bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Agricultural Practices on the
Zea mays
L. Endophytic Community
Authors: Seghers Dave , Wittebolle Lieven , Top Eva M. , Verstraete Willy , Siciliano Steven D. 
Published: March 2004

ABSTRACT

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of
Zea mays
L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.
Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic
Mollicutes
Strains by Reverse Line Blot Hybridization
Authors: Wang Hui , Kong Fanrong , Jelfs Peter , James Gregory , Gilbert Gwendolyn L. 
Published: March 2004

ABSTRACT

We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (
Mycoplasma arginini
,
Mycoplasma fermentans
,
Mycoplasma hyorhinis
,
Mycoplasma orale
, and
Acholeplasma laidlawii
) and human infection (
Mycoplasma pneumoniae, Mycoplasma hominis
,
Mycoplasma genitalium
,
Ureaplasma parvum
, and
Ureaplasma urealyticum
). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.
Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage
Authors: Van Hamme Jonathan D. , Fedorak Phillip M. , Foght Julia M. , Gray Murray R. , Dettman Heather D. 
Published: March 2004

ABSTRACT

The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C—S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C—S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound,
bis
-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C—S bonds. A unique bacterial strain,
Rhodococcus
sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C—S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including
Rhodococcus
sp. strain IGTS8, were all unable to cleave the C—S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C—S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.
Microbial Biogeography along an Estuarine Salinity Gradient: Combined Influences of Bacterial Growth and Residence Time
Authors: Crump Byron C. , Hopkinson Charles S. , Sogin Mitchell L. , Hobbie John E. 
Published: March 2004

ABSTRACT

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([
14
C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla
Proteobacteria
,
Bacteroidetes
, and
Actinobacteria
. Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.
Optimization of Procedures for Counting Viruses by Flow Cytometry
Authors: Brussaard Corina P. D. 
Published: March 2004

ABSTRACT

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at −80°C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 × 10
−5
dilution of commercial stock), incubated for 10 min in the dark at 80°C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least −80°C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.
Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification
Authors: Völlenkle Christine , Weigert Stefan , Ilk Nicola , Egelseer Eva , Weber Viktoria , Loth Fritz , Falkenhagen Dieter , Sleytr Uwe B. , Sára Margit 
Published: March 2004

ABSTRACTThe chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA fromBacillus sphaericusCCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed inEscherichia coliHMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.
Microbiological Characterization of Wet Wheat Distillers' Grain, with Focus on Isolation of Lactobacilli with Potential as Probiotics
Authors: Pedersen C. , Jonsson H. , Lindberg J. E. , Roos S. 
Published: March 2004

ABSTRACT

Wet wheat distillers' grain (WWDG), a residue from ethanol fermentation, was examined from a microbiological perspective. After storage, WWDG was characterized by a high content of lactobacilli, nondetectable levels of other bacteria, occasional occurrence of yeasts, and a pH of about 3.6 and contained a mixture of lactic acid, acetic acid, and ethanol. The composition of lactobacilli in WWDG was simple, including primarily the species
Lactobacillus amylolyticus
,
Lactobacillus panis
, and
Lactobacillus pontis
, as determined by 16S rRNA gene sequencing. Since the use of WWDG as pig feed has indicated a health-promoting function, some relevant characteristics of three strains of each of these species were examined together with basal physiological parameters, such as carbohydrate utilization and growth temperature. Seven of the strains were isolated from WWDG, and two strains from pig feces were included for comparison. It was clear that all three species could grow at temperatures of 45 to 50°C, with
L. amylolyticus
being able to grow at temperatures as high as 54°C. This finding could be the explanation for the simple microflora of WWDG, where a low pH together with a high temperature during storage would select for these organisms. Some strains of
L. panis
and
L. pontis
showed prolonged survival at pH 2.5 in synthetic stomach juice and good growth in the presence of porcine bile salt. In addition, members of all three species were able to bind to immobilized mucus material in vitro. Especially the isolates from pig feces but, interestingly, some isolates from WWDG as well possessed properties that might be of importance for colonization of the gastrointestinal tracts of pigs.
High Diversity among Environmental
Escherichia coli
Isolates from a Bovine Feedlot
Authors: Yang Hsiao-Hui , Vinopal Robert T. , Grasso Domenico , Smets Barth F. 
Published: March 2004

