bluuebunny/crossref_metadata_2025_split huggingface dataset:
Location and Survival of Leaf-Associated Bacteria in Relation to Pathogenicity and Potential for Growth within the Leaf
Authors: Wilson M. , Hirano S. S. , Lindow S. E. 
Published: December 2019

ABSTRACT

The growth and survival of pathogenic and nonpathogenic
Pseudomonas syringae
strains and of the nonpathogenic species
Pantoea agglomerans
,
Stenotrophomonas maltophilia
, and
Methylobacterium organophilum
were compared in the phyllosphere of bean. In general, the plant pathogens survived better than the nonpathogens on leaves under environmental stress. The sizes of the total leaf-associated populations of the pathogenic
P. syringae
strains were greater than the sizes of the total leaf-associated populations of the nonpathogens under dry conditions but not under moist conditions. In these studies the surface sterilants hydrogen peroxide and UV irradiation were used to differentiate cells that were fully exposed on the surface from nonexposed cells that were in “protected sites” that were inaccessible to these agents. In general, the population sizes in protected sites increased with time after inoculation of plants. The proportion of bacteria on leaves that were in protected sites was generally greater for pathogens than for nonpathogens and was greater under dry conditions than under moist conditions. When organisms were vacuum infiltrated into leaves, the sizes of the nonexposed “internal” populations were greater for pathogenic
P. syringae
strains than for nonpathogenic
P. syringae
strains. The sizes of the populations of the nonpathogenic species failed to increase or even decreased. The sizes of nonexposed populations following spray inoculation were correlated with the sizes of nonexposed, internal populations which developed after vacuum infiltration and incubation. While the sizes of the populations of the pathogenic
P. syringae
strains increased on leaves under dry conditions, the sizes of the populations of the nonpathogenic strains of
P. syringae
,
P. agglomerans
, and
S. maltophilia
decreased when the organisms were applied to plants. The sizes of the populations on dry leaves were also correlated with the sizes of the nonexposed populations that developed following vacuum infiltration. Although pathogenicity was not required for growth in the phyllosphere under high-relative-humidity conditions, pathogenicity apparently was involved in the ability to access and/or multiply in certain protected sites in the phyllosphere and in growth on dry leaves.
Detection of Cytolethal Distending Toxin Activity and
cdt
Genes in
Campylobacter
spp. Isolated from Chicken Carcasses
Authors: Eyigor Aysegul , Dawson Karl A. , Langlois Bruce E. , Pickett Carol L. 
Published: December 2019

ABSTRACT

This study was designed to determine whether isolates from chicken carcasses, the primary source of
Campylobacter jejuni
and
Campylobacter coli
in human infections, commonly carry the
cdt
genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates.
Campylobacter
spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets.
Campylobacter
spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as
C. jejuni
, and 35 (33%) were identified as
C. coli
. PCR tests amplified portions of the
cdt
genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the
C. jejuni
and
C. coli cdt
genes, but that the restriction patterns of the
cdt
genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all
C. jejuni
strains except four (94%) had mean CDT titers greater than 100. Only one
C. jejuni
strain appeared to produce no active CDT.
C. coli
isolates produced little or no toxin. These results confirm the high rate of
Campylobacter
sp. contamination of fresh chicken carcasses and indicate that
cdt
genes may be universally present in
C. jejuni
and
C. coli
isolates from chicken carcasses.
PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria
Authors: Hallin Sara , Lindgren Per-Eric 
Published: December 2019

ABSTRACTUsing consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplifycd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships ofnirgenes were also established. Thecd1primers were designed to amplify a 778 to 799-bp region ofcd1-nirin the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nirsequences. Together, the two sets of PCR primers amplifiednirgenes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, butcd1-nirfragments were only obtained in five samples. PCR products of the expected sizes were verified asnirgenes after hybridization to DNA probes, except in one case. The sequencednirfragments were related to othernirsequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nirwere conserved, while broad-range primers targeting conserved regions ofcd1-nirseem to be difficult to find. We also report on the existence of Cu-nirinParacoccus denitrificansPd1222.
Molecular Analysis of Bacterial Community Structure and Diversity in Unimproved and Improved Upland Grass Pastures
Authors: McCaig Allison E. , Glover L. Anne , Prosser James I. 
Published: December 2019

