bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymer depletion-driven cluster aggregation and initial phase separation in charged nanosized colloids
Authors: Gögelein Christoph , Nägele Gerhard , Buitenhuis Johan , Tuinier Remco , Dhont Jan K. G.
Published: May 2009

We study polymer depletion-driven cluster aggregation and initial phase separation in aqueous dispersions of charge-stabilized silica spheres, where the ionic strength and polymer (dextran) concentration are systematically varied, using dynamic light scattering and visual observation. Without polymers and for increasing salt and colloid content, the dispersions become increasingly unstable against irreversible cluster formation. By adding nonadsorbing polymers, a depletion-driven attraction is induced, which lowers the stabilizing Coulomb barrier and enhances the cluster growth rate. The initial growth rate increases with increasing polymer concentration and decreases with increasing polymer molar mass. These observations can be quantitatively understood by an irreversible dimer formation theory based on the classical Derjaguin, Landau, Verwey, and Overbeek pair potential, with the depletion attraction modeled by the Asakura–Oosawa–Vrij potential. At low colloid concentration, we observe an exponential cluster growth rate for all polymer concentrations considered, indicating a reaction-limited aggregation mechanism. At sufficiently high polymer and colloid concentrations, and lower salt content, a gas-liquidlike demixing is observed initially. Later on, the system separates into a gel and fluidlike phase. The experimental time-dependent state diagram is compared to the theoretical equilibrium phase diagram obtained from a generalized free-volume theory and is discussed in terms of an initial reversible phase separation process in combination with irreversible aggregation at later times.
Metal-oxide-semiconductor field-effect transistors on GaAs (111)A surface with atomic-layer-deposited Al2O3 as gate dielectrics
Authors: Xu M. , Wu Y. Q. , Koybasi O. , Shen T. , Ye P. D.
Published: May 2009

GaAs inversion-mode metal-oxide-semiconductor field-effect transistors (MOSFETs) with atomic-layer-deposited Al2O3 as gate dielectrics are fabricated on (111)A and (100) surfaces. With the same channel length of 0.75 μm, the maximum drain current of 15 mA/mm on n-channel MOSFET is obtained on (111)A surface, in great contrast to only 1 μA/mm on (100) surface. For p-channel MOSFETs, maximum drain currents of 0.17 mA/mm and 0.8 mA/mm are obtained on (111)A and (100) surfaces, respectively. An empirical model is proposed to correlate the experimental observation with the existing III-V MOS theories.
Perception of Subjective Contours in Fish
Authors: Sovrano Valeria Anna , Bisazza Angelo
Published: June 2009

The ability of fish to perceive subjective (or illusory) contours, ie contours that lack a physical counterpart in terms of luminance contrast gradients, was investigated. In the first experiment, redtail splitfins ( Xenotoca eiseni), family Goodeidae, were trained to discriminate between a geometric figure (a triangle or a square) on various backgrounds and a background without any figure. Thereafter, the fish performed test trials in which illusory squares or triangles were obtained by (i) interruptions of a background of diagonal lines, (ii) phase-shifting of a background of diagonal lines, and (iii) pacmen spatially arranged to induce perception of Kanizsa subjective surfaces. In all three conditions, fish seemed to generalise their responses to stimuli perceived as subjective contours by humans. Fish chose, correctly, squares or triangles made of interrupted or phase-shifted diagonal lines from uniform backgrounds of diagonal lines, as well as illusory square or triangle Kanizsa figures from figures in which the inducing pacmen were scrambled. In the second experiment, fish were trained to discriminate between a vertical and a horizontal bar with luminance contrast gradients, and then tested with vertically and horizontally oriented illusory bars, created either through interruption or spatial phase-shift of inducing diagonal lines. Fish appeared to be able to generalise the orientation discrimination to illusory contours. These results demonstrate that redtail splitfins are capable of perceiving illusory contours.
Divergent lineages in the heath mouse (Pseudomys shortridgei) are indicative of major contraction to geographically isolated refugia on the eastern and western sides of Australia during the early Pleistocene
Authors: Salinas Maria , Bunce Michael , Cancilla Damien , Alpers Deryn L. , Spencer Peter B. S.
Published: May 2009

