bluuebunny/crossref_metadata_2025 huggingface dataset:
Seasonal sex–specific energy expenditure in breeding and non‐breeding Palestine sunbirds Nectarinia osea
Authors: Hambly Catherine , Markman Shai , Roxburgh Lizanne , Pinshow Berry
Published: February 2007

The timing of the chick‐rearing phase is known to have a profound effect on the reproductive success of birds. However, little is known about the energetic costs faced by the parents during different periods of the breeding season. These costs may have vital consequences for both their survival and future reproduction. In most studies, daily energy expenditure (DEE) of breeding and non‐breeding birds has been compared, without controlling for the effect of season. In the present study, we examined the energy demands of breeding compared to non‐breeding Palestine sunbirds Nectarinia osea and whether there were sex‐specific differences in DEE within and between different seasons. We predicted that DEE would be elevated when birds rear chicks, especially at cooler ambient temperatures. Time‐energy budgets were constructed for pairs of sunbirds, rearing chicks, or not breeding, in spring and summer. There were significant seasonal differences in estimates of DEE in non‐breeders that were 21% higher in spring than in summer. We attributed these to increases in non‐flight metabolic rate rather than changes in time spent on different activities. Our estimates of DEE for the birds that were rearing chicks were higher than non‐breeding adults. In females the increase in DEE when breeding, compared to when not breeding, was similar in both spring and summer, while males increased their DEE much less when breeding in spring. The differences in estimated DEE, however, were not significant between male and female birds in any season. Between seasons, female breeders had 17.1% higher DEE in spring than in summer, while male breeders showed no difference in DEE when rearing chicks in different seasons. Accordingly, our initial prediction was supported, as DEE in chick‐rearing adults was higher than in non‐breeding adults. In addition, although temperatures are lower in spring, breeding in the spring is only more costly than breeding in summer for females. Apparently, males are more flexible in reallocating their time and energy spent on different activities.
Mechanics and Dynamics of the Circular Milling Process
Authors: Kardes N. , Altintas Y.
Published: February 2007

Circular milling operations are used to enlarge die and mold cavities, cylinder bores and machine airframe pockets. The tool attached to the rotating spindle follows a circular trajectory with a machining feed rate, while gradually moving forward in the direction of global axis with a step over feed. This paper presents the mathematical model of chip removal mechanism by predicting time varying cutter-part intersection as the cutter travels along the circular path. The vibration free cutting forces are predicted and experimentally verified. The dynamics of the process are modeled by considering vibrations of the end mill in two directions. The chatter stability of circular milling is modeled both in frequency domain and with a numerical model based on time finite element analysis which considers time varying immersion and directional factors. The resulting stability solutions are compared against experimental results.
Dynamic Modeling and Monitoring of Contour Crafting—An Extrusion-Based Layered Manufacturing Process
Authors: Bukkapatnam Satish , Clark Ben
Published: February 2007

Layered manufacturing (LM) processes have emerged as legitimate processes for manufacturing various precision microelectronic components and bio-implants. These processes are also being considered for fabricating large customized free forms like buildings, statues, reactor beds, and car bodies. Many of these applications demand high levels of quality (e.g., Ra<0.1μm) and functional performance. Among the LM processes, extrusion-based processes can potentially offer high production rates together with lower setup and operating costs. Yet process failures resulting from anomalies, such as nozzle clogging, overflow, dynamic instabilities, bambooing, and machine degradation impede a widespread applicability of these processes. Scientific principles that relate the sources of these anomalies to process dynamics seem necessary for effective quality monitoring. In this paper we present a nonlinear lumped-mass model to capture dynamics underlying contour crafting, which is an extrusion-based LM process. The two degrees-of-freedom model, developed based on experimental characterizations, captures salient features of the process dynamics including the prominent manifestations of process nonlinearity. Experimental investigations show that the model can lead to effective monitoring of process conditions including overflow and underflow of material from extrusion nozzle, as well as suboptimal (fast and slow) feed rates of the extrusion head.
Metallic Forging Using Electrical Flow as an Alternative to Warm/Hot Working
Authors: Perkins Timothy A. , Kronenberger Thomas J. , Roth John T.
Published: February 2007

Manufacturing processes (e.g., forging, rolling, extrusion, and forming) rely on heat to reduce the forces associated with fabricating parts. However, due to the negative implications associated with hot working, another more efficient means of applying energy is desired. This paper investigates material property changes of various metals (aluminum, copper, iron, and titanium based alloys) in response to the flow of electricity. Theory involving electromigration and electroplasticity is examined and the implications thereof are analyzed. It is shown that, using electrical current, flow stresses are reduced, resulting in a lower specific energy for open-die forging. It is also shown that an applied electrical current increases the forgeability of materials, allowing greater deformation prior to cracking. Moreover, the changes caused by the flow of electricity are significantly greater than those explained by resistive heating. Additionally, elastic recovery is decreased when using electrical flow during deformation. Finally, for most materials, these effects were dependent on strain rate. Overall, this work demonstrates that substantial increases in the forgeability of metals are achieved by deforming the material while applying an electrical current. These improvements exceed those achieved through comparable increases in workpiece temperature and demonstrate that this method provides a viable alternative to warm/hot working.
Comparison of Analytical Model to Experimental and Numerical Simulations Results for Tailor Welded Blank Forming
Authors: Bravar Matt , Krishnan Neil , Kinsey Brad
Published: February 2007

