bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production of Functionalized Biopolyester Granules by Recombinant
Lactococcus lactis
Authors: Mifune Jun , Grage Katrin , Rehm Bernd H. A. 
Published: May 2009

ABSTRACT

Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano-/microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. In this study, the food-grade, generally-regarded-as-safe gram-positive organism
Lactococcus lactis
was engineered to recombinantly produce the biopolyester poly(3-hydroxybutyrate) and the respective intracellular inclusions. The codon-optimized polyhydroxybutyrate biosynthesis operon
phaCAB
from
Cupriavidus necator
was expressed using the nisin-controlled gene expression system. Recombinant
L. lactis
accumulated up to 6% (wt/wt) poly(3-hydroxybutyrate) of cellular dry weight. Poly(3-hydroxybutyrate) granules were isolated and analyzed with respect to bound proteins using biochemical methods and with respect to shape/size using transmission electron microscopy. The immunoglobulin G (IgG) binding ZZ domain of
Staphylococcus aureus
protein A was chosen as an exemplary functionality to be displayed at the granule surface by fusing it to the N terminus of the granule-associated poly(3-hydroxybutyrate) synthase. The presence of the fusion protein at the surface of isolated granules was confirmed by peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight (mass spectrometry). The functionality of the ZZ domain-displaying granules was demonstrated by enzyme-linked immunosorbent assay and IgG affinity purification. In both assays, the ZZ beads from recombinant
L. lactis
performed at least equally to ZZ beads from
Escherichia coli
. Overall, in this study it was shown that recombinant
L. lactis
can be used to manufacture endotoxin-free poly(3-hydroxybutyrate) beads with surface functionalities that are suitable for biomedical applications.
Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences
Authors: Alvarado Pablo , Manjón Jose L. 
Published: May 2009

ABSTRACT
Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.
Specific In Situ Visualization of the Pathogenic Endophytic FungusAciculosporium take, the Cause of Witches’ Broom in Bamboo
Authors: Tanaka Eiji 
Published: May 2009

ABSTRACTThe endophytic fungusAciculosporium take(Ascomycota;Clavicipitaceae) causes continuous shoot growth in bamboo. The colonized shoot eventually results in witches’ broom formation but maintains normal leaf arrangement and branching pattern. To analyze the mechanism of well-regulated symptom development, the location of the fungal endophytic hyphae in host tissues was visualized. A colorimetric in situ hybridization technique using a species-specific oligonucleotide probe targeting the 18S rRNA ofA. takewas used. In situ hybridization was performed on tissue sections of diseased shoots with or without external signs of fungal colonization. Specific signals were detected in intercellular spaces of the bamboo tissues. Most signals were detected in the shoot apical meristem and the leaf primordia. In addition, fewer signals were detected in the lateral buds, juvenile leaves, and stems. These results indicate thatA. takegrows endophytically, particularly in the shoot apical meristem of the host. The location ofA. takehyphae suggests that the mechanism of symptom development can be explained by the action of exogenous fungal auxin, which continuously induces primordium initiation within the host.
Succession of Bacterial Populations during Plant Residue Decomposition in Rice Field Soil
Authors: Rui Junpeng , Peng Jingjing , Lu Yahai 
Published: May 2009

ABSTRACT

The incorporation of rice residues into paddy fields strongly enhances methane production and emissions. Although the decomposition processes of plant residues in rice field soil has been documented, the structure and dynamics of the microbial communities involved are poorly understood. The purpose of the present study was to determine the dynamics of short-chain fatty acids and the structure of bacterial communities during residue decomposition in a rice field soil. The soil was anaerobically incubated with the incorporation of rice root or straw residues for 90 days at three temperatures (15, 30, and 45°C). The dynamics of fatty acid intermediates showed an initial cumulative phase followed by a rapid consumption phase and a low-concentration quasi-steady state. Correspondingly, the bacterial populations displayed distinct successions during residue decomposition. Temperature showed a strong effect on the dynamics of bacterial populations. Members of
Clostridium
(clusters I and III) were most dominant in the incubations, particularly in the early successions.
Bacteroidetes
and
Chlorobi
were abundant in the later successions at 15 and 30°C, while
Acidobacteria
were selected at 45°C. We suggest that the early successional groups are responsible for the decomposition of the easily degradable fraction of residues, while the late successional groups become more important in decomposing the less-degradable or resistant fraction of plant residues. The bacterial succession probably is related to resource availability during residue decomposition. The fast-growing organisms are favored at the beginning, while the slow-growing bacteria are better adapted in the later stages, when substrate availability is limiting.
Viral and Flagellate Control of Prokaryotic Production and Community Structure in Offshore Mediterranean Waters
Authors: Bonilla-Findji Osana , Herndl Gerhard J. , Gattuso Jean-Pierre , Weinbauer Markus G. 
Published: May 2009

