bluuebunny/crossref_metadata_2025 huggingface dataset:
L-Arogenate Is a Chemoattractant Which Can Be Utilized as the Sole Source of Carbon and Nitrogen by Pseudomonas aeruginosa
Authors: Fischer R S , Song J , Gu W , Jensen R A
Published: January 2020

L-Arogenate is a commonplace amino acid in nature in consideration of its role as a ubiquitous precursor of L-phenylalanine and/or L-tyrosine. However, the questions of whether it serves as a chemoattractant molecule and whether it can serve as a substrate for catabolism have never been studied. We found that Pseudomonas aeruginosa recognizes L-arogenate as a chemoattractant molecule which can be utilized as a source of both carbon and nitrogen. Mutants lacking expression of either cyclohexadienyl dehydratase or phenylalanine hydroxylase exhibited highly reduced growth rates when utilizing L-arogenate as a nitrogen source. Utilization of L-arogenate as a source of either carbon or nitrogen was dependent upon (sigma)(sup54), as revealed by the use of an rpoN null mutant. The evidence suggests that catabolism of L-arogenate proceeds via alternative pathways which converge at 4-hydroxyphenylpyruvate. In one pathway, prephenate formed in the periplasm by deamination of L-arogenate is converted to 4-hydroxyphenylpyruvate by cyclohexadienyl dehydrogenase. The second route depends upon the sequential action of periplasmic cyclohexadienyl dehydratase, phenylalanine hydroxylase, and aromatic aminotransferase.
Development and application of 16S rRNA-targeted probes for detection of iron- and manganese-oxidizing sheathed bacteria in environmental samples
Authors: Siering P L , Ghiorse W C
Published: January 2020

Comparative sequence analysis of the 16S rRNA genes from several Leptothrix and Sphaerotilus strains led to the design of an oligonucleotide probe (PS-1) based on a sequence within the hypervariable region 1 specific for four Leptothrix strains and for one of the four Sphaerotilus natans strains examined. Another probe (PSP-6) was based on a sequence within the hypervariable region 2. PSP-6 was specific for one of the two evolutionary lineages previously described for Leptothrix spp. (P. L. Siering and W. C. Ghiorse, Int. J. Syst. Bacteriol. 46:173-182, 1996). Fluorescein-labeled oligonucleotide probes were synthesized, and their specificity for fluorescence in situ hybridization identification was confirmed by a laser scanning microscopy technique (W. C. Ghiorse, D. N. Miller, R. L. Sandoli, and P. L. Siering, Microsc. Res. Tech. 33:73-86, 1996) to compare whole-cell hybridizations of closely related bacteria. Probe specificity was also tested in dot blot against total RNA isolated from four Leptothrix strains, four Sphaerotilus strains, and 15 other members of the class Proteobacteria. When the probes were tested on samples from the Sapsucker Woods wetland habitat where Leptothrix spp. are thought to play a role in manganese and iron oxidation, positive signals were obtained from several sheathed filamentous bacteria including some that were morphologically similar to previously isolated strains of "Leptothrix discophora." Other unknown filamentous sheathed bacteria also gave strong positive signals. This work provides a foundation for future studies correlating the presence of members of the Leptothrix-Sphaerotilus group of sheathed bacteria with manganese and iron oxidation activity in habitats where biological iron and manganese oxidation are important environmental processes.
Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85
Authors: Maglione G , Russell J B , Wilson D B
Published: January 2020

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).
Plasmid Transfer between Spatially Separated Donor and Recipient Bacteria in Earthworm-Containing Soil Microcosms
Authors: Daane L L , Molina J , Sadowsky M J
Published: January 2020

