bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa
Authors: Yaver D S , Xu F , Golightly E J , Brown K M , Brown S H , Rey M W , Schneider P , Halkier T , Mondorf K , Dalboge H
Published: January 2020

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.
Bleaching of Hardwood Kraft Pulp with Manganese Peroxidase from Phanerochaete sordida YK-624 without Addition of MnSO(inf4)
Authors: Harazono K , Kondo R , Sakai K
Published: January 2020

In vitro bleaching of an unbleached hardwood kraft pulp was performed with partially purified manganese peroxidase (MnP) from the fungus Phanerochaete sordida YK-624 without the addition of MnSO(inf4) in the presence of oxalate, malonate, or gluconate as manganese chelator. When the pulp was treated without the addition of MnSO(inf4), the pulp brightness increased by about 10 points in the presence of 2 mM oxalate, but the brightness did not significantly increase in the presence of 50 mM malonate, a good manganese chelator. Residual MnP activity decreased faster during the bleaching with MnP without MnSO(inf4) in the presence of malonate than in the presence of oxalate. Oxalate reduced MnO(inf2) which already existed in the pulp or was produced from Mn(sup2+) by oxidation with MnP and thus supplied Mn(sup2+) to the MnP system. The presence of gluconate, produced by the H(inf2)O(inf2)-generating enzyme glucose oxidase, also improved the pulp brightness without the addition of MnSO(inf4), although treatment with gluconate was inferior to that with oxalate with regard to increase of brightness. It can be concluded that bleaching of hardwood kraft pulp with MnP, using manganese originally existing in the pulp, is possible in the presence of oxalate, a good manganese chelator and reducing reagent.
Organic carbon utilization by obligately and facultatively autotrophic beggiatoa strains in homogeneous and gradient cultures
Authors: Hagen K D , Nelson D C
Published: January 2020

Marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous gliding bacteria that use hydrogen sulfide and thiosulfate as electron donors for chemolithotrophic energy generation. They are known to be capable of chemolithoautotrophic growth in sulfide gradient media; here we report the first successful bulk cultivation of these strains in a defined liquid medium. To investigate their nutritional versatilities, strains MS-81-6 and MS-81-1c were grown in sulfide-oxygen gradient media supplemented with single organic compounds. Respiration rates and biomass production relative to those of controls grown in unsupplemented sulfide-limited media were monitored to determine whether organic compounds were utilized as sources of energy and/or cell carbon. With cells grown in sulfide gradient and liquid media, we showed that strain MS-81-6 strongly regulates two enzymes, the tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase and the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, in response to the presence of organic carbon (acetate) in the growth medium. In contrast, strain MS-81-1c lacked 2-oxoglutarate dehydrogenase activity and regulated ribulose-1,5-bisphosphate carboxylase/oxygenase activity only slightly in response to organic substrates. Tracer experiments with radiolabeled acetate showed that strain MS-81-1c did not oxidize acetate to CO(inf2) but could synthesize approximately 20% of its cell carbon from acetate. On the basis of these results, we conclude that Beggiatoa strain MS-81-1c is an obligate chemolithoautotroph, while strain MS-81-6 is a versatile facultative chemolithoautotroph.
Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4
Authors: Chee-Sanford J C , Frost J W , Fries M R , Zhou J , Tiedje J M
Published: January 2020

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.
Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR and restriction fragment length polymorphism analysis with crushed root nodules
Authors: Rouvier C , Prin Y , Reddell P , Normand P , Simonet P
Published: January 2020

