bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a sensitive chemiluminometric assay for the detection of beta-galactosidase in permeabilized coliform bacteria and comparison with fluorometry and colorimetry
Authors: Van Poucke S O , Nelis H J
Published: January 2020

We developed a chemiluminometric assay of beta-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate. Permeabilization of cells is required to ensure the efficient cellular uptake of this compound. By this method, one coliform seeded in 100 ml of sterile water can be detected after a 6- to 9-h propagation phase followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry afforded 4- and 1,000-fold increases in sensitivity and 1- and 6-h increases in the speed of detection, respectively.
Detection of viable Cryptosporidium parvum oocysts by PCR
Authors: Wagner-Wiening C , Kimmig P
Published: January 2020

PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts.
Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans
Authors: Shinjoh M , Tomiyama N , Asakura A , Hoshino T
Published: January 2020

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.
Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose
Authors: Wong T Y , Pei H , Bancroft K , Childers G W
Published: January 2020

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.
PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA
Authors: Thornhill R H , Burgess J G , Matsunaga T
Published: January 2020

PCR primers specific to the 16S ribosomal DNA (rDNA) of magnetic cocci were designed and used to amplify DNA from magnetically isolated magnetic cocci. The PCR products were subcloned by ligation into plasmid vector pCRII, and five clones containing approximately 270-bp fragments of amplified DNA were sequenced. The specific primers were also used to detect magnetic coccus 16S rDNA in environmental samples. Magnetic coccus 16S rDNA was amplified from the water column above sediment kept in an anoxic environment in the laboratory, but little was amplified from a water column kept in an oxic environment. These results suggest that magnetic cocci in the water column in an anoxic environment had migrated there from the sediment as a response to the microoxic or anoxic conditions, rather than having been present previously in a nonmagnetic form and having become magnetic due to these conditions. The specific primers were also used to detect magnetic cocci in aquatic sediment. DNA was extracted from sediment by direct lysis and purified for use as a PCR template by electrophoresis on an agarose-polyvinylpyrrolidone gel. 16S rDNA was then amplified and subcloned, and two clones were sequenced. The clones were screened for chimeric DNA by comparing sections of each with the GenBank database.
Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem
Authors: Maeda M , Chung S Y , Song E , Kudo T
Published: January 2020

Rhodococcus erythropolis TA421 was isolated from a termite ecosystem and is able to degrade a wide range of polychlorinated biphenyl (PCB) congeners. Genetic and biochemical analyses of the PCB catabolic pathway of this organism revealed that there are four different bphC genes (bphC1, bphC2, bphC3, and bphC4) which encode 2,3-dihydroxybiphenyl dioxygenases. As determined by Southern hybridization, none of the bphC genes exhibits homology to any other bphC gene. bphC1, bphC2, and bphC4 encode enzymes that have narrow substrate specificities and cleave the first aromatic ring in the meta position. In contrast, bphC3 encodes a meta cleavage dioxygenase with broad substrate specificity. Asturias et al. have shown that the closely related organism Rhodococcus globerulus P6 contains three different bphC genes (bphC1, bphC2, and bpHC3) which encode meta cleavage dioxygenases. The data suggest that there is a diverse family of bphC genes which encode PCB meta cleavage dioxygenases in members of the genus Rhodococcus.
Nonpigmented and Bacteriochlorophyll-Containing Bradyrhizobia Isolated from Aeschynomene indica
Authors: van Berkum P , Tully R E , Keister D L
Published: January 2020

