bluuebunny/crossref_metadata_2025 huggingface dataset:
An origin of DNA replication from Lactococcus lactis bacteriophage c2
Authors: Waterfield N R , Lubbers M W , Polzin K M , Le Page R W , Jarvis A W
Published: January 2020

An origin of DNA relication was identified in the intergenic region between the early and late gene regions of prolate lactococcal phage c2. A DNA fragment containing this origin, designated ori, was shown to direct DNA replication in Lactococcus lactis but not in Escherichia coli. A comparison of ori with the corresponding regions of other prolate phages revealed strict conservation of the nucleotide sequence in one half of this intergenic region. This conserved region alone would not support DNA replication. No open reading frames were identified in the ori fragment, suggesting that host factors alone are sufficient to initiate DNA replication at ori. A novel class of lactococcal vectors and E. coli-L. lactis shuttle vectors based on ori have been constructed.
Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector
Authors: Wyckoff H A , Barnes M , Gillies K O , Sandine W E
Published: January 2020

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.
Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin
Authors: Faith N G , Shere J A , Brosch R , Arnold K W , Ansay S E , Lee M S , Luchansky J B , Kaspar C W
Published: January 2020

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.
Characterization of 16S rRNA genes from oil field microbial communities indicates the presence of a variety of sulfate-reducing, fermentative, and sulfide-oxidizing bacteria
Authors: Voordouw G , Armstrong S M , Reimer M F , Fouts B , Telang A J , Shen Y , Gevertz D
Published: January 2020

Oil field bacteria were characterized by cloning and sequencing of PCR-amplified 16S rRNA genes. A variety of gram-negative, sulfate-reducing bacteria was detected (16 members of the family Desulfovibrionaceae and 8 members of the family Desulfobacteriaceae). In contrast, a much more limited number of anaerobic, fermentative, or acetogenic bacteria was found (one Clostridium sp., one Eubacterium sp., and one Synergistes sp.). Potential sulfide oxidizers and/or microaerophiles (Thiomicrospira, Arcobacter, Campylobacter, and Oceanospirillum spp.) were also detected. The first two were prominently amplified from uncultured production water DNA and represented 28 and 47% of all clones, respectively. Growth on media containing sulfide as the electron donor and nitrate as the electron acceptor and designed for the isolation of Thiomicrospira spp. gave only significant enrichment of the Campylobacter sp., which was shown to be present in different western Canadian oil fields. This newly discovered sulfide oxidizer may provide a vital link in the oil field sulfur cycle by reoxidizing sulfide formed by microbial sulfate or sulfur reduction.
Glucanolytic Actinomycetes Antagonistic to Phytophthora fragariae var. rubi, the Causal Agent of Raspberry Root Rot
Authors: Valois D , Fayad K , Barasubiye T , Garon M , Dery C , Brzezinski R , Beaulieu C
Published: January 2020

A collection of about 200 actinomycete strains was screened for the ability to grow on fragmented Phytophthora mycelium and to produce metabolites that inhibit Phytophthora growth. Thirteen strains were selected, and all produced (beta)-1,3-, (beta)-1,4-, and (beta)-1,6-glucanases. These enzymes could hydrolyze glucans from Phytophthora cell walls and cause lysis of Phytophthora cells. These enzymes also degraded other glucan substrates, such as cellulose, laminarin, pustulan, and yeast cell walls. Eleven strains significantly reduced the root rot index when inoculated on raspberry plantlets.
Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes
Authors: Harmsen H J , Kengen H M , Akkermans A D , Stams A J , de Vos W M
Published: January 2020