DNA extracted directly from nodules was used to assess the genetic diversity of Frankia strains symbiotically associated with two species of the genus Casuarina and two of the genus Allocasuarina naturally occurring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of Casuarina-infective Frankia strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the nif operon. PCR products were then analyzed by using a set of restriction endonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of the eight unisolated Frankia strains from Casuarina equisetifolia in Australia. Group 2 consisted of two unisolated Frankia strains from C. equisetifolia, whereas groups 3 to 5 comprised all unisolated strains from Casuarina cunninghamiana, Allocasuarina torulosa, and Allocasuarina littoralis, respectively. These results demonstrate that, contrary to the results of previous molecular studies of isolated strains, there is genetic diversity among Frankia strains that infect members of the family Casuarinacaeae. The apparent high homogeneity of Frankia strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia.
Vibrio mimicus diarrhea following ingestion of raw turtle eggs
Authors: Campos E , Bolaños H , Acuña M T , Díaz G , Matamoros M C , Raventós H , Sánchez L M , Sánchez O , Barquero C
Published: January 2020

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.
Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles
Authors: St Leger R J , Joshi L , Bidochka M J , Rizzo N W , Roberts D W
Published: January 2020

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.
Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation
Authors: Pizarro J , Jedlicki E , Orellana O , Romero J , Espejo R T
Published: January 2020

The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions.
A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef
Authors: Witham P K , Yamashiro C T , Livak K J , Batt C A
Published: January 2020

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0. The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.
Differentiation and grouping of isolates of the Ganoderma lucidum complex by random amplified polymorphic DNA-PCR compared with grouping on the basis of internal transcribed spacer sequences
Authors: Hseu R S , Wang H H , Wang H F , Moncalvo J M
Published: January 2020

Laccate polypores of the Ganoderma lucidum species complex are widespread white rot fungi of economic importance, but isolates cannot be identified by traditional taxonomic methods. Parsimony analysis of nucleotide sequences from the internal transcribed spacers (ITS) of the ribosomal gene (rDNA) distinguished six lineages in this species complex. Each ITS lineage may represent one or more putative species. While some isolates have identical ITS sequences, all of them could be clearly differentiated by genetic fingerprinting using random amplified polymorphic DNA (RAPD). To investigate the suitability of RAPD markers for taxonomic identification and grouping of isolates of the G. lucidum complex, RAPD fragments (RAPDs) were used as phenotypic characters in numerical and parsimony analyses. Results show that data from RAPDS do not distinguish the same clades as ITS data do. Groupings based on analysis of RAPD data were very sensitive to the choice of the grouping method used, and no consistent grouping of isolates could be proposed. However, analysis with RAPDs did resolve several robust terminal clades containing putatively conspecific isolates, suggesting that RAPDs might be helpful for systematics at the lower taxonomic levels that are unresolved by ITS sequence data. The limitations of RAPDs for systematics are briefly discussed. The conclusion of this study is that ITS sequences can be used to identify isolates of the G. lucidum complex, whereas RAPDs can be used to differentiate between isolates having identical ITS sequences. The practical implications of these results are briefly illustrated.
Oxidation of 6,7-dihydro-5H-benzocycloheptene by bacterial strains expressing naphthalene dioxygenase, biphenyl dioxygenase, and toluene dioxygenase yields homochiral monol or cis-diol enantiomers as major products
Authors: Resnick S M , Gibson D T
Published: January 2020

Bacterial strains expressing naphthalene, biphenyl, and toluene dioxygenase were examined for their abilities to oxidize 6,7-dihydro-5H-benzocycloheptene (benzocyclohept-1-ene). The major oxidation products were isolated, and their absolute configurations were determined by chiral 1H nuclear magnetic resonance analysis of diastereomeric boronate esters, chiral stationary-phase high-pressure liquid chromatography, and stereo-chemical correlation. Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae (formerly identified as a Beijerinckia sp.) B8/36 expressing naphthalene and biphenyl dioxygenases, respectively, oxidized benzocyclohept-1-ene to a major product identified as (-)-(1R,2S)-cis-dihydroxybenzocycloheptane (> 98% enantiomeric excess [ee], 50 and 90% yield, respectively). In contrast, Pseudomonas putida F39/D expressing toluene dioxygenase oxidized benzocyclohept-1-ene to (+)-(5R)-hydroxybenzocyclohept-1-ene (> 98% ee, 90% yield) as the major metabolite and to the "opposite" diol, (+)-(1S,2R)-cis-dihydroxybenzocycloheptane (> 98% ee, 10% yield). The results indicate that, for benzocyclohept-1-ene, the major reaction catalyzed by naphthalene and biphenyl dioxygenases is dioxygenation whereas toluene dioxygenase catalyzes mainly R-stereospecific benzylic monooxygenation. Although the type of reaction catalyzed by each organism was not predictable, the absolute configuration of the diol and monol products formed by naphthalene and toluene dioxygenases are consistent with the stereochemistry of the products formed by these enzymes from other benzocycloalkene substrates.
Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments
Authors: Teske A , Wawer C , Muyzer G , Ramsing N B
Published: January 2020

