bluuebunny/crossref_metadata_2025 huggingface dataset:
Experimental Escherichia coli O157:H7 carriage in calves
Authors: Brown C A , Harmon B G , Zhao T , Doyle M P
Published: January 2020

Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding.
Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes
Authors: Krueger D M , Cavanaugh C M
Published: January 2020

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins.
Further characterization of Renibacterium salmoninarum extracellular products
Authors: Barton T A , Bannister L A , Griffiths S G , Lynch W H
Published: January 2020

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.
Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin
Authors: Schwartz J L , Potvin L , Chen X J , Brousseau R , Laprade R , Dean D H
Published: January 2020

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.
Pressure-enhanced activity and stability of a hyperthermophilic protease from a deep-sea methanogen
Authors: Michels P C , Clark D S
Published: January 2020

We describe the properties of a hyperthermophilic, barophilic protease from Methanococcus jannaschii, an extremely thermophilic deep-sea methanogen. This enzyme is the first protease to be isolated from an organism adapted to a high-pressure-high-temperature environment. The partially purified enzyme has a molecular mass of 29 kDa and a narrow substrate specificity with strong preference for leucine at the P1 site of polypeptide substrates. Enzyme activity increased up to 116(deg)C and was measured up to 130(deg)C, one of the highest temperatures reported for the function of any enzyme. In addition, enzyme activity and thermostability increased with pressure: raising the pressure to 500 atm increased the reaction rate at 125(deg)C 3.4-fold and the thermostability 2.7-fold. Spin labeling of the active-site serine revealed that the active-site geometry of the M. jannaschii protease is not grossly different from that of several mesophilic proteases; however, the active-site structure may be relatively rigid at moderate temperatures. The barophilic and thermophilic behavior of the enzyme is consistent with the barophilic growth of M. jannaschii observed previously (J. F. Miller et al., Appl. Environ. Microbiol. 54:3039-3042, 1988).
Size and complexity of the nuclear genome of Colletotrichum graminicola
Authors: Randhir R J , Hanau R M
Published: January 2020

DNA reassociation was used to estimate GC content, size, and complexity of the nuclear genomes of Colletotrichum from maize and sorghum. Melting-temperature analysis indicated that the GC content of the maize pathotype DNA was 51% and that the GC content of the sorghum pathotype was 52%. DNA reassociation kinetics employing S1 nuclease digestion and an appropriately modified second-order equation indicated that the genome sizes of the maize and sorghum pathotypes were 4.8 x 10(7) bp, and 5.0 x 10(7) bp, respectively. Genomic reconstruction experiments based on Southern blot hybridization between a cloned single-copy gene, PYR1 (orotate phosphoribosyl transferase), and maize-pathotype DNA confirmed the size of the nuclear genome. The single-copy component of the genomes of both pathotypes was estimated at about 90%. For both pathotypes, ca. 7% of the genome represented repetitive DNA, and 2 to 3% was foldback DNA.
Impact of trichloroethylene and toluene on nitrogen cycling in soil
Authors: Fuller M E , Scow K M
Published: January 2020

The effects of trichloroethylene (TCE) and toluene on soil nitrogen-cycling activities were examined. Ammonium oxidation potential (AOP) was reduced after incubation with as little as 1 microgram of TCE ml-1, and the effects were generally greater when toluene was present and increased with longer exposure. Arginine ammonification potential and denitrification enzyme activity were constant regardless of TCE concentration or the presence of toluene, while nitrite oxidation potential (NOP) exhibited variable sensitivity. KCl-extractable ammonium levels increased dramatically after exposure to 30 and 60 micrograms of TCE ml-1 in the presence of toluene, whereas gamma-irradiated or sodium azide-treated soil incubated with the same concentrations of TCE and toluene showed no increase. Alfalfa-amended soils showed similar decreases in AOP and increases in extractable ammonium during incubation with 60 micrograms of TCE ml-1 and 20 micrograms of toluene ml-1, although most probable number estimates of the ammonium oxidizer population showed no difference between exposed and unexposed soil. AOP and extractable ammonium returned slowly to control levels after 28 days of incubation in the presence of TCE and toluene. Activity assays to which various TCE and toluene concentrations were added indicated that AOP and NOP were relatively more sensitive to these compounds than was arginine ammonification potential. These results indicate that the soil microbial populations responsible for nitrogen cycling exhibit different sensitivities to TCE and toluene and that they may be more susceptible to adverse effects than previously thought.
Isolation and characterization of a chlorinated-pyridinol-degrading bacterium
Authors: Feng Y , Racke K D , Bollag J
Published: January 2020