The legume genus Aeschynomene is unusual, since many species develop stem nodules and the bradyrhizobia isolated from these nodules produce bacteriochlorophyll (Bchl). Evidence is presented that the bradyrhizobia of Aeschynomene indica have wide distribution throughout the world, since A. indica was nodulated when grown in 58 soils collected in 14 different countries. Only 38 of 79 isolates tested synthesized Bchl and carotenoids during heterotrophic growth. Nine isolates produced Bchl constitutively, and cultures were pigmented after growth in the dark. The other isolates required light for Bchl production. The DNA from seven pigmented and three nonpigmented bradyrhizobia hybridized with a DNA probe containing the genes for the photosynthetic apparatus of Rhodobacter capsulatus, but DNA from two other nonpigmented isolates did not hybridize with this probe. A relationship between pigmentation in culture and symbiotic phenotype was not evident, since bradyrhizobia of both Bchl phenotypes nodulated stems of A. indica and formed nitrogen-fixing symbioses. Several isolates, which were ineffective on A. indica, probably do belong to the proposed cross-inoculation group 3 (D. Alazard, Appl. Environ. Microbiol. 50:732-734, 1985), since they did not nodulate Aeschynomene americana or Macroptilium atropurpureum. Since it has been suggested that extant rhizobia arose from photosynthetic ancestors (J. I. Sprent, p. 45-54, in P. M. Gresshoff, L. E. Roth, G. Stacey, and W. E. Newton, ed., Nitrogen Fixation: Achievements and Objectives, 1990), we propose that the nonpigmented isolates may represent an extant lineage of an intermediate evolutionary stage.
Isolation of a cold-sensitive fermentation mutant of a baker's yeast strain and its use in a refrigerated dough process
Authors: Kyogoku Y , Ouchi K
Published: January 2020

Conventional baker's yeast converts sugars in dough into CO2 and ethanol to a significant extent when the dough is stored for days at 5 degrees C. We have isolated Csf (cold-sensitive fermentation) mutants of a commercial baker's yeast by a selection method including as the critical step a nystatin treatment to mutagenized cells at 10 degrees C in the presence of antimycin A. The fermentative activity of mutant strain CSF3 was substantially zero at 5 degrees C and one-fifth that of the parent at 10 degrees C but was restored to the same level as the parental activity at 25 to 40 degrees C. In contrast with the parent, the mutant strain normally produced white bread dough and butter roll dough even after the dough was stored for a week at 5 degrees C.
Expression vectors for the use of eukaryotic luciferases as bacterial markers with different colors of luminescence
Authors: Cebolla A , Vázquez M E , Palomares A J
Published: January 2020

An easy way to identify microorganisms is to provide them with gene markers that confer a unique phenotype. Several genetic constructions were developed to use eukaryotic luciferase genes for bacterial tagging. The firefly and click bettle luciferase genes, luc and lucOR, respectively, were cloned under constitutive control and regulated control from different transcriptional units driven by P1, lambda PR, and Ptrc promoters. Comparison of the expression of each gene in Escherichia coli cells from identical promoters showed that bioluminescence produced by luc could be detected luminometrically in a more sensitive manner. In contrast, luminescence from intact lucOR-expressing cells was much more stable and resistant to high temperatures than that from luc-expressing cells. To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on broad-host-range vectors. Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indicated that both luciferases were poorly expressed from P1 in most bacterial hosts. In contrast, the lambda promoter PR yielded constitutively high levels of luciferase expression in all bacterial species tested. PR activity was not regulated by temperature when the thermosensitive repressor cI857 was present in the bacterial species tested, except for E. coli. In contrast, the regulated lacIq-Ptrc::lucOR fusion expression system behaved in a manner similar to that observed in E. coli cells. After IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, this system produced the highest levels of lucOR expression in all bacterial species tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Isolation and characterization of RNA from low-biomass deep-subsurface sediments
Authors: Ogram A , Sun W , Brockman F J , Fredrickson J K
Published: January 2020

Three methods for the isolation of microbial RNA from low-biomass deep-subsurface sediments have been developed and evaluated. RNA was isolated from samples taken from depths ranging from 173 to 217 m, and samples represented a variety of lithologies, including lacustrine, fluvial sand, and paleosol sediments. Cell numbers in these samples were estimated to be between log 4.0 and log 5.1/g on the basis of phospholipid fatty acid analysis. The most efficient method examined is based on the direct lysis of microbial cells followed by the extraction of RNA with alkaline phosphate buffers and subsequent inactivation of nucleases by extraction with guanidinium isothiocyanate. Estimated recoveries of mRNA for this method are approximately 26%. The recovered RNA included both mRNA and rRNA, as evidenced by the detection of sequences homologous to transcripts from the toluene-4-monooxygenase gene of Pseudomonas mendocina KR1 and bacterial, archaeal, and eukaryotic rRNA. An unexpectedly high relative concentration of archaeal rRNA (22 to 40%) was observed for these samples.
Phenotypic characterization of intestinal Escherichia coli of pigs during suckling, postweaning, and fattening periods
Authors: Katouli M , Lund A , Wallgren P , Kühn I , Söderlind O , Möllby R
Published: January 2020