The heath mouse (Pseudomys shortridgei) is a locally rare species; it was considered extinct in Western Australia until its rediscovery 20 years ago. It is not often detected in faunal surveys and is confined to two ecologically distinct habitats on either side of the Australian continent. An important and immediate conservation question has been to determine the genetic diversity within each of its current habitats and to determine the differences between the geographically separate populations. Measures of genetic differentiation amongst P. shortridgei populations in their eastern (Victoria) and western (Western Australia) distribution were estimated using nuclear (microsatellite loci) and partial sequence of mitochondrial DNA (427 bp Cytochrome b gene and 637 bp of the D-loop). Both markers identified differences between the east- and west-coast populations. MtDNA data showed significant divergence between populations with monophyly within them, and nuclear loci investigated also showed two clear genetic clusters based on Bayesian inference. As a result of these findings, we suggest that the heath mouse comprises two highly divergent (but genetically diverse) lineages and the aridity of the Nullarbor Plain has clearly been a barrier for dispersal since the early Pleistocene (~1.43 million years ago). The populations either side of the Nullarbor Plain are genetically differentiated and should be defined as separate Evolutionary Significant Units (ESUs).
Reversing a tree regeneration crisis in an endangered ecoregion
Authors: Fischer Joern , Stott Jenny , Zerger Andre , Warren Garth , Sherren Kate , Forrester Robert I.
Published: June 2009

Global food demand is growing rapidly. Livestock grazing can provide a valuable source of protein, but conventional grazing is often unsustainable. We studied an 800,000-ha section of a threatened ecoregion in southeastern Australia. Conventional management in the region involves continuous livestock grazing with few rest periods and regular fertilizer application. By using remotely sensed data on tree cover and extensive field data on livestock grazing regimes, soil chemistry, tree diameters, and tree regeneration, we show that the region is facing a tree regeneration crisis. Under conventional management, across the region, millions of hectares of land currently supporting tens of millions of trees will be treeless within decades from now. This would have severe negative ramifications for biodiversity and key ecosystem services, including water infiltration and shade provision for livestock. However, we identified an unexpected win–win solution for tree regeneration and commercial grazing. A relatively new practice in the region is fast-rotational grazing, characterized by prolonged rest periods in between short, intensive grazing events. The probability of regeneration under fast-rotational grazing was up to 4-fold higher than under conventional grazing, and it did not differ significantly from the probability of regeneration in ungrazed areas. In addition, trees were more likely to regenerate where soil nutrient levels were low. These findings suggest that the tree regeneration crisis can be reversed by applying low-input, fast-rotational grazing. New policy settings supporting these practices could signal a turning point for the region, from ecological decline to ecological recovery.
Anterior cingulate cortex hypoactivations to an emotionally salient task in cocaine addiction
Authors: Goldstein Rita Z. , Alia-Klein Nelly , Tomasi Dardo , Carrillo Jean Honorio , Maloney Thomas , Woicik Patricia A. , Wang Ruiliang , Telang Frank , Volkow Nora D.
Published: May 2009

Anterior cingulate cortex (ACC) hypoactivations during cognitive processing characterize drug addicted individuals as compared with healthy controls. However, impaired behavioral performance or task disengagement may be crucial factors. We hypothesized that ACC hypoactivations would be documented in groups matched for performance on an emotionally salient task. Seventeen individuals with current cocaine use disorders (CUD) and 17 demographically matched healthy controls underwent functional magnetic resonance imaging during performance of a rewarded drug cue-reactivity task previously shown to engage the ACC. Despite lack of group differences in objective or subjective task-related performance, CUD showed more ACC hypoactivations throughout this emotionally salient task. Nevertheless, intensity of emotional salience contributed to results: (
i
) CUD with the largest rostroventral ACC [Brodmann Area (BA) 10, 11, implicated in default brain function] hypoactivations to the most salient task condition (drug words during the highest available monetary reward), had the least task-induced cocaine craving; (
ii
) CUD with the largest caudal-dorsal ACC (BA 32) hypoactivations especially to the least salient task condition (neutral words with no reward) had the most frequent current cocaine use; and (
iii
) responses to the most salient task condition in both these ACC major subdivisions were positively intercorrelated in the controls only. In conclusion, ACC hypoactivations in drug users cannot be attributed to task difficulty or disengagement. Nevertheless, emotional salience modulates ACC responses in proportion to drug use severity. Interventions to strengthen ACC reactivity or interconnectivity may be beneficial in enhancing top-down monitoring and emotion regulation as a strategy to reduce impulsive and compulsive behavior in addiction.
Altered dynein-dependent transport in piRNA pathway mutants
Authors: Navarro Caryn , Bullock Simon , Lehmann Ruth
Published: May 2009

