay-particle-size fraction separated from soil (by sedimentation according to Stokes' Law), was approximately 3 ng. Antibodies (Ab) to the toxins from B. thuringiensis subsp. kurstaki and from B. thuringiensis subsp. thuringiensis were raised in goats and rabbits, respectively, and each Ab was rendered specific by adsorption onto CNBr-activated Sepharose coupled with the other toxin. The preadsorbed Ab were specific for the toxins from both subspecies, both free and bound on M, K, or the clay-particle-size fraction of soil. The toxins that were added to sterile and nonsterile soil amended with M or K or not amended were detected on the clay-particle-size fraction of the soil after various periods of incubation by the dot blot ELISA. No toxins were detected on the silt- and sand-particle-size fractions. Each dot blot, containing various amounts of toxins and/or clays, was applied to a polyvinylidene difluoride membrane in a dot blot vacuum system. The toxins were still detectable on the clay-particle-size fraction of nonsterile soil after 40 days. This agreed with preliminary results of other studies in this laboratory that when these toxins bind on clay minerals, they become resistant to utilization by microorganisms.
Evaluation of the immunofluorescence procedure for detection of Giardia cysts and Cryptosporidium oocysts in water
Authors: LeChevallier M W , Norton W D , Siegel J E , Abbaszadegan M
Published: January 2020

The accurate determination of the presence of Giardia cysts and Cryptosporidium oocysts in surface waters requires a reliable method for the detection and enumeration of these pathogenic organisms. Published methods have usually reported recovery efficiencies of less than 50% for both cysts and oocysts. Typically, the losses are greater for Cryptosporidium oocysts than they are for Giardia cysts. The purpose of this study was to examine procedures used for sample collection, elution, concentration, and clarification to determine when losses of cysts and oocysts occurred during processing. The results showed that major losses of cysts and oocysts occurred during centrifugation and clarification. Depending on the centrifugation force, oocyst losses of as high as 30% occurred for each centrifugation step. A 1.15-specific-gravity Percoll-sucrose gradient was needed to optimize recovery of oocysts from natural water samples. Minor improvements in the procedure could be accomplished by selecting a filter other than the recommended 1-micron-pore-size (nominal-porosity) polypropylene filter.
Bacterial growth on surfaces: automated image analysis for quantification of growth rate-related parameters
Authors: Moller S , Kristensen C S , Poulsen L K , Carstensen J M , Molin S
Published: January 2020

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surface-growing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surface-bound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.
Molecular cloning of the gene which encodes beta-N-acetylglucosaminidase from a marine bacterium, Alteromonas sp. strain O-7
Authors: Tsujibo H , Fujimoto K , Tanno H , Miyamoto K , Kimura Y , Imada C , Okami Y , Inamori Y
Published: January 2020

The gene encoding the periplasmic beta-N-acetylglucosaminidase (GlcNAcase B) from a marine Alteromonas sp. strain, O-7, was cloned and sequenced. The protein sequence of GlcNAcase B revealed a highly significant homology with Vibrio GlcNAcase and alpha- and beta-chains of human beta-hexosaminidase.
Comparative assessment of the aerobic and anaerobic microfloras of earthworm guts and forest soils
Authors: Karsten G R , Drake H L
Published: January 2020

Aerobic and anaerobic microbial potentials of guts from earthworms (Lumbricus rubellus Hoffmeister and Octolasium lacteum (Oerl.)) collected from a beech forest were evaluated. On the basis of enumeration studies, microbes capable of growth under both aerobic and anaerobic conditions were more numerous in the earthworm intestine than in the beech forest soil from which the worms were obtained. The intestine of worms displayed nearly equivalent aerobic and anaerobic microbial growth potentials; in comparison, soils displayed greater aerobic than anaerobic microbial growth potentials. Hence, the ratio of microbes capable of growth under obligately anaerobic conditions to those capable of growth under aerobic conditions was higher with the worm intestine than with the soil. Process level studies corroborated these population differentials: (i) under anaerobic conditions, worm gut homogenates consumed glucose, cellobiose, or ferulate more readily than did soil homogenates; and (ii) under aerobic conditions, worm gut homogenates consumed cellobiose or oxygen more readily than did soil homogenates. Collectively, these results reinforce the general concept that the earthworm gut is not microbiologically equivalent to soil and also suggest that the earthworm gut might constitute a microhabitat enriched in microbes capable of anaerobic growth and activity.
Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus
Authors: McAlpin C E , Mannarelli B
Published: January 2020

Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification. A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques. Chromosomal DNA of A. flavus var. flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector. Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A. flavus var. flavus digested with either EcoRI or PstI. One of these clones was chosen for further analysis and was subcloned into pUC19. The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K. E. Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups. The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI. The cloned probe may be species specific as it hybridized with the DNA of all isolates of A. flavus tested in addition to strains recognized as varieties of A. flavus (e.g., A. flavus var. oryzae, A. flavus var. parasiticus, and A. flavus var. sojae). pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.
Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents
Authors: Watrin L , Martin-Jezequel V , Prieur D
Published: January 2020

A minimal growth medium containing only nine amino acids and vitamins as the sole carbon and energy sources allowed the growth of Pyrococcus abyssi GE 5, a novel hyperthermophilic sulfur-metabolizing archaeon isolated from deep-sea hydrothermal vents. The generation time in this medium was about 40 min, and cell densities up to 5 x 10(sup8) cells ml(sup-1) were attained. These results are similar to those obtained previously with complex proteinaceous media.
Characterization of a Genomic Region Required for Production of the Antibiotic Pyoluteorin by the Biological Control Agent Pseudomonas fluorescens Pf-5
Authors: Kraus J , Loper J E
Published: January 2020

A 21-kb region required for the biosynthesis of the polyketide antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5 was identified and cloned. Seven previously isolated mutants deficient in pyoluteorin production (Plt(sup-)) had Tn5 insertions spanning the 21-kb region. Sequences flanking Tn5 inserts were cloned from genomic DNA of three Plt(sup-) mutants and used as probes to identify wild-type alleles of the plt loci from a genomic library of Pf-5. Five cosmids containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb region and, when introduced into a Plt(sup-) mutant, partially restored pyoluteorin production. To study the expression of the genes required for pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphotransferase gene, was introduced into the genomic plt region of Pf-5. Carbon sources that influenced pyoluteorin production by Pf-5 had parallel effects on ice nucleation activity of Pf-5 containing a genomic plt::Tn3-nice fusion, indicating that inaZ was transcribed from a promoter of the plt region. Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cucumber spermosphere was delayed in comparison with expression in the cotton spermosphere. This study demonstrates that genes required for pyoluteorin production were expressed in situ by the biological control bacterium.
Root Exudate-Induced Promoter Activity in Pseudomonas fluorescens Mutants in the Wheat Rhizosphere
Authors: van Overbeek L S , van Elsas J D
Published: January 2020

Tn5-B20 (lacZ as reporter gene) transcriptional fusion mutants of Pseudomonas fluorescens R2f were screened for their response to wheat root exudate. Several mutants showed (beta)-galactosidase activity under the influence of wheat root exudate. In one such mutant, RIWE8, gene expression was specifically induced by proline but not by 125 other substrates. This mutant also showed reporter gene induction, albeit to a lesser extent, by exudate of maize and grass roots but not by that of clover roots. In situ promoter activity of RIWE8 was found in Flevo silt loam soil amended with proline but not in water-, arginine-, glutamic acid-, or malic acid-amended soils. Reporter gene expression in RIWE8 was triggered in a model rhizosphere-soil system in the presence of wheat, maize, and grass roots but not in the presence of clover roots or in root-free (bulk) soil. The induction of expression of the reporter gene in soil, using this approach, is suggestive that promoter activity in RIWE8 may be useful for the construction of organisms with rhizosphere-controlled beneficial genes.
Activity of synchronized cells of a steady-state biofilm recirculated reactor during xenobiotic biodegradation
Authors: Ascon-Cabrera M A , Thomas D , Lebeault J M
Published: January 2020