A highly discriminatory and standardized biochemical fingerprinting method was used to monitor the persistence and colonization of intestinal Escherichia coli isolated from the feces of four sows and their litters (four piglets from each) during the suckling, postweaning, and fattening periods. Altogether, 195 fecal samples were collected and 1,827 E. coli strains were tested (mean number of isolates tested per fecal sample per pig, 9.5). Strains were divided into similarity groups on the basis of their biochemical phenotypes (BPTs). The diversity of E. coli strains in each sample was measured with Simpson's index of diversity, and similarity between E. coli floras of piglets was calculated with a population similarity index. Each fecal sample contained several BPTs of E. coli, some of which dominated that population. The intestinal colonization of piglets consisted of successive waves of different E. coli BPTs, the tenure of which varied from a few days to 2 weeks. Most of these BPTs disappeared in the succeeding samples and were not recovered again from the same piglets. On the other hand, some E. coli strains which colonized piglets early during the suckling period persisted for a long period and were referred to as resident BPTs. Each piglet carried more than one resident BPT (mean of 2.4 BPTs per pig), some of which were also found in other piglets.(ABSTRACT TRUNCATED AT 250 WORDS)
Polyclonal antibodies to chlorosome proteins as probes for green sulfur bacteria
Authors: Cahill A D , Stolz J F
Published: January 2020

We found that polyclonal antibodies raised against chlorosome polypeptides from green sulfur bacteria reacted to Chlorobium tepidum, Chlorobium limicola, and Chlorobium phaeobacteroides but not to Chloroflexus aurantiacus. These antibodies successfully labeled only green sulfur species in marine microbial mat samples. Our results suggest that these antibodies may be useful as immunohistochemical probes.
Reliability of poly B-411, a polymeric anthraquinone-based dye, in determining the rot type caused by wood-inhabiting fungi
Authors: Cookson L J
Published: January 2020

In both Basidiomycotina and Ascomycotina, Poly B-411 decolorization was an excellent indicator of the ability to cause white rot: 109 of the 110 isolates of brown rot fungi tested definitely did not decolorize Poly B-411, and 392 of the 401 mainly active isolates of white rot fungi decolorized Poly B-411. The Bavendamm (tannic acid) reaction was a less reliable test: of 74 white rot isolates examined that could not decay wood, 6 decolorized Poly B-411, but 19 gave positive Bavendamm reactions. Of 80 isolates of Ascomycotina and Deuteromycotina that do not cause white rot, only 4 decolorized Poly B-411, but 17 gave a positive Bavendamm test.
Construction of Lactose-Utilizing Xanthomonas campestris with a Mini-Mu Derivative
Authors: Drahovska H , Turna J
Published: January 2020

Xanthomonas campestris is not able to grow in lactose media. The lactose operon from Escherichia coli as part of a mini-Mu phage was integrated at random sites in the chromosome of this bacterium. Clones expressing (beta)-galactosidase were selected. The resulting strain X. campestris 204, is suitable for production of xanthan gum directly from lactose.
Possible errors in assay for beta-glycosidase activity
Authors: Chadwick R W , Allison J C , Talley D L , George S E
Published: January 2020

Cecal homogenates were assayed for the enzymes beta-glucosidase, beta-glucuronidase, and beta-galactosidase. Anaerobic incubation with the addition of excess 3,4-dichloronitrobenzene, a substrate for nitroreductase, significantly increased the detection of the beta-glycosidase enzymes' activities.
Glutathione-s-transferase activity and metabolism of glutathione conjugates by rhizosphere bacteria
Authors: Zablotowicz R M , Hoagland R E , Locke M A , Hickey W J
Published: January 2020