In situ hybridization with fluorescent oligonucleotides was used to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors that had been fed for several months with either sucrose or a mixture of volatile fatty acids. Sections of the granules were hybridized with 16S rRNA-targeted oligonucleotide probes for Bacteria, Archaea, specific phylogenetic groups of methanogens, and two syntrophic propionate-oxidizing strains, MPOB and KOPROP1. Cells of the syntrophic strain KOPROP1 were not detected in either type of sludge granules. Hybridizations of the sucrose-fed granules showed an outer layer of mainly bacterial microcolonies with different morphologies. More inwards of these granules, a layer of different methanogenic microcolonies mixed with large colonies of the syntrophic strain MPOB could be detected. The MPOB colonies were intertwined with hydrogen- or formate-consuming methanogens, indicating their syntrophic growth. The granules fed with volatile fatty acids showed an outer layer of mainly bacteria and then a thick layer of Methanosaeta-like methanogens mixed with a few bacteria and a layer of methanogens mixed with syntrophic MPOB microcolonies. The centers of both sludge types consisted of large cavities and methanogenic microcolonies. These results indicate a juxtapositioning of syntrophic bacteria and methanogens and provide additional evidence for a layered microbial architecture of anaerobic granular sludge.
Isolation of Thermus strains from hot composts (60 to 80 degrees C)
Authors: Beffa T , Blanc M , Lyon P F , Vogt G , Marchiani M , Fischer J L , Aragno M
Published: January 2020

High numbers (10(7) to 10(10) cells per g [dry weight]) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70 degrees C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80 degrees C) of the composting process.
Nisin stimulates oxygen consumption by Staphylococcus aureus and Escherichia coli
Authors: Carneiro de Melo A M , Cook G M , Miles R J , Poole R K
Published: January 2020

Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane.
Cloning and targeted disruption of enpg-1, encoding the major in vitro extracellular endopolygalacturonase of the chestnut blight fungus, Cryphonectria parasitica
Authors: Gao S , Choi G H , Shain L , Nuss D L
Published: January 2020

The gene enpg-1, encoding the major extracellular endopolygalacturonase (endoPG) purified from culture filtrates of the chestnut blight fungus, Cryphonectria parasitica, was cloned and characterized. The deduced mature enpg-1 protein product, ENPG-1, had a calculated molecular mass of 34.5 kDa and a pI of 7.2, consistent with empirically derived values for the purified enzyme, and had 66% identity with an endoPG from the maize pathogen Cochliobolus carbonum. Targeted disruption of enpg-1 was accomplished by homologous recombination with a cloned copy of the gene that contained the Escherichia coli hygromycin B phosphotransferase gene (hph) inserted into exon 1. enpg-1 disruption resulted in no reduction in canker formation on dormant American chestnut stems. Unexpectedly, the level of polygalacturonase (PG) activity measured in cankered bark tissue infected with enpg-1 disruptants was indistinguishable from that found in canker tissue infected with virulent strain EP155. Isoelectric focusing and activity gel analysis of PG activity extracted from canker bark tissue revealed ENPG-1 to be a minor (less than 5%) activity component in tissue infected with the virulent strain and to be absent in tissue infected with the disruption mutants. The predominant activity in both canker samples consisted of two previously undetected acidic PG forms that appear absent in C. parasitica culture filtrates. We conclude from these results that the major C. parasitica extracellular endoPG produced in culture, ENPG-1, does not play a significant role in fungal virulence. However, the identification of two acidic PG activities expressed predominantly, if not exclusively, in planta provides new opportunities for examining the importance of PGs in C. parasitica pathogenesis.
Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction
Authors: Moser D P , Nealson K H
Published: January 2020