The sulfate-reducing bacterial populations of a stratified marine water column, Mariager Fjord, Denmark, were investigated by molecular and culture-dependent approaches in parallel. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA and DNA encoding rRNA (rDNA) isolated from the water column indicated specific bacterial populations in different water column layers and revealed a highly differentiated pattern of rRNA- and rDNA-derived PCR amplificates, probably reflecting active and resting bacterial populations. Hybridization of DGGE patterns with rRNA probes indicated the increased presence and activity (by at least 1 order of magnitude) of sulfate-reducing bacteria within and below the chemocline. Parallel to this molecular approach, an approach involving most-probable-number (MPN) counts was used, and it found a similar distribution of cultivable sulfate-reducing bacteria in the water column of Mariager Fjord, Approximately 25 cells and 250 cells per ml above and below the chemocline, respectively, were found. Desulfovibrio- and Desulfobulbus-related strains occurred in the oxic zone. DGGE bands from MPN cultures were sequenced and compared with those obtained from nucleic acids extracted from water column samples. The MPN isolates were phylogenetically affiliated with sulfate-reducing delta subdivision proteobacteria (members of the genera Desulfovibrio, Desulfobulbus, and Desulfobacter), whereas the molecular isolates constituted an independent lineage of the delta subdivision proteobacteria. DGGE of PCR-amplified nucleic acids with general eubacterial PCR primers conceptually revealed the general bacterial population, whereas the use of culture media allowed cultivable sulfate-reducing bacteria to be selected. A parallel study of Mariager Fjord biogeochemistry, bacterial activity, and bacterial counts complementing this investigation has been presented elsewhere (N.B. Ramsing, H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup, Appl. Environ.
Priming effect of substrate addition in soil-based biodegradation tests
Authors: Shen J , Bartha R
Published: January 2020

To test whether substrate addition changes background CO2 evolution of soil, we measured both 14CO2 and net CO2 evolution from various test compounds. Glucose caused a priming effect, defined as substrate-stimulated soil organic matter mineralization. Formate, benzoate, n-hexadecane, and bis(2-ethylhexyl)phthalate caused no priming, and phenol caused only a transient one. The priming effect of glucose appears to be unusual and does not require a general rejection of net CO2 evolution measurements in biodegradability testing.
Degradation of Perennial Ryegrass Leaf and Stem Cell Walls by the Anaerobic Fungus Neocallimastix sp. Strain CS3b
Authors: Sijtsma L , Tan B
Published: January 2020

The degradation of cell walls isolated from stems and leaves of perennial ryegrass by the anaerobic fungus Neocallimastix sp. strain CS3b was studied in a defined medium. The combined cellulose and hemicellulose fraction represented 53.1 (wt/wt) and 63.3% (wt/wt) of the dry weight of control grass leaf and stem cell walls, respectively. In both leaf and stem cell walls, glucose was the major neutral monosaccharide, followed by xylose, arabinose, and galactose. After 2 days of fermentation with Neocallimastix sp. strain CS3b, treated cell walls contained smaller amounts of neutral sugars compared with those of undigested cell walls. These results were more evident for glucose, xylose, and arabinose than for galactose. Furthermore, the sugar content of leaf cell walls decreased before a decline in the sugar content of stem cell walls was observed. Data from formate and hydrogen production indicated that the growth of Neocallimastix sp. strain CS3b was completed in 4 days in the culture system used. During this period, the fungus liberated about 95% of the fermentable sugars in untreated material. On a percentage basis, no significant differences were found in final extent of degradation of glucose, xylose, and arabinose. Galactose, however, was degraded to a lesser extent.
Rapid and sensitive method for the detection of Mycobacterium chlorophenolicum PCP-1 in soil based on 16S rRNA gene-targeted PCR
Authors: Briglia M , Eggen R I , de Vos W M , van Elsas J D
Published: January 2020

