bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in
Bacillus subtilis
Authors: Kouwen Thijs R. H. M. , Dubois Jean-Yves F. , Freudl Roland , Quax Wim J. , van Dijl Jan Maarten 
Published: October 2008

ABSTRACT

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known “cell factory”
Bacillus subtilis
, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of
B. subtilis
for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from
Staphylococcus carnosus
; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule
Escherichia coli
PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were ∼3.5-fold larger than the amounts secreted by the parental strain
B. subtilis
168. Our findings indicate that
Bacillus
strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.
DNA Microarray Analysis of the Heat Shock Transcriptome of the Obligate Intracytoplasmic Pathogen
Rickettsia prowazekii
Authors: Audia Jonathon P. , Patton Mary C. , Winkler Herbert H. 
Published: October 2008

ABSTRACT

Here we present the first oligonucleotide DNA microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen
Rickettsia prowazekii
using temperature upshift as a model stress condition, and we describe a methodology for isolating highly purified rickettsial RNA. In toto, 23 transcripts were significantly increased by temperature upshift (≥2.0-fold;
P
< 0.05), and no transcripts demonstrated reproducible decreases. Array results for three heat shock-inducible mRNAs were confirmed using quantitative reverse transcription-PCR.
Relationship between Sublethal Injury and Microbial Inactivation by the Combination of High Hydrostatic Pressure and Citral or
tert
-Butyl Hydroquinone
Authors: Somolinos Maria , García Diego , Pagán Rafael , Mackey Bernard 
Published: October 2008

ABSTRACT

The aim was to investigate (i) the occurrence of sublethal injury in
Listeria monocytogenes
,
Escherichia coli
, and
Saccharomyces cerevisiae
after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds,
tert
-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In
E. coli
and
L. monocytogenes
, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in
S. cerevisiae
, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to
E. coli
or
L. monocytogenes
but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log
10
cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log
10
cycles of
E. coli
at pH 7.0 and almost 3 log
10
cycles of
L. monocytogenes
at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.
Staphylococcus aureus
-Fibronectin Interactions with and without Fibronectin-Binding Proteins and Their Role in Adhesion and Desorption
Authors: Xu Chun-Ping , Boks Niels P. , de Vries Joop , Kaper Hans J. , Norde Willem , Busscher Henk J. , van der Mei Henny C. 
Published: October 2008

ABSTRACT

Adhesion and residence-time-dependent desorption of two
Staphylococcus aureus
strains with and without fibronectin (Fn) binding proteins (FnBPs) on Fn-coated glass were compared under flow conditions. To obtain a better understanding of the role of Fn-FnBP binding, the adsorption enthalpies of Fn with staphylococcal cell surfaces were determined using isothermal titration calorimetry (ITC). Interaction forces between staphylococci and Fn coatings were measured using atomic force microscopy (AFM). The strain with FnBPs adhered faster and initially stronger to an Fn coating than the strain without FnBPs, and its Fn adsorption enthalpies were higher. The initial desorption was high for both strains but decreased substantially within 2 s. These time scales of staphylococcal bond ageing were confirmed by AFM adhesion force measurement. After exposure of either Fn coating or staphylococcal cell surfaces to bovine serum albumin (BSA), the adhesion of both strains to Fn coatings was reduced, suggesting that BSA suppresses not only nonspecific but also specific Fn-FnBP interactions. Adhesion forces and adsorption enthalpies were only slightly affected by BSA adsorption. This implies that under the mild contact conditions of convective diffusion in a flow chamber, adsorbed BSA prevents specific interactions but does allow forced Fn-FnBP binding during AFM or stirring in ITC. The bond strength energies calculated from retraction force-distance curves from AFM were orders of magnitude higher than those calculated from desorption data, confirming that a penetrating Fn-coated AFM tip probes multiple adhesins in the outermost cell surface that remain hidden during mild landing of an organism on an Fn-coated substratum, like that during convective diffusional flow.
Population Heterogeneity of
Lactobacillus plantarum
WCFS1 Microcolonies in Response to and Recovery from Acid Stress
Authors: Ingham Colin J. , Beerthuyzen Marke , van Hylckama Vlieg Johan 
Published: October 2008

ABSTRACT

Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium
Lactobacillus plantarum
grows, it acidifies the growth medium to a low pH. We have examined the growth of
L. plantarum
microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS-Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.
Transposable Element Loads in a Bacterial Symbiont of Weevils Are Extremely Variable
Authors: Dougherty Kevin M. , Plague Gordon R. 
Published: October 2008

ABSTRACTNot only are transposable elements profuse in the bacterial endosymbiont of maize weevils, but we found that their quantities also vary ∼10-fold among individual weevils. Because multicopy elements can facilitate homologous recombination, this insertion sequence (IS) load variability suggests that these essentially asexual bacteria may exhibit substantial intraspecific genomic variation.
Epsilonproteobacteria
Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas
Authors: Grote Jana , Jost Günter , Labrenz Matthias , Herndl Gerhard J. , Jürgens Klaus 
Published: October 2008