Maintenance of genome integrity in germ cells is crucial for the success of future generations. In
Drosophila
, and mammals, transposable element activity in the germline can cause DNA breakage and sterility. Recent studies have shown that proteins involved in piRNA (PIWI-interacting RNA) biogenesis are necessary for retrotransposon silencing in the
Drosophila
germline. Females mutant for genes in the piRNA biogenesis pathway produce eggs with patterning defects that result from Chk-2 (checkpoint kinase-2) DNA damage checkpoint activation. Here we show that large ribonucleoprotein aggregates form in response to DNA damage checkpoint activation in egg chambers of females defective in piRNA biogenesis. Aggregate formation is specific to piRNA biogenesis mutants, as other mutations that activate the same Chk-2-dependent checkpoint do not cause aggregate formation. These aggregates contain components of the dynein motor machinery, retrotransposon RNA, and protein and axial patterning RNAs. Disruption of the aggregates by colcemid treatment leads to increased retrotransposon RNA levels, indicating that these structures may be the destination of retrotransposon RNA transport and may be degradation or sequestration sites. We propose that aggregate formation is a cellular response to protect germ cells from DNA damage caused by elevated retrotransposon expression.
Unique features of the nitrogenase VFe protein from
Azotobacter vinelandii
Authors: Lee Chi Chung , Hu Yilin , Ribbe Markus W.
Published: May 2009

Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N
2
) to ammonia (NH
3
). The vanadium (V)-nitrogenase is very similar to the “conventional” molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of
Azotobacter vinelandii
V-nitrogenase has been focused on 2 incomplete forms of this protein: αβ
2
and α
2
β
2
, both of which contain the small δ-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from
A. vinelandii
. This VFe protein has a previously-unsuspected, α
2
β
2
δ
4
-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H
2
evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.
The Group Migration of Dictyostelium Cells Is Regulated by Extracellular Chemoattractant Degradation
Authors: Garcia Gene L. , Rericha Erin C. , Heger Christopher D. , Goldsmith Paul K. , Parent Carole A.
Published: May 2009

Starvation of Dictyostelium induces a developmental program in which cells form an aggregate that eventually differentiates into a multicellular structure. The aggregate formation is mediated by directional migration of individual cells that quickly transition to group migration in which cells align in a head-to-tail manner to form streams. Cyclic AMP acts as a chemoattractant and its production, secretion, and degradation are highly regulated. A key protein is the extracellular phosphodiesterase PdsA. In this study we examine the role and localization of PdsA during chemotaxis and streaming. We find that pdsA− cells respond chemotactically to a narrower range of chemoattractant concentrations compared with wild-type (WT) cells. Moreover, unlike WT cells, pdsA− cells do not form streams at low cell densities and form unusual thick and transient streams at high cell densities. We find that the intracellular pool of PdsA is localized to the endoplasmic reticulum, which may provide a compartment for storage and secretion of PdsA. Because we find that cAMP synthesis is normal in cells lacking PdsA, we conclude that signal degradation regulates the external cAMP gradient field generation and that the group migration behavior of these cells is compromised even though their signaling machinery is intact.
Structural Basis of Ist1 Function and Ist1–Did2 Interaction in the Multivesicular Body Pathway and Cytokinesis
Authors: Xiao Junyu , Chen Xiao-Wei , Davies Brian A. , Saltiel Alan R. , Katzmann David J. , Xu Zhaohui
Published: May 2009

The ESCRT machinery functions in several important eukaryotic cellular processes. The AAA-ATPase Vps4 catalyzes disassembly of the ESCRT-III complex and may regulate membrane deformation and vesicle scission as well. Ist1 was proposed to be a regulator of Vps4, but its mechanism of action was unclear. The crystal structure of the N-terminal domain of Ist1 (Ist1NTD) reveals an ESCRT-III subunit-like fold, implicating Ist1 as a divergent ESCRT-III family member. Ist1NTD specifically binds to the ESCRT-III subunit Did2, and cocrystallization of Ist1NTD with a Did2 fragment shows that Ist1 interacts with the Did2 C-terminal MIM1 (MIT-interacting motif 1) via a novel MIM-binding structural motif. This arrangement indicates a mechanism for intermolecular ESCRT-III subunit association and may also suggest one form of ESCRT-III subunit autoinhibition via intramolecular interaction.
Bacteriolysis by Agaricus bisporus
Authors: Fermor T. R. , Wood D. A. , Lincoln S. P. , Fenlon J. S.
Published: June 2009