Tailor welded blanks (TWBs) offer several notable benefits including decreased part weight, reduced manufacturing costs, and improved dimensional consistency. However the reduced formability and other characteristics of the forming process associated with TWBs has hindered the industrial utilization of this blank type for all possible applications. One concern with TWB forming is that weld line movement occurs, which alters the final location of the various materials in the TWB combination. In this technical brief, an analytical model to predict the initial weld line placement necessary to satisfy the desired, final weld line location and strain at the weld line is used. Results from this model are compared to an experimental, symmetric steel TWB case and a 3D numerical simulation, nonsymmetric aluminum TWB case. This analytical model is an extension of one previously presented, but eliminates a plane strain assumption that is unrealistic for most sheet metal forming applications. Good agreement between the analytical model, experimental, and numerical simulation results with respect to initial weld line location was obtained for both cases. Results for the model with a plane strain assumption are also provided, demonstrating the importance of eliminating this assumption.
Feynman’s wobbling plate
Authors: Tuleja Slavomir , Gazovic Boris , Tomori Alexander , Hanc Jozef
Published: February 2007

In the book Surely You Are Joking, Mr. Feynman! Richard Feynman tells a story of a Cornell cafeteria plate being tossed into the air. As the plate spun, it wobbled. Feynman noticed a relation between the two motions. He solved the motion of the plate by using the Lagrangian approach. This solution didn’t satisfy him. He wanted to understand the motion of the plate by analyzing the motion of its individual particles and the forces acting on them. He was successful, but he didn’t tell us how he did it. We provide an elementary explanation for the two-to-one ratio of wobble to spin frequencies, based on an analysis of the motion of the particles and the forces acting on them. We also demonstrate the power of numerical simulation and computer animation to provide insight into a physical phenomenon and guidance on how to do the analysis.
Fluctuations near a phase transition in liquid crystals
Authors: Moses Thomas , Reeves Jason , Pirondi Paulo
Published: February 2007

The increase in molecular orientational fluctuations as a phase transition is approached is an interesting and dramatic phenomenon, but associated undergraduate experiments are rare. We present an advanced undergraduate experiment on light scattering from the molecular orientational fluctuations in liquid crystals. In the high-temperature, disordered phase of a liquid crystal, small clusters of parallel-oriented molecules form spontaneously and scatter light. The size of the fluctuations and the consequent light scattering increase dramatically as the temperature is reduced approaching the weakly first-order phase transition to the ordered liquid crystal phase. We discuss a simplified theory that captures the essential physics.
Isolation of Potentially Pathogenic
Escherichia coli
O157:H7 from the Ganges River
Authors: Hamner Steve , Broadaway Susan C. , Mishra Veer Bhadra , Tripathi Anshuman , Mishra Rajesh Kumar , Pulcini Elinor , Pyle Barry H. , Ford Timothy E.
Published: February 2007

Escherichia coli
serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for
stx
1
,
stx
2
, and
eae
gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.
Nickel-Resistant Determinant fromLeptospirillum ferriphilum
Authors: Tian Jian , Wu Ningfeng , Li Jiang , Liu Yajie , Guo Jun , Yao Bin , Fan Yunliu
Published: February 2007

Leptospirillum ferriphilumstrain UBK03 isolated from a mine in Jiangxi, China, is resistant to Ni2+(30 to 40 mM). A four-gene nickel resistance cluster was identified and, when transformed intoEscherichia coli, enabled growth in 6 mM nickel. Mutation experiments revealed that the genesncrA,ncrB, andncrCcould confer nickel resistance inEscherichia coli, whereas the genencrYcould have a negative effect on nickel resistance.
Yeast Populations Residing on Healthy orBotrytis-Infected Grapes from a Vineyard in Attica, Greece
Authors: Nisiotou Aspasia A. , Nychas George-John E.
Published: February 2007

The yeast flora associated with healthy andBotrytis-infected grapes was assessed. Molecular identification methods assigned isolates to six genera and nine species. For the first timeHanseniaspora opuntiaewas encountered as an inhabitant of the grape ecosystem. By using DraI, an informative restriction fragment length polymorphism pattern was generated to distinguishH. opuntiaefrom the closely related organismHanseniaspora guilliermondii. Botrytisinfection resulted in a larger population and greater diversity of yeasts enriched with fermentative or spoilage species.
Degradation of
N
-Acyl-
l
-Homoserine Lactones by
Bacillus cereus
in Culture Media and Pork Extract
Authors: Medina-Martínez Maria Stella , Uyttendaele Mieke , Rajkovic Andreja , Nadal Pol , Debevere Johan
Published: February 2007

