bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Methylobacterium species by 16S rRNA gene-targeted PCR
Authors: Nishio T , Yoshikura T , Itoh H
Published: January 2020

We designed PCR primers for specific amplification of the 16S rRNA genes of seven species of the genus Methylobacterium. All of the pairwise species tested were successfully differentiated by PCR detection with a combination of five primer sets, with the exception of M. extorquens and M. rhodesianum. These primers did not cross-react with closely related bacteria in the alpha subclass tested.
Karyotypic Variability in Ribosomal DNA Subchromosome Size among Colpodid Ciliates, a Possible Tool To Differentiate Colpodid Species
Authors: Martin A , Palacios G , Olmo A , Martin-Gonzalez A , Ruiz-Perez L M , Gutierrez J C
Published: January 2020

Pulsed-field gel electrophoresis has been applied to analyze the karyotypic variability among colpodid ciliates. The 18S ribosomal gene was found at different locations in the electrophoretic pattern, and these size variations in the ribosomal DNA subchromosomal molecule seem to be species specific. This could potentially be a useful new tool with which to differentiate colpodid ciliates.
Comparative analysis of antibiotic resistance, immunofluorescent colony staining, and a transgenic marker (bioluminescence) for monitoring the environmental fate of rhizobacterium
Authors: Mahaffee W F , Bauske E M , van Vuurde J W , van der Wolf J M , van den Brink M , Kloepper J W
Published: January 2020

Field releases of the wild-type plant growth-promoting rhizobacterium Pseudomonas fluorescens 89B-27, its bioluminescent derivative GEM-8 (89B-27::Tn4431), and a spontaneous rifampin-resistant variant estimating the wild-type population. Seed and root samples were taken 0, 7, 14, 21, or 28, 35 or 42, and 70 days after planting in each year and processed for enumeration by spiral plating or immunofluorescent colony staining (IFC). In both years, the populations of 89B-27, R34, and GEM-8, as measured by IFC, were not significantly different (P > 0.05) from each other at each sampling time. However, the populations of R34 and GEM-8, as measured by spiral plating and differentiation based on their respective phenotypes, were significantly lower (P < 0.05) than the wild-type populations and their IFC-determined populations. These data indicate that traditional marker systems may underestimate populations and hence the survival and colonization of genetically marked bacteria.
Immunomagnetic detection of Bacillus stearothermophilus spores in food and environmental samples
Authors: Blake M R , Weimer B C
Published: January 2020

There are currently no methods for the rapid and sensitive detection of bacterial spores that could be used to direct raw materials containing high spore loads away from products that pose a food safety risk. Existing methods require an overnight incubation, cannot detect spores below 10(5) CFU/ml, or are not specific to particular species. This work describes a method to specifically detect < 10(4) CFU of bacterial spores per ml within 2 h. Polyclonal antibodies to Bacillus stearothermophilus spores were attached to 2.8-micron-diameter magnetic polystyrene beads by using a polythreonine cross-linker via the antibody carbohydrate moiety. A biotin-avidin-amplified sandwich enzyme-linked immunosorbent assay coupled to a fluorescent substrate was used to quantitate captured spores. The concentration of B. stearothermophilus spores in samples was linearly correlated to fluorescent activity (r2 = 0.99) with a lower detection limit of 8 x 10(3) CFU/ml and an upper detection limit of 8 x 10(5) CFU/ml. The detection limits are not fixed and can be changed by varying the immunomagnetic bead concentration. Several food and environmental samples were tested to demonstrate the versatility of the assay.
Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains
Authors: Pelletier C , Bouley C , Cayuela C , Bouttier S , Bourlioux P , Bellon-Fontaine M N
Published: January 2020

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.
Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales
Authors: Redecker D , Thierfelder H , Walker C , Werner D
Published: January 2020

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.
Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene
Authors: Iizumi T , Nakamura K
Published: January 2020

