bluuebunny/crossref_metadata_2025_split huggingface dataset:
Silver(I), Mercury(II), Cadmium(II), and Zinc(II) Target Exposed Enzymic Iron-Sulfur Clusters when They Toxify Escherichia coli
Authors: Xu Fang Fang , Imlay James A. 
Published: February 2012

ABSTRACT

The toxicity of soft metals is of broad interest to microbiologists, both because such metals influence the community structures in natural environments and because several metals are used as antimicrobial agents. Their potency roughly parallels their thiophilicity, suggesting that their primary biological targets are likely to be enzymes that contain key sulfhydryl moieties. A recent study determined that copper poisons
Escherichia coli
in part by attacking the exposed [4Fe-4S] clusters of dehydratases. The present investigation sought to test whether other soft metals also target these enzymes.
In vitro
experiments revealed that low-micromolar concentrations of Ag(I) and Hg(II) directly inactivated purified fumarase A, a member of the dehydratase family. The enzyme was also poisoned by higher levels of Cd(II) and Zn(II), but it was unaffected by even millimolar concentrations of Mn(II), Co(II), Ni(II), and Pb(II). Electron paramagnetic resonance analysis and measurements of released iron confirmed that damage was associated with destruction of the [4Fe-4S] cluster, and indeed, the reconstruction of the cluster fully restored activity. Growth studies were then performed to test whether dehydratase damage might underlie toxicity
in vivo
. Barely toxic doses of Ag(I), Hg(II), Cd(II), and Zn(II) inactivated all tested members of the [4Fe-4S] dehydratase family. Again, activity was recovered when the clusters were rebuilt. The metals did not diminish the activities of other sampled enzymes, including NADH dehydrogenase I, an iron-sulfur protein whose clusters are shielded by polypeptide. Thus, the data indicate that dehydratases are damaged by the concentrations of metals that initiate bacteriostasis.
Sialic Acid Catabolism and Transport Gene Clusters Are Lineage Specific in Vibrio vulnificus
Authors: Lubin Jean-Bernard , Kingston Joseph J. , Chowdhury Nityananda , Boyd E. Fidelma 
Published: February 2012

ABSTRACT

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several
Vibrio
species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In
Vibrio cholerae
on chromosome I, these genes are carried on the
Vibrio
pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced
Vibrio vulnificus
clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within
V. vulnificus
, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of
V. vulnificus
, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in
V. vulnificus
, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in
siaM
, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in
V. vulnificus
and that the TRAP transporter is essential for sialic acid uptake.
Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe
Authors: Woudstra Cedric , Skarin Hanna , Anniballi Fabrizio , Fenicia Lucia , Bano Luca , Drigo Ilenia , Koene Miriam , Bäyon-Auboyer Marie-Hélène , Buffereau Jean-Philippe , De Medici Dario , Fach Patrick 
Published: February 2012

ABSTRACT

Clostridium botulinum
types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the
C. botulinum
subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including
C. botulinum
types C (
n
= 12), C-D (
n
= 29), D (
n
= 5), and D-C (
n
= 10), other botulinum neurotoxin (BoNT)-producing
Clostridium
strains (
n
= 20), non-BoNT-producing clostridia (
n
= 20), and other bacterial species (
n
= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of
C. botulinum
in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (
n
= 108), poultry (
n
= 60), and bovines (
n
= 56). Among the 292 samples, 144 were positive for either the
bont/
C-D or the
bont/
D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.
The N-Terminal β-Sheet of the Hyperthermophilic Endoglucanase from Pyrococcus horikoshii Is Critical for Thermostability
Authors: Yang Trent C. , Legault Steve , Kayiranga Emery A. , Kumaran Jyothi , Ishikawa Kazuhiko , Sung Wing L. 
Published: February 2012

ABSTRACT

The β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeon
Pyrococcus horikoshii
(EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α)
8
-barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.
Interaction of Bifidobacterium bifidum LMG13195 with HT29 Cells Influences Regulatory-T-Cell-Associated Chemokine Receptor Expression
Authors: López Patricia , González-Rodríguez Irene , Sánchez Borja , Ruas-Madiedo Patricia , Suárez Ana , Margolles Abelardo , Gueimonde Miguel 
Published: February 2012