Summary: Agaricus bisporus, the cultivated mushroom, was able to mineralize dead 14C-labelled Bacillus subtilis and utilize the cellular components as sole source of carbon and nitrogen for growth. Consistently higher levels of bacteriolytic activity were obtained when A. bisporus was grown on lower concentrations of bacteria, 1·5 mg ml−1 as opposed to 3mg ml−1 basal liquid medium. A. bisporus also mineralized 14C-labelled bacteria in the presence of readily available alternative carbon and nitrogen sources such as glucose and ammonium sulphate. 14C-labelled bacteria were degraded to 14CO2 more efficiently than [14C]cellulose. A. bisporus was able efficiently to mineralize biomass produced in situ (>20% of total label released as 14CO2) where label was incorporated into the microbial biomass by composting rather than added as killed bacteria. This is the first time that quantitative data have been produced to show that the microbial biomass in wheat straw/animal manure compost might be significant in the nutritional strategy of basidiomycetes.
Characterization of X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp. lactis
Authors: Lloyd Richard J. , Pritchard Graham G.
Published: June 2009

A dipeptidyl aminopeptidase catalysing hydrolysis of X-prolyl amidomethylcoumarin (AMC) substrates has been purified from Lactococcus lactis subsp. lactis H1. The active enzyme has a molecular mass of approximately 150 kDa, a subunit molecular mass of 82 to 83 kDa and is inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. The K
m and k
cat values for five different dipeptidyl AMC substrates (Gly-Pro-; Leu-Pro-; Lys-Pro-; Phe-Pro- and Glu-Pro-AMC) are similar except for the K
m value for Glu-Pro-AMC, which is about threefold higher than that for the other substrates. The enzyme also catalyses hydrolysis of X-Ala-AMC substrates but with much lower k
cat and higher K
m values than the corresponding X-Pro-AMC substrates. The β-casein-derived heptapeptides Lys-Ala-Val-Pro-Tyr-Pro-Gln and Tyr-Pro-Phe-Pro-Gly-Pro-Ile were hydrolysed, but bradykinins with N-terminal sequences Arg-Pro-Pro- and Lys-Pro-Pro-were not. Dipeptidyl aminopeptidase specific activity is the same in a plasmid-free strain of L. lactis subsp. lactis H1 and in the wild-type, indicating that the enzyme is chromosomally encoded.
Acquisition of iron from host sources by mesophilic Aeromonas species
Authors: Massad George , Arceneaux Jean E. L. , Byers B. Rowe
Published: June 2009

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.
Regulation of protein phosphorylation in the cyanobacterium Anabaena strain PCC 7120
Authors: Mann Nicholas H. , Rippka Rosmarie , Herdman Michael
Published: June 2009

Protein kinase activities have been detected in cell-free extracts of the cyanobacterium Anabaena PCC 7120. At least 12 polypeptides in the soluble fraction were phosphorylated in vitro at the expense of [γ
-32P]ATP and the pattern of phosphorylation was shown to be regulated by intermediary metabolites and other effectors, at physiological concentrations. Glucose 6-phosphate exerted a regulatory effect on a phosphopolypeptide of M
r 56000 (p56) by stimulating a protein phosphatase, whereas ribulose 5-phosphate inhibited the corresponding protein kinase. In addition, DTT and the calmodulin antagonist trifluoperazine influenced the phosphorylation state of several different polypeptides, indicative of control by redox conditions and a calmodulin-like mediator, respectively. Furthermore, it was established that the phosphorylation of p56 required Mg2+ (> 100 μM) whereas that of a polypeptide of M
r 16000 occurred in the absence of Mg2+ and was inhibited by high concentrations (> 1 mm) of this cation. Several of the phosphopolypeptides detected in vitro corresponded in mobility on SDS-PAGE to species phosphorylated in vivo.
Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus β-galactosidase sequences
Authors: Schroeder Craig J. , Robert Catherine , Lenzen Gerlinde , McKay Larry L. , Mercenier Annick
Published: June 2009

ThelacZgene fromStreptococcus thermophilusA054, a commercial yogurt strain, was cloned on a 7·2 kbPstI fragment inEscherichia coliand compared with the previously clonedlacZgene fromS. thermophilusATCC 19258. Using the dideoxy chain termination method, the DNA sequences of bothlacZstructural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for thelacSgene from the initiation codon forlacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of theβ-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of theS. thermophilusamino acid sequences with those fromLactobacillus bulgaricus, E. coliandKlebsiella pneumoniaeshowed 48, 35 and 32·5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid squence were identified when theβ-galactosidase proteins fromS. thermophilus, E. coliandL. bulgaricuswere compared.
Characterization of the periplasmic cytochromes c of Paracoccus denitrificans: identification of the electron acceptor for methanol dehydrogenase, and description of a novel cytochrome c heterodimer
Authors: Long Anthony R. , Anthony Christopher
Published: June 2009