The dnaK gene of an ammonia-oxidizing bacterium, Nitrosomonas europaea, was cloned and sequenced. It was found that the dnaK gene product was highly homologous to previously analyzed dnaK gene products from other organisms at the amino acid level. Two partial open reading frames located upstream and downstream of the dnaK gene were also found and identified as grpE and dnaJ genes, respectively, by the predicted amino acid homology of their gene products to other bacterial GrpE and DnaJ proteins. Transcription of the dnaK gene was strongly induced by a heat shock from 30 to 37 degrees C. An analysis of the expression of the dnaK gene fused to the lacZ translational reporter gene also showed eightfold increase in beta-galactosidase activity after the heat shock induction. Heat-inducible transcription start sites of the dnaK gene, revealed by primer extension analysis, were located 16 and 17 nucleotides upstream from the translational start codon of the dnaK gene, and the predicted promoter sequence showed a homology to the consensus sequence of sigma 32-dependent heat shock promoters of gram-negative bacteria. The upstream region of the dnaK gene did not contain the inverted repeat structure that was involved in the regulation of the heat shock gene of several gram-negative and gram-positive bacteria. Therefore, we conclude that the heat shock regulatory mechanism of the N. europaea dnaK gene may be similar to the sigma 32-dependent mechanism observed in other gram-negative bacteria.
Clustering of trichothecene-producing Fusarium strains determined from 28S ribosomal DNA sequences
Authors: Mulè G , Logrieco A , Stea G , Bottalico A
Published: January 2020

The genus Fusarium includes several species that produce trichothecenes. We analyzed DNA sequences from a variable region at the 5' end of the large nuclear ribosomal DNA (rDNA) (28S) to determine the genetic relatedness of trichothecene-producing Fusarium species. All trichothecene-producing strains clustered together, and two monophyletic groups were resolved. The first clade included strains of F. acuminatum, F. sambucinum, F. tumidum, F. compactum, F. camptoceras (red pigment), F. sporotrichioides, and F. venenatum, which produced type A trichothecenes (T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol). The second clade consisted of strains of F. crookwellense, F. culmorum, and F. graminearum, which produced type B trichothecenes (fusarenone-X, nivalenol, and deoxynivalenol). The phylogenetic placement of the species based on rDNA correlated better with toxic secondary metabolite data rather than with the current classification system based on morphology.
Denitrifying Bacteria in the Earthworm Gastrointestinal Tract and In Vivo Emission of Nitrous Oxide (N(inf2)O) by Earthworms
Authors: Karsten G R , Drake H L
Published: January 2020

Earthworms (Lumbricus rubellus and Octolasium lacteum) and gut homogenates did not produce CH(inf4), and methanogens were not readily culturable from gut material. In contrast, the numbers of culturable denitrifiers averaged 7 x 10(sup7) and 9 x 10(sup6) per g (dry weight) of gut material for L. rubellus and O. lacteum, respectively; these values were 256- and 35-fold larger than the numbers of culturable denitrifiers in the soil from which the earthworms were obtained. Anaerobically incubated earthworm gut homogenates supplemented with nitrate produced N(inf2)O at rates exceeding that of soil homogenates. Furthermore, living earthworms emitted N(inf2)O under aerobic conditions, and N(inf2)O emission was stimulated by acetylene. For earthworms collected from a mildly acidic (pH 6) beech forest soil, the rates of N(inf2)O emission for earthworms and soil averaged 884 and 2 pmol per h per g (fresh weight), respectively. In contrast, for earthworms collected from a more acidic (pH 4.6) oak-beech forest soil, N(inf2)O emission by earthworms and soil averaged 145 and 45 pmol per h per g (fresh weight), respectively. Based on the extrapolation of this data, earthworms accounted for an estimated 16 and 0.25% of the total N(inf2)O produced at the stand level of these beech and oak-beech forest soils, respectively.
Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates
Authors: Meinander N Q , Hahn-Hägerdal B
Published: January 2020

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.
Stimulation of aryl metabolite production in the basidiomycete Bjerkandera sp. strain BOS55 with biosynthetic precursors and lignin degradation products
Authors: Mester T , Swarts H J , Romero i Sole S , de Bont J A , Field J A
Published: January 2020

