bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring of Microbial Metabolites and Bacterial Diversity in Beef Stored under Different Packaging Conditions
Authors: Ercolini Danilo , Ferrocino Ilario , Nasi Antonella , Ndagijimana Maurice , Vernocchi Pamela , La Storia Antonietta , Laghi Luca , Mauriello Gianluigi , Guerzoni M. Elisabetta , Villani Francesco
Published: July 2011

Beef chops were stored at 4°C under different conditions: in air (A), modified-atmosphere packaging (MAP), vacuum packaging (V), or bacteriocin-activated antimicrobial packaging (AV). After 0 to 45 days of storage, analyses were performed to determine loads of spoilage microorganisms, microbial metabolites (by solid-phase microextraction [SPME]-gas chromatography [GC]-mass spectrometry [MS] and proton nuclear magnetic resonance [
1
H NMR]), and microbial diversity (by PCR–denaturing gradient gel electrophoresis [DGGE] and pyrosequencing). The microbiological shelf life of meat increased with increasing selectivity of storage conditions. Culture-independent analysis by pyrosequencing of DNA extracted directly from meat showed that
Brochothrix thermosphacta
dominated during the early stages of storage in A and MAP, while
Pseudomonas
spp. took over during further storage in A. Many different bacteria, several of which are usually associated with soil rather than meat, were identified in V and AV; however, lactic acid bacteria (LAB) dominated during the late phases of storage, and
Carnobacterium divergens
was the most frequent microorganism in AV. Among the volatile metabolites, butanoic acid was associated with the growth of LAB under V and AV storage conditions, while acetoin was related to the other spoilage microbial groups and storage conditions.
1
H NMR analysis showed that storage in air was associated with decreases in lactate, glycogen, IMP, and ADP levels and with selective increases in levels of 3-methylindole, betaine, creatine, and other amino acids. The meat microbiota is significantly affected by storage conditions, and its changes during storage determine complex shifts in the metabolites produced, with a potential impact on meat quality.
Development of a Method To Produce Hemoglobin in a Bioreactor Culture of Escherichia coli BL21(DE3) Transformed with a Plasmid Containing Plesiomonas shigelloides Heme Transport Genes and Modified Human Hemoglobin Genes
Authors: Smith B. J. Z. , Gutierrez P. , Guerrero E. , Brewer C. J. , Henderson D. P.
Published: July 2011

We describe a method for production of recombinant human hemoglobin by
Escherichia coli
grown in a bioreactor.
E. coli
BL21(DE3) transformed with a plasmid containing hemoglobin genes and
Plesiomonas shigelloides
heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.
Inactivation of Escherichia coli O157:H7 on Radish Seeds by Sequential Treatments with Chlorine Dioxide, Drying, and Dry Heat without Loss of Seed Viability
Authors: Bang Jihyun , Kim Haeyoung , Kim Hoikyung , Beuchat Larry R. , Ryu Jee-Hoon
Published: July 2011

We developed and validated a treatment to inactivate
Escherichia coli
O157:H7 on radish seeds without decreasing seed viability. Treatments with aqueous ClO
2
followed by drying and dry-heat treatments were evaluated for efficacy to inactivate the pathogen. Conditions to dry radish seeds after treatment with water (control) or ClO
2
were established. When treated seeds with high water activity (a
w
) (>0.99) were stored at 45°C and 23% relative humidity (RH), the a
w
decreased to <0.30 within 24 h. Drying high-a
w
seeds before exposing them to dry-heat treatment (≥60°C) was essential to preserve seed viability. The germination rate of radish seeds which had been immersed in water for 5 min, dried at 45°C and 23% RH for 24 h, and heated at 70°C for 48 h or at 80°C for 24 h was not significantly decreased (
P
≤ 0.05) compared to that of untreated radish seeds. Sequential treatments with ClO
2
(500 μg/ml, 5 min), drying (45°C, 23% RH, 24 h), and dry heating (70°C, 23% RH, 48 h) eliminated
E. coli
O157:H7 (5.9 log CFU/g) on radish seeds and, consequently, sprouts produced from them without decreasing the germination rate. These sequential treatments are recommended for application to radish seeds intended for sprout production.
Necessity of OxyR for the Hydrogen Peroxide Stress Response and Full Virulence in Ralstonia solanacearum
Authors: Flores-Cruz Zomary , Allen Caitilyn
Published: July 2011