ABSTRACT

Recent studies have indicated that chemoautotrophic
Epsilonproteobacteria
might play an important role, especially as anaerobic or microaerophilic dark CO
2
-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO
2
-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of
Epsilonproteobacteria
to dark CO
2
fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [
14
C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10
5
14
CO
2
-fixing prokaryotic cells ml
−1
were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10
4
14
CO
2
-fixing cells ml
−1
were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance.
14
CO
2
-incorporating cells belonged exclusively to the domain
Bacteria
. Among these, members of the
Epsilonproteobacteria
were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the
Sulfurimonas
subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO
2
-fixing group. In conclusion,
Epsilonproteobacteria
were found to be mainly responsible for chemoautotrophic activity in the dark CO
2
fixation maxima of the Black Sea and central Baltic Sea redoxclines. These
Epsilonproteobacteria
might be relevant in similar habitats of the world's oceans, where high dark CO
2
fixation rates have been measured.
Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude
Escherichia coli
Cell Extracts in Response to Copper Ions
Authors: Thieme Daniel , Neubauer Peter , Nies Dietrich H. , Grass Gregor 
Published: October 2008

ABSTRACT

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from
Escherichia coli
without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of
E. coli
. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.
Rapid Classification and Identification of Salmonellae at the Species and Subspecies Levels by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry
Authors: Dieckmann Ralf , Helmuth Reiner , Erhard Marcel , Malorny Burkhard 
Published: October 2008

ABSTRACT

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing
Salmonella enterica
subsp.
enterica
(subspecies I),
S. enterica
subsp.
salamae
(subspecies II),
S. enterica
subsp.
arizonae
(subspecies IIIa),
S. enterica
subsp.
diarizonae
(subspecies IIIb),
S. enterica
subsp.
houtenae
(subspecies IV), and
S. enterica
subsp.
indica
(subspecies VI), and
Salmonella bongori
were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for
Salmonella
, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of
Salmonella
strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of
Salmonella
subspecies in minutes was established.
Control of Lipid Accumulation in the Yeast
Yarrowia lipolytica
Authors: Beopoulos Athanasios , Mrozova Zuzana , Thevenieau France , Le Dall Marie-Thérèse , Hapala Ivan , Papanikolaou Seraphim , Chardot Thierry , Nicaud Jean-Marc 
Published: October 2008

ABSTRACT

A genomic comparison of
Yarrowia lipolytica
and
Saccharomyces cerevisiae
indicates that the metabolism of
Y. lipolytica
is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the
GUT2
gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in
Y. lipolytica
in this study. This Δ
gut2
mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to β-oxidation.
Y. lipolytica
contains six acyl-coenzyme A oxidases (Aox), encoded by the
POX1
to
POX6
genes, that catalyze the limiting step of peroxisomal β-oxidation. Additional deletion of the
POX1
to
POX6
genes in the Δ
gut2
strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.
Possible Involvement of an Extracellular Superoxide Dismutase (SodA) as a Radical Scavenger in Poly(
cis
-1,4-Isoprene) Degradation
Authors: Schulte Carina , Arenskötter Matthias , Berekaa Mahmoud M. , Arenskötter Quyen , Priefert Horst , Steinbüchel Alexander 
Published: October 2008

ABSTRACT

Gordonia westfalica
Kb1 and
Gordonia polyisoprenivorans
VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(
cis
-1,4-isoprene) degradation. To investigate the function of this enzyme in
G. polyisoprenivorans
VH2, the
sodA
gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(
cis
-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(
cis
-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from
G. westfalica
Kb1 was heterologously expressed in the
sodA sodB
double mutant
Escherichia coli
QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the
G. westfalica
Kb1 SodA and indirectly protection of
G. westfalica
cells by SodA from oxidative damage. Both
sodA
from
G. polyisoprenivorans
VH2 and
sodA
from
G. westfalica
Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from
Mycobacterium smegmatis
strain MC
2
155 and
Micrococcus luteus
NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of
G. polyisoprenivorans
, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both
Gordonia
species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of
G. polyisoprenivorans
VH2, which was purified under native conditions from the cells. In
G. westfalica
Kb1 and
G. polyisoprenivorans
VH2, SodA probably provides protection against reactive oxygen intermediates which occur during degradation of poly(
cis
-1,4-isoprene).
Isolation of New
Stenotrophomonas
Bacteriophages and Genomic Characterization of Temperate Phage S1
Authors: García Pilar , Monjardín Cristina , Martín Rebeca , Madera Carmen , Soberón Nora , Garcia Eva , Meana Álvaro , Suárez Juan E. 
Published: October 2008

