bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial and Species Variations in Bacterial Communities Associated with Corals from the Red Sea as Revealed by Pyrosequencing
Authors: Lee On On , Yang Jiangke , Bougouffa Salim , Wang Yong , Batang Zenon , Tian Renmao , Al-Suwailem Abdulaziz , Qian Pei-Yuan
Published: August 2012

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which
Gammaproteobacteria
was the most dominant group, and
Chloroflexi
,
Chlamydiae
, and the candidate phylum
WS3
were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of
Vibrio
,
Pseudoalteromonas
,
Serratia
,
Stenotrophomonas
,
Pseudomonas
, and
Achromobacter
in the corals. On the other hand, bacteria such as
Chloracidobacterium
and
Endozoicomonas
were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.
Structure-Based Engineering of Methionine Residues in the Catalytic Cores of Alkaline Amylase from Alkalimonas amylolytica for Improved Oxidative Stability
Authors: Yang Haiquan , Liu Long , Wang Mingxing , Li Jianghua , Wang Nam Sun , Du Guocheng , Chen Jian
Published: August 2012

This work aims to improve the oxidative stability of alkaline amylase from
Alkalimonas amylolytica
through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H
2
O
2
at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the
k
cat
values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 10
4
to 5.6 × 10
4
min
−1
. The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.
Vip3C, a Novel Class of Vegetative Insecticidal Proteins from Bacillus thuringiensis
Authors: Palma Leopoldo , Hernï¿½ndez-Rodrï¿½guez Carmen Sara , Maeztu Mireya , Hernï¿½ndez-Martï¿½nez Patricia , Ruiz de Escudero Iï¿½igo , Escriche Baltasar , Muï¿½oz Delia , Van Rie Jeroen , Ferrï¿½ Juan , Caballero Primitivo
Published: August 2012

Three
vip3
genes were identified in two
Bacillus thuringiensis
Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm
2
.
Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum
Authors: Srisimarat Wiraya , Kaulpiboon Jarunee , Krusong Kuakarun , Zimmermann Wolfgang , Pongsawasdi Piamsook
Published: August 2012

Corynebacterium glutamicumamylomaltase (CgAM) catalyzes the formation of large-ring cyclodextrins (LR-CDs) with a degree of polymerization of 19 and higher. The clonedCgAMgene was ligated into the pET-17b vector and used to transformEscherichia coliBL21(DE3). Site-directed mutagenesis of Tyr-172 inCgAM to alanine (Y172A) was performed to determine its role in the control of LR-CD production. Both the recombinant wild-type (WT) and Y172A enzymes were purified to apparent homogeneity and characterized. The Y172A enzyme exhibited lower disproportionation, cyclization, and hydrolysis activities than the WT. Thekcat/Kmof the disproportionation reaction of the Y172A enzyme was 2.8-fold lower than that of the WT enzyme. The LR-CD product profile from enzyme catalysis depended on the incubation time and the enzyme concentration. Interestingly, the Y172A enzyme showed a product pattern different from that of the WTCgAM at a long incubation time. The principal LR-CD products of the Y172A mutated enzyme were a cycloamylose mixture with a degree of polymerization of 28 or 29 (CD28 or CD29), while the principal LR-CD product of the WT enzyme was CD25 at 0.05 U of amylomaltase. These results suggest that Tyr-172 plays an important role in determining the LR-CD product profile of this novelCgAM.
Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase
Authors: Weber Stefan S. , Polli Fabiola , Boer Rï¿½mon , Bovenberg Roel A. L. , Driessen Arnold J. M.
Published: August 2012

Intense classical strain improvement has yielded industrial
Penicillium chrysogenum
strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-
l
-cysteinyl-
d
-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.
Identification and Regulation offusA, the Polyketide Synthase Gene Responsible for Fusarin Production in Fusarium fujikuroi
Authors: Díaz-Sánchez Violeta , Avalos Javier , Limón M. Carmen
Published: August 2012