A method based on 16S rRNA gene-targeted PCR and oligonucleotide probing was developed for detecting Mycobacterium chlorophenolicum PCP-1 in soil. The primers and probe were specific for PCP-1 in DNA extracts of three soils. The method allowed for PCP-1 detection in soil with a detection limit of 3 x 10(2) cells per g.
Temporal Variation in Genetic Diversity and Structure of a Lotic Population of Burkholderia (Pseudomonas) cepacia
Authors: Wise M G , McArthur J V , Wheat C , Shimkets L J
Published: January 2020

The genetic structure and temporal patterns of genetic diversity in a population of Burkholderia (Pseudomonas) cepacia, isolated from a southeastern blackwater stream, were investigated by multilocus enzyme electrophoresis. Allelic variation in seven structural gene loci was monitored at a single stream location at 0, 6, 12, and 24 h and at 2, 4, 8, 16, and 32 days. Over the length of the study, 217 isolates were collected, from which 65 unique electrophoretic types (ETs) were identified. Most of these ETs were present at only one or two time periods and were considered transients; however, one resident ET was particularly abundant (64 of the 217 isolates [29.4%]) and was found at all time points except day 32. The mean genetic diversity of the entire population was 0.520, and the index of association (a measure of multilocus linkage disequilibrium) was 1.33. These results, taken in conjunction with a previous study focusing on spatial patterns of genetic diversity in lotic B. cepacia, show that these bacterial populations exhibit greater variability among sites than within a site over time, suggesting relative stability over short time periods.
A pressurized chemostat for the study of marine barophilic and oligotrophic bacteria
Authors: Jannasch H W , Wirsen C O , Doherty K W
Published: January 2020

A continuous culture system that allows bacteria to be grown in steady-state populations under pressures of up to 700 atm (71 MPa) was constructed and tested. With readily available or slightly modified high-pressure chromatography equipment, a continuous flow of sterile medium is pressurized and passed through a 500-ml nylon-coated titanium reactor at flow rates of 0.01 to 10 ml min(sup-1). The pressure in the reactor is controlled by a backpressure regulator with greater than 1% accuracy. In test experiments, a culture of a psychro- and barophilic marine isolate from a depth of 4,900 m (strain F1-A, identified as a Shewanella sp.) was grown at 1, 300, and 450 atm (0.1, 30.4, and 40.5 MPa) and dilution rates of 60 and 90% of the organism's maximum growth rate (determined at 1 atm) in the required complex medium at levels of 3.3 and 0.33 mg of dissolved organic carbon per liter in the reservoir. Growth limitation by carbon was assured by an appropriate C/N/P ratio of the medium. The data indicate that barophilic growth characteristics in steady-state cultures of this psychro- and barophilic deep-sea isolate were positively affected by a decreasing growth rate at the higher of two substrate concentrations in the reservoir. After a 10-fold lowering of the substrate concentration, the effect was reversed. Under these conditions, the cell viability increased significantly, especially at the higher of the two pressures tested. The basic design of the system can principally also be used for growth studies on hyperthermophilic bacteria and archaea.
Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes
Authors: Marie D , Vaulot D , Partensky F
Published: January 2020

Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.
Bacterial Dissimilatory Reduction of Arsenic(V) to Arsenic(III) in Anoxic Sediments
Authors: Dowdle P R , Laverman A M , Oremland R S
Published: January 2020