The isolation of a pure culture of bacteria able to use 3,5,6-trichloro-2-pyridinol (TCP) as a sole source of carbon and energy under aerobic conditions was achieved for the first time. The bacterium was identified as a Pseudomonas sp. and designated ATCC 700113. [2,6-(sup14)C]TCP degradation yielded (sup14)CO(inf2), chloride, and unidentified polar metabolites.
Magnification of tributyl tin toxicity to oyster larvae by bioconcentration in biofilms of Shewanella colwelliana
Authors: Labare M L , Coon S L , Matthias C , Weiner R M
Published: January 2020

The toxic effects of dissolved versus bioconcentrated tributyl tin (TBT) on oyster larvae were compared. Water column TBT levels, which had no effect in solution, inhibited natural attachment and metamorphosis of oyster larvae on bottom surfaces due to bioconcentration by biofilms. This mechanism should be considered when evaluating heavy metal toxicity in the environment.
Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6
Authors: Maltseva O , Oriel P
Published: January 2020

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.
Effects of elevated dissolved CO2 levels on batch and continuous cultures of Aspergillus niger A60: an evaluation of experimental methods
Authors: McIntyre M , McNeil B
Published: January 2020

The effects of elevated levels of dissolved carbon dioxide (dCO2), produced by gassing with CO2-enriched gas mixtures, upon an industrial strain of Aspergillus niger (strain A60) producing citrate and gluconate were quantitatively assessed. Particular attention was paid to the reliability and accuracy of the steam-sterilizable dCO2 probe, especially in the presence of high concentrations of potentially interfering acidic species. The response of the organism to elevated dCO2 levels was assessed by using both batch and chemostat cultures, and the sensitivity of the organism in different growth phases (lag, exponential, and stationary) was examined. Chemostat cultures showed markedly less inhibition (in terms of biomass and organic acid synthesis) than did batch cultures. Studies in batch culture indicated that lag-phase cultures were especially sensitive to elevated dCO2 levels. Overall, the results of this study indicate that previous experimental methods used to examine dCO2 effects in submerged cultures (continuous CO2-enriched gassing of batch cultures from time zero) have been inappropriate and have led to systematic overestimation of the inhibitory effects of dCO2 on mycelial organisms.
Genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples
Authors: Wawer C , Jetten M S , Muyzer G
Published: January 2020

The genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples were determined in order to show in parallel the existing and active members of Desulfovibrio populations. DNA and total RNA were extracted from different anaerobic bioreactor samples; RNA was transcribed into cDNA. Subsequently, PCR was performed to amplify a ca.-440-bp fragment of the [NiFe] hydrogenase large-subunit gene and its mRNA. Denaturing gradient gel electrophoresis analysis was used to separate the PCR products according to their sequence and thereby to visualize the individual community members. Desulfovibrio strains corresponding to amplified [NiFe] hydrogenase transcripts were regarded as metabolically active, because in pure cultures transcripts were detectable in exponentially growing cells but not in cultures in the stationary phase. DNA sequencing and comparative sequence analysis were used to identify the detected organisms on the basis of their [NiFe] hydrogenase sequences. The genes of characterized Desulfovibrio spp. showed a considerable extent of divergence (ca. 30%), whereas sequences obtained from bacterial populations of the bioreactors showed a low level of variation and indicated the coexistence of closely related strains probably belonging to the species Desulfovibrio sulfodismutans. Under methanogenic conditions, all detected populations were active; under denitrifying conditions, no [NiFe] hydrogenase mRNA was visible. Changes in activity and composition of Desulfovibrio populations caused by changes in the environmental conditions could be monitored by using the approach described in this study.
Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum
Authors: Salim K , Haedens V , Content J , Leblon G , Huygen K
Published: January 2020

