bluuebunny/crossref_metadata_2025 huggingface dataset:
The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces
Authors: Luz D.E. , Nepomuceno R.S.L. , Spira B. , Ferreira R.C.C.
Published: February 2012

The Pst system is a high‐affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate‐binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild‐type strain treated with an anti‐PstS antibody. Nevertheless, a reduced ability to bind to saliva‐coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild‐type strains. Taken together, the present data indicate that the S. mutans Pst system participates in phosphate uptake, cell growth and expression of virulence‐associated traits.
Fuel Property Effects on PCCI Combustion in a Heavy-Duty Diesel Engine
Authors: Dumitrescu Cosmin E. , Stuart Neill W. , Guo Hongsheng , Hosseini Vahid , Chippior Wallace L.
Published: February 2012

An experimental study was performed to investigate fuel property effects on premixed charge compression ignition (PCCI) combustion in a heavy-duty diesel engine. A matrix of research diesel fuels designed by the Coordinating Research Council, referred to as the Fuels for Advanced Combustion Engines (FACE), was used. The fuel matrix design covers a wide range of cetane numbers (30 to 55), 90% distillation temperatures (270 to 340 °C) and aromatics content (20 to 45%). The fuels were tested in a single-cylinder Caterpillar diesel engine equipped with a common-rail fuel injection system. The engine was operated at 900 rpm, a relative air/fuel ratio of 1.2 and 60% exhaust gas recirculation (EGR) for all fuels. The study was limited to a single fuel injection event starting between −30° and 0 °CA after top dead center (aTDC) with a rail pressure of 150 MPa. The brake mean effective pressure (BMEP) ranged from 2.6 to 3.1 bar depending on the fuel and its injection timing. The experimental results show that cetane number was the most important fuel property affecting PCCI combustion behavior. The low cetane number fuels had better brake specific fuel consumption (BSFC) due to more optimized combustion phasing and shorter combustion duration. They also had a longer ignition delay period available for premixing, which led to near-zero soot emissions. The two fuels with high cetane number and high 90% distillation temperature produced significant soot emissions. The two fuels with high cetane number and high aromatics produced the highest brake specific NOx emissions, although the absolute values were below 0.1 g/kW-h. Brake specific HC and CO emissions were primarily a function of the combustion phasing, but the low cetane number fuels had slightly higher HC and lower CO emissions than the high cetane number fuels.
A Hybrid Reliability Approach Based on Probability and Interval for Uncertain Structures
Authors: Jiang C. , Han X. , Li W. X. , Liu J. , Zhang Z.
Published: February 2012

Traditional reliability analysis generally uses probability approach to quantify the uncertainty, while it needs a great amount of information to construct precise distributions of the uncertain parameters. In this paper, a new reliability analysis technique is developed based on a hybrid uncertain model, which can deal with problems with limited information. All uncertain parameters are treated as random variables, while some of their distribution parameters are not given precise values but variation intervals. Due to the existence of the interval parameters, a limit-state strip enclosed by two bounding hyper-surfaces will be resulted in the transformed normal space, instead of a single hyper-surface as we usually obtain in conventional reliability analysis. All the limit-state strips are then summarized into two different classes and corresponding reliability analysis models are proposed for them. A monotonicity analysis is carried out for probability transformations of the random variables, through which effects of the interval distribution parameters on the limit state can be well revealed. Based on the monotonicity analysis, two algorithms are then formulated to solve the proposed hybrid reliability models. Three numerical examples are investigated to demonstrate the effectiveness of the present method.
Design and Analysis of a Foldable/Unfoldable Corrugated Architectural Curved Envelop
Authors: Gioia Francesco , Dureisseix David , Motro René , Maurin Bernard
Published: February 2012