ABSTRACT

Twenty-two phages that infect
Stenotrophomonas
species were isolated through sewage enrichment and prophage induction. Of them, S1, S3, and S4 were selected due to their wide host ranges compared to those of the other phages. S1 and S4 are temperate siphoviruses, while S3 is a virulent myovirus. The genomes of S3 and S4, about 33 and 200 kb, were resistant to restriction digestion. The lytic cycles lasted 30 min for S3 and about 75 min for S1 and S4. The burst size for S3 was 100 virions/cell, while S1 and S4 produced about 75 virus particles/cell. The frequency of bacteriophage-insensitive host mutants, calculated by dividing the number of surviving colonies by the bacterial titer of a parallel, uninfected culture, ranged between 10
−5
and 10
−6
for S3 and 10
−3
and 10
−4
for S1 and S4. The 40,287-bp genome of S1 contains 48 open reading frames (ORFs) and 12-bp 5′ protruding cohesive ends. By using a combination of bioinformatics and experimental evidence, functions were ascribed to 21 ORFs. The morphogenetic and lysis modules are well-conserved, but no lysis-lysogeny switch or DNA replication gene clusters were recognized. Two major clusters of genes with respect to transcriptional orientation were observed. Interspersed among them were lysogenic conversion genes encoding phosphoadenosine phosphosulfate reductase and GspM, a protein involved in the general secretion system II. The
attP
site of S1 may be located within a gene that presents over 75% homology to a
Stenotrophomonas
chromosomal determinant.
Phaeobacter
and
Ruegeria
Species of the
Roseobacter
Clade Colonize Separate Niches in a Danish Turbot (
Scophthalmus maximus
)-Rearing Farm and Antagonize
Vibrio anguillarum
under Different Growth Conditions
Authors: Porsby Cisse Hedegaard , Nielsen Kristian Fog , Gram Lone 
Published: October 2008

ABSTRACT

Members of the
Roseobacter
clade colonize a Spanish turbot larval unit, and one isolate (
Phaeobacter
strain 27-4) is capable of disease suppression in in vivo challenge trials. Here, we demonstrate that roseobacters with antagonistic activity against
Vibrio anguillarum
also colonize a Danish turbot larval farm that relies on a very different water source (the Danish fiord Limfjorden as opposed to the Galician Atlantic Ocean). Phylogenetic analyses based on 16S rRNA and gyrase B gene sequences revealed that different species colonized different niches in the larval unit.
Phaeobacter inhibens-
and
Phaeobacter gallaeciensis
-like strains were primarily found in the production sites, whereas strains identified as
Ruegeria mobilis
or
Ruegeria pelagia
were found only in the algal cultures.
Phaeobacter
spp. were more inhibitory against the general microbiota from the Danish turbot larval unit than were the
Ruegeria
spp.
Phaeobacter
spp. produced tropodithietic acid (TDA) and brown pigment and antagonized
V. anguillarum
when grown under shaking (200 rpm) and stagnant (0 rpm) conditions, whereas
Ruegeria
spp. behaved similarly to
Phaeobacter
strain 27-4 and expressed these three phenotypes only during stagnant growth. Both genera attached to an inert surface and grew in multicellular rosettes after stagnant growth, whereas shaking conditions led to single cells with low attachment capacity. Bacteria from the
Roseobacter
clade appear to be universal colonizers of marine larval rearing units, and since the Danish
Phaeobacter
spp. displayed antibacterial activity under a broader range of growth conditions than did
Phaeobacter
strain 27-4, these organisms may hold greater promise as fish probiotic organisms.
Loss of Gibberellin Production in
Fusarium verticillioides
(
Gibberella fujikuroi
MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster
Authors: Bömke Christiane , Rojas Maria C. , Hedden Peter , Tudzynski Bettina 
Published: October 2008

ABSTRACT

Fusarium verticillioides
(
Gibberella fujikuroi
mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the
Gibberella fujikuroi
species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to
Fusarium fujikuroi
(
G. fujikuroi
MP-C) and
Fusarium konzum
(MP-I), although most members of the
G. fujikuroi
species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in
F. verticillioides
(MP-A) is due to the loss of a majority of the GA gene cluster as found in
F. fujikuroi
. The remaining part of the cluster consists of the full-length
F. verticillioides des
gene (Fv
des
), encoding the GA
4
desaturase, and the coding region of Fv
P450-4
, encoding the
ent
-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of
F. fujikuroi
. The GA production capacity of
F. verticillioides
was restored by transforming a cosmid with the entire GA gene cluster from
F. fujikuroi
, indicating the existence of an active regulation system in
F. verticillioides
. Furthermore, the GA
4
desaturase gene
des
from
F. verticillioides
encodes an active enzyme which was able to restore the GA production in a corresponding
des
deletion mutant of
F. fujikuroi
.