By using appropriate Corynebacterium glutamicum-Escherichia coli shuttle plasmids, the gene encoding the fibronectin-binding protein 85A (85A) from Mycobacterium tuberculosis was expressed in C. glutamicum, also an actinomycete and nonsporulating gram-positive rod bacterium, which is widely used in industrial amino acid production. The 85A gene was weakly expressed in C. glutamicum under the control of the ptac promoter from E. coli, but it was produced efficiently under the control of the promoter of the cspB gene encoding PS2, one of the two major secreted proteins from C. glutamicum. The 85A protein was produced in various forms, with or without its own signal sequence and with or without the signal sequence and the NH2-terminal (18-amino-acid) mature sequence of PS2. Western blot analysis with monoclonal antibodies raised against the M. tuberculosis antigen 85 complex showed that recombinant 85A protein was present in the corynebacterial cell wall extract and also released in extracellular culture medium. NH2-terminal microsequencing of recombinant 85A secreted by C. glutamicum showed that signal peptide was effectively cleaved off at the predicted site. The recombinant 85A protein was biologically active in vitro, inducing significant secretion of Th1 T-cell cytokines, particularly interleukin-2 and gamma interferon, in spleen cell cultures from mice vaccinated with live Mycobacterium bovis BCG. Heterologous expression of mycobacterial antigens in C. glutamicum now offers a potent tool for further immunological characterization and large scale preparation of these recombinant proteins.
Evidence That Ceriporiopsis subvermispora Degrades Nonphenolic Lignin Structures by a One-Electron-Oxidation Mechanism
Authors: Srebotnik E , Jensen K A , Kawai S , Hammel K E
Published: January 2020

The white-rot fungus Ceriporiopsis subvermispora is able to degrade nonphenolic lignin structures but appears to lack lignin peroxidase (LiP), which is generally thought to be responsible for these reactions. It is well established that LiP-producing fungi such as Phanerochaete chrysosporium degrade nonphenolic lignin via one-electron oxidation of its aromatic moieties, but little is known about ligninolytic mechanisms in apparent nonproducers of LiP such as C. subvermispora. To address this question, C. subvermispora and P. chrysosporium were grown on cellulose blocks and given two high-molecular-weight, polyethylene glycol-linked model compounds that represent the major nonphenolic arylglycerol-(beta)-aryl ether structure of lignin. The model compounds were designed so that their cleavage via one-electron oxidation would leave diagnostic fragments attached to the polyethylene glycol. One model compound was labeled with (sup13)C at C(inf(alpha)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(alpha))-C(inf(beta)) cleavage after one-electron oxidation. The other model compound was labeled with (sup13)C at C(inf(beta)) of its propyl side chain and carried ring alkoxyl substituents that favor C(inf(beta))-O-aryl cleavage after one-electron oxidation. To assess fungal degradation of the models, the high-molecular-weight metabolites derived from them were recovered from the cultures and analyzed by (sup13)C nuclear magnetic resonance spectrometry. The results showed that both C. subvermispora and P. chrysosporium degraded the models by routes indicative of one-electron oxidation. Therefore, the ligninolytic mechanisms of these two fungi are similar. C. subvermispora might use a cryptic LiP to catalyze these C(inf(alpha))-C(inf(beta)) and C(inf(beta))-O-aryl cleavage reactions, but the data are also consistent with the involvement of some other one-electron oxidant.
Development and application of monoclonal antibodies for in situ detection of indigenous bacterial strains in aquatic ecosystems
Authors: Faude U C , Höfle M G
Published: January 2020