Fusarins are a class of mycotoxins of the polyketide family produced by differentFusariumspecies, including the gibberellin-producing fungusFusarium fujikuroi. Based on sequence comparisons between polyketide synthase (PKS) enzymes for fusarin production in otherFusariumstrains, we have identified theF. fujikuroiorthologue, calledfusA. The participation offusAin fusarin biosynthesis was demonstrated by targeted mutagenesis. Fusarin production is transiently stimulated by nitrogen availability in this fungus, a regulation paralleled by thefusAmRNA levels in the cell. Illumination of the cultures results in a reduction of the fusarin content, an effect partially explained by a high sensitivity of these compounds to light. Mutants of thefusAgene exhibit no external phenotypic alterations, including morphology and conidiation, except for a lack of the characteristic yellow and/or orange pigmentation of fusarins. Moreover, thefusAmutants are less efficient than the wild type at degrading cellophane on agar cultures, a trait associated with pathogenesis functions inFusarium oxysporum. ThefusAmutants, however, are not affected in their capacities to grow on plant tissues.
Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid
Authors: Angermayr S. Andreas , Paszota Michal , Hellingwerf Klaas J.
Published: August 2012

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO
2
has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an
ldh
gene from
Bacillus subtilis
(encoding an
l
-lactate dehydrogenase) into the genome of
Synechocystis
sp. strain PCC6803 leads to
l
-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single
ldh
mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.
Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1
Authors: Lapteva Y. S. , Zolova O. E. , Shlyapnikov M. G. , Tsfasman I. M. , Muranova T. A. , Stepnaya O. A. , Kulaev I. S. , Granovsky I. E.
Published: August 2012

Lytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of the
Lysobacter
sp. strain XL1
alpA
and
alpB
genes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB).
In silico
analysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of the
alpA
and
alpB
open reading frames (ORFs) in
Escherichia coli
confirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. The
alpA
and
alpB
mRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of the
alpA
and
alpB
mRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount of
alpA
mRNA in cells of
Lysobacter
sp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.
Genotyping of Present-Day and Historical Geobacillus Species Isolates from Milk Powders by High-Resolution Melt Analysis of Multiple Variable-Number Tandem-Repeat Loci
Authors: Seale R. Brent , Dhakal Rajat , Chauhan Kanika , Craven Heather M. , Deeth Hilton C. , Pillidge Christopher J. , Powell Ian B. , Turner Mark S.
Published: August 2012

Spores of thermophilic
Geobacillus
species are a common contaminant of milk powder worldwide due to their ability to form biofilms within processing plants. Genotyping methods can provide information regarding the source and monitoring of contamination. A new genotyping method was developed based on multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in conjunction with high-resolution melt analysis (MLV-HRMA) and compared to the currently used method, randomized amplified polymorphic DNA PCR (RAPD-PCR). Four VNTR loci were identified and used to genotype 46
Geobacillus
isolates obtained from retailed powder and samples from 2 different milk powder processing plants. These 46 isolates were differentiated into 16 different groups using MLV-HRMA (
D
= 0.89). In contrast, only 13 RAPD-PCR genotypes were identified among the 46 isolates (
D
= 0.79). This new method was then used to analyze 35 isolates obtained from powders with high spore counts (>10
4
spores ï¿½ g
−1
) from a single processing plant together with 27 historical isolates obtained from powder samples processed in the same region of Australia 17 years ago. Results showed that three genotypes can coexist in a single processing run, while the same genotypes observed 17 years ago are present today. While certain genotypes could be responsible for powders with high spore counts, there was no correlation to specific genotypes being present in powder plants and retailed samples. In conclusion, the MLV-HRMA method is useful for genotyping
Geobacillus
spp. to provide insight into the prevalence and persistence of certain genotypes within milk powder processing plants.
Protection of Wood from Microorganisms by Laccase-Catalyzed Iodination
Authors: Schubert M. , Engel J. , Thöny-Meyer L. , Schwarze F. W. M. R. , Ihssen J.
Published: August 2012