ABSTRACTProbiotics play an important role in the maintenance of the gastrointestinal barrier. In addition to direct effects on mucosal integrity, the interaction with the intestinal mucosa may have an active immunoregulatory effect. In the present work, we exposed HT29 intestinal epithelial cells to twoBifidobacteriumspecies to determine their effect on gene expression profile, enterocyte monolayer integrity, and T-cell response.Bifidobacterium breveIPLA 20004 triggered a more pronounced increase in the transepithelial resistance of the enterocyte monolayer thanBifidobacterium bifidumLMG13195. The transcriptome profile of HT29 cells cultured in the presence ofB. bifidumLMG13195 showed an increased expression of immune mediators and, interestingly, chemotactic molecules (CXCL10, CCL20, CXCL11 and CCL22) able to recruit lymphocytes. Since regulatory T cells (Treg cells) may express receptors for specific chemokines, we cultured peripheral blood mononuclear cells with supernatants of HT29 cells previously treated withBifidobacteriumstrains and analyzed FOXP3 and CD25 Treg markers and CCR6, CXCR3, CCR4, and CCR3 expression on CD4+lymphocytes. The proportion of CD25highFOXP3+cells was significantly increased after culture withB. bifidumLMG13195-conditioned HT29 supernatant. Moreover, this treatment led to the largest amount of CCR6+CXCR3−CCR4+CCR3+CD4+cells expressing high levels of CD25, corresponding to the Treg population. These results suggest that soluble factors secreted afterB. bifidumLMG13195 contact with intestinal epithelial cells favored the generation of CD4+CD25highlymphocytes expressing chemokine receptor Treg markers, thus making possible their recruitment to the intestinal mucosa.
Thermus thermophilus Nucleoside Phosphorylases Active in the Synthesis of Nucleoside Analogues
Authors: Almendros Marcos , Berenguer José , Sinisterra Jose-Vicente 
Published: February 2012

ABSTRACT

Cells extracts from
Thermus thermophilus
HB27 express phosphorolytic activities on purines and pyrimidine nucleosides. Five putative encoding genes were cloned and expressed in
Escherichia coli
, and the corresponding recombinant proteins were purified and studied. Two of these showed phosphorolytic activities against purine nucleosides, and third one showed phosphorolytic activity against pyrimidine nucleosides
in vitro
, and the three were named TtPNPI, TtPNPII, and TtPyNP, respectively. The optimal temperature for the activity of the three enzymes was beyond the water boiling point and could not be measured accurately, whereas all of them exhibited a wide plateau of optimal pHs that ranged from 5.0 to 7.0. Analytical ultracentrifugation experiments revealed that TtPNPI was a homohexamer, TtPNPII was a monomer, and TtPyNP was a homodimer. Kinetic constants were determined for the phosphorolysis of the natural substrates of each enzyme. Reaction tests with nucleoside analogues revealed critical positions in the nucleoside for its recognition. Activities with synthetic nucleobase analogues, such as 5-iodouracil or 2,6-diaminopurine, and arabinosides were detected, supporting that these enzymes could be applied for the synthesis of new nucleoside analogs with pharmacological activities.
Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria
Authors: Wagh Ashwini , Shen Shujie , Shen Fen Ann , Miller Charles D. , Walsh Marie K. 
Published: February 2012

ABSTRACT

The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for
Listeria monocytogenes
isolates and 0.2 to 2 mM for
Mycobacterium
isolates.
Rearrangement of Gene Order in the
phaCAB
Operon Leads to Effective Production of Ultrahigh-Molecular-Weight Poly[(
R
)-3-Hydroxybutyrate] in Genetically Engineered Escherichia coli
Authors: Hiroe Ayaka , Tsuge Kenji , Nomura Christopher T. , Itaya Mitsuhiro , Tsuge Takeharu 
Published: February 2012