Glutathione-S-transferase (GST) activity was determined in 36 species of rhizosphere bacteria with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) and in 18 strains with the herbicide alachlor. Highest levels of CDNB-GST activity (60 to 222 nmol (middot) h(sup-1) (middot) mg(sup-1)) were found in gram-negative bacteria: Enterobacter cloacae, Citrobacter diversus, Klebsiella planticola, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, and Xanthomonas campestris. There was very low CDNB-GST activity in the gram-positive strains. Rapid metabolism of CDNB-glutathione conjugates, attributable to high levels of (gamma)-glutamyltranspeptidase, also occurred in the gram-negative bacteria, especially pseudomonads. Alachlor-GST activity detected in cell extracts and whole-cell suspensions of some strains of the families Enterobacteriaceae and Pseudomonaceae was 50- to 100-fold lower than CDNB-GST activity (0.5 to 2.5 nmol (middot) h(sup-1) (middot) mg(sup-1)) and was, for the most part, constitutive. The glutathione-alachlor conjugate was rarely detected. Cysteineglycine and/or cysteine conjugates were the major products of alachlor-GST metabolism. Whole-cell suspensions of certain Pseudomonas spp. dechlorinated from 20 to 75% of 100 (mu)M alachlor in 24 h. Results indicate that rhizosphere bacteria, especially fluorescent pseudomonads, may play an important role in the degradation of xenobiotics such as alachlor via GST-mediated reactions.
Effect of substrate and cell surface hydrophobicity on phosphate utilization in bacteria
Authors: Lemke M J , Churchill P F , Wetzel R G
Published: January 2020

We measured the rates of utilization of hydrophobic and hydrophilic phosphate compounds in gram-negative bacteria with different surface hydrophobicities, isolated from wetland habitats. Three hydrophobic and two hydrophilic bacterial species were selected for study by measuring cell adherence to hydrocarbons. The bacteria were grown under phosphorus-limited conditions with P(infi), hydrophilic (beta)-glycerophosphate, or hydrophobic phosphatidic acid as the phosphate source. Hydrophilic bacteria grew most rapidly on P(infi), followed by (beta)-glycerophosphate. Phosphatidic acid did not support growth or did so at a much later time (40 h) than did the other phosphate treatments. Although all hydrophobic species grew well on these substrates, the rate of growth of two Acinetobacter baumannii isolates on phosphatidic acid exceeded the rate of growth on phosphate or (beta)-glycerophosphate. A membrane phospholipid and lipopolysaccharide were used as a source of phosphorus by hydrophobic species, whereas hydrophilic species could not use the membrane phospholipids and used lipopolysaccharide to a lesser extent. Besides hydrophobic interaction between cells and substrate, phosphatase activity, which was cell bound in hydrophilic species but 30 to 50% unbound in hydrophobic species, affected cell growth. Dialyzed culture supernatant containing phosphatase from hydrophobic species increased the phosphate availability to hydrophilic species. Additionally, cellular extracts from a hydrophilic species, when added to hydrophilic cells, permitted growth on hydrophobic phosphate sources. Naturally occurring amphiphilic humic acids affected the utilization of P(infi) and (beta)-glycerophosphate in bacteria with hydrophilic surfaces but did not affect hydrophobic bacteria. Our results indicate that hydrophobic phosphate sources can be used by bacteria isolated from aquatic environments as the sole phosphorus source for growth. This utilization, in part, appears to be related to cell surface hydrophobicity and extracellular enzyme production.
Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia
Authors: Adhikari T B , Cruz C , Zhang Q , Nelson R J , Skinner D Z , Mew T W , Leach J E
Published: January 2020

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.
Solution (sup13)C Nuclear Magnetic Resonance Spectroscopic Analysis of the Amino Acids of Methanosphaera stadtmanae: Biosynthesis and Origin of One-Carbon Units from Acetate and Carbon Dioxide
Authors: Miller T L , Chen X , Yan B , Bank S
Published: January 2020

We found that general pathways for amino acid synthesis of Methanosphaera stadtmanae, a methanogen that forms CH(inf4) from H(inf2) and methanol, resembled those of methanogens that form CH(inf4) from CO(inf2) or from the methyl group of acetate. We determined the incorporation of (sup14)C-labeled CO(inf2), formate, methanol, methionine, serine, and acetate into cell macromolecules. Labeling of amino acid carbons was determined by solution nuclear magnetic resonance spectroscopy after growth with (sup13)C-labeled acetate, CO(inf2), serine, and methanol. The (alpha) and (beta) carbons of serine and alanine were formed from carboxyl and methyl carbons of acetate, respectively, and the amino acid carboxyl groups were formed from CO(inf2). This indicates that pyruvate was formed by reductive carboxylation of acetate. Labeling of the methyl carbon of methionine indicated that the major route of synthesis was from the hydroxymethyl carbon of serine that arises from the methyl carbon of acetate. Methanol was a minor source of the methyl of methionine. Unambiguous assignment was made of the sources of all carbons of histidine. Labeling of the histidine 7 position ((epsilon) carbon) was consistent with formation from the C-2 of the purine ring of ATP and the origin of the C-2 from a formyl unit derived from the hydroxymethyl carbon of serine.
Sequence analysis of a gene cluster involved in metabolism of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia AC1100
Authors: Daubaras D L , Hershberger C D , Kitano K , Chakrabarty A M
Published: January 2020