The plant pathogen
Ralstonia solanacearum
, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The
R. solanacearum
genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An
R. solanacearum
oxyR
mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that
oxyR
is essential for the hydrogen peroxide stress response. Unexpectedly, the
oxyR
mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that
katE
,
kaG
,
ahpC1
,
grxC
, and
oxyR
itself were each differentially expressed in the
oxyR
mutant background and in response to hydrogen peroxide, suggesting that
oxyR
is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the
oxyR
mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that
R. solanacearum
is exposed to inhibitory concentrations of ROS
in planta
and that OxyR-mediated responses to ROS during plant pathogenesis are important for
R. solanacearum
host adaptation and virulence.
Recombinant Protein Production by
In Vivo
Polymer Inclusion Display
Authors: Grage Katrin , Peters Verena , Rehm Bernd H. A.
Published: July 2011

A novel approach to produce purified recombinant proteins was established. The target protein is produced as polyhydroxyalkanoate (PHA) synthase fusion protein, which mediates intracellular formation of PHA inclusions displaying the target protein. After isolation of the PHA inclusions, the pure target protein was released by simple enterokinase digestion.
The Chlamydomonas reinhardtii Molybdenum Cofactor Enzyme crARC Has a Zn-Dependent Activity and Protein Partners Similar to Those of Its Human Homologue
Authors: Chamizo-Ampudia Alejandro , Galvan Aurora , Fernandez Emilio , Llamas Angel
Published: July 2011

The ARC (amidoxime reducing component) proteins are molybdenum cofactor (Moco) enzymes named hmARC1 and hmARC2 (human ARCs [hmARCs]) in humans and YcbX in
Escherichia coli.
They catalyze the reduction of a broad range of N-hydroxylated compounds (NHC) using reducing power supplied by other proteins. Some NHC are prodrugs or toxic compounds. YcbX contains a ferredoxin (Fd) domain and requires the NADPH flavin reductase CysJ to reduce NHC. In contrast, hmARCs lack the Fd domain and require a human cytochrome
b5
(hCyt
b5
) and a human NADH Cyt
b5
reductase (hCyt
b5-
R) to reduce NHC. The ARC proteins in the plant kingdom are uncharacterized. We demonstrate that
Chlamydomonas reinhardtii
mutants defective in Moco biosynthesis genes are sensitive to the NHC
N
6
-hydroxylaminopurine (HAP). The
Chlamydomonas reinhardtii
ARC protein crARC has been purified and characterized. The six
Chlamydomonas
Fds were isolated, but none of them are required by crARC to reduce HAP. We have also purified and characterized five
C. reinhardtii
Cyt
b5
(crCyt
b5
) and two flavin reductases, one that is NADPH dependent (crCysJ) and one that is NADH dependent (crCyt
b5
-R). The data show that crARC uses crCyt
b5
-
1
and crCyt
b5
-R to reduce HAP. The crARC has a Zn-dependent activity, and the presence of Zn increases its
V
max
more than 14-fold. In addition, all five cysteines of crARC were substituted by alanine, and we demonstrate that the fully conserved cysteine 252 is essential for both Moco binding and catalysis. Therefore, it is proposed that crARC belongs to the sulfite oxidase family of Moco enzymes.
CCR4-Associated Factor 1 Coordinates the Expression of Plasmodium falciparum Egress and Invasion Proteins
Authors: Balu Bharath , Maher Steven P. , Pance Alena , Chauhan Chitra , Naumov Anatoli V. , Andrews Robert M. , Ellis Peter D. , Khan Shahid M. , Lin Jing-wen , Janse Chris J. , Rayner Julian C. , Adams John H.
Published: July 2011

Coordinated regulation of gene expression is a hallmark of the
Plasmodium falciparum
asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for
Plasmodium
development within the human host.
RNA-Mediated Silencing in Algae: Biological Roles and Tools for Analysis of Gene Function
Authors: Cerutti Heriberto , Ma Xinrong , Msanne Joseph , Repas Timothy
Published: July 2011