This paper describes periplasmic c-type cytochromes from two strains of Paracoccus denitrificans NCIB 8944 grown in heterotrophic or methylotrophic conditions. It is shown that the functions of two monomeric, monohaem cytochromes induced during growth on methanol have been wrongly designated in previous work. The CO-reactive cytochrome c
553 (30 kDa) is not the electron acceptor for methanol dehydrogenase; this is shown to be the role of the cytochrome c
552 (22 kDa). The monomeric 45 kDa cytochrome induced in conditions of oxygen insufficiency is a dihaem c-type cytochrome and does not contain haem b as previously assumed. In addition to these cytochromes, the Oxford strain of NCIB 8944 contains two cytochrome c complexes. One of these (150 kDa), produced in relatively small amounts, consists of a non-haem protein plus four haemoproteins (28, 33, 41 and 47 kDa). The second complex is a novel dimeric multi-haem cytochrome c (46 kDa) which constitutes about 25% of the periplasmic c-type cytochrome. It reacts with CO and has no methionine ligands. One subunit (16 kDa) has two low-spin haems; the larger subunit (30 kDa) has three haems which have low-spin characteristics in the oxidized state and are high-spin in the reduced state. The subunits were readily separated at pH 12 and could be subsequently reconstituted into a complex indistinguishable from the original. The 30 kDa subunit was denatured on prolonged exposure to high pH, which also converted it to a low-spin cytochrome. No function could be designated for these novel c-type cytochrome complexes.
Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli
Authors: Reverbel-Leroy Corinne , Belaich Anne , Bernadac Alain , Gaudin Christian , Belaich Jean-Pierre , Tardif Chantal
Published: June 2009

The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique. The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other ß-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in
Escherichia coli
using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 °. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.
Fatty acid adaptation in an Antarctic bacterium - changes in primer utilization
Authors: Nichols David S. , Russell Nicholas J.
Published: June 2009

The fatty acid composition and temperature/growth characteristics of a psychrophilic bacterium, strain ACAM 456, isolated from Antarctic sea-ice is reported. The bacterium produced acyl components that may be grouped in three different carbon chain types: even-chain, odd-chain and iso-branched odd-chain. The proportions of these chain types varied according to growth temperature, and were manipulated by growth on L-serine, t-leucine or propionic acid as sole carbon sources. De novo fatty acid synthesis was investigated using sodium [1-14C]acetate, L-[U-14C]leucine and L-[U-14C]serine as radioactive precursors. Compared with a control culture, resuspension of midexponential phase cells in artificial seawater led to a change in the selection and/or intracellular availability of acyl chain primer molecules. The proportion of radiolabel incorporated into even-chain length components from cells declined, whereas the percentage of radiolabel present in odd-chain length components increased. An increase in incubation temperature augmented this effect, and also elicited a rise in the proportion of label present in branchedchain products. ACAM 456 manipulated the utilization of acyl chain primer molecules as an adaptive response to changes in environmental conditions. In particular, the regulation of odd-chain length fatty acids is described as a novel adaptational response.
The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum
Authors: Mura G. M. , Pedroni P. , Pratesi C. , Galli G. , Serbolisca L. , Grandi G.
Published: June 2009

Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an a2ß2 tetramer, with the a and ß subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 μmol min-1 (mg protein)-1 a K
m for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T
50 of about 70 †C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in
Escherichia coli
at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.
Bacteriophage T4 Development Depends on the Physiology of its Host Escherichia Coli
Authors: Hadas Hilla , Einav Monica , Fishov Itzhak , Zaritsky Arieh
Published: June 2009

Several parameters of phage T4 adsorption to and growth inEscherichia coliB/r were determined. All changed monotonously with the bacterial growth rate (μ), which was modified by nutritional conditions. Adsorption rate was faster at higher μ values, positively correlated to cell size, and increased by pretreatment with low penicillin (Pn) concentrations; it was directly proportional to total cellular surface area, indicating a constant density of T4 receptors on cell envelopes irrespective of growth conditions. Parameters of phage development and cell lysis were μ-dependent. The rate of phage release and burst size increased, while the eclipse and latent periods decreased with increasing μ. Differentiation between the contribution of several physiological parameters to the development of T4 was performed by manipulating the host cells. A competitive inhibitor of glucose uptake, methyl α-D-glucoside, was exploited to reduce the growth rate in the same effective carbon source. Synchronous cells were obtained by the ‘baby-machine’ and large cells were obtained by pretreatment with low Pn concentrations. Lysis was delayed by superinfection, and DNA content and concentration were modified by growing athymutant in limiting thymine concentrations. The results indicate that burst size is not limited by cell size or DNA composition, nor directly by the rate of metabolism, but rather by the rates of synthesis and assembly of phage components and by lysis time. The rates of synthesis and assembly of phage components seem to depend on the content of the protein-synthesizing system and lysis time seems to depend on cellular dimensions.
High-osmolarity signalling in Saccharomyces cerevisiae is modulated in a carbon-source-dependent fashion
Authors: Siderius Marco , Rots Eveline , Mager Willem H.
Published: June 2009