The maintenance of a steady-state biofilm in a continuous-flow fixed-bed reactor, as a consequence of the reproduction-detachment of cells (an interfacial cell physiology phenomenon of steady-state biofilm) during the biodegradation of 2,4,6-trichlorophenol by Pseudomonas cells, was determined. After cell adhesion on an open-pore glass support, the biofilm was formed in a packed-bed recirculated reactor. After the steady-state biofilm was reached, the mechanisms of the interfacial cell detachment (at the biofilm-liquid interface) were determined. It was established that (i) the hydrophobicity of immobilized sessile cells (parent cells) increased (from 50 to 80%) as the dilution rate increased, while the hydrophobicity of detached suspended cells (daughter cells) remained constant (about 45%); and (ii) the immediately detached suspended cells showed a synchronized growth in about three generations. These results indicate that (i) the immobilized sessile and suspended detached cells grew synchronically at the end and at the beginning of the cell cycle, respectively; and (ii) the hydrophobicity difference of immobilized sessile and suspended detached cells permitted the cells detachment. Therefore, it is probable that independent of shear stress (due to recirculated flow), the synchronized growth and hydrophobicity of cells (which vary during the cell cycle) are the main factors permitting the maintenance of a steady-state xenobiotic-degrading biofilm reactor (in which the overall accumulation of biofilm is determined by the average growth rate of the biofilm cells minus the rate of detachment of cells from the biofilm).
Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution
Authors: Kreader C A
Published: January 2020

Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS)
Survival and activity of Pseudomonas sp. strain B13(FR1) in a marine microcosm determined by quantitative PCR and an rRNA-targeting probe and its effect on the indigenous bacterioplankton
Authors: Leser T D , Boye M , Hendriksen N B
Published: January 2020

Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.
Legionella contamination of dental-unit waters
Authors: Atlas R M , Williams J F , Huntington M K
Published: January 2020

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)
Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds
Authors: Zhao T , Doyle M P , Shere J , Garber L
Published: January 2020

The prevalence of Escherichia coli O157:H7 in dairy herds is poorly understood, even though young dairy animals have been reported to be a host. From February to May 1993, 662 fecal samples from 50 control herds in 14 states, and from June to August 1993, 303 fecal samples from 14 case herds in 11 states were collected for isolation of E. coli O157:H7. Case herds were those in which E. coli O157:H7 was isolated from preweaned calves in a previous U.S. Department of Agriculture study, whereas control herds from which E. coli O157:H7 had not been isolated previously were randomly selected from the same states as case herds. Among the control herds, E. coli O157:H7 was isolated from 6 of 399 calves (1.5%) that were between 24 h old and the age of weaning and from 13 of 263 calves (4.9%) that were between the ages of weaning and 4 months. Eleven of 50 control herds (22%) were positive. Among the case herds, E. coli O157:H7 was isolated from 5 of 171 calves (2.9%) that were between 24 h old and the age of weaning and from 7 of 132 calves (5.3%) that were between the ages of weaning and 4 months. Seven of 14 case herds (50%) were positive. Sixteen of 31 isolates were obtained by direct plating, with populations ranging from 10(3) to 10(5) CFU/g. Fifteen of 31 isolates were isolated by enrichment only. Nineteen of the isolates produced both verocytotoxin 1 (VT-1) and VT-2, whereas 12 produced VT-2 only.
Biosynthesis of l-Phenylalanine and l-Tyrosine in the Actinomycete Amycolatopsis methanolica
Authors: Abou-Zeid A , Euverink G , Hessels G I , Jensen R A , Dijkhuizen L
Published: January 2020