Strain-specific monoclonal antibodies (MAbs) were developed for three different bacterial isolates obtained from a freshwater environment (Lake Plusssee) in the spring of 1990. The three isolates, which were identified by molecular methods, were as follows: Cytophaga johnsonae PX62, Comamonas acidovorans PX54, and Aeromonas hydrophila PU7718. These strains represented three species that were detected in high abundance during a set of mesocosm experiments in Lake Plusssee by the direct analysis of low-molecular-weight RNAs from bacterioplankton. We developed one MAb each for the bacterial isolates PX54 and PU7718 that did not show any cross-reactivity with other bacterial strains by immunofluorescence microscopy. Each MAb recognized the general lipopolysaccharide fraction of the homologous strain. These MAbs were tested successfully for their ability to be used for the in situ detection and counting of bacteria in lake water by immunofluorescence microscopy. During the spring of 1993, A. hydrophila PU7718 showed a depth distribution in Lake Plusssee with a pronounced maximum abundance at 6 m, whereas Comamonas acidovorans PX54 showed a depth distribution with a maximum abundance at the surface. The application of these MAbs to the freshwater samples enabled us to determine the cell morphologies and microhabitats of these strains within their natural environment. The presence of as many as 8,000 cells of these strains per ml in their original habitats 3 years after their initial isolation demonstrated the persistence of individual strains of heterotrophic bacteria over long time spans in pelagic habitats.
Inhibition of methanogenesis by methyl fluoride: studies of pure and defined mixed cultures of anaerobic bacteria and archaea
Authors: Janssen P H , Frenzel P
Published: January 2020

Methyl fluoride (fluoromethane [CH(inf3)F]) has been used as a selective inhibitor of CH(inf4) oxidation by aerobic methanotrophic bacteria in studies of CH(inf4) emission from natural systems. In such studies, CH(inf3)F also diffuses into the anaerobic zones where CH(inf4) is produced. The effects of CH(inf3)F on pure and defined mixed cultures of anaerobic microorganisms were investigated. About 1 kPa of CH(inf3)F, similar to the amounts used in inhibition experiments, inhibited growth of and CH(inf4) production by pure cultures of aceticlastic methanogens (Methanosaeta spp. and Methanosarcina spp.) and by a methanogenic mixed culture of anaerobic microorganisms in which acetate was produced as an intermediate. With greater quantities of CH(inf3)F, hydrogenotrophic methanogens were also inhibited. At a partial pressure of CH(inf3)F of 1 kPa, homoacetogenic, sulfate-reducing, and fermentative bacteria and a methanogenic mixed culture of anaerobic microorganisms based on hydrogen syntrophy were not inhibited. The inhibition by CH(inf3)F of the growth and CH(inf4) production of Methanosarcina mazei growing on acetate was reversible. CH(inf3)F inhibited only acetate utilization by Methanosarcina barkeri, which is able to use acetate and hydrogen simultaneously, when both acetate and hydrogen were present. These findings suggest that the use of CH(inf3)F as a selective inhibitor of aerobic CH(inf4) oxidation in undefined systems must be interpreted with great care. However, by a careful choice of concentrations, CH(inf3)F may be useful for the rapid determination of the role of acetate as a CH(inf4) precursor.
In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria)
Authors: Pernthaler J , Alfreider A , Posch T , Andreatta S , Psenner R
Published: January 2020

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.
Studies of the catabolic pathway of degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45: removal of the amino group from 2-aminomuconic semialdehyde
Authors: He Z , Spain J C
Published: January 2020

Pseudomonas pseudoalcaligenes JS45 utilizes nitrobenzene as the sole source of nitrogen, carbon, and energy. Previous studies have shown that degradation of nitrobenzene involves the reduction of nitrobenzene to nitrosobenzene and hydroxylaminobenzene, followed by rearrangement to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. In the present paper, we report the enzymatic reactions responsible for the release of ammonia after ring cleavage. 2-Aminomuconic semialdehyde was oxidized to 2-aminomuconate in the presence of NAD by enzymes in crude extracts. 2-Aminomuconate was subsequently deaminated stoichiometrically to 4-oxalocrotonic acid. No cofactors are required for the deamination. Two enzymes, 2-aminomuconic semialdehyde dehydrogenase and a novel 2-aminomuconate deaminase, distinguished by partial purification of the crude extracts, catalyzed the two reactions. 4-Oxalocrotonic acid was further degraded to pyruvate and acetaldehyde. The key enzyme, 2-aminomuconate deaminase, catalyzed the hydrolytic deamination that released ammonia, which served as the nitrogen source for growth of the organism.
Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater
Authors: Kasuya K , Inoue Y , Tanaka T , Akehata T , Iwata T , Fukui T , Doi Y
Published: January 2020