Burkholderia cepacia AC1100 utilizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. PT88 is a chromosomal deletion mutant of B. cepacia AC1100 and is unable to grow on 2,4,5-T. The nucleotide sequence of a 5.5-kb chromosomal fragment from B. cepacia AC1100 which complemented PT88 for growth on 2,4,5-T was determined. The sequence revealed the presence of six open reading frames, designated ORF1 to ORF6. Five polypeptides were produced when this DNA region was under control of the T7 promoter in Escherichia coli; however, no polypeptide was produced from the fourth open reading frame, ORF4. Homology searches of protein sequence databases were performed to determine if the proteins involved in 2,4,5-T metabolism were similar to other biodegradative enzymes. In addition, complementation studies were used to determine which genes were essential for the metabolism of 2,4,5-T. The first gene of the cluster, ORF1, encoded a 37-kDa polypeptide which was essential for complementation of PT88 and showed significant homology to putative trans-chlorodienelactone isomerases. The next gene, ORF2, was necessary for complementation and encoded a 47-kDa protein which showed homology to glutathione reductases. ORF3 was not essential for complementation; however, both the 23-kDa protein encoded by ORF3 and the predicted amino acid sequence of ORF4 showed homology to glutathione S-transferases. ORF5, which encoded an 11-kDa polypeptide, was essential for growth on 2,4,5-T, but the amino acid sequence did not show homology to those of any known proteins. The last gene of the cluster, ORF6, was necessary for complementation of PT88, and the 32-kDa protein encoded by this gene showed homology to catechol and chlorocatechol-1,2-dioxygenases.
Characterization of a new pathway for epichlorohydrin degradation by whole cells of xanthobacter strain py2
Authors: Small F J , Tilley J K , Ensign S A
Published: January 2020

The degradation of epichlorohydrin (3-chloropropylene oxide or 1-chloro-2,3-epoxypropane) by whole-cell suspensions of Xanthobacter strain Py2 was investigated. Cell suspensions prepared from cultures grown with propylene as the carbon source readily degraded epichlorohydrin. The ability to degrade epichlorohydrin correlated with the expression of enzymes involved in alkene and epoxide metabolism, since cell suspensions prepared from cultures grown with glucose or acetone, in which the enzymes of alkene and epoxide oxidation are not expressed, did not degrade epichlorohydrin. The alkene monooxygenase-specific inhibitor propyne had no effect on the degradation of epichlorohydrin, demonstrating that alkene monooxygenase is not involved in epichlorohydrin conversion. The interaction of epichlorohydrin and epibromohydrin with the epoxidase which catalyzes aliphatic epoxide conversions was established by showing that the epihalohydrins were specific and potent inhibitors of propylene oxide-dependent O(inf2) consumption by cell suspensions. The rates of degradation of epoxides in whole-cell suspensions decreased in the series propylene oxide > epifluorohydrin > epichlorohydrin > epibromohydrin. The pathway of epichlorohydrin degradation was investigated and found to proceed with stoichiometric dechlorination of epichlorohydrin. The first detectable product of epichlorohydrin degradation was chloroacetone. Chloroacetone was further degraded by the cell suspensions, and in the process, acetone was formed as a nonstoichiometric product. Acetone was further degraded by the cell suspensions with enzymes apparently induced by the accumulation of acetone. The metabolism of allyl chloride (3-chloropropylene) by propylene-grown cells was initiated by alkene monooxygenase and proceeded through epichlorohydrin, chloroacetone, and acetone as intermediate degradation products. These studies reveal a new pathway for halogenated epoxide degradation which involves halogenated and aliphatic ketones as well as other unidentified intermediates and which is unique from previously characterized hydrolytic degradative pathways.
Experimental infection of calves and adult cattle with Escherichia coli O157:H7
Authors: Cray W C , Moon H W
Published: January 2020