High-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possibly via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.
The pncA gene from naturally pyrazinamide-resistant Mycobacterium avium encodes pyrazinamidase and confers pyrazinamide susceptibility to resistant M. tuberculosis complex organisms
Authors: Sun Zhonghe , Scorpio Angelo , Zhang Ying
Published: June 2009

The antituberculosis drug pyrazinamide (PZA) needs to be converted into pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) in order to show bactericidal activity against Mycobacterium tuberculosis. M. avium is naturally resistant to PZA. To investigate whether this natural resistance to PZA is due to inability of the M. avium PZase to convert PZA to bactericidal POA, the M. avium PZase gene (pncA) was cloned by using the M. tuberculosis pncA gene as a probe. Sequence analysis showed that the M. avium pncA gene is 561 bp long, encoding a protein with a predicted size of about 19·8 kDa; but Western blotting showed that the M. avium PZase migrated as a 24 kDa band when expressed in M. bovis BCG and Escherichia coli. Sequence comparison revealed that M. avium PZase has 67·7% and 32·8% amino acid identity with the corresponding enzymes from M. tuberculosis and E. coli, respectively. Southern blot analysis with the M. avium pncA gene as a probe showed that M. terrae, M. gastri, M. marinum, M. fortuitum, M. xenopi, M. gordonae, M. szulgai, M. celatum and M. kansasii have close pncA homologues, whereas M. chelonae and M. smegmatis did not give significant hybridization signals. Transformation with the M. avium pncA gene conferred PZA susceptibility to PZA-resistant M. tuberculosis complex organisms, indicating that the nonsusceptibility of M. avium to PZA is not due to an ineffective PZase enzyme, but appears to be related to other factors such as transport of POA.
Physical map of the Bacillus subtilis 166 genome: evidence for the inversion of an approximately 1900 kb continuous DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment
Authors: Itaya Mitsuhiro
Published: June 2009

An I-Ceul-Notl-Sfil endonuclease map of the Bacillus subtilis 166 genome was constructed. It was almost identical to that of B. subtilis 168 except for the inversion of an approximately 1900 kb DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment. Continuity of the inverted segment was investigated by direct measurement of the distances between the two genomic loci where I-Scel recognition sites were created in the 168 and the 166 genomes. Size difference of the I-Scel fragments between the two strains fully demonstrated the inversion of an approximately 1900 kb long continuous DNA segment and the location of the two inversion junctions in the genome. The 100 kb DNA segment including the lysogenic SP prophage was translocated close to one of the inversion junctions and was probably associated with the duplication of a 5 kb segment These rearrangements are consistent with those indicated by genetic analyses
Heterologous expression of heterotrophic nitrification genes
Authors: Crossman Lisa C. , Moir James W. B. , Enticknap Julie J. , Richardson David J. , Spiro Stephen
Published: June 2009

Paracoccus denitrificans is a heterotrophic organism capable of oxidizing ammonia to nitrite during growth on an organic carbon and energy source. This pathway, termed heterotrophic nitrification, requires the concerted action of an ammonia monooxygenase (AMO) and hydroxylamine oxidase (HAO). The genes required for heterotrophic nitrification have been isolated by introducing a Pa. denitrificans genomic library into Pseudomonas putida and screening for the accumulation of nitrite. In contrast to the situation in chemolithoautotrophic ammonia oxidizers, the genes encoding AMO and HAO are present in single linked copies in the genome of Pa. denitrificans. AMO from Pa. denitrificans expressed in Ps. putida is capable of oxidizing ethene (ethylene) to epoxyethane (ethylene oxide), which is indicative of a relaxed substrate specificity. Further, when expressed in the methylotroph Methylobacterium extorquens AM1, the AMO endows on this organism the ability to grow on ethene and methane. Thus, the Pa. denitrificans AMO is capable of oxidizing methane to methanol, as is the case for the AMO from Nitrosomonas europaea. The heterotrophic nitrification genes are moderately toxic in M. extorquens, more toxic in Ps. putida, and non-toxic in Escherichia coli. Toxicity is due to the activity of the gene products in M. extorquens, and both expression and activity in Ps. putida. This is the first time that the genes encoding an active AMO have been expressed in a heterologous host.
Exponential-phase expression of spvA of the Salmonella typhimurium virulence plasmid: induction in intracellular salts medium and intracellularly in mice and cultured mammalian cells
Authors: Wilson Julie A. , Doyle Thomas J. , Gulig Paul A.
Published: June 2009