In the present work, Norway spruce wood (
Picea abies
L.) was reacted with a commercial
Trametes versicolor
laccase in the presence of potassium iodide salt or the phenolic compounds thymol and isoeugenol to impart an antimicrobial property to the wood surface. In order to assess the efficacy of the wood treatment, a leaching of the iodinated and polymerized wood and two biotests including bacteria, a yeast, blue stain fungi, and wood decay fungi were performed. After laccase-catalyzed oxidation of the phenols, the antimicrobial effect was significantly reduced. In contrast, the enzymatic oxidation of iodide (I
−
) to iodine (I
2
) in the presence of wood led to an enhanced resistance of the wood surface against all microorganisms, even after exposure to leaching. The efficiency of the enzymatic wood iodination was comparable to that of a chemical wood preservative, VP 7/260a. The modification of the lignocellulose by the laccase-catalyzed iodination was assessed by the Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) technique. The intensities of the selected lignin-associated bands and carbohydrate reference bands were analyzed, and the results indicated a structural change in the lignin matrix. The results suggest that the laccase-catalyzed iodination of the wood surface presents an efficient and ecofriendly method for wood protection.
Insight into the Evolution of Magnetotaxis in Magnetospirillum spp., Based on
mam
Gene Phylogeny
Authors: Lefèvre Christopher T. , Schmidt Marian L. , Viloria Nathan , Trubitsyn Denis , Schüler Dirk , Bazylinski Dennis A.
Published: August 2012

Vibrioid- to helical-shaped magnetotactic bacteria phylogenetically related to the genus
Magnetospirillum
were isolated in axenic cultures from a number of freshwater and brackish environments located in the southwestern United States. Based on 16S rRNA gene sequences, most of the new isolates represent new
Magnetospirillum
species or new strains of known
Magnetospirillum
species, while one isolate appears to represent a new genus basal to
Magnetospirillum
. Partial sequences of conserved
mam
genes, genes reported to be involved in the magnetosome and magnetosome chain formation, and form II of the ribulose-1,5-bisphosphate carboxylase/oxygenase gene (
cbbM
) were determined in the new isolates and compared. The
cbbM
gene was chosen for comparison because it is not involved in magnetosome synthesis; it is highly conserved and is present in all but possibly one of the genomes of the magnetospirilla and the new isolates. Phylogenies based on 16S rRNA,
cbbM
, and
mam
gene sequences were reasonably congruent, indicating that the genes involved in magnetotaxis were acquired by a common ancestor of the
Magnetospirillum
clade. However, in one case, magnetosome genes might have been acquired through horizontal gene transfer. Our results also extend the known diversity of the
Magnetospirillum
group and show that they are widespread in freshwater environments.
Regulation of the Cell Division Cycle in Trypanosoma brucei
Authors: Li Ziyin
Published: August 2012

The cell division cycle is tightly regulated by the activation and inactivation of a series of proteins that control the replication and segregation of organelles to the daughter cells. During the past decade, we have witnessed significant advances in our understanding of the cell cycle in
Trypanosoma brucei
and how the cycle is regulated by various regulatory proteins. However, many other regulators, especially those unique to trypanosomes, remain to be identified, and we are just beginning to delineate the signaling pathways that drive the transitions through different cell cycle stages, such as the G
1
/S transition, G
2
/M transition, and mitosis-cytokinesis transition. Trypanosomes appear to employ both evolutionarily conserved and trypanosome-specific molecules to regulate the various stages of its cell cycle, including DNA replication initiation, spindle assembly, chromosome segregation, and cytokinesis initiation and completion. Strikingly, trypanosomes lack some crucial regulators that are well conserved across evolution, such as Cdc6 and Cdt1, which are involved in DNA replication licensing, the spindle motor kinesin-5, which is required for spindle assembly, the central spindlin complex, which has been implicated in cytokinesis initiation, and the actomyosin contractile ring, which is located at the cleavage furrow. Conversely, trypanosomes possess certain regulators, such as cyclins, cyclin-dependent kinases, and mitotic centromere-associated kinesins, that are greatly expanded and likely play diverse cellular functions. Overall, trypanosomes apparently have integrated unique regulators into the evolutionarily conserved pathways to compensate for the absence of those conserved molecules and, additionally, have evolved certain cell cycle regulatory pathways that are either different from its human host or distinct between its own life cycle forms.
cis
- and
trans
-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae
Authors: Subramanian Shoba , Woolford Carol A. , Desai Jigar V. , Lanni Frederick , Mitchell Aaron P.
Published: August 2012