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.
Purification and partial characterization of a tripeptidase from Pediococcus pentosaceus K9.2
Authors: Simitsopoulou M , Vafopoulou A , Choli-Papadopoulou T , Alichanidis E
Published: January 2020

A tripeptidase was purified from the cytoplasm of Pediococcus pentosaceus K9.2 by anion-exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography. The molecular mass of the enzyme was estimated by gel filtration at 100,000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peptidase showed one protein band of 45,000 Da. Optimal enzyme activity was obtained at pH 7.0 and at 50 degrees C. The peptidase hydrolyzed all tripeptides tested. Cleavage was not observed with dipeptides, oligopeptides, or amino acid-p-nitroanilide derivatives. Strong inhibition of activity was caused by EDTA, 1,10-phenanthroline, dithiothreitol, and beta-mercaptoethanol, whereas phenylmethylsulfonyl fluoride and sulfur-reactive reagents had no effect on peptidase activity. Mg2+, Mn2+, and Ca2+ stimulated the hydrolyzing activity of the enzyme. The 20 N-terminal amino acids of the tripeptidase from P. pentosaceus had 84% identity with those from the corresponding N-terminal region of the tripeptidase from Lactococcus lactis subsp. cremoris Wg2.
A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis
Authors: Sanders J W , Venema G , Kok J
Published: January 2020

A chloride-inducible promoter previously isolated from the chromosome of Lactococcus lactis (J. W. Sanders, G. Venema, J. Kok, and K. Leenhouts, Mol. Gen. Genet., in press) was exploited for the inducible expression of homologous and heterologous genes. An expression cassette consisting of the positive-regulator gene gadR, the chloride-inducible promoter Pgad, and the translation initiation signals of gadC was amplified by PCR. The cassette was cloned upstream of Escherichia coli lacZ, the holin-lysin cassette (lytPR) of the lactococcal bacteriophage r1t, and the autolysin gene of L. lactis, acmA. Basal activity of Pgad resulted in a low level of expression of all three proteins. Growth in the presence of 0.5 M NaCl of a strain containing the gadC::lacZ fusion resulted in a 1,500-fold increase of beta-galactosidase activity. The background activity levels of LytPR and AcmA had no deleterious effects on cell growth, but induction of lysin expression by addition of 0.5 M NaCl resulted in inhibition of growth. Lysis was monitored by following the release of the cytoplasmic marker enzyme PepX. Released PepX activity was maximal at 1 day after induction of lytPR expression with 0.1 M NaCl. Induction of acmA expression resulted in slower release of PepX from the cells. The presence of the inducing agent NaCl resulted in the stabilization of osmotically fragile cells.
Distribution of metabolic activity and phosphate starvation response of lux-tagged Pseudomonas fluorescens reporter bacteria in the barley rhizosphere
Authors: Kragelund L , Hosbond C , Nybroe O
Published: January 2020

The purpose of this study was to determine the metabolic activity of Pseudomonas fluorescens DF57 in the barley rhizosphere and to assess whether sufficient phosphate was available to the bacterium. Hence, two DF57 reporter strains carrying chromosomal luxAB gene fusions were introduced into the rhizosphere. Strain DF57-40E7 expressed luxAB constitutively, making bioluminescence dependent upon the metabolic activity of the cells under defined assay conditions. The DF57-P2 reporter strain responded to phosphate limitation, and the luxAB gene fusion was controlled by a promoter containing regulatory sequences characteristic of members of the phosphate (Pho) regulon. DF57 generally had higher metabolic activity in a gnotobiotic rhizosphere than in the corresponding bulk soil. Within the rhizosphere the distribution of metabolic activity along the root differed between the rhizosphere soil and the rhizoplane, suggesting that growth conditions may differ between these two habitats. The DF57-P2 reporter strain encountered phosphate limitation in a gnotobiotic rhizosphere but not in a natural rhizosphere. This difference in phosphate availability seemed to be due to the indigenous microbial population, as DF57-P2 did not report phosphate limitation when established in the rhizosphere of plants in sterilized soil amended with indigenous microorganisms.
Heat inactivation of Mycobacterium paratuberculosis in raw milk: are current pasteurization conditions effective?
Authors: Stabel J R , Steadham E M , Bolin C A
Published: January 2020