ABSTRACT

Bacterial community structure and diversity in rhizospheres in two types of grassland, distinguished by both plant species and fertilization regimen, were assessed by performing a 16S ribosomal DNA (rDNA) sequence analysis of DNAs extracted from triplicate soil plots. PCR products were cloned, and 45 to 48 clones from each of the six libraries were partially sequenced. Phylogenetic analysis of the resultant 275 clone sequences indicated that there was considerable variation in abundance in replicate unfertilized, unimproved soil samples and fertilized, improved soil samples but that there were no significant differences in the abundance of any phylogenetic group. Several clone sequences were identical in the 16S rDNA region analyzed, and the clones comprised eight pairs of duplicate clones and two sets of triplicate clones. Many clones were found to be most closely related to environmental clones obtained in other studies, although three clones were found to be identical to culturable species in databases. The clones were clustered into operational taxonomic units at a level of sequence similarity of >97% in order to quantify diversity. In all, 34 clusters containing two or more sequences were identified, and the largest group contained nine clones. A number of diversity, dominance, and evenness indices were calculated, and they all indicated that diversity was high, reflecting the low coverage of rDNA libraries achieved. Differences in diversity between sample types were not observed. Collector’s curves, however, indicated that there were differences in the underlying community structures; in particular, there was reduced diversity of organisms of the α subdivision of the class
Proteobacteria
(α-proteobacteria) in improved soils.
Effects of Nickel and Cobalt on Kinetics of Methanol Conversion by Methanogenic Sludge as Assessed by On-Line CH
4
Monitoring
Authors: Gonzalez-Gil Graciela , Kleerebezem Robbert , Lettinga Gatze 
Published: December 2019

ABSTRACT
When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds.
Production of Manganese Peroxidase and Organic Acids and Mineralization of
14
C-Labelled Lignin (
14
C-DHP) during Solid-State Fermentation of Wheat Straw with the White Rot Fungus
Nematoloma frowardii
Authors: Hofrichter Martin , Vares Tamara , Kalsi Mika , Galkin Sari , Scheibner Katrin , Fritsche Wolfgang , Hatakka Annele 
Published: December 2019

ABSTRACT

The basidiomycetous fungus
Nematoloma frowardii
produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic
14
C-ring-labelled lignin (
14
C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as
14
CO
2
, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the
14
C-DHP to
14
CO
2
(which accounted for up to 8% of the initial radioactivity added) and
14
C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate).
Construction and Analysis of a Library for Random Insertional Mutagenesis in
Streptococcus pneumoniae
: Use for Recovery of Mutants Defective in Genetic Transformation and for Identification of Essential Genes
Authors: Lee Myeong S. , Dougherty Brian A. , Madeo Anne C. , Morrison Donald A. 
Published: December 2019

ABSTRACT

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of
Streptococcus pneumoniae
, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in
Escherichia coli
. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the
Bacillus subtilis
competence gene
comFA
. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless
lacZ
reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.
Potential of Conjugal Transfer as a Strategy for the Introduction of Recombinant Genetic Material into Strains of
Lactobacillus helveticus
Authors: Thompson J. K. , McConville K. J. , McReynolds C. , Collins M. A. 
Published: December 2019

ABSTRACT

Cointegrates generated between a plasmid pIP501 deletion derivative (pVA797) and nonconjugative shuttle vector pSA3 were confirmed as capable of exconjugation from lactococci into a range of strains of
Lactobacillus helveticus
with the concomitant expression of a recombinant gene. The plasmid cointegrate that was formed appeared to be segregationally stable at 37°C in some host strains. In all strains, however, the plasmid became increasingly unstable as the incubation temperature was raised. The technique offers not only a generalized method for the introduction of novel genetic material into this important industrial microbe but also the possibility of exploiting the thermal sensitivity of the plasmid to enable it to act as a delivery system for the integration of cloned genes into the bacterial chromosome, at restrictive temperatures, by recombination at regions of homology.
Expression of an
Aspergillus niger
Phytase Gene (
phyA
) in
Saccharomyces cerevisiae
Authors: Han Yanming , Wilson David B. , Lei Xin gen 
Published: December 2019