Thespvgenes ofSalmonella typhimuriumand other non-typhoidalSalmonellaserovars are essential for efficient systemic infection beyond the intestines in orally inoculated mice as a model for enteric fever. These virulence genes are not significantly expressed by salmonellae during exponential growth inLbroth but are induced when the bacteria enter the stationary phase of growth. Using RNase protection analysis to directly measurespvAmRNA from the virulence plasmid ofS. typhimurium,we found thatspvAwas maximally induced in an SpvR- and RpoS-dependent manner during exponential growth in Intracellular Salts Medium, which mimics the intracellular environment of mammalian cells. A clonedspvA-lacZoperon fusion inS. typhimuriumwas induced intracellularly in peritoneal cells of mice, correlatingin vivointracellular gene expression with intracellular function of thespvgenes in infected mice.spvAwas also induced intracellularlyin vitrowithin both Henle-407 intestinal epithelial cells and J774.A1 macrophage-like cells when the bacteria were replicating with exponential kinetics. Prevention of invasion of salmonellae with cytochalasinDinhibitedspvAinduction within tissue culture cells, indicating that salmonellae must be internalized forspvAto be induced. ThespvA-lacZfusion was not induced by salmonellae in extracellular fluid of the peritoneal cavity or in serum. Since induction of the spv genes occurs intracellularly during exponential growth of salmonellae, cessation of growth may not be the most relevant inducing signal for spv gene expression.
Growth competition between Halobacterium salinarium strain PHH1 and mutants affected in gas vesicle synthesis
Authors: Beard Steven J. , Hayes Paul K. , Walsby Anthony E.
Published: June 2009

To investigate the role of the buoyancy provided by gas vesicles in the facultative anaerobe Halobacterium salinarium PHH1, the growth of a gas-vacuolate (Gv+) strain in competition with two gas-vesicle-defective (Gvdef) mutants was examined. The Gv+ strain synthesized gas vesicles throughout its growth cycle, and floated up to form a thick surface scum during the exponential growth phase in static culture. Mutant Gvdef1 produced significantly fewer gas vesicles than the Gv+ strain in corresponding stages of growth, although in late stationary phase a small proportion of cells floated up to the surface of static cultures. Mutant Gvdef2 had a much lower gas vesicle content in shaken culture and produced negligible amounts of gas vesicles in static culture. The Gv+ and the two Gvdef strains grew equally well in shaken cultures, but in static cultures, where steep vertical gradients of oxygen concentration were established, Gvdef1 was outgrown by the Gv+ strain. Gvdef2 outcompeted the Gv+ strain in shallow static cultures, perhaps because Gvdef2 carried a smaller protein burden, which offset the benefits of buoyancy. This selection for Gvdef2 was lost in deeper static cultures, although it could be restored by aerating static cultures from below. The results support the hypothesis that the role of buoyancy in halobacteria is to maintain cells at the more aerated surface of brine pools.
Polythionate degradation by tetrathionate hydrolase of Thiobacillus ferrooxidans
Authors: de Jong Govardus A. H. , Hazeu Wim , Bos Piet , Kuenen J. Gijs
Published: June 2009

Cell-free extracts of Thiobacillus ferrooxidans grown with thiosulfate as energy source and prepared at high ammonium sulfate concentrations and at low pH are capable of polythionate hydrolysis. The enzyme responsible for the hydrolysis of tetrathionate (S4O2-
6) and pentathionate (S4O2-
6) was purified to homogeneity. Enzyme activity during the purification procedure was based on a continuous spectrophotometric method that detects soluble intermediates that absorb in the UV region. The end products of hydrolysis of both polythionates by the pure enzyme were thiosulfate, sulfur and sulfate. The purified enzyme has a pH optimum of around 4 and a temperature optimum of 65 �. The activity is strongly influenced by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of molecular mass 52 kDa. During purification of tetrathionate hydrolase, fractions able to hydrolyse trithionate and tetrathionate were separated, indicating that the two substrates are hydrolysed by different enzymes.
Oligonucleoticle-tnediated genetic transformation of Borrelia burgdorferi
Authors: Samuels D. Scott , Garon Claude F.
Published: June 2009