Origami and paperfolding techniques may inspire the design of structures that have the ability to be folded and unfolded: their geometry can be changed from an extended, servicing state to a compact one, and back-forth. In traditional origami, folds are introduced in a sheet of paper (a developable surface) for transforming its shape, with artistic, or decorative intent; in recent times the ideas behind origami techniques were transferred in various design disciplines to build developable foldable/unfoldable structures, mostly in aerospace industry (Miura, 1985, “Method of Packaging and Deployment of Large Membranes in Space,” Inst. Space Astronaut. Sci. Rep., 618, pp. 1–9; Ikema et al., 2009, “Deformation Analysis of a Joint Structure Designed for Space Suit With the Aid of an Origami Technology,” 27th International Symposium on Space Technology and Science (ISTS)). The geometrical arrangement of folds allows a folding mechanism of great efficiency and is often derived from the buckling patterns of simple geometries, like a plane or a cylinder (e.g., Miura-ori and Yoshimura folding pattern) (Wu et al., 2007, “Optimization of Crush Characteristics of the Cylindrical Origami Structure,” Int. J. Veh. Des., 43, pp. 66–81; Hunt and Ario, 2005, “Twist Buckling and the Foldable Cylinder: An Exercise in Origami,” Int. J. Non-Linear Mech., 40(6), pp. 833–843). Here, we interest ourselves to the conception of foldable/unfoldable structures for civil engineering and architecture. In those disciplines, the need for folding efficiency comes along with the need for structural efficiency (stiffness); for this purpose, we will explore nondevelopable foldable/unfoldable structures: those structures exhibit potential stiffness because, when unfolded, they cannot be flattened to a plane (nondevelopability). In this paper, we propose a classification for foldable/unfoldable surfaces that comprehend non fully developable (and also non fully foldable) surfaces and a method for the description of folding motion. Then, we propose innovative geometrical configurations for those structures by generalizing the Miura-ori folding pattern to nondevelopable surfaces that, once unfolded, exhibit curvature.
Involute-Based Screw Driver Profiles With High Load Capacity
Authors: Tong Shih-Hsi , Yang Daniel C. H.
Published: February 2012

In this paper, involute profiles are used for the design of screw driver and screw heads instead of traditional straight line profiles to increase their load capacity and durability. New screw driver profiles have lower contact stress than traditional screw drivers due to the surface contact between screw driver and screw head. This enables the involute screw drivers to sustain higher load capacity than traditional screw drivers and reduce the material failure at the tip portions for both screw drivers and screw heads. Four different designs of screw driver profiles are provided for illustration.
Relative Catalytic Efficiency of
ldhL
- and
ldhD
-Encoded Products Is Crucial for Optical Purity of Lactic Acid Produced by Lactobacillus Strains
Authors: Zheng Zhaojuan , Sheng Binbin , Ma Cuiqing , Zhang Haiwei , Gao Chao , Su Fei , Xu Ping
Published: February 2012

NAD-dependent
l
- and
d
-lactate dehydrogenases coexist in
Lactobacillus
genomes and may convert pyruvic acid into
l
-lactic acid and
d
-lactic acid, respectively. Our findings suggest that the relative catalytic efficiencies of
ldhL
- and
ldhD
-encoded products are crucial for the optical purity of lactic acid produced by
Lactobacillus
strains.
Use of a Coculture To Enable Current Production by Geobacter sulfurreducens
Authors: Qu Youpeng , Feng Yujie , Wang Xin , Logan Bruce E.
Published: February 2012

Microbial fuel cells often produce more electrical power with mixed cultures than with pure cultures. Here, we show that a coculture of a nonexoelectrogen (
Escherichia coli
) and
Geobacter sulfurreducens
improved system performance relative to that of a pure culture of the exoelectrogen due to the consumption of oxygen leaking into the reactor.
Characterization of Bacterial Polysaccharide Capsules and Detection in the Presence of Deliquescent Water by Atomic Force Microscopy
Authors: Su Hai-Nan , Chen Zhi-Hua , Liu Sheng-Bo , Qiao Li-Ping , Chen Xiu-Lan , He Hai-Lun , Zhao Xian , Zhou Bai-Cheng , Zhang Yu-Zhong
Published: February 2012

We detected polysaccharide capsules from
Zunongwangia profunda
SM-A87 with atomic force microscopy (AFM). The molecular organization of the capsules at the single-polysaccharide-chain level was reported. Furthermore, we found that with ScanAsyst mode the polysaccharide capsules could be detected even in the presence of deliquescent water covering the capsule.
Characterization of a Novel Lytic Protein Encoded by the Bacillus cereus E33L Gene
ampD
as a Bacillus anthracis Antimicrobial Protein
Authors: Bourguet Feliza A. , Souza Brian E. , Hinz Angela K. , Coleman Matthew A. , Jackson Paul J.
Published: February 2012