ABSTRACT

Ultrahigh-molecular-weight poly[(
R
)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered
Escherichia coli
is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as
phaCAB
) encodes the essential proteins for the production of P(3HB) in the native producer,
Ralstonia eutropha
. The three genes of the
phaCAB
operon are
phaC
, which encodes the polyhydroxyalkanoate (PHA) synthase,
phaA
, which encodes a 3-ketothiolase, and
phaB
, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the
phaCAB
operon (
phaABC
,
phaACB
,
phaBAC
,
phaBCA
,
phaCAB
, and
phaCBA
) on an expression plasmid in genetically engineered
E. coli
was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the
pha
genes relative to the promoter. The most balanced production result was achieved in the strain harboring the
phaBCA
expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.
Self-Assembled Amyloid-Like Oligomeric-Cohesin Scaffoldin for Augmented Protein Display on the Saccharomyces cerevisiae Cell Surface
Authors: Han Zhenlin , Zhang Bei , Wang Yi E. , Zuo Yi Y. , Su Wei Wen 
Published: February 2012

ABSTRACT

In this study, a molecular self-assembly strategy to develop a novel protein scaffold for amplifying the extent and variety of proteins displayed on the surface of
Saccharomyces cerevisiae
is presented. The cellulosomal scaffolding protein cohesin and its upstream hydrophilic domain (HD) were genetically fused with the yeast Ure2p N-terminal fibrillogenic domain consisting of residues 1 to 80 (Ure2p
1-80
). The resulting Ure2p
1-80
-HD-cohesin fusion protein was successfully expressed in
Escherichia coli
to produce self-assembled supramolecular nanofibrils that serve as a novel protein scaffold displaying multiple copies of functional cohesin domains. The amyloid-like property of the nanofibrils was confirmed via thioflavin T staining and atomic force microscopy. These cohesin nanofibrils attached themselves, via a green fluorescent protein (GFP)-dockerin fusion protein, to the cell surface of
S. cerevisiae
engineered to display a GFP-nanobody. The excess cohesin units on the nanofibrils provide ample sites for binding to dockerin fusion proteins, as exemplified using an mCherry-dockerin fusion protein as well as the
Clostridium cellulolyticum
CelA endoglucanase. More than a 24-fold increase in mCherry fluorescence and an 8-fold increase in CelA activity were noted when the cohesin nanofibril scaffold-mediated yeast display was used, compared to using yeast display with GFP-cohesin that contains only a single copy of cohesin. Self-assembled supramolecular cohesin nanofibrils created by fusion with the yeast Ure2p fibrillogenic domain provide a versatile protein scaffold that expands the utility of yeast cell surface display.
Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase
Authors: Moon Hee-Jung , Tiwari Manish Kumar , Singh Ranjitha , Kang Yun Chan , Lee Jung-Kul 
Published: February 2012

ABSTRACT

Ribitol dehydrogenase from
Zymomonas mobilis
(ZmRDH) catalyzes the conversion of ribitol to
d
-ribulose and concomitantly reduces NAD(P)
+
to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD
+
(S156D, [
k
cat
/

K
m

,NAD
]/[
k
cat
/

K
m

,NADP
] = 10.9, where

K
m

,NAD
is the

K
m

for NAD
+
and

K
m

,NADP
is the

K
m

for NADP
+
). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP
+
as the cofactor (S156H, [
k
cat
/

K
m

,NAD
]/[
k
cat
/

K
m

,NADP
] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.
d
-Xylose Concentration-Dependent Hydrolase Expression Profiles and the Function of CreA and XlnR in Aspergillus niger
Authors: Mach-Aigner Astrid R. , Omony Jimmy , Jovanovic Birgit , van Boxtel Anton J. B. , de Graaff Leo H. 
Published: February 2012

ABSTRACT

Aspergillus niger
is an important organism for the production of industrial enzymes such as hemicellulases and pectinases. The xylan-backbone monomer,
d
-xylose, is an inducing substance for the coordinate expression of a large number of polysaccharide-degrading enzymes. In this study, the responses of 22 genes to low (1 mM) and high (50 mM)
d
-xylose concentrations were investigated. These 22 genes encode enzymes that function as xylan backbone-degrading enzymes, accessory enzymes, cellulose-degrading enzymes, or enzymes involved in the pentose catabolic pathway in
A. niger
. Notably, genes encoding enzymes that have a similar function (e.g., xylan backbone degradation) respond in a similar manner to different concentrations of
d
-xylose. Although low
d
-xylose concentrations provoke the greatest change in transcript levels, in particular, for hemicellulase-encoding genes, transcript formation in the presence of high concentrations of
d
-xylose was also observed. Interestingly, a high
d
-xylose concentration is favorable for certain groups of genes. Furthermore, the repressing influence of CreA on the transcription and transcript levels of a subset of these genes was observed regardless of whether a low or high concentration of
d
-xylose was used. Interestingly, the decrease in transcript levels of certain genes on high
d
-xylose concentrations is not reflected by the transcript level of their activator, XlnR. Regardless of the
d
-xylose concentration applied and whether CreA was functional,
xlnR
was constitutively expressed at a low level.
Microbial Functioning and Community Structure Variability in the Mesopelagic and Epipelagic Waters of the Subtropical Northeast Atlantic Ocean
Authors: Baltar Federico , Arístegui Javier , Gasol Josep M. , Herndl Gerhard J. 
Published: February 2012