ABSTRACT

Approximately 280
Escherichia coli
isolates were isolated from a bovine feedlot at the University of Connecticut campus via enrichment in lauryl tryptose broth and random selection from MacConkey plates. The
E. coli
subspecies diversity was estimated by employing whole-cell BOX-PCR genomic fingerprints. A total of 89 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 85% fingerprint similarity as a surrogate for an OTU, while the Chao1 index estimated the
E. coli
population richness at 128 OTUs. One genotype (at a similarity level of 60%) dominated the population at 66% regardless of sampling depth or location, while no significant vertical distribution pattern was observed in terms of genotype, mobility, antibiotic resistance profile, or biofilm-forming ability. Motility, measured by a soft agar assay, had a very broad range among the
E. coli
population and was positively correlated with biofilm-forming ability in minimal medium (Spearman's rank correlation coefficient
r
= 0.619,
P
< 10
−4
) but not in Luria broth. Only an estimated 48% of the population possessed gene
agn43
, which encodes Ag43, a phase-variable outer membrane protein that has been implicated in biofilm formation in minimal medium. We observed significantly more biofilm formation in both minimal medium and Luria broth for

agn43
+

strains, with a larger effect in minimal medium. This study represents an exhaustive inventory of extant
E. coli
population diversity at a bovine feedlot and reveals significant subspecies heterogeneity in interfacial behavior.
2-O-Methylation of Fucosyl Residues of a Rhizobial Lipopolysaccharide Is Increased in Response to Host Exudate and Is Eliminated in a Symbiotically Defective Mutant
Authors: Noel K. Dale , Box Jodie M. , Bonne Valerie J. 
Published: March 2004

ABSTRACT

When
Rhizobium etli
CE3 was grown in the presence of
Phaseolus vulgaris
seed extracts containing anthocyanins, its lipopolysaccharide (LPS) sugar composition was changed in two ways: greatly decreased content of what is normally the terminal residue of the LPS, di-
O
-methylfucose, and a doubling of the 2-O-methylation of other fucose residues in the LPS O antigen.
R. etli
strain CE395 was isolated after Tn
5
mutagenesis of strain CE3 by screening for mutant colonies that did not change antigenically in the presence of seed extract. The LPS of this strain completely lacked 2-
O
-methylfucose, regardless of whether anthocyanins were present during growth. The mutant gave only pseudonodules in association with
P. vulgaris
. Interpretation of this phenotype was complicated by a second LPS defect exhibited by the mutant: its LPS population had only about 50% of the normal amount of O-antigen-containing LPS (LPS I). The latter defect could be suppressed genetically such that the resulting strain (CE395α395) synthesized the normal amount of an LPS I that still lacked 2-
O
-methylfucose residues. Strain CE395α395 did not elicit pseudonodules but resulted in significantly slower nodule development, fewer nodules, and less nitrogenase activity than
lps
+
strains. The relative symbiotic deficiency was more severe when seeds were planted and inoculated with bacteria before they germinated. These results support previous conclusions that the relative amount of LPS I on the bacterial surface is crucial in symbiosis, but LPS structural features, such as 2-O-methylation of fucose, also may facilitate symbiotic interactions.
Morphological and Physiological Changes Induced by High Hydrostatic Pressure in Exponential- and Stationary-Phase Cells of
Escherichia coli
: Relationship with Cell Death
Authors: Mañas Pilar , Mackey Bernard M. 
Published: March 2004

ABSTRACT

The relationship between a loss of viability and several morphological and physiological changes was examined with
Escherichia coli
strain J1 subjected to high-pressure treatment. The pressure resistance of stationary-phase cells was much higher than that of exponential-phase cells, but in both types of cell, aggregation of cytoplasmic proteins and condensation of the nucleoid occurred after treatment at 200 MPa for 8 min. Although gross changes were detected in these cellular structures, they were not related to cell death, at least for stationary-phase cells. In addition to these events, exponential-phase cells showed changes in their cell envelopes that were not seen for stationary-phase cells, namely physical perturbations of the cell envelope structure, a loss of osmotic responsiveness, and a loss of protein and RNA to the extracellular medium. Based on these observations, we propose that exponential-phase cells are inactivated under high pressure by irreversible damage to the cell membrane. In contrast, stationary-phase cells have a cytoplasmic membrane that is robust enough to withstand pressurization up to very intense treatments. The retention of an intact membrane appears to allow the stationary-phase cell to repair gross changes in other cellular structures and to remain viable at pressures that are lethal to exponential-phase cells.
Prevalence and Molecular Characterization of Tetracycline Resistance in
Enterococcus
Isolates from Food
Authors: Huys Geert , D'Haene Klaas , Collard Jean-Marc , Swings Jean 
Published: March 2004