ABSTRACT

A dilution and size fractionation approach was used to study the separate and combined effects of viruses and flagellates on prokaryotic production ([
3
H]leucine incorporation) and community composition (16S rRNA gene PCR and denaturing gradient gel electrophoresis [DGGE]) in the upper mixed layer and the deep chlorophyll maximum in the offshore Mediterranean Sea. Four experiments were established using differential filtration: a resource control without predators (C treatment), treatment in the presence of viruses (V treatment), treatment in the presence of flagellates (F treatment), and treatment in the presence of both predators (VF treatment). The V and VF treatments increased prokaryotic abundance (1.4- to 2.3-fold) and the number of DGGE bands (by up to 43%) and decreased prokaryotic production compared to the level for the C treatment (by 22 to 99%). For the F treatment, significant differences compared to the level for the C treatment were found as well, but trends were not consistent across experiments. The relative abundances of the high-nucleic-acid subgroups of prokaryotes with high scatter (HNAhs) in flow cytometer settings were lower in the V and VF treatments than in the C and F treatments. These differences were probably due to lysis of very active HNA prokaryotes in the V and VF treatments. Our results indicate that the presence of viruses or viruses plus flagellates sustains prokaryotic diversity and controls prokaryotic production by regulating the proportion of the highly active members of the community. Our data also suggest that lysis and grazing control influences the relationship between bacterial community composition and prokaryotic production.
Community Structure Evolution and Enrichment of Glycogen-Accumulating Organisms Producing Polyhydroxyalkanoates from Fermented Molasses
Authors: Pisco Ana R. , Bengtsson Simon , Werker Alan , Reis Maria A. M. , Lemos Paulo C. 
Published: May 2009

ABSTRACT

An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly “
Candidatus
Competibacter phosphatis” to mainly
Defluviicoccus vanus
-related organisms. The production of 3HHx was confirmed by
13
C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of
D. vanus
-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.
Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences
Authors: Valster Rinske M. , Wullings Bart A. , Bakker Geo , Smidt Hauke , van der Kooij Dick 
Published: May 2009

ABSTRACT

Free-living protozoan communities in water supplies may include hosts for
Legionella pneumophila
and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a
Hartmannella vermiformis
-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly.
H. vermiformis
was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to
Acanthamoeba polyphaga
, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of
H. vermiformis
in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.
Rapid Methods for High-Throughput Detection of Sulfoxides
Authors: Shainsky Janna , Derry Netta-Lee , Leichtmann-Bardoogo Yael , Wood Thomas K. , Fishman Ayelet 
Published: May 2009

ABSTRACT

Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene
ortho
-monooxygenase in a saturation mutagenesis library, using methyl-
p
-tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.
Cultivation of Methanogens under Low-Hydrogen Conditions by Using the Coculture Method
Authors: Sakai Sanae , Imachi Hiroyuki , Sekiguchi Yuji , Tseng I-Cheng , Ohashi Akiyoshi , Harada Hideki , Kamagata Yoichi 
Published: May 2009

ABSTRACT

We previously reported the isolation of novel methanogens by using a new cultivation method, referred to as the coculture method. Here, we extended our coculture method to various anaerobic environmental samples. As a result, we successfully cultivated some uncharacterized methanogens in coculture enrichments and eventually isolated a new methanogen, within the order
Methanomicrobiales
.
Protein Phosphatase Type 1-Interacting Protein Ysw1 Is Involved in Proper Septin Organization and Prospore Membrane Formation during Sporulation
Authors: Ishihara Makoto , Suda Yasuyuki , Inoue Ichiro , Tanaka Takayuki , Takahashi Tetsuo , Gao Xiao-Dong , Fukui Yasuhisa , Ihara Sayoko , Neiman Aaron M. , Tachikawa Hiroyuki 
Published: May 2009