ABSTRACT
We analyzed the regional distribution of bulk heterotrophic prokaryotic activity (leucine incorporation) and selected single-cell parameters (cell viability and nucleic acid content) as parameters for microbial functioning, as well as bacterial and archaeal community structure in the epipelagic (0 to 200 m) and mesopelagic (200 to 1,000 m) subtropical Northeast Atlantic Ocean. We selectively sampled three contrasting regions covering a wide range of surface productivity and oceanographic properties within the same basin: (i) the eddy field south of the Canary Islands, (ii) the open-ocean NE Atlantic Subtropical Gyre, and (iii) the upwelling filament off Cape Blanc. In the epipelagic waters, a high regional variation in hydrographic parameters and bacterial community structure was detected, accompanied, however, by a low variability in microbial functioning. In contrast, mesopelagic microbial functioning was highly variable between the studied regions despite the homogeneous abiotic conditions found therein. More microbial functioning parameters indicated differences among the three regions within the mesopelagic (i.e., viability of cells, nucleic acid content, cell-specific heterotrophic activity, nanoflagellate abundance, prokaryote-to-nanoflagellate abundance ratio) than within the epipelagic (i.e., bulk activity, nucleic acid content, and nanoflagellate abundance) waters. Our results show that the mesopelagic realm in the Northeast Atlantic is, in terms of microbial activity, more heterogeneous than its epipelagic counterpart, probably linked to mesoscale hydrographical variations.
The SUMO E3 Ligase Siz2 Exerts a Locus-Dependent Effect on Gene Silencing in Saccharomyces cerevisiae
Authors: Pasupala Nagesh , Easwaran Sreesankar , Hannan Abdul , Shore David , Mishra Krishnaveni 
Published: February 2012

ABSTRACT

In the yeast
Saccharomyces cerevisiae
, the two silent mating-type loci and subtelomeric regions are subjected to a well-characterized form of gene silencing. Establishment of silencing involves the formation of a distinct chromatin state that is refractory to transcription. This structure is established by the action of silent information regulator proteins (Sir2, Sir3, and Sir4) that bind to nucleosomes and initiate the deacetylation of multiple lysine residues in histones H3 and H4. Sir2 protein is a conserved histone deacetylase that is critical for mating-type and telomeric silencing, as well as a Sir3/4-independent form of silencing observed within the ribosomal DNA (rDNA) repeat locus. We report here that sumoylation plays an important role in regulating gene silencing. We show that increased dosage of
SIZ2
, a SUMO (
s
mall
u
biquitin-related
mo
difier) ligase, is antagonistic to gene silencing and that this effect is enhanced by mutation of
ESC1
, whose product is involved in tethering telomeres to the nuclear periphery. We present evidence indicating that an elevated
SIZ2
dosage causes reduced binding of Sir2 protein to telomeres. These data support the idea that sumoylation of specific substrates at the nuclear periphery regulates the availability of Sir2 protein at telomeres.
Bestatin Inhibits Cell Growth, Cell Division, and Spore Cell Differentiation in Dictyostelium discoideum
Authors: Poloz Yekaterina , Catalano Andrew , O'Day Danton H. 
Published: February 2012

ABSTRACT

Bestatin methyl ester (BME) is an inhibitor of Zn
2+
-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used
Dictyostelium
as a model organism to study the effects of BME. Only two Zn
2+
-binding aminopeptidases have been identified in
Dictyostelium
to date, puromycin-sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we show that PsaA is differentially expressed throughout growth and development of
Dictyostelium
, and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP (where GFP is green fluorescent protein) also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaAΔNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division, and differentiation in
Dictyostelium
likely through inhibition of PsaA.