Preweaned calves and adult cattle were inoculated with 10(10) CFU of Escherichia coli O157:H7 strain 3081, a calf isolate which produces Shiga-like toxin, to define the magnitude and duration of fecal shedding of E. coli O157:H7 for each age group. Fecal samples of eight of eight, eight of eight, three of eight, and two of eight calves were positive at 2, 7, 14, and 20 weeks, respectively. In contrast, nine of nine, two of nine, and one of nine steers were positive at 2, 7, and 14 weeks, respectively. The magnitude of shedding (CFU per gram) by individual animals at any one time postinoculation varied widely within each age group but was greater for calves as a group. The differences in shedding patterns between adults and calves were statistically significant. After inoculation, 25 of 29 animals remained healthy and 4 of 17 calves had transient diarrhea. Histologic sections of the brain, kidney, jejunum, ileum, cecum, and colon taken at necropsy from nine calves either 3, 14, or 18 days postinoculation or three adults either 2, 3, or 4 days postinoculation were normal. E. coli O157:H7 was recovered from the alimentary tracts of all of the animals necropsied, and there was no evidence of spread to the liver, spleen, or kidneys. Four calves that had ceased shedding were reinfected when inoculated again with the same strain. E. coli O157:H7 was recovered from none of five and two of five adults inoculated with 10(4) and 10(7) CFU, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing
Authors: Gilot P , Andre P
Published: January 2020

Esterases from Listeria monocytogenes strains isolated from cheeses were analyzed by starch gel electrophoresis. Five esterases, numbered from EST 1 to EST 5 in order of decreasing anodal migration, were identified. The EST 1, EST 3, EST 4, and EST 5 set was most active toward alpha-naphthyl propionate, while EST 2 was most active toward alpha-naphthyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed in the population. Twelve esterase patterns were defined and used to subdivide serotypes.
Comparison of two methods for detection of Giardia cysts and Cryptosporidium oocysts in water
Authors: Nieminski E C , Schaefer F W , Ongerth J E
Published: January 2020

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.
Influence of different chemical treatments on transport of Alcaligenes paradoxus in porous media
Authors: Gross M J , Logan B E
Published: January 2020

Seven chemicals, three buffers, and a salt solution known to affect bacterial attachment were tested to quantify their abilities to enhance the penetration of Alcaligenes paradoxus in porous media. Chemical treatments included Tween 20 (a nonionic surfactant that affects hydrophobic interactions), sodium dodecyl sulfate (an anionic surfactant), EDTA (a cell membrane permeabilizer that removes outer membrane lipopolysaccharides), sodium PPi (a surface charge modifier), sodium periodate (an oxidizer that cleaves surface polysaccharides), lysozyme (an enzyme that cleaves cell wall components), and proteinase K (a nonspecific protease that cleaves peptide bonds). Buffers included MOPS [3-(N-morpholino)propanesulfonic acid], Tris, phosphate, and an unbuffered solution containing only NaCl. Transport characteristics in the porous media were compared by using a sticking coefficient, alpha, defined as the rate at which particles stick to a grain of medium divided by the rate at which they strike the grain. Tween 20 reduced alpha by 2.5 orders of magnitude, to alpha = 0.0016, and was the most effective chemical treatment for decreasing bacterial attachment to glass beads in buffered solutions. Similar reductions in alpha were achieved in unbuffered solutions by reducing the solution ionic strength to 0.01 mM. EDTA, protease, and other treatments designed to alter cell structures did not reduce alpha by more than an order of magnitude. The number of bacteria retained by the porous media was decreased by treatments that made A. paradoxus more hydrophobic and less electrostatically charged, although alpha was poorly correlated with electrophoretic mobility and hydrophobicity index measurements at lower alpha values.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of C-terminal direct repeating units of the Streptococcus mutans glucosyltransferase-S in glucan binding
Authors: Lis M , Shiroza T , Kuramitsu H K
Published: January 2020