Most gene transfer studies have been performed with relatively homogeneous soil systems in the absence of soil macrobiota, including invertebrates. In this study we examined the influence of earthworm activity (burrowing, casting, and feeding) on transfer of plasmid pJP4 between spatially separated donor (Alcaligenes eutrophus) and recipient (Pseudomonas fluorescens) bacteria in nonsterile soil columns. A model system was designed such that the activity of earthworms would act to mediate cell contact and gene transfer. Three different earthworm species (Aporrectodea trapezoides, Lumbricus rubellus, and Lumbricus terrestris), representing each of the major ecological categories (endogeic, epigeic, and anecic), were evaluated. Inoculated soil microcosms, with and without added earthworms, were analyzed for donor, recipient, and transconjugant bacteria at 5-cm-depth intervals by using selective plating techniques. Transconjugants were confirmed by colony hybridization with a mer gene probe. The presence of earthworms significantly increased dispersal of the donor and recipient strains. In situ gene transfer of plasmid pJP4 from A. eutrophus to P. fluorescens was detected only in earthworm-containing microcosms, at a frequency of (symbl)10(sup2) transconjugants per g of soil. The depth of recovery was dependent on the burrowing behavior of each earthworm species; however, there was no significant difference in the total number of transconjugants among the earthworm species. Donor and recipient bacteria were recovered from earthworm feces (casts) of all three earthworm species, with numbers up to 10(sup6) and 10(sup4) bacteria per g of cast, respectively. A. trapezoides egg capsules (cocoons) formed in the inoculated soil microcosms contained up to 10(sup7) donor and 10(sup6) recipient bacteria per g of cocoon. No transconjugant bacteria, however, were recovered from these microhabitats. To our knowledge, this is the first report of gene transfer between physically isolated bacteria in nonsterile soil, using burrowing earthworms as a biological factor to facilitate cell-to-cell contact.
Plasmid Transfer between Marine Vibrio Strains during Predation by the Heterotrophic Microflagellate Cafeteria roenbergensis
Authors: Otto K , Weichart D , Kjelleberg S
Published: January 2020

The effect of grazing by the heterotrophic microflagellate Cafeteria roenbergensis on plasmid transfer between marine Vibrio S14 strains was studied by using artificial seawater. Several factors of potential importance for regulation of the plasmid transfer, such as nutrient release, production of a flagellate-derived substance(s) that may affect plasmid transfer, and the presence of surfaces, were investigated. Only living flagellates gave rise to, at instances, plasmid transfer enhanced more than 2 orders of magnitude. We propose that the activity of grazing flagellates allows for the significant increase in plasmid transfer observed under predation conditions. This may be due to a localized increase in bacterial numbers through filter feeding, thus providing high cell densities with increased possibility for cell-to-cell-contact and hence plasmid transfer.
Growth of Vibrio anguillarum in Salmon Intestinal Mucus
Authors: Garcia T , Otto K , Kjelleberg S , Nelson D R
Published: January 2020

The physiological changes of Vibrio anguillarum in response to growth in salmon intestinal mucus were investigated. Growth, survival, and changes in protein expression during growth in media supplemented with mucus were compared to growth and starvation in the identical media without mucus. V. anguillarum exhibited a rapid decline in CFU following growth in mucus as the sole carbon source. No such decline was observed in Luria broth with a 2% NaCl concentration, in glucose-minimal broth (3M), or during starvation in a carbon-, nitrogen-, and phosphorus-free salt solution (NSS). The changes in protein expression during growth in mucus were examined by labeling cells with [(sup35)S]methionine and analyzing the labeled proteins by one- and two-dimensional gel electrophoresis and autoradiography. Comparison of [(sup35)S]methionine-labeled proteins from mucus-grown cells with 3M-grown cells and NSS-starved cells revealed four de novo mucus-inducible proteins (Mips). These Mips were localized in the membrane fraction of V. anguillarum. Additionally, at least one other membrane protein was found to have increased expression in response to growth in mucus.
Molecular Identification and Localization of Filamentous Symbiotic Bacteria Associated with the Hydrothermal Vent Annelid Alvinella pompejana
Authors: Cary S C , Cottrell M T , Stein J L , Camacho F , Desbruyeres D
Published: January 2020

Alvinella pompejana is a polychaetous annelid that inhabits high-temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.
Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant
Authors: Morais P V , Mesquita C , Andrade J L , da Costa M S
Published: January 2020

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.
Organic solutes in hyperthermophilic archaea
Authors: Martins L O , Huber R , Huber H , Stetter K O , Da Costa M S , Santos H
Published: January 2020