Auxotrophic mutants of the actinomycete Amycolatopsis methanolica requiring l-Phe or l-Tyr were isolated and identified as strains lacking prephenate dehydratase (strain GH71) or arogenate dehydrogenase (strain GH70), respectively. A. methanolica thus employs single pathways only for the biosynthesis of these aromatic amino acids. Anion-exchange chromatography of extracts revealed two peaks with Phe as well as Tyr aminotransferase (AT) activity (Phe/Tyr ATI and Phe/Tyr ATII) and three peaks with prephenate AT activity (Ppa ATI to Ppa ATIII). Phe/Tyr ATI and Ppa ATI coeluted and appear to function as the A. methanolica branched-chain amino acid AT. Ppa ATII probably functions as the aspartate AT. Mutant studies showed that Phe/Tyr ATII is the dominant AT in l-Phe biosynthesis and in l-Tyr catabolism but not in l-Tyr biosynthesis. Biochemical studies showed that Ppa ATIII is highly specific for prephenate and provided evidence that Ppa ATIII is the dominant AT in l-Tyr biosynthesis.
Phosphorus-31 and carbon-13 nuclear magnetic resonance study of glucose and xylose metabolism in agarose-immobilized Candida tropicalis
Authors: Lohmeier-Vogel E M , Hahn-Hägerdal B , Vogel H J
Published: January 2020

Candida tropicalis can ferment both hexose and pentose sugars. Here, we have used 31P and 13C nuclear magnetic resonance spectroscopy to study the capacity of this yeast species to metabolize glucose or xylose when immobilized in small (< 1-mm-diameter) agarose beads. Immobilized C. tropicalis metabolizing glucose showed rapid initial growth within the beads. A corresponding drop in the intracellular pH (from 7.8 to 7.25) and hydrolysis of intracellular polyphosphate stores were observed. Although the initial rate of glucose metabolism with immobilized C. tropicalis was similar to the rate observed previously in cell suspensions, a decrease by a factor of 2.5 occurred over 24 h. In addition to ethanol, a significant amount of glycerol was also produced. When immobilized C. tropicalis consumed xylose, cell growth within the beads was minimal. The intracellular pH dropped rapidly by 1.05 pH units to 6.4. Intracellular ATP levels were lower and intracellular Pi levels were higher than observed with glucose-perfused cells. Consumption of xylose by immobilized C. tropicalis was slower than was previously observed for oxygen-limited cell suspensions, and xylitol was the only fermentation product.
Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools
Authors: Balayeva N M , Eremeeva M E , Tissot-Dupont H , Zakharov I A , Raoult D
Published: January 2020

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.
Detection of ammonium-oxidizing bacteria of the beta-subclass of the class Proteobacteria in aquatic samples with the PCR
Authors: Voytek M A , Ward B B
Published: January 2020

The PCR was used as the basis for the development of a sensitive and specific assay for the detection of ammonium-oxidizing bacteria belonging to the beta-subclass of the class Proteobacteria. PCR primers were selected on the basis of nucleic acid sequence data available for seven species of nitrifiers in this subclass. The specificity of the ammonium oxidizer primers was evaluated by testing known strains of nitrifiers, several serotyped environmental nitrifier isolates, and other members of the Proteobacteria, including four very closely related, nonnitrifying species (as determined by rRNA sequence analysis). DNA extracts from 19 bacterio-plankton samples collected from Lake Bonney, Antarctica, and the Southern California Bight were assayed for the presence of ammonium oxidizers. By using a two-stage amplification procedure, ammonium oxidizers were detected in samples collected from both sites. Chemical data collected simultaneously support the occurrence of nitrification and the presence of nitrifiers. This is the first report describing PCR primers specific for ammonium-oxidizing bacteria and the successful amplification of nitrifier genes coding for rRNA from DNA extracts from natural samples. This application of PCR is of particular importance for the detection and study of microbes, such as autotrophic nitrifiers, which are difficult or impossible to isolate from indigenous microbial communities.
Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1
Authors: Lin X , Kelemen D W , Miller E S , Shih J C
Published: January 2020

Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers. By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease. The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined. A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA. Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers. PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions. The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria. The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases. Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.
Biochemical analysis of starch degradation by Ruminobacter amylophilus 70
Authors: Anderson K L
Published: January 2020

Ruminobacter amylophilus is an obligate anaerobe that uses only alpha-linked glucose molecules (i.e., maltose, maltodextrins, and starch) as a source of energy, making it an excellent model for the study of bacterial starch degradation. Constitutive amylase, amylopectinase, and pullulanase activities were found in intracellular and extracellular fractions of R. amylophilus. However, extracellular activities apparently resulted from cell lysis. Both soluble and membrane-bound polysaccharidase activities were detected. Most of the soluble polysaccharidase activity partitioned with the periplasmic cell fraction. No alpha-glucosidase or maltase activity was detected in either the cellular or extracellular fraction. In addition, intact cells of R. amylophilus bound U-14C-starch. This binding could be saturated and was constitutive and sensitive to proteinase K, indicating protein or protein complex mediation. Competition experiments showed that these starch-binding sites had equally high affinities for starch and maltodextrins larger than maltotriose. The sites had a reduced affinity for maltose and virtually no affinities for glucose and nonstarch polysaccharides. These findings suggest that R. amylophilus binds starch molecules to the cell surface as an initial step in transporting the molecule through the outer membrane and into the periplasmic space. Extracellular polysaccharides do not appear to be involved in starch degradation.
Airborne Bacteria in the Atmospheric Surface Layer: Temporal Distribution above a Grass Seed Field
Authors: Lighthart B , Shaffer B T
Published: January 2020

Temporal airborne bacterial concentrations and meteorological conditions were measured above a grass seed field in the Willamette River Valley, near Corvallis, Oreg., in the summer of 1993. The concentration of airborne bacteria had a maximum of 1,368.5 CFU/m(sup3), with a coefficient of variation of 90.5% and a mean of 121.3 CFU/m(sup3). The lowest concentration of bacteria occurred during the predawn hours, with an average of 32.2 CFU/m(sup3), while sunrise and early evening hours had the highest averages (164.7 and 158.1 CFU/m(sup3), respectively). The concentrations of bacteria in the atmosphere varied greatly, with a maximum difference between two 2-min samples of 1,995 CFU/m(sup3). The concentrations of bacteria in the atmosphere could be divided into five time periods during the day that were thought to be related to the local diurnal sea breeze and Pacific Coast monsoon weather conditions as follows: (i) the nighttime minimum concentration, i.e., 2300 to 0600 h; (ii) the sunrise peak concentration, i.e., 0600 to 0800 h; (iii) the midday accumulating concentration, i.e., 0800 to 1515 h; (iv) the late-afternoon sea breeze trough concentration, i.e., 1515 to 1700 h; and (v) the evening decrease to the nighttime minimum concentration, i.e., 1700 to 2300 h. The sunrise peak concentration (period ii) is thought to be a relatively general phenomenon dependent on ground heating by the sun, while the afternoon trough concentration is thought to be a relatively local phenomenon dependent on the afternoon sea breeze. Meteorological conditions are thought to be an important regulating influence on airborne bacterial concentrations in the outdoor atmosphere in the Willamette River Valley.
Nitrate Respiration in Chemoautotrophic Symbionts of the Bivalve Lucinoma aequizonata Is Not Regulated by Oxygen
Authors: Hentschel U , Felbeck H
Published: January 2020

The marine bivalve Lucinoma aequizonata has intracellular chemoautotrophic symbionts residing in the gill tissue. These bacteria are capable of nitrate respiration even under fully saturated oxygen conditions. Nitrate reductase in the symbionts of L. aequizonata appears to be constitutively expressed and without significant regulation by oxygen or nitrate. We discuss the stationary-phase growth state of the symbionts as an explanation for the lack of enzyme induction.
Acid tolerance of enterohemorrhagic Escherichia coli
Authors: Benjamin M M , Datta A R
Published: January 2020