ABSTRACT

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an
Aspergillus niger
phytase gene (
phyA
) in
Saccharomyces cerevisiae
and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the
phyA
gene was inserted into the expression vector pYES2 and was expressed in
S. cerevisiae
as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the
phyA
gene was expressed as an active, extracellular phytase in
S. cerevisiae
, and its thermostability was affected by glycosylation.
Selective Isolation and Distribution of
Sporichthya
Strains in Soil
Authors: Suzuki Shin-ichi , Okuda Toru , Komatsubara Saburo 
Published: December 2019

ABSTRACT

A simplified enrichment method in which centrifugation is used for selective isolation of
Sporichthya
strains from soil is described. Gellan gum plus 2 mM CaCl
2
stimulated growth of
Sporichthya polymorpha
KCC A0089 so that this organism was readily recognized on an isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 10 mM MOPS (morpholinepropanesulfonic acid) (pH 8.0) and then incubating the preparation at 27°C for 60 min and centrifuging it at 1,000 ×
g
for 10 min. Dry heat treatment at 80°C for 60 min increased the ratio of
Sporichthya
colonies to nonfilamentous bacteria on a gellan gum plate. Since
S. polymorpha
was sensitive to 14 antibiotics, including nalidixic acid, addition of these antibiotics was not suitable for isolating
Sporichthya
strains. Our isolates were identified as
Sporichthya
strains on the basis of their morphological and chemotaxonomic characteristics. By combining the techniques described above, we isolated a number of
Sporichthya
strains from 21 soil samples, which were collected in Belgium, France, India, Japan, Papua New Guinea, Spain, Taiwan, the United Kingdom, and the United States.
Sporichthya
strains are widely distributed in the world. To our knowledge, this is the first time that
Sporichthya
strains have been isolated from locations other than the United States or Japan.
Molecular Analyses of the Methane-Oxidizing Microbial Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane Monooxygenase, and Methanol Dehydrogenase
Authors: Henckel Thilo , Friedrich Michael , Conrad Ralf 
Published: December 2019

ABSTRACT

Rice field soil with a nonsaturated water content induced CH
4
consumption activity when it was supplemented with 5% CH
4
. After a lag phase of 3 days, CH
4
was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH
4
mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the γ subdivision of the class
Proteobacteria
[γ-
Proteobacteria
]) and type II methylotrophs (members of the α-
Proteobacteria
) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (
pmoA
) and methanol dehydrogenase (
mxaF
), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH
4
was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera
Flavobacterium
and
Bacillus
and was not influenced by CH
4
. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera
Methylobacter
,
Methylococcus
, and
Methylocystis
. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the
mxaF
and
pmoA
genes revealed that there were pronounced community shifts when CH
4
oxidation began. High CH
4
concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.
Accumulation of Trehalose by Overexpression of
tps1
, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast
Schizosaccharomyces pombe
Authors: Soto Teresa , Fernández Juana , Vicente-Soler Jero , Cansado Jose , Gacto Mariano 
Published: December 2019

ABSTRACT

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast
Schizosaccharomyces pombe
. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of
S. pombe
transformed with a plasmid bearing the
tps1
gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in
S. pombe
.
Biodiversity of
Clostridium botulinum
Type E Strains Isolated from Fish and Fishery Products
Authors: Hyytiä Eija , Hielm Sebastian , Björkroth Johanna , Korkeala Hannu 
Published: December 2019

ABSTRACT

The genetic biodiversity of
Clostridium botulinum
type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (
Sma
I-
Xma
I and
Xho
I) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.