Algae are a large group of aquatic, typically photosynthetic, eukaryotes that include species from very diverse phylogenetic lineages, from those similar to land plants to those related to protist parasites. The recent sequencing of several algal genomes has provided insights into the great complexity of these organisms. Genomic information has also emphasized our lack of knowledge of the functions of many predicted genes, as well as the gene regulatory mechanisms in algae. Core components of the machinery for RNA-mediated silencing show widespread distribution among algal lineages, but they also seem to have been lost entirely from several species with relatively small nuclear genomes. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have now been identified by high-throughput sequencing in green, red, and brown algae. However, the natural roles of RNA-mediated silencing in algal biology remain poorly understood. Limited evidence suggests that small RNAs may function, in different algae, in defense mechanisms against transposon mobilization, in responses to nutrient deprivation and, possibly, in the regulation of recently evolved developmental processes. From a practical perspective, RNA interference (RNAi) is becoming a promising tool for assessing gene function by sequence-specific knockdown. Transient gene silencing, triggered with exogenously synthesized nucleic acids, and/or stable gene repression, involving genome-integrated transgenes, have been achieved in green algae, diatoms, yellow-green algae, and euglenoids. The development of RNAi technology in conjunction with system level “omics” approaches may provide the tools needed to advance our understanding of algal physiological and metabolic processes.
The Iron-Responsive Regulator Irr Is Required for Wild-Type Expression of the Gene Encoding the Heme Transporter BhuA in Brucella abortus 2308
Authors: Anderson Eric S. , Paulley James T. , Martinson David A. , Gaines Jennifer M. , Steele Kendra H. , Roop R. Martin
Published: July 2011

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in
Brucella
strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in
Brucella abortus
2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of
bhuA
expression is mediated by the iron-responsive regulator Irr. Specifically, a
bhuA-lacZ
transcriptional fusion does not produce elevated levels of β-galactosidase in response to iron deprivation in the isogenic
irr
mutant BEA5, and, unlike the parental strain,
B. abortus
BEA5 cannot utilize heme as an iron source
in vitro
and is attenuated in mice. A derivative of the
bhuA-lacZ
transcriptional fusion lacking the predicted Irr binding site upstream of the
bhuA
promoter does not produce elevated levels of β-galactosidase in response to iron deprivation in the parental
B. abortus
2308 strain, and a direct and specific interaction between a recombinant version of the
Brucella
Irr and the
bhuA
promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in
Bradyrhizobium japonicum
, readily detectable levels of Irr were found only in
B. abortus
2308 cells by Western blot analysis following growth under iron-deprived conditions.
Protection and Attachment of Vibrio cholerae Mediated by the Toxin-Coregulated Pilus in the Infant Mouse Model
Authors: Krebs Shelly J. , Taylor Ronald K.
Published: July 2011

Colonization of the human small intestine by
Vibrio cholerae
is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTXΦ receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to Caco-2 cells and found that mutants lacking TCP were defective in attachment compared to the wild type. Overexpression of ToxT, the activator of TCP, significantly increased attachment of wild-type
V. cholerae
to Caco-2 cells. Using field-emission scanning electron microscopy (FESEM), we also observed TCP-mediated attachment to the small intestines of infected infant mice by using antibodies specific to TCP and
V. cholerae
. Remarkably, we also visualized matrices comprised of TCP appearing to engulf
V. cholerae
during infection, and we demonstrated that these matrices protected the bacteria from a component of bile, disclosing a possible new role of this pilus in protection of the bacterial cells from antimicrobial agents. This study provides new insights into TCP's function in
V. cholerae
colonization of the small intestine, describing additional roles in mediating attachment and protection of
V. cholerae
bacterial cells.
O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide
Authors: Egge-Jacobsen Wolfgang , Salomonsson Emelie Näslund , Aas Finn Erik , Forslund Anna-Lena , Winther-Larsen Hanne C. , Maier Josef , Macellaro Anna , Kuoppa Kerstin , Oyston Petra C. F. , Titball Richard W. , Thomas Rebecca M. , Forsberg Åke , Prior Joann L. , Koomey Michael
Published: July 2011

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genusFrancisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation inFrancisella tularensisand its sufficiency for PilA glycosylation inEscherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified fromFrancisella tularensissubsp.tularensisandF. tularensissubsp.holarcticaand demonstrated that the protein undergoes multisite,O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains ofFrancisellasubspecies thus express anO-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved inFrancisellaspecies,O-linked protein glycosylation may be a feature common to members of this genus.
Elucidation of Structural and Antigenic Properties of Pneumococcal Serotype 11A, 11B, 11C, and 11F Polysaccharide Capsules
Authors: Calix Juan J. , Nahm Moon H. , Zartler Edward R.
Published: July 2011

