bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel ATP-Binding Cassette Transporter Involved in Multidrug Resistance in the Phytopathogenic Fungus
Penicillium digitatum
Authors: Nakaune Ryoji , Adachi Kiichi , Nawata Osamu , Tomiyama Masamitsu , Akutsu Katsumi , Hibi Tadaaki 
Published: December 2019

ABSTRACT

Demethylation inhibitor (DMI)-resistant strains of the plant pathogenic fungus
Penicillium digitatum
were shown to be simultaneously resistant to cycloheximide, 4-nitroquinoline-
N
-oxide (4NQO), and acriflavine. A
PMR1
(
Penicillium
multidrug resistance) gene encoding an ATP-binding cassette (ABC) transporter (P-glycoprotein) was cloned from a genomic DNA library of a DMI-resistant strain (LC2) of
Penicillium digitatum
by heterologous hybridization with a DNA fragment containing an ABC-encoding region from
Botrytis cinerea
. Sequence analysis revealed significant amino acid homology to the primary structures of PMR1 (protein encoded by the
PMR1
gene) and ABC transporters of
Saccharomyces cerevisiae
(PDR5 and SNQ2),
Schizosaccharomyces pombe
(HBA2),
Candida albicans
(CDR1), and
Aspergillus nidulans
(AtrA and AtrB). Disruption of the
PMR1
gene of
P. digitatum
DMI-resistant strain LC2 demonstrated that PMR1 was an important determinant of resistance to DMIs. The effective concentrations inhibiting radial growth by 50% (EC
50
s) and the MICs of fenarimol and bitertanol for the
PMR1
disruptants (Δ
pmr1
mutants) were equivalent to those for DMI-sensitive strains. Northern blot analysis indicated that severalfold more
PMR1
transcript accumulated in the DMI-resistant strains compared with those in DMI-sensitive strains in the absence of fungicide. In both DMI-resistant and -sensitive strains, transcription of
PMR1
was strongly enhanced within 10 min after treatment with the DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participates directly in the DMI resistance of the fungus.
Molecular Diversity of Rhizobia Occurring on Native Shrubby Legumes in Southeastern Australia
Authors: Lafay Bénédicte , Burdon Jeremy J. 
Published: December 2019

ABSTRACTThe structure of rhizobial communities nodulating native shrubby legumes in open eucalypt forest of southeastern Australia was investigated by a molecular approach. Twenty-one genomic species were characterized by small-subunit ribosomal DNA PCR-restriction fragment length polymorphism and phylogenetic analyses, among 745 rhizobial strains isolated from nodules sampled on 32 different legume host species at 12 sites. Among these rhizobial genomic species, 16 belonged to theBradyrhizobiumsubgroup, 2 to theRhizobium leguminosarumsubgroup, and 3 to theMesorhizobiumsubgroup. Only one genomic species corresponded to a known species (Rhizobium tropici). The distribution of the various genomic species was highly unbalanced among the 745 isolates, legume hosts, and sites.Bradyrhizobiumspecies were by far the most abundant, andRhizobium tropicidominated among theRhizobiumandMesorhizobiumisolates in the generally acid soils where nodules were collected. Although a statistically significant association occurred between the eight most common genomic species and the 32 hosts, there was sufficient overlap in distributions that no clear specificity between rhizobial genomic species and legume taxa was observed. However, for three legume species, some preference for particular genomic species was suggested. Similarly, no geographical partitioning was found.
Polymorphisms in
Phytophthora infestans
: Four Mitochondrial Haplotypes Are Detected after PCR Amplification of DNA from Pure Cultures or from Host Lesions
Authors: Griffith Gareth W. , Shaw David S. 
Published: December 2019

ABSTRACT

Four pairs of primers were designed for PCR amplification of known polymorphic regions of the mitochondrial genome of
Phytophthora infestans
. Digestion of the amplified products with restriction enzymes allows identification of previously identified haplotypes. Product P2 cut with
Msp
I uniquely identifies haplotypes Ib and IIa, while types Ia and IIb are differentiated by digestion of product P4 with
Eco
RI. Digestion of products P1 and P3 gave results similar to that with digestion of P4, but amplification of these products was less robust. Thus, all four common haplotypes are identified by amplifying and digesting products P2 and P4. Identification of haplotypes was also possible from DNA extracted directly from small, late-blight lesions on both tomato and potato leaves, making isolation of the fungus unnecessary. A rapid and efficient method of monitoring changes in the pathogen population is facilitated. These PCR primers were also useful for differentiating other
Phytophthora
species.
Spatial Physiological Heterogeneity in
Pseudomonas aeruginosa
Biofilm Is Determined by Oxygen Availability
Authors: Xu Karen D. , Stewart Philip S. , Xia Fuhu , Huang Ching-Tsan , McFeters Gordon A. 
Published: December 2019