Lytic proteins encoded by bacterial genomes have been implicated in cell wall biosynthesis and recycling. The
Bacillus cereus
E33L
ampD
gene encodes a putative
N
-acetylmuramoyl-
l
-alanine amidase. This gene, expressed
in vitro
, produced a very stable, highly active lytic protein. Very low concentrations rapidly and efficiently lyse vegetative
Bacillus anthracis
cells.
Nonribosomal Peptide Synthetase Genes
pesL
and
pes1
Are Essential for Fumigaclavine C Production in Aspergillus fumigatus
Authors: O'Hanlon Karen A. , Gallagher Lorna , Schrettl Markus , Jöchl Christoph , Kavanagh Kevin , Larsen Thomas O. , Doyle Sean
Published: February 2012

The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,
Aspergillus fumigatus
, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in
A. fumigatus
. Deletion of either
pesL
(Δ
pesL
) or
pes1
(Δ
pes1
) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H
2
O
2
, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of
pesL
resulted in severely reduced virulence in an invertebrate infection model (
P
< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for
A. fumigatus
. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both
A. fumigatus
wild-type and Δ
pesL
strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis
in vitro
. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.
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

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

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

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

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

Probiotics 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

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

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

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

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

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

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

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

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

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.
Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population
Authors: Williamson Kerry S. , Richards Lee A. , Perez-Osorio Ailyn C. , Pitts Betsey , McInnerney Kathleen , Stewart Philip S. , Franklin Michael J.
Published: February 2012

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of
Pseudomonas aeruginosa
growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the
rmf
and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a
P. aeruginosa
Δ
rmf
strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick
P. aeruginosa
biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.
RNase III Controls the Degradation ofcorAmRNA in Escherichia coli
Authors: Lim Boram , Sim Se-Hoon , Sim Minji , Kim Kyungsub , Jeon Che Ok , Lee Younghoon , Ha Nam-Chul , Lee Kangseok
Published: February 2012

InEscherichia coli, thecorAgene encodes a transporter that mediates the influx of Co2+, Mg2+, and Ni2+into the cell. During the course of experiments aimed at identifying RNase III-dependent genes inE. coli, we observed that steady-state levels ofcorAmRNA as well as the degree of cobalt influx into the cell were dependent on cellular concentrations of RNase III. In addition, changes incorAexpression levels by different cellular concentrations of RNase III were closely correlated with degrees of resistance ofE. colicells to Co2+and Ni2+.In vitroandin vivocleavage analyses ofcorAmRNA identified RNase III cleavage sites in the 5′-untranslated region of thecorAmRNA. The introduction of nucleotide substitutions at the identified RNase III cleavage sites abolished RNase III cleavage activity oncorAmRNA and resulted in prolonged half-lives of the mRNA, which demonstrates that RNase III cleavage constitutes a rate-determining step forcorAmRNA degradation. These findings reveal an RNase III-mediated regulatory pathway that functions to modulatecorAexpression and, in turn, the influx of metal ions transported by CorA inE. coli.
Levels of Germination Proteins in Dormant and Superdormant Spores of Bacillus subtilis
Authors: Ghosh Sonali , Scotland Michelle , Setlow Peter
Published: February 2012

Bacillus subtilis
spores that germinated poorly with saturating levels of nutrient germinants, termed superdormant spores, were separated from the great majority of dormant spore populations that germinated more rapidly. These purified superdormant spores (1.5 to 3% of spore populations) germinated extremely poorly with the germinants used to isolate them but better with germinants targeting germinant receptors not activated in superdormant spore isolation although not as well as the initial dormant spores. The level of β-galactosidase from a
gerA-lacZ
fusion in superdormant spores isolated by germination via the GerA germinant receptor was identical to that in the initial dormant spores. Levels of the germination proteins GerD and SpoVAD were also identical in dormant and superdormant spores. However, levels of subunits of a germinant receptor or germinant receptors activated in superdormant spore isolation were 6- to 10-fold lower than those in dormant spores, while levels of subunits of germinant receptors not activated in superdormant spore isolation were only ≤2-fold lower. These results indicate that (i) levels of β-galactosidase from
lacZ
fusions to operons encoding germinant receptors may not be an accurate reflection of actual germinant receptor levels in spores and (ii) a low level of a specific germinant receptor or germinant receptors is a major cause of spore superdormancy.
The PduM Protein Is a Structural Component of the Microcompartments Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation by Salmonella enterica
Authors: Sinha Sharmistha , Cheng Shouqiang , Fan Chenguang , Bobik Thomas A.
Published: February 2012