We examined the accumulation of organic solutes under optimum growth conditions in 12 species of thermophilic and hyperthermophilic Archaea belonging to the Crenarchaeota and Euryarchaeota. Pyrobaculum aerophilum, Thermoproteus tenax, Thermoplasma acidophilum, and members of the order Sulfolobales accumulated trehalose. Pyrococcus furiosus accumulated di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate and (beta)-mannosylglycerate, Methanothermus fervidus accumulated cyclic-2,3-bisphosphoglycerate and (beta)-mannosylglycerate, while the only solute detected in Pyrodictium occultum was di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate. Methanopyrus kandleri accumulated large concentrations of cyclic-2,3-bisphosphoglycerate. On the other hand, Archaeoglobus fulgidus accumulated three phosphorylated solutes; prominent among them was a compound identified as di-glycerol-phosphate. This solute increased in concentration as the salinity of the medium and the growth temperature were raised, suggesting that this compound serves as a general stress solute. Di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate accumulated at supraoptimal temperature only. The relationship between the accumulation of unusual solutes and high temperatures is also discussed.
Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR
Authors: Kaufmann P , Pfefferkorn A , Teuber M , Meile L
Published: January 2020

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.
Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures
Authors: Coakley M , Fitzgerald G , Ros R P
Published: January 2020

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.
Chlorella virus PBCV-1 encodes a homolog of the bacteriophage T4 UV damage repair gene denV
Authors: Furuta M , Schrader J O , Schrader H S , Kokjohn T A , Nyaga S , McCullough A K , Lloyd R S , Burbank D E , Landstein D , Lane L , Van Etten J L
Published: January 2020

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photodamaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.
The nodular microsymbionts of Gymnostoma spp. are Elaeagnus-infective Frankia strains
Authors: Navarro E , Nalin R , Gauthier D , Normand P
Published: January 2020

The phylogenetic relationships of Frankia strains infective on Gymnostoma with other Frankia strains was analyzed. Partial sequencing of the 16S rDNA and use of specific primers showed that the Frankia strains present in Gymnostoma are phylogenetically close to Elaeagnus-infective strains. This finding was confirmed by using the sequences of the hypervariable nifDK intergenic spacer. The strains present in Gymnostoma nodules were close to one another. Clustered with Elaeagnus-infective strains, and distantly related to Casuarina and Alnus-infective strains. Morphological observations of strains and cross-inoculation trials showed that Gymnostoma-infective strains are indistinguishable from Elaeagnus-infective strains. Results of both phenotypic and genotypic approaches indicate that Gymnostoma-infective strains are Elaeagnus infective and not Casuarina infective.
A rapid and simple method for estimating sulfate reduction activity and quantifying inorganic sulfides
Authors: Ulrich G A , Krumholz L R , Suflita J M
Published: January 2020

A simplified passive extraction procedure for quantifying reduced inorganic sulfur compounds from sediments and water is presented. This method may also be used for the estimation of sulfate reduction rates. Efficient extraction of FeS, FeS(inf2), and S(sup2-) was obtained with this procedure; however, the efficiency for S(sup0) depended on the form that was tested. Passive extraction can be used with samples containing up to 20 mg of reduced sulfur. We demonstrated the utility of this technique in a determination of both sulfate reduction rates and reduced inorganic sulfur pools in marine and freshwater sediments. A side-by-side comparison of the passive extraction method with the established single-step distillation technique yielded comparable results with a fraction of the effort.
Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain from Soil by Using Markers Associated with an Engineered Catabolic Pathway
Authors: Hwang I , Farrand S K
Published: January 2020

Volume 63, no. 2, p. 602: the article title should read as shown above. [This corrects the article on p. 602 in vol. 63.].
Deletions in the carboxyl-terminal region of Streptococcus gordonii glucosyltransferase affect cell-associated enzyme activity and sucrose-associated accumulation of growing cells
Authors: Vickerman M M , Clewell D B
Published: January 2020