The Rim101/PacC pathway governs adaptation to alkaline pH in many fungi. Output of the pathway is mediated by transcription factors of the Rim101/PacC family, which are activated by proteolytic cleavage. The proteolytic complex includes scaffold protein Rim20 and endosome-associated subunits of the endosomal sorting complex required for transport (ESCRT). We provide here evidence that
Saccharomyces cerevisiae
Rim13, the protease that is implicated in Rim101 cleavage, is associated with the Rim20-ESCRT complex, and we investigate its regulation. Rim13-GFP is dispersed in cells grown in acidic medium but forms punctate foci when cells encounter alkaline conditions. A
vps4Δ
mutant, which accumulates elevated levels of endosomal ESCRT, also accumulates elevated levels of Rim13-GFP foci, independently of external pH. In the
vps4Δ
background, mutation of ESCRT subunit Snf7 or of Rim20 blocks the formation of Rim13 foci, and we found that Rim13 and Rim20 are colocalized. The Rim13 ortholog PalB of
Aspergillus nidulans
has been shown to undergo ESCRT and membrane association through an N-terminal MIT domain, but Rim13 orthologs in the
Saccharomyces
clade lack homology to this N-terminal region. Instead, there is a clade-limited C-terminal region, and we show that point mutations in this region prevent punctate localization and impair Rim13 function. We suggest that
RIM13
arose from its ancestral gene through two genome rearrangements. The ancestor lost the coding region for its MIT domain through a 5′ rearrangement and acquired the coding region for the
Saccharomyces
-specific functional equivalent through a 3′ rearrangement.
The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803
Authors: Dutheil Jérémy , Saenkham Panatda , Sakr Samer , Leplat Christophe , Ortega-Ramos Marcia , Bottin Hervé , Cournac Laurent , Cassier-Chauvat Corinne , Chauvat Franck
Published: August 2012

We have thoroughly investigated the
abrB2
gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium
Synechocystis
strain PCC6803. We report that
abrB2
is expressed from an active but atypical promoter that possesses an extended −10 element (TGTAATAT) that compensates for the absence of a −35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended −10 promoter box and the absence of a −35 element are two well-conserved features in
abrB2
genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the
hox
operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the
hox
operon is weakly expressed even though it possesses the two sequences resembling canonical −10 and −35 promoter boxes. In both the AbrB2-repressed promoters of the
abrB2
gene and the
hox
operon, we found a repeated DNA motif [TT-(N
5
)-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the
abrB2
promoter. We think that our
abrB2
-deleted mutant with increased expression of the
hox
operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the
abrB2
gene and the
hox
operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in
Synechocystis
.
Origin and Fate of the 3′ Ends of Single-Stranded DNA Generated by Conjugal Transfer of Plasmid R1162
Authors: Becker Eric C. , Meyer Richard
Published: August 2012

During conjugation, a single strand of DNA is cleaved at the origin of transfer (
oriT
) by the plasmid-encoded relaxase. This strand is then unwound from its complement and transferred in the 5′-to-3′ direction, with the 3′ end likely extended by rolling-circle replication. The resulting, newly synthesized
oriT
must then be cleaved as well, prior to recircularization of the strand in the recipient. Evidence is presented here that the R1162 relaxase contains only a single nucleophile capable of cleaving at
oriT
, with another molecule therefore required to cleave at a second site. An assay functionally isolating this second cleavage shows that this reaction can take place in the donor cell. As a result, there is a flux of strands with free 3′ ends into the recipient. These ends are susceptible to degradation by exonuclease I. The degree of susceptibility is affected by the presence of an uncleaved
oriT
within the strand. A model is presented where these internal
oriT
s bind and trap the relaxase molecule covalently bound to the 5′ end of the incoming strand. Such a mechanism would result in the preferential degradation of transferred DNA that had not been properly cleaved in the donor.
Function and Regulation of Isoforms of Carbon Monoxide Dehydrogenase/Acetyl Coenzyme A Synthase in Methanosarcina acetivorans
Authors: Matschiavelli Nicole , Oelgeschläger Ellen , Cocchiararo Berardino , Finke Johannes , Rother Michael
Published: August 2012