Aryl metabolites are known to have an important role in the ligninolytic system of white rot fungi. The addition of known precursors and aromatic acids representing lignin degradation products stimulated the production of aryl metabolites (veratryl alcohol, veratraldehyde, p-anisaldehyde, and 3-chloro-p-anisaldehyde) in the white rot fungus Bjerkandera sp. strain BOS55. The presence of manganese (Mn) is known to inhibit the biosynthesis of veratryl alcohol (T. Mester, E. de Jong, and J.A. Field, Appl. Environ. Microbiol. 61:1881-1887, 1995). A new finding of this study was that the production of the other aryl metabolites, p-anisaldehyde and 3-chloro-p-anisaldehyde, was also inhibited by Mn. We attempted to bypass the Mn-inhibited step in the biosynthesis of aryl metabolites by the addition of known and suspected precursors. Most of these compounds were not able to bypass the inhibiting effect of Mn. Only the fully methylated precursors (veratrate, p-anisate, and 3-chloro-p-anisate) provided similar concentrations of aryl metabolites in the presence and absence of Mn, indicating that Mn does not influence the reduction of the benzylic acid group. The addition of deuterated benzoate and 4-hydroxybenzoate resulted in the formation of deuterated aryl metabolites, indicating that these aromatic acids entered into the biosynthetic pathway and were common intermediates to all aryl metabolites. Only deuterated chlorinated anisyl metabolites were produced when the cultures were supplemented with deuterated 3-chloro-4-hydroxybenzoate. This observation combined with the fact that 3-chloro-4-hydroxybenzoate is a natural product of Bjerkandera spp. (H. J. Swarts, F. J. M. Verhagen, J. A. Field, and J. B. P. A. Wijnberg, Phytochemistry 42:1699-1701, 1996) suggest that it is a possible intermediate in chlorinated anisyl metabolite biosynthesis.
An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors
Authors: Sørensen A H , Ahring B K
Published: January 2020

An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test. The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make the test well suited for routine analysis of large numbers of samples and thus for process surveillance during operation of biogas digesters.
Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera
Authors: Schloter M , Wiehe W , Assmus B , Steindl H , Becke H , Höflich G , Hartmann A
Published: January 2020

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.
Vegetable-Associated Pediococcus parvulus Produces Pediocin PA-1
Authors: Bennik M , Smid E J , Gorris L
Published: January 2020

Two bacteriocin-producing strains of Pediococcus parvulus were isolated from minimally processed vegetables. Recombinant DNA techniques revealed the presence of the pediocin PA-1 gene in both strains. Biochemical analysis confirmed the production of pediocin PA-1 and excluded the presence of other bacteriocins.
Transformation of Escherichia coli with a large plasmid of Acidiphilium multivorum AIU 301 encoding arsenic resistance
Authors: Suzuki K , Wakao N , Sakurai Y , Kimura T , Sakka K , Ohmiya K
Published: January 2020

Acidiphilium multivorum AIU 301 isolated from acid mineral water had strong arsenic resistance. This bacterium harbored a number of plasmids with different molecular sizes. A plasmid of 56 kbp, named pKW301, was isolated from A. multivorum AIU 301. When pKW301 was transferred into Escherichia coli JM109 by electroporation, an E. coli transformant carrying pKW301 exhibited resistance to sodium arsenite, sodium arsenate, and mercuric (II) chloride.
Evolutionary relationships of cultivated psychrophilic and barophilic deep-sea bacteria
Authors: Delong E F , Franks D G , Yayanos A A
Published: January 2020

Evolutionary relationships of cultivated barophilic bacteria were determined. All psychrophilic and barophilic isolates were affiliated with one of five genera of the gamma subdivision of the class Proteobacteria ((gamma)-Proteobacteria): Shewanella, Photobacterium, Colwellia, Moritella, and a new group containing strain CNPT3. The data indicate that the barophilic phenotype has evolved independently in different (gamma)-Proteobacteria genera.
Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes
Authors: Leloup L , Ehrlich S D , Zagorec M , Morel-Deville F
Published: January 2020

Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.
The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation
Authors: Proctor L M , Lai R , Gunsalus R P
Published: January 2020

Methanogenic Archaea are found in a wide range of environments and use several strategies to adjust to changes in extracellular solute concentrations. One methanogenic archaeon, Methanosarcina thermophila TM-1, can adapt to various osmotic conditions by synthesis of alpha-glutamate and a newly discovered compatible solute, Ne-acetyl-beta-lysine, or by accumulation of glycine betaine (betaine) and potassium ions from the environment. Since betaine transport has not been characterized for any of the methanogenic Archaea, we examined the uptake of this solute by M. thermophila TM-1. When cells were grown in mineral salts media containing from 0.1 to 0.8 M NaC1, M. thermophila accumulated betaine in concentrations up to 140 times those of a concentration gradient within 10 min of exposure to the solute. The betaine uptake system consisted of a single, high-affinity transporter with an apparent K3 of 10 microM and an apparent maximum transport velocity of 1.15 nmol/min/mg of protein. The transporter appeared to be specific for betaine, since potential substrates, including glycine, sarcosine, dimethyl glycine, choline, and proline, did not significantly inhibit betaine uptake. M. thermophila TM-1 cells can also regulate the capacity for betaine accumulation, since the rate of betaine transport was reduced in cells pregrown in a high-osmolarity medium when 500 microM betaine was present. Betaine transport appears to be H+ and/or Na+ driven, since betaine transport was inhibited by several types of protonophores and sodium ionophores.
Pristine environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria
Authors: Kamagata Y , Fulthorpe R R , Tamura K , Takami H , Forney L J , Tiedje J M
Published: January 2020

2,4-Dichlorophenoxyacetic acid (2,4-D)-degrading bacteria were isolated from pristine environments which had no history of 2,4-D exposure. By using 2,4-D dye indicator medium or 14C-labeled 2,4-D medium, six strains were isolated from eight enrichment cultures capable of degrading 2,4-D. Phylogenetic analyses based on 16S ribosomal DNA (rDNA) sequencing and physiological properties revealed that one isolate from Hawaiian volcanic soil could be classified in the genus Variovorax (a member of the beta subdivision of the class Proteobacteria) and that the other five isolates from Hawaiian volcanic soils, Saskatchewan forest soil, and Chilean forest soil have 16S rDNAs with high degrees of similarity to those of the Bradyrhizobium group (a member of the alpha subdivision of the class Proteobacteria). All the isolates grow slowly on either nutrient media (0.1 x Bacto Peptone-tryptone-yeast extract-glucose [PTYG] or 0.1 x Luria broth [LB] medium) or 2,4-D medium, with mean generation times of 16 to 30 h, which are significantly slower than previously known 2,4-D degraders. Nutrient-rich media such as full-strength PTYG and LB medium did not allow their growth. PCR amplification using internal consensus sequences of tfdA (a gene encoding an enzyme for the first step of 2,4-D mineralization, found in pJP4 of Alcaligenes eutrophus JMP134 and some other 2,4-D-degrading bacteria) as primers and Southern hybridization with pJP4-tfdA as a probe revealed that the isolate belonging to the genus Variovorax carried the tfdA gene. This gene was transmissible to A. eutrophus JMP228 carrying a plasmid with a mutant tfdA gene. The other five isolates did not appear to carry tfdA, and 2,4-D-specific alpha-ketoglutarate-dependent dioxygenase activity could not be detected in cell lysates. These results indicate that 2,4-D-degrading bacteria in pristine environments are slow-growing bacteria and that most of their phylogenies and catabolic genes differ from those of 2,4-D degraders typically isolated from agricultural soils or contaminated environments.
A new computational method for detection of chimeric 16S rRNA artifacts generated by PCR amplification from mixed bacterial populations
Authors: Komatsoulis G A , Waterman M S
Published: January 2020