Incubation of anoxic salt marsh sediment slurries with 10 mM As(V) resulted in the disappearance over time of the As(V) in conjunction with its recovery as As(III). No As(V) reduction to As(III) occurred in heat-sterilized or formalin-killed controls or in live sediments incubated in air. The rate of As(V) reduction in slurries was enhanced by addition of the electron donor lactate, H(inf2), or glucose, whereas the respiratory inhibitor/uncoupler dinitrophenol, rotenone, or 2-heptyl-4-hydroxyquinoline N-oxide blocked As(V) reduction. As(V) reduction was also inhibited by tungstate but not by molybdate, sulfate, or phosphate. Nitrate inhibited As(V) reduction by its action as a preferred respiratory electron acceptor rather than as a structural analog of As(V). Nitrate-respiring sediments could reduce As(V) to As(III) once all the nitrate was removed. Chloramphenicol blocked the reduction of As(V) to As(III) in nitrate-respiring sediments, suggesting that nitrate and arsenate were reduced by separate enzyme systems. Oxidation of [2-(sup14)C]acetate to (sup14)CO(inf2) by salt marsh and freshwater sediments was coupled to As(V). Collectively, these results show that reduction of As(V) in sediments proceeds by a dissimilatory process. Bacterial sulfate reduction was completely inhibited by As(V) as well as by As(III).
Mechanism by which gamma irradiation increases the sensitivity of Salmonella typhimurium ATCC 14028 to heat
Authors: Kim A Y , Thayer D W
Published: January 2020

Effects of irradiation and heating on survival of Salmonella typhimurium ATCC 14028 were examined by measuring DNA damage and the integrity of the cytoplasmic membrane. S. typhimurium cells fell into two distinct groups following heating: (i) heat-sensitive cells, which were rapidly inactivated at 65 degrees C and (ii) heat-resistant cells, which were only slowly inactivated at 65 degrees C. Radiation sensitivity of S. typhimurium was greater in the presence of air than in the presence of N2 gas (radiation doses required to inactivate 90% of the cells, 0.394 +/- 0.029 in air and 0.561 +/- 0.035 in N2). Recovery of the covalently closed circular form of plasmid pBR322 from S. typhimurium transformants (Ampr Tetr) was decreased by irradiation but not by heating. Heating prior to irradiation significantly decreased the recovery of plasmid DNA without affecting survival of S. typhimurium. Transformability of the recovered plasmid pBR322 was affected by neither irradiation nor heating, and mutation of antibiotic resistance genes was not detected in S. typhimurium. Heating, but not irradiation, caused destabilization of the cytoplasmic membrane, allowing penetration of hydrophobic dye. These results suggest that lethality of heating followed by irradiation for S. typhimurium was additive, reflecting irradiation-induced DNA damage and heat-induced membrane destabilization. When irradiation preceded heating in the absence of air, more cells were inactivated than was expected, because of heat-inactivating radiation-damaged DNA.
Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli
Authors: Graupner S , Wackernagel W
Published: January 2020

Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid. Evidence that plasmid-like structures are formed in H. polymorpha and that their formation is stimulated by DNA damage is presented in this study.
Defective site-specific integration elements are present in the genome of virulent bacteriophage LL-H of Lactobacillus delbrueckii
Authors: Mikkonen M , Dupont L , Alatossava T , Ritzenthaler P
Published: January 2020

The phage attachment site, attP, and the integrase-encoding gene, int, are sufficient to promote site-specific integration of the temperate phage mv4 genome into the chromosome of the Lactobacillus delbrueckii host (L. Dupont, B. Boizet-Bonhoure, M. Coddeville, F. Auvray, and P. Ritzenthaler, J. Bacteriol. 177:586--595, 1995). The mv4 genome region containing these elements was compared at the nucleotide and amino acid levels with that of the closely related virulent phage LL-H. Complex DNA rearrangements were identified; a truncated integrase gene and two sites homologous to the mv4 attP site were detected in the genome of the virulent phage LL-H. These observations suggest that the two phages derive from a common temperate ancestor.
Community Structure of Filamentous, Sheath-Building Sulfur Bacteria, Thioploca spp., off the Coast of Chile
Authors: Schulz H N , Jorgensen B B , Fossing H A , Ramsing N B
Published: January 2020