ABSTRACT

The role of oxygen availability in determining the local physiological activity of
Pseudomonas aeruginosa
growing in biofilms was investigated. Biofilms grown in an ambient-air environment expressed approximately 1/15th the alkaline phosphatase specific activity of planktonic bacteria subjected to the same phosphate limitation treatment. Biofilms grown in a gaseous environment of pure oxygen exhibited 1.9 times the amount of alkaline phosphatase specific activity of air-grown biofilms, whereas biofilms grown in an environment in which the air was replaced with pure nitrogen prior to the inducing treatment did not develop alkaline phosphatase activity. Frozen cross sections of biofilms stained for alkaline phosphatase activity with a fluorogenic stain demonstrated that alkaline phosphatase activity was concentrated in distinct bands adjacent to the gaseous interfaces. These bands were approximately 30 μm thick with biofilms grown in air, 2 μm thick with biofilms grown in pure nitrogen, and 46 μm thick with biofilms grown in pure oxygen. Overall biofilm thickness ranged from approximately 117 to approximately 151 μm. Measurements with an oxygen microelectrode indicated that oxygen was depleted locally within the biofilm and that the oxygen-replete zone was of a dimension similar to that of the biologically active zone, as indicated by alkaline phosphatase induction. These experiments revealed marked spatial physiological heterogeneity within
P. aeruginosa
biofilms in which active protein synthesis was restricted by oxygen availability to the upper 30 μm of the biofilm. Such physiological heterogeneity has implications for microbial ecology and for understanding the reduced susceptibilities of biofilms to antimicrobial agents.
Specific Cell Wall Proteins Confer Resistance to Nisin upon Yeast Cells
Authors: Dielbandhoesing S. K. , Zhang H. , Caro L. H. P. , van der Vaart J. M. , Klis F. M. , Verrips C. T. , Brul S. 
Published: December 2019

ABSTRACT

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type
Saccharomyces cerevisiae
. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104–3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both
CWP1
and
CWP2
was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.
Possible Transmission of
Streptococcus iniae
from Wild Fish to Cultured Marine Fish
Authors: Zlotkin Amir , Hershko Hannah , Eldar Avi 
Published: December 2019

ABSTRACT

Streptococcus iniae
was isolated from diseased wild fish collected near a mariculture facility where gilthead sea bream and European sea bass exhibited a similar infection. Species-specific PCR and ribotyping confirmed that wild and cultured fish were infected by a single
S. iniae
clone. Wild fish are therefore potential amplifiers of pathogenic
S. iniae
strains.
A Gliding Bacterium Strain Inhibits Adhesion and Motility of Another Gliding Bacterium Strain in a Marine Biofilm
Authors: Burchard Robert P. , Sorongon Maria L. 
Published: December 2019

ABSTRACTTwo species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genusCytophaga. One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.
Effective Recovery of Bacterial DNA and Percent-Guanine-Plus-Cytosine-Based Analysis of Community Structure in the Gastrointestinal Tract of Broiler Chickens
Authors: Apajalahti Juha H. A. , Särkilahti Laura K. , Mäki Brita R. E. , Heikkinen J. Pekka , Nurminen Päivi H. , Holben William E. 
Published: December 2019

ABSTRACT
A DNA-based, direct method for initial characterization of the total bacterial community in ileum and cecum of the chicken gastrointestinal (GI) tract was developed. The efficiencies of bacterial extraction and lysis were >95 and >99%, respectively, and therefore the DNA recovered should accurately reflect the bacterial communities of the ileal and cecal digesta. Total bacterial DNA samples were fractionated according to their percent G+C content. The profiles reflecting the composition of the bacterial community were reproducible within each compartment, but different between the compartments of the GI tract. This approach is independent of the culturability of the bacteria in the consortium and can be used to improve our understanding of how diet and other variables modulate the microbial communities of the GI tracts of animals.
PCR-Ribotyping of
Xenorhabdus
and
Photorhabdus
Isolates from the Caribbean Region in Relation to the Taxonomy and Geographic Distribution of Their Nematode Hosts
Authors: Fischer-Le Saux Marion , Mauléon Hervé , Constant Philippe , Brunel Brigitte , Boemare Noël 
Published: December 2019