Despite the emerging impact of serogroup 11 serotypes in
Streptococcus pneumoniae
epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of
O
-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of
O
-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the
O
-acetyltransferases encoded by
wcwC
,
wcwR
,
wcwT
, and
wcjE
. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of
S. pneumoniae
serogroup 11.
Peptide-Regulated Gene Depletion System Developed for Use in Streptococcus pneumoniae
Authors: Berg Kari Helene , Biørnstad Truls Johan , Straume Daniel , Håvarstein Leiv Sigve
Published: July 2011

To facilitate the study of pneumococcal genes that are essential for viability or normal cell growth, we sought to develop a tightly regulated, titratable gene depletion system that interferes minimally with normal cellular functions. A possible candidate for such a system is the recently discovered signal transduction pathway regulating competence for natural transformation inStreptococcus thermophilus. This pathway, which is unrelated to the ComCDE pathway used for competence regulation inStreptococcus pneumoniae, has not been fully elucidated, but it is known to include a short unmodified signaling peptide, ComS*, an oligopeptide transport system, Ami, and a transcriptional activator, ComR. The transcriptional activator is thought to bind to an inverted repeat sequence termed the ECom box. We introduced the ComR protein and the ECom box into the genome ofS. pneumoniaeR6 and demonstrated that addition of synthetic ComS* peptide induced the transcription of a luciferase gene inserted downstream of the ECom box. To determine whether the ComRS system could be used for gene depletion studies, thelicD1gene was inserted behind the chromosomally located ECom box promoter by using the Janus cassette. Then, the native versions oflicD1andlicD2were deleted, and the resulting mutant was recovered in the presence of ComS*. Cultivation of thelicD1 licD2double mutant in the absence of ComS* gradually affected its ability to grow and propagate, demonstrating that the ComRS system functions as intended. In the present study, the ComRS system was developed for use inS. pneumoniae. In principle, however, it should work equally well in many other Gram-positive species.
CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola
Authors: Cai Shichun , Zheng Xin , Dong Xiuzhu
Published: July 2011

Previously, we found that exoglucanase Cel48A from
Cellulosilyticum ruminicola
H1 bound intensively to Avicel; however, no known carbohydrate-binding module (CBM) was observed in the protein. Bioinformatics suggested that a C-terminal fragment of 127 amino acids, named the
Cellulosilyticum
-specific paralogous module (CPM), could function in binding. CPM-appended proteins are all putative (hemi)cellulases from
Cellulosilyticum
spp. In the present work, we demonstrated that Cel48A without the CPM retained only exoglucanase activity and lost the Avicel-binding ability, while the isolated CPM exhibited a high affinity for Avicel. In addition, the CPM bound to chitin, but not to soluble polysaccharides, making it a type A CBM, which binds only insoluble polysaccharides. Phylogenetic analysis clustered the CPM and its homologs as a separate branch that was distantly related to CBM subfamilies 3a (28% identity), 3b (24% identity), and 3c (21% identity). Sequence alignment revealed distinct secondary structures of the new CBM 3 group, in particular, a conserved Pro66-Trp67 insert preceding strand β4′, a deletion preceding strand β6, and incomplete strands β8 and β9. An alanine scan for six aromatic and three nonaromatic amino acid residues (D66, P66, and R111) by site-directed mutagenesis determined that Phe62, Pro66, Trp67, Tyr68, Arg111, and Trp117 were the functional residues for binding. Among them, Phe62, Pro66, and Trp67 were the newly determined key sites in the CPM for binding. Three-dimensional homolog modeling revealed two types of substrate-binding sites, planar and groove, in the CPM. Thus, a new subfamily, CBM family 3d, is proposed.
Competence-Dependent Endogenous DNA Rearrangement and Uptake of Extracellular DNA Give a Natural Variant of Streptococcus mutans without Biofilm Formation
Authors: Narisawa Naoki , Kawarai Taketo , Suzuki Naomi , Sato Yutaka , Ochiai Kuniyasu , Ohnishi Makoto , Watanabe Haruo , Senpuku Hidenobu
Published: July 2011