The filamentous sulfur bacteria Thioploca spp. produce dense bacterial mats in the shelf area off the coast of Chile and Peru. The mat consists of common sheaths, shared by many filaments, that reach 5 to 10 cm down into the sediment. The structure of the Thioploca communities off the Bay of Concepcion was investigated with respect to biomass, species distribution, and three-dimensional orientation of the sheaths. Thioploca sheaths and filaments were found across the whole shelf area within the oxygen minimum zone. The maximum wet weight of sheaths, 800 g m(sup-2), was found at a depth of 90 m. The bacterial filaments within the sheaths contributed about 10% of this weight. The highest density of filaments was found within the uppermost 1 cm of the mat. On the basis of diameter classes, it was possible to distinguish populations containing only Thioploca spp. from mixed populations containing Beggiatoa spp. Three distinct size classes of Thioploca spp. were found, two of which have been described previously as Thioploca araucae and Thioploca chileae. Many Thioploca filaments did not possess a visible sheath, and about 20% of the sheaths contained more than one Thioploca species. The three-dimensional sheath structure showed that Thioploca filaments can move from the surface and deep into the sediment.
Dipstick enzyme immunoassay to detect Fusarium T-2 toxin in wheat
Authors: De Saeger S , Van Peteghem C
Published: January 2020

A dipstick enzyme immunoassay for the rapid detection of Fusarium T-2 toxin in wheat was developed. An Immunodyne ABC membrane was precoated with rabbit anti-mouse immunoglobulins. After the strips were immersed in a solution of monoclonal anti-T-2 toxin antibodies, a direct competitive enzyme immunoassay was performed. This assay included the incubation of the antibody-coated dipsticks in a mixture of sample and T-2 toxin-horseradish peroxidase conjugate. Afterwards, the strips were placed in a chromogen-containing substrate solution (H202-3,3',5,5'-tetramethylbenzidine) for color reaction. The dot color intensity of toxin-positive dipsticks was visually distinguishable from that of the negative control. A portable colorimeter was used to confirm and quantify the visual observations. With coated strips, the tests could be performed in 45 min. The visual detection limit for T-2 toxin in buffer solution was 0.25 ng/ml. Artificially infected wheat samples were extracted with 80% methanol-water. A dilution of the raw extract of 1:8 was sufficient to avoid matrix effects. It was possible to make visually a clear distinction between the negative control and a wheat extract spiked with 12 ng/g.
Ribulose-1,5-bisphosphate carboxylase/oxygenase gene expression and diversity of Lake Erie planktonic microorganisms
Authors: Xu H H , Tabita F R
Published: January 2020