Conversion of acetate to methane (aceticlastic methanogenesis) is an ecologically important process carried out exclusively by methanogenic archaea. An important enzyme for this process as well as for methanogenic growth on carbon monoxide is the five-subunit archaeal CO dehydrogenase/acetyl coenzyme A (CoA) synthase multienzyme complex (CODH/ACS) catalyzing both CO oxidation/CO
2
reduction and cleavage/synthesis of acetyl-CoA.
Methanosarcina acetivorans
C2A contains two very similar copies of a six-gene operon (
cdh
genes) encoding two isoforms of CODH/ACS (Cdh1 and Cdh2) and a single CdhA subunit, CdhA3. To address the role of the CODH/ACS system in
M. acetivorans
, mutational as well as promoter/reporter gene fusion analyses were conducted. Phenotypic characterization of
cdh
disruption mutants (three single and double mutants, as well as the triple mutant) revealed a strict requirement of either Cdh1 or Cdh2 for acetotrophic or carboxidotrophic growth, as well as for autotrophy, which demonstrated that both isoforms are
bona fide
CODH/ACS. While expression of the Cdh2-encoding genes was generally higher than that of genes encoding Cdh1, both appeared to be regulated differentially in response to growth phase and to changing substrate conditions. While dispensable for growth, CdhA3 clearly affected expression of
cdh1
, suggesting that it functions in signal perception and transduction rather than in catabolism. The data obtained argue for a functional hierarchy and regulatory cross talk of the CODH/ACS isoforms.
Minor Pilins of the Type IV Pilus System Participate in the Negative Regulation of Swarming Motility
Authors: Kuchma S. L. , Griffin E. F. , O'Toole G. A.
Published: August 2012

Pseudomonas aeruginosaexhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either thepilWorpilXgene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations inpilWorpilXalso fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) orbifAmutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute toP. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.
Phenotype Switching Is a Natural Consequence of Staphylococcus aureus Replication
Authors: Edwards Andrew M.
Published: August 2012

The pathogen
Staphylococcus aureus
undergoes phenotype switching
in vivo
from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse
S. aureus
strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that
S. aureus
phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the
S. aureus
life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.
Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum
Authors: Jin Huanan , Nikolau Basil J.
Published: August 2012

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the
Rhodospirillum rubrum
genome revealed by the occurrence of three homologous PHA polymerase genes (
phaC1
,
phaC2
, and
phaC3
).
In vitro
biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple
phaC
deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that
phaC2
contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast,
phaC3
is an insignificant contributor to PHA productivity, and
phaC1
, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C
5
and C
6
monomers. The
in vitro
characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of
R. rubrum
was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.
Reduced Na
+
Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB
Authors: Jakkula S. Vivek , Guan Lan
Published: August 2012

The melibiose permease of
Salmonella enterica
serovar Typhimurium (MelB
St
) catalyzes symport of melibiose with Na
+
, Li
+
, or H
+
. Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na
+
-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (
V
max
) for melibiose transport in
Escherichia coli
DW2 and greatly decrease Na
+
affinity, indicating that intracellular release of Na
+
is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop
4-5
and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases
V
max
with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.
Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea
Authors: Tomita Hiroya , Yokooji Yuusuke , Ishibashi Takuya , Imanaka Tadayuki , Atomi Haruyuki
Published: August 2012

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4′-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon
Thermococcus kodakarensis
utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from
T. kodakarensis
. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable
k
cat
/

K
m

values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg
2+
, respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.
Practical management of sudden cardiac arrest on the football field
Authors: Kramer Efraim Benjamin , Botha Martin , Drezner Jonathan , Abdelrahman Yasser , Dvorak Jiri
Published: August 2012

Sudden cardiac arrest (SCA) remains a tragic occurrence on the football field. The limits of preparticipation cardiovascular screening make it compulsory that prearranged emergency medical services be available at all football matches to immediately respond to any collapsed player. Management of SCA involves prompt recognition, immediate cardiopulmonary resuscitation (CPR) and early defibrillation. Any football player who collapses without contact with another player or obstacle should be regarded as being in SCA until proven otherwise. An automated external defibrillator (AED), or manual defibrillator if an AED is not available, should be immediately accessible on the field during competitions. This study presents guidelines for a practical and systematic approach to the management of SCA on the football field.
Development of a generic working definition of ‘supportive care’
Authors: Cramp Fiona , Bennett Michael I
Published: August 2012

Context

The term ‘supportive care’, despite everyday and widespread use, remains an ambiguous concept that lacks clarity. A generic definition would underpin the development of services and provide a basis for disease specific approaches to care.

Objectives

To develop a generic working definition of supportive care that could be applied to a range of diseases and chronic illnesses.