The production of water-insoluble glucan (WIG) enables
Streptococcus mutans
to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous
gtfB
and
gtfC
genes. Conversely, a single hybrid gene from the endogenous recombination of
gtfB
and
gtfC
is easily generated using RecA, resulting in
S. mutans
UA159 WIG
−
(rate of ∼1.0 × 10
−3
). The pneumococcus
recA
gene is regulated as a late competence gene.
comX
gene mutations did not lead to the appearance of WIG
−
cells. The biofilm collected from the flow cell had more WIG
−
cells than among the planktonic cells. Among the planktonic cells, WIG
−
cells appeared after 16 h and increased ∼10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG
−
cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG
+
and recipient WIG
−
cells, the recipient cells reverted to WIG
+
and acquired an intact
gtfBC
region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG
−
cells and that both are induced by the same signal transducer, the
com
system. Our findings may help in understanding how
S. mutans
can adapt to the oral environment and may explain the evolution of
S. mutans
.
Genomes and Characterization of Phages Bcep22 and BcepIL02, Founders of a Novel Phage Type in Burkholderia cenocepacia
Authors: Gill Jason J. , Summer Elizabeth J. , Russell William K. , Cologna Stephanie M. , Carlile Thomas M. , Fuller Alicia C. , Kitsopoulos Kate , Mebane Leslie M. , Parkinson Brandi N. , Sullivan David , Carmody Lisa A. , Gonzalez Carlos F. , LiPuma John J. , Young Ry
Published: July 2011

Within theBurkholderia cepaciacomplex,B. cenocepaciais the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeuticB. cenocepaciapodophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.
UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and Their Prevalence in Bacteria
Authors: Coyne Michael J. , Fletcher C. Mark , Reinap Barbara , Comstock Laurie E.
Published: July 2011

Xylose is rarely described as a component of bacterial glycans. UDP-xylose is the nucleotide-activated form necessary for incorporation of xylose into glycans and is synthesized by the decarboxylation of UDP-glucuronic acid (UDP-GlcA). Enzymes with UDP-GlcA decarboxylase activity include those that lead to the formation of UDP-xylose as the end product (Uxs type) and those synthesizing UDP-xylose as an intermediate (ArnA and RsU4kpxs types). In this report, we identify and confirm the activities of two Uxs-type UDP-GlcA decarboxylases of
Bacteroides fragilis
, designated BfUxs1 and BfUxs2.
Bfuxs1
is located in a conserved region of the
B. fragilis
genome, whereas
Bfuxs2
is in the heterogeneous capsular polysaccharide F (PSF) biosynthesis locus. Deletion of either gene separately does not result in the loss of a detectable phenotype, but deletion of both genes abrogates PSF synthesis, strongly suggesting that they are functional paralogs and that the
B. fragilis
NCTC 9343 PSF repeat unit contains xylose. UDP-GlcA decarboxylases are often annotated incorrectly as NAD-dependent epimerases/dehydratases; therefore, their prevalence in bacteria is underappreciated. Using available structural and mutational data, we devised a sequence pattern to detect bacterial genes encoding UDP-GlcA decarboxylase activity. We identified 826 predicted UDP-GlcA decarboxylase enzymes in diverse bacterial species, with the ArnA and RsU4kpxs types confined largely to proteobacterial species. These data suggest that xylose, or a monosaccharide requiring a UDP-xylose intermediate, is more prevalent in bacterial glycans than previously appreciated. Genes encoding BfUxs1-like enzymes are highly conserved in
Bacteroides
species, indicating that these abundant intestinal microbes may synthesize a conserved xylose-containing glycan.
Most Mutant OccR Proteins That Are Defective in Positive Control Hold Operator DNA in a Locked High-Angle Bend
Authors: Tsai Ching-Sung , Chen Chia-Sui , Winans Stephen C.
Published: July 2011

OccR is a LysR-type transcriptional regulator of
Agrobacterium tumefaciens
that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the
occ
genes occurs in response to octopine, a nutrient released from crown gall tumors. OccR also functions as an autorepressor in the presence or absence of octopine. OccR binds to a site between
occQ
and
occR
in the presence or absence of octopine, although octopine triggers a conformational change that shortens the DNA footprint and relaxes a DNA bend. In order to determine the roles of this conformational change in transcriptional activation, we isolated 11 OccR mutants that were defective in activation of the
occQ
promoter but were still capable of autorepression. The mutations in these mutants spanned most of the length of the protein. Two additional positive-control mutants were isolated using site-directed mutagenesis. Twelve mutant proteins displayed a high-angle DNA bend in the presence or absence of octopine. One mutant, the L26A mutant, showed ligand-responsive DNA binding similar to that of wild-type OccR and therefore must be impaired in a subsequent step in activation.
Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter
Authors: Aktas Meriyem , Jost Kathinka A. , Fritz Christiane , Narberhaus Franz
Published: July 2011