The single glucosyltransferase (GTF) of Streptococcus gordonii Challis CH1 makes alpha 1,3- and alpha 1,6-linked glucans from sucrose. The GTF carboxyl-terminal region has six direct repeats thought to be involved in glucan binding. Strains with defined mutations in this region have been described recently (M. M. Vickerman, M. C. Sulavik, P. E. Minick, and D. B. Clewell, Infect. Immun. 64:5117-5128, 1996). Strain CH107 GTF has three internal direct repeats deleted; the 59 carboxyl-terminal amino acids are identical to those of the parental strain. This deletion resulted in decreased enzyme activity but did not affect the amount of cell-associated GTF protein. The GTFs of strains CH2RPE and CH4RPE have six and eight direct repeats, respectively, but are both missing the 14 carboxyl-terminal amino acids. Strain CH2RPE had significantly decreased levels of cell-associated GTF; this decrease was not obviated by the increased number of direct repeats in strain CH4RPE. Thus, the carboxyl-terminal amino acids appeared to influence the amount of cell-associated GTF more than the direct repeats. The qualitative and quantitative differences in the GTFs did not affect the abilities of these strains to accumulate on hydroxyapatite beads in the absence of sucrose. However, when sucrose was added as a substrate for GTF, the mutant strains were unable to accumulate on these surfaces to the same extent as the parent. These differences in sucrose-associated accumulation may be due to changes in the nature of the glucans produced by the different enzymes and/or cohesive interactions between these glucans and the GTF on the surfaces of the growing streptococci.
Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus
Authors: Pasamontes L , Haiker M , Wyss M , Tessier M , van Loon A P
Published: January 2020

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.
Subcuticular bacteria from the brittle star Ophiactis balli (Echinodermata: Ophiuroidea) represent a new lineage of extracellular marine symbionts in the alpha subdivision of the class Proteobacteria
Authors: Burnett W J , McKenzie J D
Published: January 2020

Many species of echinoderms, in all five extant classes, contain subcuticular bacterial symbionts (SCB). The role of these extracellular symbionts and the nature of the relationship remain unclear. We have sequenced 16S rRNA genes from symbionts to determine their phylogenetic affinities. Symbionts of an ophiuroid, Ophiactis balli, appear closely related to bacteria within the alpha group of the class Proteobacteria, including intracellular endosymbionts and pathogens. SCB are clearly of separate origin from other documented major groups of marine symbiotic bacteria.
Manganese Is Not Required for Biobleaching of Oxygen-Delignified Kraft Pulp by the White Rot Fungus Bjerkandera sp. Strain BOS55
Authors: Moreira M T , Feijoo G , Sierra-Alvarez R , Lema J , Field J A
Published: January 2020

The white rot fungus Bjerkandera sp. strain BOS55 extensively delignified and bleached oxygen-delignified eucalyptus kraft pulp handsheets. Biologically mediated brightness gains of up to 14 ISO (International Standards Organization units) were obtained, providing high final brightness values of up to 80% ISO. In nitrogen-limited cultures (2.2 mM N), manganese (Mn) greatly improved manganese-dependent peroxidase (MnP) production. However, the biobleaching was not affected by the Mn nutrient regimen, ranging from 1,000 (mu)M added Mn to below the detection limit of 0.26 (mu)M Mn in EDTA-extracted pulp medium. The lowest Mn concentration tested was at least several orders of magnitude lower than the K(infm) known for MnP. Consequently, it was concluded that Mn is not required for biobleaching in Bjerkandera sp. strain BOS55. Nonetheless, fast protein liquid chromatography profiles indicated that MnP was the predominant oxidative enzyme produced even under culture conditions in the near absence of manganese. High nitrogen (22 mM N) and exogenous veratryl alcohol (2 mM) repressed biobleaching in Mn-deficient but not in Mn-sufficient culture medium. No correlation was observed between the titers of extracellular peroxidases and the biobleaching. However, the decolorization rate of the polyaromatic dye Poly R-478 was moderately correlated to the biobleaching under a wide range of Mn and N nutrient regimens.
A Change in a Single Midgut Receptor in the Diamondback Moth (Plutella xylostella) Is Only in Part Responsible for Field Resistance to Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai
Authors: Wright D J , Iqbal M , Granero F , Ferre J
Published: January 2020