The growth of bacteria by dissimilatory elemental sulfur reduction is generally associated with obligate anaerobes and thermophiles in particular. Here we describe the sulfur-dependent growth of the facultatively anaerobic mesophile Shewanella putrefaciens. Six of nine representative S. putrefaciens isolates from a variety of environments proved able to grow by sulfur reduction, and strain MR-1 was chosen for further study. Growth was monitored in a minimal medium (usually with 0.05% Casamino Acids added as a growth stimulant) containing 30 mM lactate and limiting concentrations of elemental sulfur. When mechanisms were provided for the removal of the metabolic end product, H2S, measurable growth was obtained at sulfur concentrations of from 2 to 30 mM. Initial doubling times were ca. 1.5 h and substrate independent over the range of sulfur concentrations tested. In the cultures with the highest sulfur concentrations, cell numbers increased by greater than 400-fold after 48 h, reaching a maximum density of 6.8 x 10(8) cells ml-1. Yields were determined as total cell carbon and ranged from 1.7 to 5.9 g of C mol of S(0) consumed-1 in the presence of the amino acid supplement and from 0.9 to 3.4 g of C mol of S(0-1) in its absence. Several lines of evidence indicate that cell-to-sulfur contact is not required for growth. Approaches for the culture of sulfur-metabolizing bacteria and potential ecological implications of sulfur reduction in Shewanella-like heterotrophs are discussed.
Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody
Authors: Lynch N A , Jiang H , Gibson D T
Published: January 2020

A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.
Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage
Authors: Jaquette C B , Beuchat L R , Mahon B E
Published: January 2020

The efficacy of chlorine and hot water treatments in killing Salmonella stanley inoculated onto alfalfa seeds was determined. Treatment of seeds containing 10(2) to 10(3) CFU/g in 100-micrograms/ml active chlorine solution for 5 or 10 min caused a significant (P < or = 0.05) reduction in population, and treatment in 290-micrograms/ml chlorine solution resulted in a significant reduction compared with treatment in 100 micrograms of chlorine per ml. However, concentrations of chlorine of up to 1,010 micrograms/ml failed to result in further significant reductions. Treatment of seeds containing 10(1) to 10(2) CFU of S. stanley per g for 5 min in a solution containing 2,040 micrograms of chlorine per ml reduced the population to undetectable levels (< 1 CFU/g). Treatment of seeds in water for 5 or 10 min at 54 degrees C caused a significant reduction in the S. stanley population, and treatment at > or = 57 degrees C reduced populations to < or = 1 CFU/g. However, treatment at > or = 54 degrees C for 10 min caused a substantial reduction in viability of the seeds. Treatment at 57 or 60 degrees C for 5 min appears to be effective in killing S. stanley without substantially decreasing germinability of seeds. Storage of seeds for 8 to 9 weeks at 8 and 21 degrees C resulted in reductions in populations of S. stanley of about 1 log10 and 2 log10 CFU/g, respectively. The behavior of S. stanley on seeds during soaking germination, sprouting, and refrigerated storage of sprouts was determined. An initial population of 3.29 log10 CFU/g increased slightly during 6 h of soaking, by about 10(3) CFU/g during a 24-h germination period, and by an additional 10 CFU/g during a 72-h sprouting stage. A population of 10(7) CFU/g of mature alfalfa sprouts was detected throughout a subsequent 10-day storage period at 5 degrees C. These studies indicate that while populations of S. stanley can be greatly reduced, elimination of this organism from alfalfa seeds may not be reliably achieved with traditional disinfection procedures. If S. stanley is present on seeds at the initiation of the sprout production process, populations exceeding 10(7) CFU/g can develop and survive on mature sprouts exposed to handling practices used in commercial production and marketing.
Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity
Authors: Biosca E G , Amaro C
Published: January 2020

In this work, the enzymatic activities of selected strains of biotypes 1 and 2 of Vabrio vulnificus were analyzed by using conventional methods and the API ZYM system. The toxic activities of extracellular products (ECPs) were further evaluated by in vitro and in vivo experiments. The ECPs of both biotypes (i) showed high-level hydrolytic activities, (ii) displayed cytotoxicity for fish cell lines, and (iii) were lethal for eels. Exotoxins seem to be proteinaceous since heat treatment of ECP samples destroyed their toxicity. Only biotype 2 strains were virulent for cels, suggesting that host specificity must be related to differences in cell surface properties. Infectivity trials with other fish species also revealed that only biotype 2 strains were virulent.
Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris
Authors: Aamand J , Ahl T , Spieck E
Published: January 2020

Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da. The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR. By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions. The highest level of NOR was observed in N. hamburgensis when cells were growing mixotrophically. Analysis of whole-cell extracts of N. hamburgensis, N. winogradskyi, and N. vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter. The immunological analysis enables detection of the key enzyme of the genus Nitrobacter.
Screening of microorganisms for biodegradation of poly(lactic-acid) and lactic acid-containing polymers
Authors: Torres A , Li S M , Roussos S , Vert M
Published: January 2020

The ability of some microorganisms to use lactic acid stereocopolymers and copolymers with glycolic acid as sole carbon and energy sources was studied under controlled or natural conditions. First, 14 filamentous fungal strains were tested in liquid cultures, adopting total lactic acid consumption, nitrogen source exhaustion, and maximal biomass production as selection criteria. Two strains of Fusarium moniliforme and one strain of Penicillium roqueforti were able to totally assimilate DL-lactic acid, partially soluble racemic oligomers (MW = 1,000), and the nitrogen source. Only one strain of F. moniliforme was able to grow on a poly(lactic acid)-glycolic acid copolymer (MW = 150,000) after 2 months of incubation at 28 degrees C on synthetic agar medium. Mycelium development was examined by scanning electron microscopy. F. moniliforme filaments were observed to grow not only at the copolymer surface but also through the bulk of the copolymer. In a second approach, plates made of a racemic poly(lactic acid) were buried in the soil before being incubated in petri dishes containing mineral agar medium under controlled conditions. Five strains of different filamentous fungi were isolated, and their ability to assimilate racemic poly(lactic acid) oligomers was tested in liquid cultures.
Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis
Authors: Flahaut S , Frere J , Boutibonnes P , Auffray Y
Published: January 2020

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.
Gene transfer of Alcaligenes eutrophus JMP134 plasmid pJP4 to indigenous soil recipients
Authors: DiGiovanni G D , Neilson J W , Pepper I L , Sinclair N A
Published: January 2020

This study evaluated the potential for gene transfer of a large catabolic plasmid from an introduced organism to indigenous soil recipients. The donor organism Alcaligenes eutrophus JMP134 contained the 80-kb plasmid pJP4, which contains genes that code for mercury resistance. Genes on this plasmid plus chromosomal genes also allow degradation of 2,4-dichloruphenoxyacetic acid (2,4-D). When JMP134 was inoculated into a nonsterile soil microcosm amended with 1,000 micrograms of 2,4-D g-1, significant (10(6) g of soil-1) populations of indigenous recipients or transconjugants arose. These transconjugants all contained an 80-kb plasmid similar in size to pJP4, and all degraded 2,4-D. In addition, all transconjugants were resistant to mercury and contained the tfdB gene of pJP4 as detected by PCR. No mercury-resistant, 2,4-D-degrading organisms with large plasmids or the tfdB gene were found in the 2,4-D-amended but uninoculated control microcosm. These data clearly show that the plasmid pJP4 was transferred to indigenous soil recipients. Even more striking is the fact that not only did the indigenous transconjugant population survive and proliferate but also enhanced rates of 2,4-D degradation occurred relative to microcosms in which no such gene transfer occurred. Overall, these data indicate that gene transfer from introduced organisms is an effective means of bioaugmentation and that survival of the introduced organism is not a prerequisite for biodegradation that utilizes introduced biodegradative genes.
Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase
Authors: Williams M D , Rahn J A , Sherman D H
Published: January 2020

A novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (Spodoptera frugiperda) by use of individual baculovirus vectors. This system includes expression of a dehydrase-domain mutant rat fatty acid synthase cDNA and the phbC gene encoding polyhydroxyalkanoate synthase from Alcaligenes eutrophus. The dehydrase-deficient fatty acid synthase provides de novo synthesis of R-(-)-3-hydroxybutyryl-coenzyme A as a premature termination product rather than palmityl-coenzyme A, the normal product of wild-type rat fatty acid synthase. High levels of this mutant multifunctional protein provide a suitable precursor pool of R-(-)-3-hydroxybutyryl-coenzyme A for conversion to poly-3-hydroxybutyrate in insect cells coexpressing the phbC gene product. This strategy for redesigning a poly-3-hydroxybutyrate biosynthetic pathway suggests a new method for generating structurally diverse polyhydroxyalkanoates by metabolic engineering.
Citrate and Sugar Cofermentation in Leuconostoc oenos, a (sup13)C Nuclear Magnetic Resonance Study
Authors: Ramos A , Santos H
Published: January 2020

(sup13)C nuclear magnetic resonance spectroscopy was used to investigate citrate-glucose cometabolism in nongrowing cell suspensions of the wine lactic acid bacterium Leuconostoc oenos. The use of isotopically enriched substrates allowed us to identify and quantify in the end products the carbon atoms derived from each of the substrates supplied; furthermore, it was possible to differentiate between products derived from the metabolism of endogenous carbon reserves and those derived from external substrates. Citrate-sugar cometabolism was also monitored in dilute cell suspensions for comparison with the nuclear magnetic resonance results. A clear metabolic shift of the end products from glucose metabolism was observed when citrate was provided along with glucose: ethanol was replaced by acetate, and 2,3-butanediol was produced. Reciprocally, the production of lactate and 2,3-butanediol from citrate was increased in the presence of glucose. When citrate was cometabolized with glucose, a 10-fold reduction in the intracellular concentration of glucose-6-phosphate was observed, a result in line with the observed citrate-induced stimulation of glucose consumption. The presence of citrate provided additional pathways for NADP(sup+) regeneration and allowed the diversion of sugar carbon to reactions in which ATP was synthesized. The increased growth rates and maximal biomass yields of L. oenos growing on citrate-glucose mixtures resulted from increased ATP synthesis both by substrate-level phosphorylation and by a chemiosmotic mechanism.
Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature
Authors: Graham A F , Mason D R , Peck M W
Published: January 2020

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.
Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments
Authors: Murray A E , Hollibaugh J T , Orrego C
Published: January 2020

The phylogenetic compositions of bacterioplankton assemblages from San Francisco Bay and Tomales Bay, Calif., differed substantially when analyzed by PCR-denaturing gradient gel electrophoresis; these differences are consistent with the results of previous studies demonstrating differences in their metabolic capabilities. PCR-denaturing gradient gel electrophoresis analysis of complex microbial assemblages was sensitive and reliable, and the results were reproducible as shown by experiments with constructed and naturally occurring assemblages.
Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene
Authors: Kawai S , Suzuki H , Yamamoto K , Inui M , Yukawa H , Kumagai H
Published: January 2020

Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined. The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed. The enzymatic properties of the S. bovis malic enzyme were almost identical to those of other malic enzymes previously reported. However, we found that the S. bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate. The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity. A sequence analysis of the cloned fragment revealed the presence of two open reading frames that were 1,299 and 1,170 nucleotides long. The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis.
Studies of Giardia spp. and Cryptosporidium spp. in two adjacent watersheds
Authors: Ong C , Moorehead W , Ross A , Isaac-Renton J
Published: January 2020