A new computational method (chimeric alignment) has been developed to detect chimeric 16S rRNA artifacts generated during PCR amplification from mixed bacterial populations. In contrast to other nearest-neighbor methods (e.g., CHECK_CHIMERA) that define sequence similarity by k-tuple matching, the chimeric alignment method uses the score from dynamic programming alignments. Further, the chimeric alignments are displayed to the user to assist in sequence classification. The distribution of improvement scores for 500 authentic, nonchimeric sequences and 300 artificial chimeras (constructed from authentic sequences) was used to study the sensitivity and accuracy of both chimeric alignment and CHECK_CHIMERA. At a constant rate of authentic sequence misclassification (5%), chimeric alignment incorrectly classified 13% of the artificial chimeras versus 14% for CHECK_CHIMERA. Interestingly, only 1% of nonchimeras and 10% of chimeras were misclassified by both programs, suggesting that optimum performance is obtained by using the two methods to assign sequences to three classes: high-probability nonchimeras, high-probability chimeras, and sequences that need further study by other means. This study suggests that k-tuple-based matching methods are more sensitive than alignment-based methods when there is significant parental sequence similarity, while the opposite becomes true as the sequences become more distantly related. The software and a World Wide Web-based server are available at http://www-hto.usc.edu/software/mglobal CHI.
Gene Expression Analysis during Conidial Germ Tube and Appressorium Development in Colletotrichum trifolii
Authors: Buhr T L , Dickman M B
Published: January 2020

Preinfection development in Colletotrichum spp. exhibits three morphologies (conidia, germ tubes, and appressoria) and is directed by a complex interplay of environmental signals. Germ tube morphogenesis for Colletotrichum trifolii and the related fungus Colletotrichum gloeosporioides f. sp. aeschynomene was shown to be partially dependent on a balance between self-germination inhibitors and environmental nutrients or cutin. The degree of responsiveness to these environmental signals was strikingly different between the two fungal species. A solid contact surface stimulated germ tube morphogenesis and was the only apparent requirement for appressorium morphogenesis in both fungi. A population of C. trifolii conidia was incubated on a solid surface in the presence of cutin to stimulate nearly synchronous preinfection morphogenesis for gene expression analysis. RNA analysis of signal-transducing genes from C. trifolii, including genes for a serine-threonine kinase (TB3), calmodulin, and protein kinase C, showed that maximum transcription of all three genes occurred in conidia prior to or during germ tube morphogenesis. Transcription of melanin biosynthetic genes THR1 and SCD1 (Y. Kubo, Y. Takano, and I. Furusawa, Colletotrichum Newsl. II:5-10, 1996; N. S. Perpetua, Y. Kubo, N. Yasuda, Y. Takano, and I. Furusawa, Mol. Plant-Microbe Interact. 9:323-329, 1996) was highest prior to and during appressorium morphogenesis.
Structure of a laccase-mediated product of coupling of 2,4-diamino-6-nitrotoluene to guaiacol, a model for coupling of 2,4,6-trinitrotoluene metabolites to a humic organic soil matrix
Authors: Dawel G , Kastner M , Michels J , Poppitz W , Gunther W , Fritsche W
Published: January 2020