ABSTRACT

In the present study, a collection of 187
Enterococcus
food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species
Enterococcus faecalis
(
n
= 33),
E. durans
(
n
= 7),
E. faecium
(
n
= 3),
E. casseliflavus
(
n
= 1), and
E. gallinarum
(
n
= 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 μg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the
tet
(M) (
n
= 43),
tet
(L) (
n
= 16), and
tet
(S) (
n
= 1) genes. In 15 isolates, including all of those for which the MIC was 256 μg/ml, both
tet
(M) and
tet
(L) were found. Furthermore, all
tet
(M)-containing enterococci also harbored a member of the Tn
916
-Tn
1545
conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the
erm
(B) gene. Filter mating experiments revealed that 10
E. faecalis
isolates, 3
E. durans
isolates, and 1
E. faecium
isolate could transfer either
tet
(M),
tet
(L), or both of these genes to
E. faecalis
recipient strain JH2-2. In most cases in which only
tet
(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn
916
-Tn
1545
family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable
tet
(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of ≥99.6%. Two of these SHGs contained
tet
(M) mosaic structures previously found in Tn
916
elements and on
Lactobacillus
and
Neisseria
plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.
Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of
Clostridium thermocellum
Authors: Zhang Yi-Heng Percival , Lynd Lee R. 
Published: March 2004

ABSTRACT

Rates of phosphorolytic cleavage of β-glucan substrates were determined for cell extracts from
Clostridium thermocellum
ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by ≥20-fold for both cellobiose and cellopentaose over a 10-fold range of β-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of β-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of
V
max
values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent
K

m

for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).
Characteristics of a New Enantioselective Thermostable Dipeptidase from
Brevibacillus borstelensis
BCS-1 and Its Application to Synthesis of a
d
-Amino-Acid-Containing Dipeptide
Authors: Baek Dae Heoun , Song Jae Jun , Kwon Seok-Joon , Park Chung , Jung Chang-Min , Sung Moon-Hee 
Published: March 2004

ABSTRACT

A new thermostable dipeptidase gene was cloned from the thermophile
Brevibacillus borstelensis
BCS-1 by genetic complementation of the
d
-Glu auxotroph
Escherichia coli
WM335 on a plate containing
d
-Ala-
d
-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an
M
r
of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from
Listeria monocytogenes
. The enzyme was purified to homogeneity from recombinant
E. coli
WM335 harboring the dipeptidase gene from
B. borstelensis
BCS-1. Investigation of the enantioselectivity (
E
) to the P
1
and P
1
′ site of Ala-Ala revealed that the ratio of the specificity constant (
k
cat

/K
m

) for
l
-enantioselectivity to the P
1
site of Ala-Ala was 23.4 ± 2.2 [
E
= (
k
cat

/K
m

)

l,d

/(
k
cat

/K
m

)

d,d

], while the
d
-enantioselectivity to the P
1
′ site of Ala-Ala was 16.4 ± 0.5 [
E
= (
k
cat

/K
m

)

l,d

/(
k
cat

/K
m

)

l,l

] at 55°C. The enzyme was stable up to 55°C, and the optimal pH and temperature were 8.5 and 65°C, respectively. The enzyme was able to hydrolyze
l
-Asp-
d
-Ala,
l
-Asp-
d
-AlaOMe,
Z
-
d
-Ala-
d
-AlaOBzl, and
Z
-
l
-Asp-
d
-AlaOBzl, yet it could not hydrolyze
d
-Ala-
l
-Asp,
d
-Ala-
l
-Ala,
d
-AlaNH
2
, and
l
-AlaNH
2.
The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener
Z
-
l
-Asp-
d
-AlaOBzl.