A population (SERD3) of the diamondback moth (Plutella xylostella L.) with field-evolved resistance to Bacillus thuringiensis subsp. kurstaki HD-1 (Dipel) and B. thuringiensis subsp. aizawai (Florbac) was collected. Laboratory-based selection of two subpopulations of SERD3 with B. thuringiensis subsp. kurstaki (Btk-Sel) or B. thuringiensis subsp. aizawai (Bta-Sel) increased resistance to the selecting agent with little apparent cross-resistance. This result suggested the presence of independent resistance mechanisms. Reversal of resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai was observed in the unselected SERD3 subpopulation. Binding to midgut brush border membrane vesicles was examined for insecticidal crystal proteins specific to B. thuringiensis subsp. kurstaki (Cry1Ac), B. thuringiensis subsp. aizawai (Cry1Ca), or both (Cry1Aa and Cry1Ab). In the unselected SERD3 subpopulation (ca. 50- and 30-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai), specific binding of Cry1Aa, Cry1Ac, and Cry1Ca was similar to that for a susceptible population (ROTH), but binding of Cry1Ab was minimal. The Btk-Sel (ca. 600-and 60-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) and Bta-Sel (ca. 80-and 300-fold resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai) subpopulations also showed reduced binding to Cry1Ab. Binding of Cry1Ca was not affected in the Bta-Sel subpopulation. The results suggest that reduced binding of Cry1Ab can partly explain resistance to B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai. However, the binding of Cry1Aa, Cry1Ac, and Cry1Ca and the lack of cross-resistance between the Btk-Sel and Bta-Sel subpopulations also suggest that additional resistance mechanisms are present.
Metabolic and Genotypic Fingerprinting of Fluorescent Pseudomonads Associated with the Douglas Fir-Laccaria bicolor Mycorrhizosphere
Authors: Frey P , Frey-Klett P , Garbaye J , Berge O , Heulin T
Published: January 2020

A collection of 300 isolates of fluorescent pseudomonads was established from Douglas fir-Laccaria bicolor mycorrhizas and mycorrhizosphere and from adjacent bulk soil. These isolates were first phenotypically characterized with the Biolog method. Taxonomic identification assigned 90% of the isolates to the different biovars of Pseudomonas fluorescens, with inverted frequencies of biovars V and I from the bulk soil to the mycorrhizas, suggesting that the mycorrhizas exert a selective stimulation of the P. fluorescens bv. I and a counterselection of the P. fluorescens bv. V present in the soil. Multivariate analyses of the carbon source utilization data led to the definition of homogenous metabolic groups and to the identification of the most discriminating substrates for each group. The isolates from the mycorrhizosphere and from the mycorrhizas seem to preferentially utilize carbohydrates, in particular trehalose, which is the most abundant carbohydrate accumulated in the mycelium of L. bicolor. The results suggest that L. bicolor exerts a trehalose-mediated selection on the fluorescent pseudomonads present in the vicinity of the mycorrhizas. Isolates of P. fluorescens from the mycorrhizosphere and mycorrhizas were then genotypically characterized by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes and enterobacterial repetitive intergenic consensus-PCR DNA fingerprinting. Both methods revealed a high genetic polymorphism within the population studied, which was well correlated with the phenotypic characterization.
Thiocarbamate herbicide-inducible nonheme haloperoxidase of Rhodococcus erythropolis NI86/21
Authors: De Schrijver A , Nagy I , Schoofs G , Proost P , Vanderleyden J , van Pée K H , De Mot R
Published: January 2020

During biodegradation of thiocarbamate herbicides by Rhodococcus erythropolis NI86/21, a protein with an M(r) of 30,000 is induced (I. Nagy, G. Schoofs, F. Compernolle, P. Proost, J. Vanderleyden, and R.De Mot, J. Bacteriol. 177:676-687, 1995). Based on N-terminal sequence data for the protein purified by two-dimensional electrophoresis, the corresponding structural gene, thcF, was cloned and sequenced. The deduced protein sequence of ThcF is homologous to those of nonheme haloperoxidases. A particularly high level of sequence identity (72.6%) was observed for the chloroperoxidase from Pseudomonas pyrrocinia. A polyclonal antibody against the latter enzyme cross-reacted with ThcF either produced by the original Rhodococcus cells or overexpressed heterologously in Escherichia coli. In both thiocarbamate-grown Rhodococcus cells and E. coli cells expressing thcF, the haloperoxidase activity of ThcF was demonstrated. The thiocarbamate-inducible R. erythropolis ThcF protein represents the first (nonheme) haloperoxidase to be identified in a nocardioform actinomycete.
Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea
Authors: Behrendt C J , Blanchette R A
Published: January 2020