This work presents laccase-mediated model reactions for coupling of reduced 2,4,6-trinitrotoluene (TNT) metabolites to an organic soil matrix. The structure of an isolated coupling product of 2,4-diamino-6-nitrotoluene (2,4-DANT) to guaiacol as humic constituent was determined. Among several structures, the compound was identified conclusively to be the trinuclear coupling product 5-(2-amino-3-methyl-4-nitroanilino)-3,3(prm1)-dimethoxy-4,4(prm1)-diphenoqu inone. The compound has a weight of 409 g mol(sup-1) and may serve as a model reaction for the biogenic formation of bound residues in soil from TNT by coupling aminotoluenes (reduced TNT metabolites) to humic constituents. A linear correlation of the substrate consumption to the enzyme activity was detected. Based on this observation, the described reaction of 2,4-DANT coupling to guaiacol may be used for determination of laccase activity since the reaction was not inhibited by other compounds of culture supernatants. We propose a two-step mechanism for the coupling reaction because 2,4-DANT was not transformed by laccases in the absence of guaiacol and guaiacol oxidation was independent of the presence of 2,4-DANT. The first reaction step is a laccase-mediated dimerization of two guaiacol monomers with subsequent oxidation to a diphenoquinone. The second step is the nucleophilic addition of 2,4-DANT to the ortho position of the carbonyl group of the diphenoquinone structure.
Growth of Thiobacillus ferrooxidans: a Novel Experimental Design for Batch Growth and Bacterial Leaching Studies
Authors: Harvey P I , Crundwell F K
Published: January 2020

The concentrations of ferrous and ferric ions change dramatically during the course of the batch experiments usually performed to study the kinetics of the bacterial oxidation of ferrous ions and sulfide minerals. This change in concentration of the iron species during the course of the experiment often makes it difficult to interpret the results of these experiments, as is evidenced by the lack of consensus concerning the mechanism of bacterial leaching. If the concentrations of ferrous and ferric ions were constant throughout the course of the batch experiment, then the role of the bacteria could be easily established, because the rate of the chemical leaching should be the same at a given redox potential in the presence and in the absence of bacteria. In this paper we report an experiment designed to obtain kinetic data under these conditions. The redox potential is used as a measure of the concentrations of ferrous and ferric ions, and the redox potential of the leaching solution is controlled throughout the experiment by electrolysis. The effects of ferrous, ferric, and arsenite ions on the rate of growth of Thiobacillus ferrooxidans on ferrous ions in this redox-controlled reactor are presented. In addition, the growth of this bacterium on ferrous ions in batch culture was also determined, and it is shown that the parameters obtained from the batch culture and the redox-controlled batch culture are the same. An analysis of the results from the batch culture indicates that the initial number of bacteria that are adapted to the solution depends on the concentrations of ferrous and arsenite ions.
Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae
Authors: Zhang J H , Quigley N B , Gross D C
Published: January 2020

Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv. syringae on multienzymatic peptide synthetases. Sequence analysis of the interval between the syrB and syrD genes of P. syringae pv. syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP. The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues. Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli. The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA. The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems. Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media. The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media. Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits. These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P. syringae pv. syringae.
Unique regulation of crystal protein production in Bacillus thuringiensis subsp. yunnanensis is mediated by the cry protein-encoding 103-megadalton plasmid
Authors: Srinivas G , Vennison S J , Sudha S N , Balasubramanian P , Sekar V
Published: January 2020

In sporulating cultures of Bacillus thuringiensis subsp. yunnanensis HD977, two cell types are observed: cells forming only spores and cells forming only crystals. Curing analysis suggested that the crystal proteins are plasmid encoded. Through plasmid transfer experiments, it was established that a 103-MDa plasmid is involved in the crystal production. Conjugal transfer of this plasmid to Cry- recipient cells of Bacillus thuringiensis subsp. kurstaki HD73-26 conferred the ability to produce crystals exclusively on asporogenous cells of the recipient, indicating that the 103-MDa plasmid mediates the unique regulation of Cry protein production. When the dipteran-specific cryIVB gene was introduced into wild-type (Cry+) and Cry- backgrounds of B. thuringiensis subsp. yunnanensis by phage CP51ts45-mediated transduction, similar to all other B. thuringiensis strains, irregular crystals of CryIVB protein were produced by spore-forming cells in both backgrounds. However, the synthesis of the bipyramidal inclusions of B. thuringiensis subsp. yunnanensis was still limited only to asporogenous cells of the transductant. Thus, it appears that the unique property of exclusive crystal formation in asporogenous cells of B. thuringiensis subsp. yunnanensis is associated with the crystal protein gene(s) per se or its cis acting elements. As the crystals in B. thuringiensis subsp. yunnanensis were formed only in asporogenous cells, attempts were made to find out whether crystal formation had any inhibitory effect on sporulation. It was observed that both Cry+ and Cry- strains of B. thuringiensis subsp. yunnanensis (HD977 and HD977-1, respectively) exhibited comparable sporulation efficiencies. In addition, the Cry- B. thuringiensis subsp. kurstaki host (HD73-26) and its Cry+ transconjugant (HD73-26-16), expressing the B. thuringiensis subsp. yunnanensis crystal protein, were also comparable in their sporulation efficiencies, indicating that production of the crystal proteins of B. thuringiensis subsp. yunnanensis does not affect the process of sporulation.
Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme
Authors: Savijoki K , Palva A
Published: January 2020