ABSTRACT

The genetic diversity of symbiotic
Xenorhabdus
and
Photorhabdus
bacteria associated with entomopathogenic nematodes was examined by a restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes (rDNAs). A total of 117 strains were studied, most of which were isolated from the Caribbean basin after an exhaustive soil sampling. The collection consisted of 77 isolates recovered from entomopathogenic nematodes in 14 Caribbean islands and of 40 reference strains belonging to
Xenorhabdus
and
Photorhabdus
spp. collected at various localities worldwide. Thirty distinctive 16S rDNA genotypes were identified, and cluster analysis was used to distinguish the genus
Xenorhabdus
from the genus
Photorhabdus
. The genus
Xenorhabdus
appears more diverse than the genus
Photorhabdus
, and for both genera the bacterial genotype diversity is in congruence with the host-nematode taxonomy. The occurrence of symbiotic bacterial genotypes was related to the ecological distribution of host nematodes.
Method To Immobilize the Aphid-Pathogenic FungusErynia neoaphidisin an Alginate Matrix for Biocontrol
Authors: Shah P. A. , Aebi M. , Tuor U. 
Published: December 2019

ABSTRACTErynia neoaphidisis an important fungal pathogen of aphid pests worldwide. There have been few reported attempts to formulate this natural agent for use in biocontrol. In the current study, factors involved in the immobilization ofE. neoaphidishyphae in an alginate matrix were investigated. Hyphae of two isolates cultured in liquid medium were 220 to 620 μm in length and 7 to 19 μm in diameter with a 74 to 83% cytoplasmic content. The optimal concentration of low-viscosity sodium alginate for production of conidia from entrapped hyphae was 1.5% (wt/vol), and 0.1 and 0.25 M calcium chloride were equally suitable for use as the gelling solution. Alginate beads were rinsed with 10% sucrose after gelling. However, beads should not be left for longer than 40 min in 0.1 M calcium chloride or 10% sucrose to prevent a 10% loss in conidial production. A 40% (vol/vol) concentration of fungal biomass produced significantly more conidia than either 20% or the standard concentration of 10%. This effect persisted even after beads were dried overnight in a laminar flow hood and stored at 4°C for 4 days. Conidia from freshly produced alginate beads caused 27 to 32% infection in Pea aphids as determined by standardized laboratory bioassays. This finding was not significantly different from infections in aphids inoculated with fresh mycelial mats or plugs from Petri dish cultures. In conclusion, algination appears to be a promising technique for utilizingE. neoaphidisin the biocontrol of aphid pests.
Cloning and Expression of the
Lactococcus lactis purDEK
Genes, Required for Growth in Milk
Authors: Nilsson Dan , Kilstrup Mogens 
Published: December 2019

ABSTRACT

An operon containing the genes
purD
and
purE
and part of the
purK
gene was cloned from the facultative anaerobic gram-positive bacterium
Lactococcus lactis
by complementation of the
purD
mutation in
Escherichia coli
SØ609. The genes encode enzymes in the de novo pathway of purine nucleotides. The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of
purD
indicated that a region of 145 bp is enough to give regulated expression of the reporter
lacLM
genes, which encode β-galactosidase. Deletion of a region 79 bp upstream of the transcription start point reduced the promoter activity 33-fold when incubated in a purine-free medium and to values below the detection limit when incubated in a purine-containing medium. No secondary transcription start points were mapped in or close to this region, indicating that a putative activator site and not a promoter was deleted or partly destroyed.
Construction of a Proline-Producing Mutant of the Extremely Thermophilic Eubacterium
Thermus thermophilus
HB27
Authors: Kosuge Takehide , Hoshino Takayuki 
Published: December 2019

ABSTRACT

Growth of
Thermus thermophilus
HB27 was inhibited by a proline analog, 3,4-dehydroproline (DHP). This result suggested that the γ-glutamyl kinase (the product of the
proB
gene) was inhibited by feedback inhibition in
T. thermophilus
. DHP-resistant mutants were reported previously for
Escherichia coli
(A. M. Dandekar and S. L. Uratsu, J. Bacteriol. 170:5943–5945, 1988) and
Serratia marcescens
(K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol. 138:693–699, 1992), and their mutated sites in the
proB
gene were identified. Comparison of the amino acid sequence of
T. thermophilus
γ-glutamyl kinase with those of
E. coli
and
S. marcescens
mutants revealed that the DHP resistance mutations occurred in the amino acids conserved among the three organisms. For eliminating the feedback inhibition, we first constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis at the
proB
gene as reported for the proline-producing mutant of
S. marcescens
. The mutant, TH401, excreted about 1 mg of
l
-proline per liter at 70°C after 12 h of incubation. It was also suggested that
T. thermophilus
had a proline degradation and transport pathway since it was able to grow in minimal medium containing
l
-proline as sole nitrogen source. In order to disrupt the proline degradation or transport genes, TH401 was mutated by UV irradiation. Seven mutants unable to utilize
l
-proline for their growth were isolated. One of the mutants, TH4017, excreted about 2 mg of
l
-proline per liter in minimal medium at 70°C after 12 h of incubation.
Naturally Occurring Deletion Mutants Are Parasitic Genotypes in a Wild-Type Nucleopolyhedrovirus Population of
Spodoptera exigua
Authors: Muñoz Delia , Castillejo Juan I. , Caballero Primitivo 
Published: December 2019