Currently, it is not known whether commercial pasteurization effectively kills Mycobacterium paratuberculosis in contaminated raw milk. Results from holder test tube experiments indicated that a residual population of viable bacteria remained after treatment at 65, 72, 74, or 76 degrees C for 0 to 30 min. Use of a laboratory-scale pasteurizer unit demonstrated that treatment of raw milk at 72 degrees C for 15 s effectively killed all M. paratuberculosis.
Bacterial transformations of naphthothiophenes
Authors: Kropp K G , Andersson J T , Fedorak P M
Published: January 2020

Vol. 63, no. 9, p. 3468, Table 1, subheading under heading "Total recovery": "((delta)mol)" should read "((mu)mol)." [This corrects the article on p. 3463 in vol. 63.].
Cloning of cellobiose phosphoenolpyruvate-dependent phosphotransferase genes: functional expression in recombinant Escherichia coli and identification of a putative binding region for disaccharides
Authors: Lai X , Davis F C , Hespell R B , Ingram L O
Published: January 2020

Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.
Evidence that Formation of Protoanemonin from Metabolites of 4-Chlorobiphenyl Degradation Negatively Affects the Survival of 4-Chlorobiphenyl-Cometabolizing Microorganisms
Authors: Blasco R , Mallavarapu M , Wittich R , Timmis K N , Pieper D H
Published: January 2020

A rapid decline in cell viability of different PCB-metabolizing organisms was observed in soil microcosms amended with 4-chlorobiphenyl. The toxic effect could not be attributed to 4-chlorobiphenyl but was due to a compound formed from the transformation of 4-chlorobiphenyl by the natural microflora. Potential metabolites of 4-chlorobiphenyl, 4-chlorobenzoate and 4-chlorocatechol, caused similar toxic effects. We tested the hypothesis that the toxic effects are due to the formation of protoanemonin, a plant-derived antibiotic, which is toxic to microorganisms and which has been shown to be formed from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. Consistent with our hypothesis, addition to soil microcosms of strains able to reroute intermediary 4-chlorocatechol from the 3-oxoadipate pathway and into the meta-cleavage pathway or able to mineralize 4-chlorocatechol by a modified ortho-cleavage pathway resulted in reversal of this toxic effect. Surprisingly, while direct addition of protoanemonin influenced both the viability of fungi and the microbial activity of the soil microcosm, there was little effect on bacterial viability due to its rapid degradation. This rapid degradation accounts for our inability to detect this compound in soils amended with 4-chlorocatechol. However, significant accumulation of protoanemonin was observed by a mixed bacterial community enriched with benzoate or a mixture of benzoate and 4-methylbenzoate, providing the metabolic potential of the soil to form protoanemonin. The effects of soil heterogeneity and microcosm interactions are discussed in relation to the different effects of protoanemonin when applied as a shock load and when it is produced in small amounts from precursors over long periods.
Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp. medellin and expression in a crystal-negative B. thuringiensis strain
Authors: Thiery I , Delécluse A , Tamayo M C , Orduz S
Published: January 2020

A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp. medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene. The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B. thuringiensis subsp. kyushuensis. The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B. thuringiensis subsp. israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence. The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B. thuringiensis subsp. israelensis in which large inclusions of the Cyt1Ab1 protein were produced. Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain. Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed. The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B. thuringiensis subsp. medellin.
Contrasting bacterial strategies to coexist with a flagellate predator in an experimental microbial assemblage
Authors: Pernthaler J , Posch T , Simek K , Vrba J , Amann R , Psenner R
Published: January 2020