Agrobacterium tumefaciens
is a facultative phytopathogen that causes crown gall disease. For successful plant transformation
A. tumefaciens
requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in
A. tumefaciens
, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in
A. tumefaciens
. The ABC-type choline transporter is encoded by the chromosomally located
choXWV
operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [
14
C]choline uptake is severely reduced in
A. tumefaciens choX
mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [

K
D

] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine,

K
D

of ≈80 μM; betaine,

K
D

of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on
Sinorhizobium meliloti
ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding.
Role for Escherichia coli YidD in Membrane Protein Insertion
Authors: Yu Zhong , Lavèn Mariëlle , Klepsch Mirjam , de Gier Jan-Willem , Bitter Wilbert , van Ulsen Peter , Luirink Joen
Published: July 2011

YidC has an essential but poorly defined function in membrane protein insertion and folding in bacteria. The
yidC
gene is located in a gene cluster that is highly conserved in Gram-negative bacteria, the gene order being
rpmH
,
rnpA
,
yidD
,
yidC
, and
trmE
. Here, we show that
Escherichia coli yidD
, which overlaps with
rnpA
and is only 2 bp upstream of
yidC
, is expressed and localizes to the inner membrane, probably through an amphipathic helix. Inactivation of
yidD
had no discernible effect on cell growth and viability. However, compared to control cells, Δ
yidD
cells were affected in the insertion and processing of three YidC-dependent inner membrane proteins. Furthermore,
in vitro
cross-linking showed that YidD is in proximity of a nascent inner membrane protein during its localization in the Sec-YidC translocon, suggesting that YidD might be involved in the insertion process.
FliZ Acts as a Repressor of the
ydiV
Gene, Which Encodes an Anti-FlhD
4
C
2
Factor of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium
Authors: Wada Takeo , Tanabe Yasushi , Kutsukake Kazuhiro
Published: July 2011

YdiV acts as an anti-FlhD
4
C
2
factor, which negatively regulates the class 2 flagellar operons in poor medium in
Salmonella enterica
serovar Typhimurium. On the other hand, one of the class 2 flagellar genes,
fliZ
, encodes a positive regulator of the class 2 operons. In this study, we found that the FliZ-dependent activation of class 2 operon expression was more profound in poor medium than in rich medium and not observed in the
ydiV
mutant background. Transcription of the
ydiV
gene was shown to increase in the
fliZ
mutant. Purified FliZ protein was shown
in vitro
to bind to the promoter region of the
nlpC
gene, which is located just upstream of the
ydiV
gene, and to repress its transcription. These results indicate that FliZ is a repressor of the
nlpC-ydiV
operon and activates the class 2 operons by repressing
ydiV
expression. Therefore, the
fliZ
and
ydiV
genes form a regulatory loop.
Rsp Inhibits Attachment and Biofilm Formation by Repressing
fnbA
in Staphylococcus aureus MW2
Authors: Lei Mei G. , Cue David , Roux Christelle M. , Dunman Paul M. , Lee Chia Y.
Published: July 2011

Biofilms contribute to virulence of
Staphylococcus aureus
. Formation of biofilms is multifactorial, involving polysaccharide, protein, and DNA components, which are controlled by various regulators. Here we report that deletion of the
rsp
gene resulted in an increase in biofilm formation in strain MW2, suggesting that Rsp is a repressor of biofilm formation. Using SDS-PAGE, we found that Rsp profoundly affected cell surface and secreted proteins. The
rsp
gene was transcribed monocistronically, and the transcripts were most abundant at the exponential growth phase. Microarray analyses revealed that Rsp represses 75 genes, including 9 genes encoding cell wall-anchored proteins, and activates 22 genes, including 5 genes encoding secreted proteases. Among these genes,
fnbA
,
fnbB
,
sasG
, and
spa
(which encode cell wall-anchored proteins) and
splABCD
(which encode secreted proteases) have been implicated in biofilm formation. To deconvolute Rsp's contribution to biofilm formation, we analyzed deletion mutants of these genes either in the wild-type or in the
rsp
mutant background. We found that
fnbA
deletion in the
rsp
mutant restored biofilm formation to the wild-type level, indicating that FnbA plays a major role in Rsp regulation of biofilm formation. Further studies revealed that Rsp inhibited biofilm formation at the stage of primary attachment through repressing
fnbA
. Rsp belongs to the AraC/XylS family of regulatory proteins. We expressed the putative Rsp DNA binding domain (Rsp
DBD
) in
Escherichia coli
and showed that Rsp
DBD
was able to specifically bind to a short DNA fragment containing the
fnbA
promoter, suggesting that Rsp represses
fnbA
expression by direct DNA binding.
Interaction of Mycobacterium tuberculosis Elongation Factor Tu with GTP Is Regulated by Phosphorylation
Authors: Sajid Andaleeb , Arora Gunjan , Gupta Meetu , Singhal Anshika , Chakraborty Kausik , Nandicoori Vinay Kumar , Singh Yogendra
Published: July 2011