The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.
Phylogenetic analysis and in situ identification of bacteria in activated sludge
Authors: Snaidr J , Amann R , Huber I , Ludwig W , Schleifer K H
Published: January 2020

The bacterial community structure of activated sludge of a large municipal wastewater treatment plant was investigated by use of the rRNA approach. Almost-full-length genes coding for the small-subunit rRNA (rDNA) were amplified by PCR and subsequently cloned into the pGEM-T vector. Clones were screened by dot blot hybridization with group-specific oligonucleotide probes. The phylogenetic affiliations of clones were compared with the results obtained with the original sample by in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes and found to be in general agreement. Twenty-five 16S rDNA clones were fully sequenced, 11 were almost fully (> 80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the majority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclasses of Proteobacteria were represented by 13 and 4 clones, respectively. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G+C content. The 16S rDNA of two clones showed similarity with 16S rDNA genes of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targeted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic representatives of the genus Arcobacter were present in significant numbers (4%) in the activated sludge sample examined. Pairs of probes targeted to the 5' and 3' regions were used for detection of chimeric sequences by in situ hybridization. Two clones could be identified as chimera by applying such a pair of probes.
Are Bacteria Omnipresent on Phanerochaete chrysosporium Burdsall?
Authors: Janse B , Gaskell J , Cullen D , Zapanta L , Dougherty M J , Tien M
Published: January 2020

Cultures of Phanerochaete chrysosporium were examined for the presence of bacteria as previously described (F. Seigle-Murandi, P. Guiraud, C. Falsen, and K.-E. Eriksson, Appl. Environ. Microbiol. 62:2477-2481, 1996). Under no conditions could bacteria be isolated from cultures of P. chrysosporium. With PCR primers corresponding to small-subunit rRNA genes, no bacterium-like product could be amplified from cultures of the widely used P. chrysosporium strain BKM-F-1767. Thus, we could find no evidence of bacteria in association with P. chrysosporium BKM-F-1767.
Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands
Authors: Graczyk T K , Balazs G H , Work T , Aguirre A A , Ellis D M , Murakawa S , Morris R
Published: January 2020

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.
Stress-Induced Production of Biofilm in the Hyperthermophile Archaeoglobus fulgidus
Authors: Lapaglia C , Hartzell P L
Published: January 2020

Archaeoglobus fulgidus, an anaerobic marine hyperthermophile, forms a biofilm in response to environmental stresses. The biofilm is a heterogeneous, morphologically variable structure containing protein, polysaccharide, and metals. Production of the biofilm can be induced by nonphysiological extremes of pH and temperature, by high concentrations of metals, and by addition of antibiotics, xenobiotics, or oxygen. Cells within the biofilm show an increased tolerance to otherwise toxic environmental conditions. Metals sequestered within the biofilm stimulate growth of A. fulgidus cells in metal-depleted medium. These data suggest that cells may produce biofilm as a mechanism for concentrating cells and attaching to surfaces, as a protective barrier, and as a reserve nutrient. Because similar biofilms are formed by Archaeoglobus profundus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, biofilm formation might be a common stress response mechanism among the archaea.
Thermophilic carbon-sulfur-bond-targeted biodesulfurization
Authors: Konishi J , Ishii Y , Onaka T , Okumura K , Suzuki M
Published: January 2020