The C-terminal glucan-binding domain of the glucosyltransferase-S of Streptococcus mutans GS-5 contains five 65-amino-acid direct repeating units. A series of deletion derivatives of both the glucosyltransferase-S and its glucan-binding domain were constructed and analyzed. The results demonstrated that the four C-terminal direct repeating units constituted part of the minimum domain required for glucan binding.
Characterization of Listeria monocytogenes isolated from poultry products and from the poultry-processing environment by random amplification of polymorphic DNA and multilocus enzyme electrophoresis
Authors: Lawrence L M , Gilmour A
Published: January 2020

A total of 289 Listeria monocytogenes strains isolated from a poultry-processing environment and poultry products over a 6-month period were characterized by random amplification of polymorphic DNA, (RAPD) to pinpoint sources of contamination within the plant and gain some measure of the persistence of individual genotypes within this environment. Eighteen RAPD profiles (A through R) were identified within this group, with 64% (184 of 289) of all strains displaying a single RAPD profile, RAPD type A. This genotype was more prevalent in the raw-poultry-processing environment, where, although its origin within this environment appeared to be the incoming birds, it was also widespread on food contact surfaces, floors, and drains. This was the only genotype which persisted throughout the entire 6-month period, and it and RAPD type B were the only two genotypes found in both the raw- and cooked-poultry-processing environments. L. monocytogenes strains isolated from cooked poultry products and the cooked-poultry-processing environment up to 1 year later (17 strains) contained only RAPD types A and B, highlighting the potential which exists for persistent strains to cross-contaminate foods processed in that environment. The other genotypes (C through R) occurred more sporadically, suggesting varied sources of contamination. These were confined to either the raw- or the cooked-poultry-processing environment and were relatively short-lived. Further characterization of a selection of RAPD type A strains, together with strains of RAPD types B through R, was carried out by multilocus enzyme electrophoresis. Strains of RAPD type A contained two electrophoretic types, one of which was serotype 1/2a and the other was 1/2c.(ABSTRACT TRUNCATED AT 250 WORDS)
Nickel-resistant bacteria from anthropogenically nickel-polluted and naturally nickel-percolated ecosystems
Authors: Stoppel R , Schlegel H G
Published: January 2020

DNA fragments harboring the nickel resistance determinants from bacteria isolated from anthropogenically polluted ecosystems in Europe and Zaire were compared with those harboring the nickel resistance determinants from bacteria isolated from naturally nickel-percolated soils from New Caledonia by DNA-DNA hybridization. The biotinylated DNA probes were derived from the previously described Alcaligenes eutrophus CH34, Alcaligenes xylosoxidans 31A, Alcaligenes denitrificans 4a-2, and Klebsiella oxytoca CCUG 15788 and four new nickel resistance-determining fragments cloned from strains isolated from soils under nickel-hyperaccumulating trees. Nine probes were hybridized with endonuclease-cleaved plasmid and total DNA samples from 56 nickel-resistant strains. Some of the New Caledonian strains were tentatively identified as Acinetobacter, Pseudomonas mendocina, Comamonas, Hafnia alvei, Burkholderia, Arthrobacter aurescens, and Arthrobacter ramosus strains. The DNA of most strains showed homologies to one or several of the following nickel resistance determinants: the cnr and ncc operons of the strains A. eutrophus CH34 and A. xylosoxidans 31A, respectively, the nre operon of strain 31A, and the nickel resistance determinants of K. oxytoca. On the basis of their hybridization reactions the nickel resistance determinants of the strains could be assigned to four groups: (i) cnr/ncc type, (ii) cnr/ncc/nre type, (iii) K. oxytoca type, and (iv) others. The majority of the strains were assigned to the known groups. Among the strains from Belgium and Zaire, exclusively the cnr/ncc and the cnr/ncc/nre types were found. Among the New Caledonian strains all four types were represented. Homologies to the nre operon were found only in combination with the cnr/ncc operon. The homologies to the cnr/ncc operon were the most abundant and were detected alone or together with homologies to the nre operon. Only the DNA of the strains isolated from soil in Scotland and the United States and that of five of the New Caledonian strains did not show any detectable homologies to any of our probes. The nickel resistance fragment isolated from Burkholderia strain 32W-2 was studied in some detail. This 15-kb BamHI fragment conferred resistance to 1 to 5 mM NiCl(inf2) to Escherichia coli and resistance to up to 25 mM NiCl(inf2) to A. eutrophus. It showed strong homologies to both the cnr/ncc operon and the nre operon and conferred strictly regulated (inducible) nickel resistance to A. eutrophus.
Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes
Authors: Pedersen K , Larsen J L
Published: January 2020