Methods

An indepth review of the literature was carried out to identify existing generic and disease specific definitions and descriptions of supportive care. Following the review, a modified two-phase Delphi study was performed. Participants included experts in the area of supportive care identified during the review in addition to charities or their representatives. A draft definition was developed based upon the outcome of the Delphi study.

Results

The literature review identified one brief generic definition of supportive care with no explanation of its origin. A further 17 disease specific definitions were identified, all relating to cancer. The review led to the development of 100 statements for inclusion in phase I of the modified Delphi study. 26 responses were received to phase I of the Delphi study and 17 responses to phase II. General agreement was received during the second stage and a final draft definition subsequently developed.

Conclusions

The resulting definition of supportive care might provide a basis for service development and could be further adapted to disease specific contexts. It would benefit from broader consultation to determine acceptability among a wider range of health professionals and service users.
Late effects and healthcare needs of survivors of allogeneic stem cell transplantation: a qualitative study
Authors: Hwang Jessica P , Roundtree Aimee K , Giralt Sergio A , Suarez-Almazor Maria
Published: August 2012

Purpose

This qualitative study of survivors of allogeneic stem cell transplantation (SCT) for haematological malignancy explored attitudes about late effects of therapy, healthcare issues and information needs.

Methods

We conducted 12 indepth cognitive interviews and three focus groups of patients who had previously had SCT and were without recurrence of their primary disease. We used grounded theory methods, where themes emerged from consensus between cocoders. Health-related quality of life was assessed with the short-form 36 (SF-36).

Results

The study included 22 patients (50% female; 95% white; mean age 47 years). The mean time from SCT was 5.2 years (±1.4 years). Most had low SF-36 scores. Participants discussed late effects of therapy, most commonly graft-versus-host disease, and how they impacted their quality of life. They reported frequent healthcare use and cancer screening after SCT and discussed problems affording care and interacting with insurance companies. Participants shared sources of health information (eg, preferring providers as their primary sources of information, but also learnt from websites, medical journals and peer experiences) and identified information barriers (eg, feeling ‘on their own’ insofar as they did not have targeted care for their needs), and expressed importance of anticipatory guidance regarding infertility. Overall, participants' personal issues and social influences impacted survivors' needs and attitudes.

Conclusions

SCT survivors face continuing and lasting health effects. The factors impacting survivorship needs are complex and may be interrelated. Future research should study the affect of incorporating personal and social issues into existing clinical SCT programmes on survivors' quality of life.
A REPRESENTATION FORMULA FOR THE p-ENERGY OF METRIC SPACE-VALUED SOBOLEV MAPS
Authors: LOGARITSCH PHILIPPE , SPADARO EMANUELE
Published: August 2012

We give an explicit representation formula for the p-energy of Sobolev maps with values in a metric space as defined by Korevaar and Schoen [Comm. Anal. Geom.1(3–4) (1993) 561–659]. The formula is written in terms of the Lipschitz compositions introduced by Ambrosio [Ann. Scuola Norm. Sup. Pisa Cl. Sci. (4)3(17) (1990) 439–478], thus further relating the two different definitions considered in the literature.
THE PROPORTIONAL COLORING PROBLEM: OPTIMIZING BUFFERS IN RADIO MESH NETWORKS
Authors: HUC FLORIAN , SALES CLÁUDIA LINHARES , RIVANO HERVÉ
Published: August 2012

In this paper, we consider a new edge coloring problem to model call scheduling optimization issues in wireless mesh networks: the proportional coloring. It consists in finding a minimum cost edge coloring of a graph which preserves the proportion given by the weights associated to each of its edges. We show that deciding if a weighted graph admits a proportional coloring is pseudo-polynomial while determining its proportional chromatic index is NP-hard. We then give lower and upper bounds for this parameter that can be computed in pseudo-polynomial time. We finally identify a class of graphs and a class of weighted graphs for which the proportional chromatic index can be exactly determined.
HOW TO MEASURE THE IMPORTANCE OF CLIMATE RISK FOR DETERMINING OPTIMAL GLOBAL ABATEMENT POLICIES?
Authors: LORENZ ALEXANDER , KRIEGLER ELMAR , HELD HERMANN , SCHMIDT MATTHIAS G. W.
Published: August 2012