Phlebiopsis gigantea (=Phanerochaete gigantea) is a white rot fungus that rapidly colonizes cut stumps, stems, and branches of pine. Two laboratory and several field studies showed that inoculation of red pine logs, Pinus resinosa, with P. gigantea reduced the pitch content of wood, facilitated bark removal, modified wood cells, and controlled detrimental sapstain. Isolations from inoculated logs revealed up to 100 and 80% colonization of the sapwood by P. gigantea after 8 weeks in the field and 32 days in the laboratory, respectively. Logs colonized by P. gigantea in both the laboratory and field showed a 9 to 71% reduction in pitch content, as well as a significant enhancement of bark removal. Examination with Simons' stain of refined wood fibers from inoculated logs revealed an increase in cell wall porosity. Blue stain fungi that cause dark discoloration of the sapwood were inhibited by inoculation with P. gigantea. These studies demonstrate that biological processing of logs with P. gigantea can result in substantial benefits to the pulp and papermaking process.
Anaerobic Transformation of Furfural by Methanococcus deltae (Delta)LH
Authors: Belay N , Boopathy R , Voskuilen G
Published: January 2020

Methanococcus deltae (Delta)LH was grown on H(inf2)-CO(inf2) in the presence of various concentrations of furfural. Furfural at higher concentrations, namely, 20 and 25 mM, inhibited growth of this organism. At concentration of 5 and 10 mM, no inhibition of growth was observed. The other methanogens in this study were not inhibited by 10 mM furfural. Among the methanogens tested, M. deltae was capable of transforming furfural, whereas Methanobacterium thermoautotrophicum Marburg, Methanosarcina barkeri 227, Methanococcus thermolithotrophicus, and Methanobrevibacter ruminantium lacked this capability. One hundred percent removal of furfural was observed within 48 h of incubation in M. deltae cultures. The end product observed during furfural metabolism was furfuryl alcohol. An almost stoichiometric amount of furfuryl alcohol was produced by M. deltae. This transformation is likely to be of value in the detoxification of furfural and in its ultimate conversion to methane and CO(inf2) by anaerobic digestion.
Isolation and characterization of fimbriae from a sparsely fimbriated strain of Porphyromonas gingivalis
Authors: Sojar H T , Hamada N , Genco R J
Published: January 2020

Porphyromonas gingivalis W50 (ATCC 53978) possesses the gene for fimbriae; however, the surface-expressed fimbriae are sparse and have not been previously isolated and characterized. We purified fimbriae from strain W50 to homogeneity by ammonium sulfate precipitation and reverse-phase high-performance liquid chromatography [H. T. Sojar, N. Hamada, and R. J. Genco, Protein Expr. Purif. 9(1):49-52, 1997]. Negative staining of purified fimbriae viewed by electron microscopy revealed that the fimbriae were identical in diameter to fimbriae of other P. gingivalis strains, such as 2561, but were shorter in length. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the apparent molecular weight of isolated fimbrillin from strain W50 was found to be identical to that of the fimbrillin molecule of strain 2561. Unlike 2561 fimbriae, W50 fimbriae, under reducing condition, exhibited a monomeric structure on SDS-PAGE at room temperature. However, under nonreduced conditions, even at 100 degrees C, no monomer was observed. In immunoblot analysis as well as immunogold labeling of isolated fimbriae, polyclonal antibodies against 2561 fimbriae, as well as antibodies against peptide I (V-V-M-A-N-T-G-A-M-E-V-G-K-T-L-A-E-V-K-Cys) and peptide J (A-L-T-T-E-L-T-A-E-N-Q-E-A-A-G-L-I-M-T-A-E-P-Cys), reacted. However, antifimbrial antibodies against strain 2561 reacted very weakly compared to anti-peptide I and anti-peptide J. Negative staining of whole W50 cells, as well as immunogold electron microscopy with anti-peptide I and anti-peptide J, showed fimbriae shorter in length and very few in number compared to those of strain 2561. Purified fimbriae showed no hemagglutinating activity. Amino acid composition was very similar to that of previously reported fimbriae of the 2561 strain.
Potassium Salts Inhibit Growth of the Cyanobacteria Microcystis spp. in Pond Water and Defined Media: Implications for Control of Microcystin-Producing Aquatic Blooms
Authors: Parker D L , Kumar H D , Rai L C , Singh J B
Published: January 2020