Diverse bacteria use proteinaceous microcompartments (MCPs) to optimize metabolic pathways that have toxic or volatile intermediates. MCPs consist of metabolic enzymes encased within a protein shell that provides a defined environment. InSalmonella enterica, a MCP is involved in B12-dependent 1,2-propanediol utilization (Pdu MCP). In this report, we show that the protein PduM is required for the assembly and function of the Pdu MCP. The results of tandem mass spectrometry and Western blot analyses show that PduM is a component of the Pdu MCP. Electron microscopy shows that apduMdeletion mutant forms MCPs with abnormal morphology. Growth tests and metabolite measurements establish that apduMdeletion mutant is unable to form functional MCPs. PduM is unrelated in sequence to proteins of known function and hence may represent a new class of MCP structural proteins. We also report a modified protocol for the purification of Pdu MCP fromSalmonellawhich allows isolation of milligram amounts of MCPs in about 4 h. We believe that this protocol can be extended or modified for the purification of MCPs from diverse bacteria.
CovR Alleviates Transcriptional Silencing by a Nucleoid-Associated Histone-Like Protein in Streptococcus mutans
Authors: Biswas Indranil , Mohapatra Saswat Sourav
Published: February 2012

In
Streptococcus mutans
, the global response regulator CovR plays an important role in biofilm formation, stress tolerance response, and caries production. We have previously demonstrated that CovR activates a large gene cluster, which is a part of a genomic island, TnSmu2. In this article, we have further characterized CovR at the molecular level to understand the gene activation mechanism. Toward this end, we mapped the transcription start site of the operon that lies upstream of the
SMU.1348
gene (P

SMU.1348

), the first gene of the cluster. We constructed a transcriptional reporter fusion and showed that CovR induces expression from P

SMU.1348

. We also demonstrated that purified CovR protects the sequence surrounding the −10 region of P

SMU.1348

. In an
in vitro
transcription assay, we showed that histone-like protein (HLP), a homologue of
Escherichia coli
HU protein, represses transcription from P

SMU.1348

.
In vivo
overexpression of HLP in
trans
also represses transcription from P

SMU.1348

. Addition of CovR to the HLP-repressed P

SMU.1348

resulted in increased transcription from the promoter, suggesting a role for CovR in countering HLP silencing. Moreover, addition of SMU.1349, a transcriptional activator of the operon, to the
in vitro
assay further stimulated the transcription. Based on our
in vivo
and
in vitro
results, we propose a model for transcriptional activation of the operon.
Zusammenhang zwischen polieren, ӓtzen und Lochfrasskorrosion der Metalle
Authors: Toušek J.
Published: February 2012

Auf den potentiodynamischen Polarisationskurven von Eisen und Nickel und Legierungen des Kobalts mit Nickel werden die Bereiche der Lochfraßkorrosion, des Polierens und Ätzens aufgezeigt und der Zusammenhang zwischen diesen drei Typen des Metallösens demonstriert. Die Ergebnisse der potentiodynamischen Messungen sind im Diagramm E vs log aA'/aH2O zusammengefaßt. Das Diagramm wird eingehend mit Rücksicht auf die Lochfraßkorrosion diskutiert.
MEASURING INCONSISTENCY IN A NETWORK INTRUSION DETECTION RULE SET BASED ON SNORT
Authors: MCAREAVEY KEVIN , LIU WEIRU , MILLER PAUL , MU KEDIAN
Published: February 2012

In this preliminary study, we investigate how inconsistency in a network intrusion detection rule set can be measured. To achieve this, we first examine the structure of these rules which are based on Snort and incorporate regular expression (Regex) pattern matching. We then identify primitive elements in these rules in order to translate the rules into their (equivalent) logical forms and to establish connections between them. Additional rules from background knowledge are also introduced to make the correlations among rules more explicit. We measure the degree of inconsistency in formulae of such a rule set (using the Scoring function, Shapley inconsistency values and Blame measure for prioritized knowledge) and compare the informativeness of these measures. Finally, we propose a new measure of inconsistency for prioritized knowledge which incorporates the normalized number of atoms in a language involved in inconsistency to provide a deeper inspection of inconsistent formulae. We conclude that such measures are useful for the network intrusion domain assuming that introducing expert knowledge for correlation of rules is feasible.
Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves
Authors: Lacerda Carla M. R. , Kisiday John , Johnson Brennan , Orton E. Christopher
Published: February 2012

This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.