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.
Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus
Authors: Shin B S , Park S H , Choi S K , Koo B T , Lee S T , Kim J I
Published: January 2020

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.
Rapid large-scale growth of Helicobacter pylori in flasks and fermentors
Authors: Deshpande M , Calenoff E , Daniels L
Published: January 2020

We developed procedures for large-scale cultivation of Helicobacter pylori in flasks and fermentors. Flasks incubated closed under a microaerophilic gas phase with a cotton plug covered by a plastic bag, followed by removal of the bag after 8 h, gave excellent growth. Growth in a 10-liter fermentor led to excessive foaming if the medium was sparged with gas; silicone- or polyglycol-based antifoaming agents were severely inhibitory. Use of fermentor surface gassing, first with a microaerophilic 6% oxygen gas mixture, then with air, and then with 95% oxygen, allowed the culture to grow to an A600 of 2.5 in < 24 h. This method was modified for scale-up to a 100-liter fermentor.
Comparison of Pulsed-Field Gel Electrophoresis DNA Fingerprints of Field Isolates of the Entomopathogen Bacillus popilliae
Authors: Macdonald R , Kalmakoff J
Published: January 2020

Eight strains of the entomopathogen Bacillus popilliae were analyzed by pulsed-field gel electrophoresis, and genomic size estimates of ;sim2,600 to 3,500 kb were obtained. The type strain, ATCC 14706, had a genomic size of 3,395 kb. For the six New Zealand isolates, the degree of similarity in the pulsed-field gel electrophoresis fingerprints may correlate with the geographical closeness of the sites of isolation. The plasmid profiles of the New Zealand isolates were also compared; four of the six strains carry plasmids in the 3.6- to 9.7-kb size range.
Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1
Authors: Latus M , Seitz H , Eberspacher J , Lingens F
Published: January 2020

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.
Metabolism of Hydroxydibenzofurans, Methoxydibenzofurans, Acetoxydibenzofurans, and Nitrodibenzofurans by Sphingomonas sp. Strain HH69
Authors: Harms H , Wilkes H , Wittich R , Fortnagel P
Published: January 2020

The metabolism of 11 substituted dibenzofurans by the dibenzofuran-degrading Sphingomonas sp. strain HH69 was investigated. Strain HH69 utilizes 2-, 3-, and 4-acetoxydibenzofuran as well as 2-, 3-, and 4-hydroxydibenzofuran as sole sources of carbon and energy. The degradation of acetoxydibenzofurans is initiated by hydrolysis of the ester bonds, yielding the corresponding hydroxydibenzofurans and acetate. Strain HH69 grew on 2-methoxydibenzofuran only after it was adapted to the utilization of 5-methoxysalicylic acid, whereas 3- and 4-methoxydibenzofuran as well as 2- and 3-nitrodibenzofuran were only cooxidized. During the breakdown of all eight hydroxy-, methoxy-, and nitrodibenzofurans studied here, the corresponding substituted salicylic acids accumulated in the culture broth. In the cases of 2- and 3-hydroxydibenzofuran as well as 2- and 3-nitrodibenzofuran, salicylic acid was also formed. Those four dibenzofurans which did not serve as carbon sources for strain HH69 were converted to a nonutilizable salicylic acid derivative. From turnover experiments with the mutant HH69/II, which is deficient in meta-cleavage, 2,2(prm1),3,4(prm1)-tetrahydroxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-methoxybiphenyl, 2,2(prm1),3-trihydroxy-5(prm1)-nitrobiphenyl, and 2,2(prm1),3-trihydroxy-4(prm1)-nitrobiphenyl were isolated as the main products formed from 3-hydroxydibenzofuran, 2-methoxydibenzofuran, and 2- and 3-nitrodibenzofuran, respectively. These results indicate significant regioselectivity for the dioxygenolytic cleavage of the ether bond of these monosubstituted dibenzofurans, with a preference for the nonsubstituted aromatic nucleus. Substituted trihydroxybiphenyls are converted further by meta-cleavage followed by the removal of the side chain of the resulting product. A stepwise degradation of this side chain was found to be involved in the metabolism of 2-hydroxydibenzofuran.