Carbon dioxide fixation is carried out primarily through the Calvin-Benson-Bassham reductive pentose phosphate cycle, in which ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme. The primary structure of the large subunit of form I RubisCO is well conserved; however, four distinct types, A, B, C, and D, may be distinguished, with types A and B and types C and D more closely related to one another. To better understand the environmental regulation of RubisCO in Lake Erie phytoplanktonic microorganisms, we have isolated total RNA and DNA from four Lake Erie sampling sites. Probes prepared from RubisCO large-subunit genes (rbcL) of the freshwater cyanobacterium Synechococcus sp. strain PCC6301 (representative of type IB) and the diatom Cylindrotheca sp. strain N1 (representative of type ID) were hybridized to the isolated RNA and DNA. To quantitate rbcL gene expression for each sample, the amount of gene expression per gene dose (i.e., the amount of mRNA divided by the amount of target DNA) was determined. With a limited number of sampling sites, it appeared that type ID (diatom) rbcL gene expression per gene dose decreased as the sampling sites shifted toward open water. By contrast, a similar trend was not observed for cyanobacterial (type IB) rbcL gene expression per gene dose. Complementary DNA specific for rbcL was synthesized from Lake Erie RNA samples and used as a template for PCR amplification of portions of various rbcL genes. Thus far, a total of 21 clones of rbcL genes derived from mRNA have been obtained and completely sequenced from the Ballast Island site. For surface water samples, deduced amino acid sequences of five of six clones appeared to be representative of green algae. In contrast, six of nine sequenced rbcL clones from 10-m-deep samples were of chromophytic and rhodophytic lineages. At 5 m deep, the active CO2-fixing planktonic organisms represented a diverse group, including organisms related to Chlorella ellipsoidea, Cylindrotheca sp. strain N1, and Olisthodiscus luteus. Although many more samplings at diverse sites must be accomplished, the discovery of distinctly different sequences of rbcL mRNA at different water depths suggests that there is a stratification of active CO2-fixing organisms in western Lake Erie.
Molecular polymorphism distribution in phenotypically distinct populations of wine yeast strains
Authors: Nadal D , Colomer B , Piña B
Published: January 2020

Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to analyze natural yeast populations from fermenting musts in El Penedès, Spain. Both analyses revealed a considerable degree of polymorphism, indicating heterogeneous natural populations. By specifically designed genetic selection protocols, strains showing potentially interesting phenotypes, such as high tolerance to ethanol and temperature or the ability to grow and to ferment in wine-water-sugar mixtures, were isolated from these natural populations. Genetic analysis showed a strong correlation between the selected phenotypes and mitochondrial DNA polymorphisms. Karyotype analysis revealed several genetically similar yeast lineages in the natural yeast microflora, which we interpret as genetically isolated subpopulations of yeast strains with distinct genetic traits, which may correspond to specific microenvironments. Thus, molecular polymorphism analysis may be useful not only to study the geographical distribution of natural yeast strains but also to identify strains with specific phenotypic properties.
Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses
Authors: Stoner D L , Browning C K , Bulmer D K , Ward T E , Macdonell M T
Published: January 2020

This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the genera Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed.
Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes
Authors: Blouin K , Walker S G , Smit J , Turner R
Published: January 2020

Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.
Automated systems for identification of heterotrophic marine bacteria on the basis of their Fatty Acid composition
Authors: Bertone S , Giacomini M , Ruggiero C , Piccarolo C , Calegari L
Published: January 2020

The fatty acid methyl ester composition of a total of 71 marine strains representing the genera Alteromonas, Deleya, Oceanospirillum, and Vibrio was determined by gas-liquid chromatographic analysis. Over 70 different fatty acids were found. The predominant fatty acids were 16:0, 16:1 cis 9, summed-in-feature (SIF) 4 (15:0 iso 2OH and/or 16:1 trans 9) and SIF 7 (18:1 cis 11, 18:1 trans 9, and/or 18:1 trans 6) for all the strains considered, but minor quantitative variations could be used to distinguish the different genera. In addition to a conventional statistical processing method to analyze the data and draw comparison between species and genera, an approach involving neutral network-based elaboration is applied. The statistical analysis and dendrogram representation gave a comparison of the species considered, while the neural network computation provided a more accurate assignment of species to their genera. Moreover, by using neural networks, it was possible to conclude that only 22 fatty acids were important for the identification of the marine genera considered. A database of Alteromonas, Deleya, Oceanospirillum, and Vibrio fatty acid methyl ester profiles was generated and is now routinely used to identify fresh marine isolates.
Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge
Authors: Harmsen H J , Akkermans A D , Stams A J , de Vos W M
Published: January 2020

Laboratory-scale upflow anaerobic sludge-bed reactors were inoculated with industrial granular sludge and fed with either propionate or propionate and sulfate. The population dynamics of the propionate-oxidizing bacteria Desulfobulbus sp. and the syntrophically growing strain SYN7 were studied in reactors by dot blot and in situ hybridization with 16S rRNA-based oligonucleotide probes.