We investigate the importance of explicitly accounting for uncertainty in the determination of optimal global climate policy. We demonstrate that the marginal risk premium determines the importance of adapting the optimal policy to uncertainty. Common integrated assessment models (IAM) of climate change suggest uncertainty has little effect because the marginal risk premium in these models is small. A rigorous investigation of the marginal risk premium and the marginal functional relationships within IAMs allows understanding the non-significance of (thin-tailed) uncertainty as a result of compensating factors in the climate cause-effect chain.
Interactive spacetime control of deformable objects
Authors: Hildebrandt Klaus , Schulz Christian , von Tycowicz Christoph , Polthier Konrad
Published: August 2012

Creating motions of objects or characters that are physically plausible and follow an animator's intent is a key task in computer animation. The
spacetime constraints
paradigm is a valuable approach to this problem, but it suffers from high computational costs. Based on spacetime constraints, we propose a framework for controlling the motion of deformable objects that offers interactive response times. This is achieved by a model reduction of the underlying variational problem, which combines dimension reduction, multipoint linearization, and decoupling of ODEs. After a preprocess, the cost for creating or editing a motion is reduced to solving a number of one-dimensional spacetime problems, whose solutions are the
wiggly splines
introduced by Kass and Anderson [2008]. We achieve interactive response times through a new fast and robust numerical scheme for solving the one-dimensional problems that is based on a closed-form representation of the wiggly splines.
Fields on symmetric surfaces
Authors: Panozzo Daniele , Lipman Yaron , Puppo Enrico , Zorin Denis
Published: August 2012

Direction fields, line fields and cross fields are used in a variety of computer graphics applications ranging from non-photorealistic rendering to remeshing. In many cases, it is desirable that fields adhere to symmetry, which is predominant in natural as well as man-made shapes. We present an algorithm for designing smooth N-symmetry fields on surfaces respecting
generalized symmetries
of the shape, while maintaining alignment with local features. Our formulation for constructing symmetry fields is based on global symmetries, which are given as input to the algorithm, with no isometry assumptions. We explore in detail the properties of generalized symmetries (reflections in particular), and we also develop an algorithm for the robust computation of such symmetry maps, based on a small number of correspondences, for surfaces of genus zero.
Pharmacological inhibition of soluble epoxide hydrolase provides cardioprotection in hyperglycemic rats
Authors: Guglielmino Kathleen , Jackson Kaleena , Harris Todd R. , Vu Vincent , Dong Hua , Dutrow Gavin , Evans James E. , Graham James , Cummings Bethany P. , Havel Peter J. , Chiamvimonvat Nipavan , Despa Sanda , Hammock Bruce D. , Despa Florin
Published: August 2012

Glycemic regulation improves myocardial function in diabetic patients, but finding optimal therapeutic strategies remains challenging. Recent data have shown that pharmacological inhibition of soluble epoxide hydrolase (sEH), an enzyme that decreases the endogenous levels of protective epoxyeicosatrienoic acids (EETs), improves glucose homeostasis in insulin-resistant mice. Here, we tested whether the administration of sEH inhibitors preserves cardiac myocyte structure and function in hyperglycemic rats. University of California-Davis-type 2 diabetes mellitus (UCD-T2DM) rats with nonfasting blood glucose levels in the range of 150–200 mg/dl were treated with the sEH inhibitor 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU) for 6 wk. Administration of APAU attenuated the progressive increase of blood glucose concentration and preserved mitochondrial structure and myofibril morphology in cardiac myocytes, as revealed by electron microscopy imaging. Fluorescence microscopy with Ca2+ indicators also showed a 40% improvement of cardiac Ca2+ transients in treated rats. Sarcoplasmic reticulum Ca2+ content was decreased in both treated and untreated rats compared with control rats. However, treatment limited this reduction by 30%, suggesting that APAU may protect the intracellular Ca2+ effector system. Using Western blot analysis on cardiac myocyte lysates, we found less downregulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), the main route of Ca2+ reuptake in the sarcoplasmic reticulum, and lower expression of hypertrophic markers in treated versus untreated UCD-T2DM rats. In conclusion, APAU enhances the therapeutic effects of EETs, resulting in slower progression of hyperglycemia, efficient protection of myocyte structure, and reduced Ca2+ dysregulation and SERCA remodeling in hyperglycemic rats. The results suggest that sEH/EETs may be an effective therapeutic target for cardioprotection in insulin resistance and diabetes.