We studied predator-induced changes within a slowly growing mixed microbial assemblage that was sustained by algal exudates in a continuous cultivation system. In situ hybridization with fluorescent monolabeled oligonucleotide probes was used for a tentative community analysis. This method also allowed us to quantify the proportions of predators with ingested bacteria of different taxonomic groups. In addition, we determined grazing rates on bacteria with fluorescently labelled prey. Bacteria belonging to the alpha and beta subdivisions of the phylum Proteobacteria ((alpha)- and (beta)-Proteobacteria, respectively) showed very different responses to the addition of a bacterivorous flagellate, Bodo saltans. Within one day, filamentous protist-inedible bacteria developed; these belonged to the (beta)-Proteobacteria and constituted between 8.7 and 34% of bacteria from this subgroup. Total abundance of (beta)-Proteobacteria decreased from 3.05 x 10(sup6) to 0.23 x 10(sup6) cells ml(sup-1), and estimated cell division rates were low. Other morphologically inconspicuous protist-edible bacteria belonging to the (alpha)-Proteobacteria were found to respond to predation by an increase in growth rate. Although these bacteria were heavily grazed upon, as on average >85% of flagellate cells had ingested (alpha)-Proteobacteria, they numerically dominated after the addition of B. saltans (mean, 1.35 x 10(sup6) cells ml(sup-1)). It was thus mainly those fast-dividing strains of (alpha)-Proteobacteria that supported the growth of the flagellate population. We conclude that bacteria in mixed assemblages can adopt at least two distinct strategies as a reaction to intense flagellate predation: to outgrow predation pressure or to develop inedible, inactive filaments. Since these strategies occurred within 24 h after the addition of the flagellate, we hypothesize that chemical stimuli released by the predator may have triggered bacterial responses.
Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains
Authors: Kuhnert P , Hacker J , Mühldorfer I , Burnens A P , Nicolet J , Frey J
Published: January 2020

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.
Molecular evidence for association between the sphingobacterium-like organism "Candidatus comitans" and the myxobacterium Chondromyces crocatus
Authors: Jacobi C A , Assmus B , Reichenbach H , Stackebrandt E
Published: January 2020

Seven strains of the myxobacterium Chondromyces crocatus, isolated from widely separated geographic regions, were investigated for the presence of an associate gram-negative, rod-shaped companion bacterium that is phylogenetically related to the genus Sphingobacterium and has been named "Candidatus comitans" (C. A. Jacobi, E. Stackebrandt, H. Reichenbach, and B. J. Tindall, Int. J. Syst. Bacteriol. 46:119-122, 1996). Five of the Chondromyces strains were found to be associated with a companion bacterium, and one strain lost its companion during the study. A 16S ribosomal DNA (16S rDNA) clone library was generated for each Chondromyces culture. Sequence similarity was > 99.1% for all but one strain of C. crocatus and all but one strain of "Candidatus comitans". The three analyzed 16S rDNA clone sequences of the companion of Cm c7 indicated that this companion strain is slightly less related to the other companion strains. The association between the companion and the myxobacterium including the sporangioles was determined by in situ hybridization with fluorescently labeled rRNA probes and scanning confocal laser microscopy. Based on these results, there are indications that the companion strains may survive environmental stress by inclusion in the aggregates and in the sporangioles of the myxobacterium.
Reduction of Disulfide Bonds by Streptomyces pactum during Growth on Chicken Feathers
Authors: Bockle B , Muller R
Published: January 2020

For disintegration of chicken feathers by Streptomyces pactum, keratinolytic proteinases and extracellular reduction of disulfide bonds were necessary. Conditions for disulfide reduction were examined with oxidized glutathione as model substrate. The reduction of glutathione depended on the presence of metabolically active cells. The mycelium also reduced tetrazolium dyes and cystine.
Effect of UV Radiation on the Bacterivory of a Heterotrophic Nanoflagellate
Authors: Sommaruga R , Oberleiter A , Psenner R
Published: January 2020

Volume 62, no. 12, p. 4397: Figure 3 should appear as follows. FIG. 3 [This corrects the article on p. 4395 in vol. 62.].
Effects of Temperature, Water Activity, and Incubation Time on Production of Aflatoxins and Cyclopiazonic Acid by an Isolate of Aspergillus flavus in Surface Agar Culture
Authors: Gqaleni N , Smith J E , Lacey J , Gettinby G
Published: January 2020

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.