During protein synthesis, translation elongation factor Tu (Ef-Tu) is responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. The activity of Ef-Tu is dependent on its interaction with GTP. Posttranslational modifications, such as phosphorylation, are known to regulate the activity of Ef-Tu in several prokaryotes. Although a study of the
Mycobacterium tuberculosis
phosphoproteome showed Ef-Tu to be phosphorylated, the role of phosphorylation in the regulation of Ef-Tu has not been studied. In this report, we show that phosphorylation of
M. tuberculosis
Ef-Tu (
Mtb
Ef-Tu) by PknB reduced its interaction with GTP, suggesting a concomitant reduction in the level of protein synthesis. Overexpression of PknB in
Mycobacterium smegmatis
indeed reduced the level of protein synthesis.
Mtb
Ef-Tu was found to be phosphorylated by PknB on multiple sites, including Thr
118
, which is required for optimal activity of the protein. We found that kirromycin, an Ef-Tu-specific antibiotic, had a significant effect on the nucleotide binding of unphosphorylated
Mtb
Ef-Tu but not on the phosphorylated protein. Our results show that the modulation of the
Mtb
Ef-Tu–GTP interaction by phosphorylation can have an impact on cellular protein synthesis and growth. These results also suggest that phosphorylation can change the sensitivity of the protein to the specific inhibitors. Thus, the efficacy of an inhibitor can also depend on the posttranslational modification(s) of the target and should be considered during the development of the molecule.
Ribozyme Stability, Exon Skipping, and a Potential Role for RNA Helicase in Group I Intron Splicing by Coxiella burnetii
Authors: Hicks Linda D. , Warrier Indu , Raghavan Rahul , Minnick Michael F.
Published: July 2011

The 23S rRNA gene of
Coxiella burnetii
, the agent of Q fever in humans, contains an unusually high number of conserved, selfish genetic elements, including two group I introns, termed Cbu.L1917 (L1917) and Cbu.L1951 (L1951). To better understand the role that introns play in
Coxiella
's biology, we determined the intrinsic stability time periods (
in vitro
half-lives) of the encoded ribozymes to be ∼15 days for L1917 and ∼5 days for L1951, possibly due to differences in their sizes (551 and 1,559 bases, respectively), relative degrees of compactness of the respective RNA structures, and amounts of single-stranded RNA.
In vivo
half-lives for both introns were also determined to be ∼11 min by the use of RNase protection assays and an
Escherichia coli
model. Intron RNAs were quantified in synchronous cultures of
C. burnetii
and found to closely parallel those of 16S rRNA; i.e., ribozyme levels significantly increased between days 0 and 3 and then remained stable until 8 days postinfection. Both 16S rRNA and ribozyme levels fell during the stationary and death phases (days 8 to 14). The marked stability of the
Coxiella
intron RNAs is presumably conferred by their association with ribosomes, a stoichiometric relationship that was determined to be one ribozyme, of either type, per 500 ribosomes. Inaccuracies in splicing (exon 2 skipping) were found to increase during the first 5 days in culture, with a rate of approximately one improperly spliced 23S rRNA per 1.3 million copies. The
in vitro
efficiency of L1917 intron splicing was significantly enhanced in the presence of a recombinant
Coxiella
RNA DEAD-box helicase (CBU_0670) relative to that of controls, suggesting that this enzyme may serve as an intron RNA splice facilitator
in vivo
.
Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA
Authors: Allen Courtni E. , Schmitt Michael P.
Published: July 2011

The human pathogen
Corynebacterium diphtheriae
utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in
C. diphtheriae
involves the
dtxR
- and iron-regulated
hmu
hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione
S
-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various
Corynebacterium
species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in
C. diphtheriae
in which hemin is initially obtained from Hb by HtaA and then transferred between surface-anchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.
Mutational Analysis of the Control Cable That Mediates Transmembrane Signaling in the Escherichia coli Serine Chemoreceptor
Authors: Kitanovic Smiljka , Ames Peter , Parkinson John S.
Published: July 2011