ABSTRACT

A wild-type nucleopolyhedrovirus (NPV) isolate from
Spodoptera exigua
from Florida (Se-US2) is a variant of the Se
M
NPV type strain since it has a unique DNA profile but is closely related to other known geographical isolates of Se
M
NPV. It consists of several genotypic variants, of which seven were identified in a Se-US2 virus stock by a modification of the in vivo cloning method developed by Smith and Crook (Virology 166:240–244, 1988). The US2A variant was the most prevalent genotype, and it was designated the prototype Se-US2 variant, while four of the variants (US2B, US2D, US2F, and US2H) were found at low frequency. US2C and US2E were also very abundant, and their diagnostic bands were easily observed in wild-type isolate restriction endonuclease patterns. The analysis of each variant, compared to the prototype US2A, showed that US2B and US2H presented minor differences, while US2D and US2F contained slightly larger insertions or deletions. Variants US2C and US2E contained major deletions of 21.1 and 14 kb, respectively, mapping at the same genomic region (between 14.5 and 30.2 map units [m.u.] and between 12.8 and 23 m.u., respectively). This is the first report of such deletion mutants in a natural baculovirus population. Variants US2A, US2B, US2D, US2F, and US2H were isolated as pure genotypes, but we failed to clone US2C and US2E in vivo. When these two variants appeared without apparent contamination with any other variant, they lost their pathogenicity for
Spodoptera exigua
larvae. A further biological characterization showed evidence that these two naturally occurring deletion mutants act as parasitic genotypes in the virus population. Bioassay data also demonstrated that pure US2A is significantly more pathogenic against second-instar
S. exigua
larvae than the wild-type isolate. The need for precise genotypic characterization of a baculovirus prior to its development as a bioinsecticide is discussed.
Cloning and High-Level Expression of α-Galactosidase cDNA fromPenicillium purpurogenum
Authors: Shibuya Hajime , Nagasaki Hiroaki , Kaneko Satoshi , Yoshida Shigeki , Park Gwi Gun , Kusakabe Isao , Kobayashi Hideyuki 
Published: December 2019

ABSTRACTThe cDNA coding forPenicillium purpurogenumα-galactosidase (αGal) was cloned and sequenced. The deduced amino acid sequence of the α-Gal cDNA showed that the mature enzyme consisted of 419 amino acid residues with a molecular mass of 46,334 Da. The derived amino acid sequence of the enzyme showed similarity to eukaryotic αGals from plants, animals, yeasts, and filamentous fungi. The highest similarity observed (57% identity) was toTrichoderma reeseiAGLI. The cDNA was expressed inSaccharomyces cerevisiaeunder the control of the yeastGAL10promoter. Almost all of the enzyme produced was secreted into the culture medium, and the expression level reached was approximately 0.2 g/liter. The recombinant enzyme purified to homogeneity was highly glycosylated, showed slightly higher specific activity, and exhibited properties almost identical to those of the native enzyme fromP. purpurogenumin terms of the N-terminal amino acid sequence, thermoactivity, pH profile, and mode of action on galacto-oligosaccharides.
Generation of Polyclonal Antibodies of Predetermined Specificity against Pediocin PA-1
Authors: Martínez José M. , Martínez María I. , Suárez Ana M. , Herranz Carmen , Casaus Pilar , Cintas Luis M. , Rodríguez Juan M. , Hernández Pablo E. 
Published: December 2019

ABSTRACTPolyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)—a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)—and by immunodotting. All immunoassays indicated the existence of pedA1-specific antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 μg/ml by immunodotting and 1 μg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 μg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 μg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 μg/ml, and the 50% binding inhibition value was reduced to 0.5 μg of pedA1/ml. All immunoassays and the slot dot assay detected the presence of pedA1 in the supernatant of the producing strainPediococcus acidilactici347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches taken for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the food industry.
Detection of
Ralstonia solanacearum
, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes
Authors: Wullings B. A. , Van Beuningen A. R. , Janse J. D. , Akkermans A. D. L. 
Published: December 2019

ABSTRACT

During the past few years,
Ralstonia
(
Pseudomonas
)
solanacearum
race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five
R. solanacearum
strains and one
Ralstonia pickettii
strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which
R. solanacearum
strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for
R. solanacearum
and the closely related
Ralstonia syzygii
and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of
R. solanacearum
are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host
Solanum dulcamara
(bittersweet) in contaminated areas.