Two adjacent British Columbia, Canada, watersheds with similar topographical features were studied. Both the Black Mountain Irrigation District (BMID) and the Vernon Irrigation District (VID) serve rural agricultural communities which are active in cattle ranching. The present study was carried out in five phases, during which a total of 249 surface water samples were tested in the study watersheds. The aims of these phases were to determine levels of parasite contamination in raw water samples collected from the intakes as well as from other sites in each watershed and to investigate cattle in the watersheds as potential sources of parasite contamination of surface drinking water supplies. Giardia cysts were not detected in the raw water samples collected from lake sources at the headwaters of both watersheds but were found in 100% (70 or 70) of water samples collected at the BMID intake and 97% (68 of 70) of water samples collected at the VID intake. Significantly higher levels (P < 0.05) of Giardia cysts were found at the BMID intake (phase 1, 7 to 2,215 cysts per 100 liters; phase 3, 4.6 to 1,880 cysts per 100 liters) when compared with that of the VID intake (2 to 114 cysts per 100 liters). The BMID watershed has a more complex system of surface water sources than the VID watershed. Cattle have access to creeks in the BMID watershed, whereas access is restricted in the VID watershed. Collection of raw water samples from a creek upstream and downstream of a cattle ranch in the BMID watershed showed that the downstream location had significantly higher (P < 0.05) levels (0.6 to 42.9 cysts per 100 liters and 1.4 to 300.0 oocysts per 100 liters) of both Giardia cysts and Cryptosporidium oocysts than those of the upstream location (0.5 to 34.4 cysts per 100 liters and 0.5 to 34.4 oocysts per 100 liters). Peak concentrations of both parasites coincided with calving activity. Fecal samples, collected from cattle in both watersheds, showed 10% (3 of 30) in the BMID and 50% (5 of 10) in the VID watersheds to be Giardia positive. No Cryptosporidium-positive fecal samples were found. Giardia cysts isolated from the BMID watershed were repeatedly infective to gerbils in contrast to those from the VID watershed. The 10 BMID drinking water Giardia isolates retrieved into culture and biotyped showed zymodeme and karyotype heterogeneity. The differences in patterns of parasite contamination and cattle management practices contribute to the unique watershed characteristics observed between two areas which are topographically similar and geographically adjacent.
Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2
Authors: Fouz B , Mazoy R , Lemos M L , del Olmo M J , Amaro C
Published: January 2020

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.
Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin
Authors: Lee M K , You T H , Young B A , Cotrill J A , Valaitis A P , Dean D H
Published: January 2020

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.
Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation
Authors: Townsend G T , Suflita J M
Published: January 2020

We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates.
Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria
Authors: Hovanec T A , DeLong E F
Published: January 2020

Three nucleic acid probes, two for autotrophic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria and one for alpha subdivision nitrite-oxidizing bacteria, were developed and used to study nitrifying bacterial phylotypes associated with various freshwater and seawater aquarium biofilters. Nitrosomonas europaea and related species were detected in all nitrifying seawater systems and accounted for as much as 20% of the total eubacterial rRNA. In contrast, nitrifying bacteria belonging to the beta-proteobacterial subdivision were detected in only two samples from freshwater aquaria showing vigorous nitrification rates. rRNA originating from nitrite-oxidizing alpha subdivision proteobacteria was not detected in samples from either aquarium environment. The data obtained indicate that chemolithotrophic ammonia oxidation in the freshwater aquaria was not due to beta-proteobacterial phylotypes related to members of the genus Nitrosomonas and their close relatives, the organisms usually implicated in freshwater nitrification. It is likely that nitrification in natural environments is even more complex than nitrification in these simple systems and is less well characterized with regard to the microorganisms responsible.
Single-strand conformation polymorphism analysis of PCR-amplified ribosomal DNA internal transcribed spacers to differentiate species of Aspergillus section Flavi
Authors: Kumeda Y , Asao T
Published: January 2020