Enterohemorrhagic Escherichia coli (EHEC) strains were tested for their ability to survive in acid pH at 37 degrees C. No loss of viability was observed in an O157:H7 EHEC strain (ATCC 43895) at pH levels of 3.0 and 2.5 for at least 5 h. The level of acid tolerance of most EHEC isolates was very high, similar to that of Shigella flexneri strains. The acid tolerance was dependent on the growth phase and pH of the growth medium.
Effect of heat treatment on survival of, and growth from, spores of nonproteolytic Clostridium botulinum at refrigeration temperatures
Authors: Peck M W , Lund B M , Fairbairn D A , Kaspersson A S , Undeland P C
Published: January 2020

Spores of five type B, five type E, and two type F strains of nonproteolytic Clostridium botulinum were inoculated into tubes of an anaerobic meat medium plus lysozyme to give approximately 10(6) spores per tube. Sets of tubes were then subjected to a heat treatment, cooled, and incubated at 6, 8, 10, 12, and 25 degrees C for up to 60 days. Treatments equivalent to heating at 65 degrees C for 364 min, 70 degrees C for 8 min, and 75 degrees C for 27 min had little effect on growth and toxin formation. After a treatment equivalent to heating at 85 degrees C for 23 min, growth occurred at 6 and 8 degrees C within 28 to 40 days. After a treatment equivalent to heating at 80 degrees C for 19 min, growth occurred in some tubes at 6, 8, 10, or 12 degrees C within 28 to 53 days and at 25 degrees C in all tubes within 15 days. Following a treatment equivalent to heating at 95 degrees C for 15 mine, growth was detected in some tubes incubated at 25 degrees C for fewer than 60 days but not in tubes incubated at 6 to 12 degrees C. The results indicate that heat treatment of processed foods equivalent to maintenance at 85 degrees C for 19 min combined with storage below 12 degrees C and a shelf life of not more than 28 days would reduce the risk of growth from spores of nonproteolytic C. botulinum by a factor of 10(6).(ABSTRACT TRUNCATED AT 250 WORDS)
Purification and structural determination of an inhibitor of starfish oocyte maturation from a Bacillus species
Authors: Toraya T , Maoka T , Tsuji H , Kobayashi M
Published: January 2020

Inhibitors of bacterial origins of starfish oocyte maturation were sought to obtain biologically active substances which act on either hormonal signal transduction or cell cycle regulation. An oocyte maturation-inhibiting substance found in culture fluid of a Bacillus species was purified to homogeneity. This substance possessed the nature of a detergent and specifically inhibited 1-methyladenine-induced oocyte maturation (50% inhibitory concentration, 3.3 microM) but not dithiothreitol-induced maturation. Its total structure was established to be the lactone of 3-hydroxy-13-methyltetradecanoyl-Glu-Leu-Leu-Val-Asp-Leu -Leu through COOH of the carboxy-terminal Leu. This structure is identical to surfactin, although although the configuration of the substance's amino acid residues has not yet been determined. Surfactin was shown to be identical with this substance in its inhibitory effect on starfish oocyte maturation as well as its chromatographic and electrophoretic properties. Therefore, it was concluded that the oocyte maturation-inhibiting substance produced by a Bacillus species is surfactin.
A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides
Authors: Buyer J S
Published: January 2020

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.
Further Evidence that the N(inf2)-Fixing Endophytic Bacterium from the Intercellular Spaces of Sugarcane Stems Is Acetobacter diazotrophicus
Authors: Dong Z , Heydrich M , Bernard K , McCully M E
Published: January 2020

Nitrogen-fixing bacteria, isolated from the sugar solution in intercellular spaces of sugarcane stems, were compared with the type strain of Acetobacter diazotrophicus (PAL-5) and found to be congruent with it in all characters studied. These characters were 37 morphological and biochemical tests, cellular fatty acid composition, and nitrogenase activity. The nitrogenase activity was measured by acetylene reduction and H(inf2) evolution and found to be unusual in that the H(inf2) evolution was suppressed much less than expected by high concentrations of acetylene.