During transmembrane signaling by
Escherichia coli
Tsr, changes in ligand occupancy in the periplasmic serine-binding domain promote asymmetric motions in a four-helix transmembrane bundle. Piston displacements of the signaling TM2 helix in turn modulate the HAMP bundle on the cytoplasmic side of the membrane to control receptor output signals to the flagellar motors. A five-residue control cable joins TM2 to the HAMP AS1 helix and mediates conformational interactions between them. To explore control cable structural features important for signal transmission, we constructed and characterized all possible single amino acid replacements at the Tsr control cable residues. Only a few lesions abolished Tsr function, indicating that the chemical nature and size of the control cable side chains are not individually critical for signal control. Charged replacements at I214 mimicked the signaling consequences of attractant or repellent stimuli, most likely through aberrant structural interactions of the mutant side chains with the membrane interfacial environment. Prolines at residues 214 to 217 also caused signaling defects, suggesting that the control cable has helical character. However, proline did not disrupt function at G213, the first control cable residue, which might serve as a structural transition between the TM2 and AS1 helix registers. Hydrophobic amino acids at S217, the last control cable residue, produced attractant-mimic effects, most likely by contributing to packing interactions within the HAMP bundle. These results suggest a helix extension mechanism of Tsr transmembrane signaling in which TM2 piston motions influence HAMP stability by modulating the helicity of the control cable segment.
Morphology of traumatic cataract: does it play a role in final visual outcome?
Authors: Shah Mehul A , Shah Shreya M , Shah Shashank B , Patel Chintan G , Patel Utsav A
Published: July 2011

Aim

To study the morphology of traumatic cataract as an important predictor for final visual outcome after treatment of traumatic cataracts.

Setting

Tertiary eye care centre in Dahod at the trijunction of Gujarat, Madhya Pradesh, and Rajasthan states in central western India.

Methods

This was a prospective observational cohort study among all patients presenting at the hospital with traumatic cataracts between January 2003 and December 2009. All information regarding demographic and ocular trauma was collected on a pretested World Eye Trauma Registry form for both the first visit and follow-up. In particular, the authors collected specific information about the morphology of traumatic cataracts; the surgical technique was determined accordingly. Data were entered and analysed with regard to the relationship between type of trauma and resulting injury, results achieved with particular surgical techniques, and the relationship between morphology and final visual outcome.

Outcome measures

Final visual outcome.

Results

Traumatic cataracts of different morphologies showed significant differences in the final visual outcome (χ2test, p=0.014).

Conclusion

The morphology of traumatic cataract plays an important role in the final visual outcome.
Personalised medicine in Canada: a survey of adoption and practice in oncology, cardiology and family medicine
Authors: Bonter Katherine , Desjardins Clarissa , Currier Nathan , Pun Jason , Ashbury Fredrick D
Published: July 2011

Introduction

In order to provide baseline data on genetic testing as a key element of personalised medicine (PM), Canadian physicians were surveyed to determine roles, perceptions and experiences in this area. The survey measured attitudes, practice, observed benefits and impacts, and barriers to adoption.

Methods

A self-administered survey was provided to Canadian oncologists, cardiologists and family physicians and responses were obtained online, by mail or by fax. The survey was designed to be exploratory. Data were compared across specialties and geography.

Results

The overall response rate was 8.3%. Of the respondents, 43%, 30% and 27% were family physicians, cardiologists and oncologists, respectively. A strong majority of respondents agreed that genetic testing and PM can have a positive impact on their practice; however, only 51% agreed that there is sufficient evidence to order such tests. A low percentage of respondents felt that they were sufficiently informed and confident practicing in this area, although many reported that genetic tests they have ordered have benefited their patients. Half of the respondents agreed that genetic tests that would be useful in their practice are not readily available. A lack of practice guidelines, limited provider knowledge and lack of evidence-based clinical information were cited as the main barriers to practice. Differences across provinces were observed for measures relating to access to testing and the state of practice. Differences across specialties were observed for the state of practice, reported benefits and access to testing.

Conclusions

Canadian physicians recognise the benefits of genetic testing and PM; however, they lack the education, information and support needed to practice effectively in this area. Variability in practice and access to testing across specialties and across Canada was observed. These results support a need for national strategies and resources to facilitate physician knowledge, training and practice in PM.