ABSTRACT

Sporulation of
Saccharomyces cerevisiae
is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of
YSW1
partially suppresses the sporulation defect of a temperature-sensitive allele of
gip1
. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in
ysw1
Δ mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in
ysw1
Δ cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.
The
Helicobacter pylori
Anti-Sigma Factor FlgM Is Predominantly Cytoplasmic and Cooperates with the Flagellar Basal Body Protein FlhA
Authors: Rust Melanie , Borchert Sophie , Niehus Eike , Kuehne Sarah A. , Gripp Eugenia , Bajceta Afrodita , McMurry Jonathan L. , Suerbaum Sebastian , Hughes Kelly T. , Josenhans Christine 
Published: May 2009

ABSTRACT

Helicobacter pylori
requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most
Helicobacter
species including
H. pylori
is a membraneous flagellar sheath. The anti-sigma factor FlgM of
H. pylori
is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of
Salmonella
spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short
H. pylori
FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation.
H. pylori
FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in
flhA
mutants and was less soluble and differentially localized in bacterial fractions of the
flhA
mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in
H. pylori
. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhA
C
). We suggest that, in
H. pylori
, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM.
Translational Control of the Antibiotic Inducibility of the PA5471 Gene Required for
mexXY
Multidrug Efflux Gene Expression in
Pseudomonas aeruginosa
Authors: Morita Yuji , Gilmour Christie , Metcalf Devon , Poole Keith 
Published: May 2009

ABSTRACT

The PA5471 gene required for induction of the MexXY multidrug efflux system in response to ribosome-targeting antimicrobials was itself shown to be inducible by ribosome-targeting antimicrobials (Y. Morita, M. L. Sobel, and K. Poole, J. Bacteriol. 188:1847-1855, 2006). Using a
lacZ
transcriptional reporter, drug inducibility of PA5471 was shown to require the entirety of the 367-bp PA5472-PA5471 intergenic region. A constitutive promoter activity was, however, localized to the first 75 bp of this region, within which a single PA5471 transcription initiation site was mapped. That 3′ sequences of the intergenic region blocked PA5471 expression and made it antibiotic dependent was suggestive of an attenuation mechanism of control. A 13-amino-acid leader peptide (LP)-encoding open reading frame preceded by a Shine-Dalgarno sequence was identified ca. 250 bp upstream of the PA5471 coding sequence, and its expression and translation were confirmed using a
lacZ
translational reporter. Alteration of the initiation codon (M1T) or introduction of translational stop signals at codons 3 (Q3Am) and 8 (C8Op) of this LP sequence (PA5471.1) yielded high-level constitutive expression of PA5471, suggesting that interference with LP translation was linked to PA5471 gene expression. Consistent with this, a Q3K mutation in the LP sequence maintained the drug inducibility of PA5471 expression. Introduction of the LP Q3Am mutation into the chromosome of
Pseudomonas aeruginosa
yielded stronger expression of PA5471 than did antibiotic (chloramphenicol) exposure of wild-type
P. aeruginosa
, in agreement with
lacZ
transcriptional fusion data. Still, the Q3Am mutation yielded modest expression of
mexXY
, less than that seen for antibiotic-treated wild-type
P. aeruginosa
. These data suggest that PA5471 is not sufficient for MexXY recruitment in response to antibiotic exposure and that additional antibiotic-dependent effects are needed.
Expression of
Kingella kingae
Type IV Pili Is Regulated by σ
54
, PilS, and PilR
Authors: Kehl-Fie Thomas E. , Porsch Eric A. , Miller Sara E. , StGeme Joseph W. 
Published: May 2009

ABSTRACT

Kingella kingae
is a member of the
Neisseriaceae
and is being recognized increasingly as an important cause of serious disease in children. Recent work has demonstrated that
K. kingae
expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells and are selected against during invasive disease. In the current study, we examined the genome of
K. kingae
strain 269-492 and identified homologs of the
rpoN
and the
pilS
and
pilR
genes that are essential for pilus expression in
Pseudomonas aeruginosa
but not in the pathogenic
Neisseria
species. The disruption of either
rpoN
or
pilR
in
K. kingae
resulted in a marked reduction in the level of transcript for the major pilus subunit (
pilA1
) and eliminated piliation. In contrast, the disruption of
pilS
resulted in only partial reduction in the level of
pilA1
transcript and a partial decrease in piliation. Furthermore, the disruption of
pilS
in colony variants with high-density piliation resulted in variants with low-density piliation. Mutations in the promoter region of
pilA1
and gel shift analysis demonstrated that both σ
54
and PilR act directly at the
pilA1
promoter, with PilR binding to two repetitive elements. These data suggest that the regulation of
K. kingae
type IV pilus expression is complex and multilayered, influenced by both the genetic state and environmental cues.