A novel genetic approach for classifying the species of Aspergillus Section Flavi is described here. This approach consists of PCR amplification of the 5.8S ribosomal DNA-intervening internal transcribed spacer regions (ITS I-5.8S-ITS II) with universal primers and of analysis of the PCR product by the principle of single-strand conformation polymorphism (SSCP). The approximately 570- to 590-bp PCR products were denatured and subjected to electrophoresis on a polyacrylamide gel supplemented with 20% formamide. The SSCP patterns of these species became more distinct by the addition of formamide to the gel and by visualization with ethidium bromide staining. A little interspecific length polymorphism among amplified ribosomal DNAs was enhanced to be detected by PCR-SSCP analysis. This analysis was capable of classifying 67 of the 68 Aspergillus Section Flavi strains tested into the following four groups, regardless of origin: A. flavus/A. oryzae, A. parasiticus/A. sojae, A. tamarii, and A. nomius. The results of restriction fragment length polymorphism analysis with PCR products of the ITS regions were consistent with those of PCR-SSCP analysis, except for A. nomius, which was not clearly differentiated from A. parasiticus/A. sojae. Nonradiolabelled PCR-SSCP analysis is inexpensive and practical to perform without special apparatus or skill and should assist in fungal morphological identification.
Anabolic Incorporation of Oxalate by Oxalobacter formigenes
Authors: Cornick N A , Allison M J
Published: January 2020

Cell-free lysates of the strict anaerobe Oxalobacter formigenes contained the following enzymatic activities: oxalyl coenzyme A reductase, glyoxylate carboligase, tartronic semialdehyde reductase, and glycerate kinase. NAD(P)-linked formate dehydrogenase, serine-glyoxylate aminotransferase, and NAD(P) transhydrogenase activities were not detected. These results support the hypothesis that O. formigenes assimilates carbon from oxalate by using the glycerate pathway, whereby oxalate is reduced to 3-phosphoglycerate before entering common biosynthetic pathways.
Production of thermostable direct hemolysin by Vibrio parahaemolyticus enhanced by conjugated bile acids
Authors: Osawa R , Yamai S
Published: January 2020

The effects of conjugated bile acids, glycocholic acid, and taurocholic acid (TC) on production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus were determined by a reversed passive latex agglutination assay against TDH. The amount of TDH excreted in growth medium containing either glycocholic acid or taurocholic acid (5 mM/liter) was, on a per-cell basis, 4- to 16-fold greater than that excreted in medium without the bile acids. The amounts of TDH released from lysed cells grown with the bile acids (5 mM/liter) were 4- to 32-fold greater than those from lysed cells grown without, suggesting that the bile acids enhanced synthesis of TDH within bacterial cells. These data imply that the conjugated bile acids play a key role in the pathogenicity of V. parahaemolyticus.
PCR-restriction fragment length polymorphism identification and host range of single-spore isolates of the flexible Frankia sp. strain UFI 132715
Authors: Lumini E , Bosco M
Published: January 2020

Twelve single-spore isolates of the flexible Elaeagnus-Frankia strain UFI 132715 fulfilled the third and the fourth of Koch's postulates on both Alnus and Elaeagnus axenic plants. Seminested nifD-nifK PCR-restriction fragment length polymorphisms provided evidence for the genetic uniformity of the single-spore frankiae with the mother strain and its plant reisolates and allowed their molecular identification directly inside Alnus and Elaeagnus nodules. The clonal nature of these single-spore-purified frankiae should allow safe mutagenesis programs, while their flexible phenotype makes them a powerful tool for understanding the molecular interactions between Frankia strains and actinorhizal plants and for identifying Frankia nodulation genes.
Analysis of partial sequences of genes coding for 16S rRNA of actinomycetes isolated from Casuarina equisetifolia nodules in Mexico
Authors: Niner B M , Brandt J P , Villegas M , Marshall C R , Hirsch A M , Valdés M
Published: January 2020

Filamentous bacteria isolated from surface-sterilized nodules of Casuarina equisetifolia trees in México were capable of reducing acetylene, a diagnostic test for nitrogenase, but were unable to nodulate their host. Analysis of partial 16S rRNA gene sequences suggests that the Mexican isolates are not Frankia strains but members of a novel clade.
Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata
Authors: Saha B C , Bothast R J
Published: January 2020

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).
Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay
Authors: Douwes J , Doekes G , Montijn R , Heederik D , Brunekreef B
Published: January 2020

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.