Petroleum contains many heterocyclic organosulfur compounds refractory to conventional hydrodesulfurization carried out with chemical catalysts. Among these, dibenzothiophene (DBT) and DBTs bearing alkyl substitutions are representative compounds. Two bacterial strains, which have been identified as Paenibacillus strains and which are capable of efficiently cleaving carbon-sulfur (C--S) bonds in DBT at high temperatures, have been isolated for the first time. Upon attacking DBT and its various methylated derivatives at temperatures up to 60(deg)C, both growing and resting cells of these bacteria can release sulfur atoms as sulfate ions and leave the monohydroxylated hydrocarbon moieties intact. Moreover, when either of these paenibacilli was incubated at 50(deg)C with light gas oil previously processed through hydrodesulfurization, the total sulfur content in the oil phase clearly decreased.
Physiological and biochemical characterization of Trichoderma harzianum, a biological control agent against soilborne fungal plant pathogens
Authors: Grondona I , Hermosa R , Tejada M , Gomis M D , Mateos P F , Bridge P D , Monte E , Garcia-Acha I
Published: January 2020

Monoconidial cultures of 15 isolates of Trichoderma harzianum were characterized on the basis of 82 morphological, physiological, and biochemical features and 99 isoenzyme bands from seven enzyme systems. The results were subjected to numerical analysis which revealed four distinct groups. Representative sequences of the internal transcribed spacer 1 (ITS 1)-ITS 2 region in the ribosomal DNA gene cluster were compared between groups confirming this distribution. The utility of the groupings generated from the morphological, physiological, and biochemical data was assessed by including an additional environmental isolate in the electrophoretic analysis. The in vitro antibiotic activity of the T. harzianum isolates was assayed against 10 isolates of five different soilborne fungal plant pathogens: Aphanomyces cochlioides, Rhizoctonia solani, Phoma betae, Acremonium cucurbitacearum, and Fusarium oxysporum f. sp. radicis lycopersici. Similarities between levels and specificities of biological activity and the numerical characterization groupings are both discussed in relation to antagonist-specific populations in known and potential biocontrol species.
Reduction of aerobic acetate production by Escherichia coli
Authors: Farmer W R , Liao J C
Published: January 2020

Acetate excretion by Escherichia coli during aerobic growth on glucose is a major obstacle to enhanced recombinant protein production. We report here that the fraction of carbon flux through the anaplerotic pathways is one of the factors influencing acetate excretion. Flux analysis of E. coli central metabolic pathways predicts that increasing the fraction of carbon flux through the phosphoenolpyruvate carboxylase (PPC) pathway and the glyoxylate bypass reduces acetate production. We tested this prediction by overexpressing PPC and deregulating the glyoxylate bypass by using a fadR strain. Results show that the acetate yield by the fadR strain with PPC overexpression is decreased more than fourfold compared to the control, while the biomass yield is relatively unaffected. Apparently, the fraction of carbon flux through the anaplerotic pathways is one of the factors that influence acetate excretion. These results confirm the prediction of our flux analysis and further suggest that E. coli is not fully optimized for efficient utilization of glucose.
Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons
Authors: Lloyd-Jones G , Lau P C
Published: January 2020

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria.
Expression and secretion of a thermostable bacterial xylanase in Kluyveromyces lactis
Authors: Walsh D J , Bergquist P L
Published: January 2020

The xynA structural gene from the extremely thermophilic anaerobe Dictyoglomus thermophilum Rt46B.1 was fused in frame with the secretion signal of the Kluyveromyces lactis killer toxin in episomal expression vectors based on the Kluyveromyces plasmid pKD1. XynA was secreted predominantly as an unglycosylated 35-kDa protein which comprised up to 90% of the total extracellular proteins and reached a concentration of 130 micrograms/ml in shake-flask cultures grown under selective conditions.
Interference of peptone and tyrosine with the lignin peroxidase assay
Authors: ten Have R , Hartmans S , Field J A
Published: January 2020

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.