We have used short oligonucleotides to genetically transform the Lyme disease spirochaete Borrelia burgdorferi. The oligonucleotides are derived from the sequence of an Arg-133 to He mutant gyrB (chromosomal) gene that confers resistance to the antibiotic coumermycin A1. Oligonucleotides were about 10000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their gyrB genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.
The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response
Authors: Nyvold Charlotte , Birkelund Svend , Christiansen Gunna
Published: June 2009

In the antigenically heterogeneous speciesMycoplasma hominisa monoclonal antibody, mAb 26.7D, was previously found to recognize a 120 kDa polypeptide fromM. hominis7488. This antibody did not react with the type strain PG21. The homologous gene fromM. hominisPG21 was cloned and sequenced and found to have a sequence identity of 91% with the gene of strain 7488. One hypervariable and two semivariable regions were detected. The epitope for mAb 26.7D was mapped to the hypervariable domain by expression of various parts of this domain inEscherichia coliusing expression vector systems. A polyclonal antiserum (pAb 121) generated against the hypervariable region of P120 from PG21 identified the P120 homologue inM. hominisPG21. Fusion proteins of the hypervariable and constant parts of the proteins were constructed and tested for reactivity with 21 human sera. Twelve sera reacted with the 7488 hypervariable fusion protein, but only four reacted with the PG21 hypervariable fusion protein. No reactivity was seen with a fusion protein containing part of the constant region of P120. Gene fragments amplified from 18M. hominisisolates by PCR confirmed the heterogeneity of the hypervariable domain. Based on restriction endonuclease cleavage patterns of the hypervariable domain the 18 isolates could be divided into four classes. Reactivity with both mAb 26.7D and pAb 121 confirmed these classes. The hypervariable, but not the constant, part of P120 was recognized by the human humoral immune response. Such a variable domain may be important in evasion of the host's immune response, and thus aid survival of the micro-organism.
A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1 as an immunological phosphate-starvation marker
Authors: Leopold Kristine , Jacobsen Susanne , Nybroe Ole
Published: June 2009

A phosphate-starvation-inducible outer-membrane protein of Pseudomonas fluorescens Ag1, expressed at phosphate concentrations below 0.08—0.13 mM, was purified and characterized. The purification method involved separation of outer-membrane proteins by SDS-PAGE and extraction of the protein from nitrocellulose or PVDF membranes after electrotransfer of proteins to the membranes. The N-terminal amino acid sequence of the purified protein, called Psi1, did not show homology to any known proteins, and in contrast to the phosphate-specific porin OprP of P. aeruginosa its mobility in SDS-PAGE was not affected by solubilization temperature. An antiserum against Psi1 recognized a protein of M
r 55000 in four other P. fluorescens strains among 24 tested strains representing Pseudomonas rRNA homology group I, showing antigenic heterogeneity within this group. A method for immunofluorescence microscopy involving cell permeabilization was adapted to visualize cell-specific expression of Psi1 in P. fluorescens exposed to limiting amounts of phosphate. This approach should be useful for further exploration of Psi1 as a marker to study the availability of phosphate to P. fluorescens in natural environments.
Gene disruption and replacement in the rapamycin-producing Streptomyces hygroscopicus strain ATCC 29253
Authors: Lomovskaya Natalie , Fonstein Leonid , Ruan Xiaoan , Stassi Diane , Katz Leonard , Hutchinson C. Richard
Published: June 2009

A system for gene disruption and replacement based on a streptomycete temperate phage vector was developed to introduce DNA in the rapamycin-producingStreptomyces hygroscopicusstrain ATCC 29253. This will be useful in attempts to produce, through genetic manipulation, novel forms of the therapeutically important immunosuppressive drug rapamycin. Recombinant phages were constructed from the ?31 phage derivative KC515 (c+attP) carrying a thiostrepton or viomycin resistance gene along with segments of theS. hygroscopicuschromosome. Each of the cloned segments also contained theaphllneomycin/kanamycin resistance gene to enable gene replacement by loss of the phage-derived DNA. Specific deletion of the entire polyketide synthase (PKS) believed to govern rapamycin biosynthesis resulted in the loss of rapamycin production. In contrast, disruption or deletion of a region predicted to encode four PKS open reading frames, or another region predicted to encode another PKS plus a cytochrome P450 hydroxylase and ferredoxin, had no effect on the production of rapamycin or nigericin, a polyether antibiotic also produced byS. hygroscopicus. Therefore,S. hygroscopicusmay have the capacity to produce polyketides additional to rapamycin and nigericin.