Ten metals were assayed in 21 Indian ponds which comprised three groups: (i) eutrophic alkaline ponds containing <2.5 mM potassium and thick growths of Microcystis aeruginosa or Microcystis flos-aquae during most of the year, (ii) equally eutrophic alkaline ponds containing >2.8 mM potassium and no detectable Microcystis growth, and (iii) oligo- or mesotrophic ponds with various potassium and hydrogen ion concentrations and no persistent Microcystis blooms. The effects of potassium on Microcystis growth were examined in filter-sterilized pond water and in defined culture media. A 50% reduction in the 10-day yield of cultured M. aeruginosa was observed in DP medium and pond water supplemented with 1 and 3 mM KCl, respectively. In contrast, the addition of 2 to 30 mM NaCl did not suppress the growth of M. aeruginosa in either DP medium or pond water. Both 5 mM KCl and 20 mM KHCO(inf3) in J medium strongly inhibited the growth of M. flos-aquae C3-9, whereas 5 to 30 mM NaCl had no effect and 20 mM NaHCO(inf3) was stimulatory. For pond water cultured with a mixture of M. aeruginosa and the duckweed Wolffia arrhiza, M. aeruginosa dominated in unsupplemented water and W. arrhiza dominated in water supplemented with 4.8 mM KCl. Implications for the ecology and control of Microcystis blooms are discussed.
Construction of specific erythromycin resistance mutations in the temperate lactococcal bacteriophage TP901-1 and their use in studies of phage biology
Authors: Koch B , Christiansen B , Evison T , Vogensen F K , Hammer K
Published: January 2020

A method for the construction and isolation of specifically designed mutations of the temperate lactococcal phage TP901-1 has been developed. Two different erm-labeled mutants were isolated. One was shown to be defective in lysogenization and excision. The other, showing normal lysogenization, was used for host range studies.
Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus
Authors: Liljeqvist S , Samuelson P , Hansson M , Nguyen T N , Binz H , Ståhl S
Published: January 2020

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.
Autolysis of Lactococcus lactis caused by induced overproduction of its major autolysin, AcmA
Authors: Buist G , Karsens H , Nauta A , van Sinderen D , Venema G , Kok J
Published: January 2020

The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells became subject to autolysis, resulting in the release of intracellular proteins.
Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains
Authors: Medina K , Carrau F M , Gioia O , Bracesco N
Published: January 2020

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.
A spontaneous change in the intracellular cyclic AMP level in Aspergillus niger is influenced by the sucrose concentration in the medium and by light
Authors: Gradisnik-Grapulin M , Legisa M
Published: January 2020

A spontaneous rise in intracellular cyclic AMP (cAMP) levels was observed in the early stages of Aspergillus niger growth under conditions yielding large amounts of citric acid. The amount of cAMP formed was found to depend on the initial concentration of sucrose in the medium. Under higher-sucrose conditions, the cAMP peak appeared earlier and was higher, while in lower-sucrose media a flattened peak was observed later in fermentation. Since in media with higher concentrations of sucrose intracellular citric acid starts to accumulate earlier and more rapidly, cAMP synthesis may be triggered by intracellular acidification, which is caused by the dissociation of citric acid. No spontaneous increase in cAMP concentrations could be detected when the cells were grown in continuously illuminated cultures, suggesting that A. niger phosphodiesterase (PDE) is photoregulated. More evidence for the activation of PDE by light was obtained from morphological studies under light and dark conditions in the presence of cAMP or N6,O2'-dibutyryl cAMP, and this idea was additionally supported by experiments in which PDE inhibitors were tested.
A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor
Authors: Délye C , Laigret F , Corio-Costet M F
Published: January 2020

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.
Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation
Authors: Howlett N G , Avery S V
Published: January 2020

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.