Degradation of the quorum-sensing signal molecule
N
-acyl-
l
-homoserine lactone (AHL) in cocultures was verified with
Bacillus cereus
and
Yersinia enterocolitica
in culture medium and in pork extract. Results showed evidence of microbial interaction when the AHL-degrading bacterium and AHL-producing bacterium were cocultured in a food-simulating condition.
Effects of Bacterial Prey Species and Their Concentration on Growth of the Amoebae
Acanthamoeba castellanii
and
Hartmannella vermiformis
Authors: Pickup Zoë L. , Pickup Roger , Parry Jacqueline D.
Published: February 2007

Two amoebae were presented with six bacterial prey at a range of concentrations, and the growth parameters of the amoebae were deduced. All but one bacterium (
Synechococcus
) resulted in a positive growth response, but the gram-positive bacterium
Staphylococcus aureus
proved to be difficult to digest and the heavily pigmented bacterium
Klebsiella ozaenae
induced unusual amoebic behavior prior to ingestion.
Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor
Authors: Ahn Johwan , Schroeder Sarah , Beer Michael , McIlroy Simon , Bayly Ronald C. , May John W. , Vasiliadis George , Seviour Robert J.
Published: February 2007

All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter
−1
to less than 0.1 mg P liter
−1
(when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-β-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that “
Candidatus
Accumulibacter phosphatis” bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial
Dechloromonas
and alphaproteobacterial tetrad-forming organisms related to
Defluviicoccus
in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.
Genotypic and Physiological Characterization of
Saccharomyces boulardii
, the Probiotic Strain of
Saccharomyces cerevisiae
Authors: Edwards-Ingram Laura , Gitsham Paul , Burton Nicola , Warhurst Geoff , Clarke Ian , Hoyle David , Oliver Stephen G. , Stateva Lubomira
Published: February 2007

Saccharomyces boulardii
, a yeast that was isolated from fruit in Indochina, has been used as a remedy for diarrhea since 1950 and is now a commercially available treatment throughout Europe, Africa, and South America. Though initially classified as a separate species of
Saccharomyces
, recent publications have shown that the genome of
S. boulardii
is so similar to
Saccharomyces cerevisiae
that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in
S. boulardii
that make it perform more effectively as a probiotic organism compared to other strains of
S. cerevisiae
. This investigation reports some of these distinguishing characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the
S. boulardii
genome such as trisomy of chromosome IX, altered copy number of a number of individual genes, and sporulation deficiency have been revealed by comparative genome hybridization using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of
S. boulardii
to its probiotic nature are discussed.
New Mathematical Modeling Approach for Predicting Microbial Inactivation by High Hydrostatic Pressure
Authors: Klotz Bernadette , Pyle D. Leo , Mackey Bernard M.
Published: February 2007

A new primary model based on a thermodynamically consistent first-order kinetic approach was constructed to describe non-log-linear inactivation kinetics of pressure-treated bacteria. The model assumes a first-order process in which the specific inactivation rate changes inversely with the square root of time. The model gave reasonable fits to experimental data over six to seven orders of magnitude. It was also tested on 138 published data sets and provided good fits in about 70% of cases in which the shape of the curve followed the typical convex upward form. In the remainder of published examples, curves contained additional shoulder regions or extended tail regions. Curves with shoulders could be accommodated by including an additional time delay parameter and curves with tails shoulders could be accommodated by omitting points in the tail beyond the point at which survival levels remained more or less constant. The model parameters varied regularly with pressure, which may reflect a genuine mechanistic basis for the model. This property also allowed the calculation of (a) parameters analogous to the decimal reduction time
D
and
z
, the temperature increase needed to change the
D
value by a factor of 10, in thermal processing, and hence the processing conditions needed to attain a desired level of inactivation; and (b) the apparent thermodynamic volumes of activation associated with the lethal events. The hypothesis that inactivation rates changed as a function of the square root of time would be consistent with a diffusion-limited process.
Helicobacter pylori
EstV: Identification, Cloning, and Characterization of the First Lipase Isolated from an Epsilon-Proteobacterium
Authors: Ruiz Cristian , Falcocchio Serena , Pastor F. I. Javier , Saso Luciano , Diaz Pilar
Published: February 2007

Bacterial lipases are attracting an enormous amount of attention due to their wide biotechnological applications and due to their roles as virulence factors in some bacteria.
Helicobacter pylori
is a significant and widespread pathogen which produces a lipase(s) and phospholipases that seem to play a role in mucus degradation and the release of proinflammatory and cytotoxic compounds. However, no
H. pylori
lipase(s) has been isolated and described previously. Therefore, a search for putative lipase-encoding genes was performed by comparing the amino acid sequences of 53 known lipolytic enzymes with the deduced proteome of
H. pylori
. As a result, we isolated, cloned, purified, and characterized EstV, a novel lipolytic enzyme encoded by open reading frame HP0739 of
H. pylori
26695, and classified it in family V of the bacterial lipases. This enzyme has the properties of a small, cell-bound carboxylesterase (EC 3.1.1.1) that is active mostly with short-chain substrates and does not exhibit interfacial activation. EstV is stable and does not require additional cofactors, and the maximum activity occurs at 50°C and pH 10. This unique enzyme is the first lipase isolated from
H. pylori
that has been described, and it might contribute to ulcer development, as inhibition by two antiulcer substances (β-aescin and glycyrrhizic acid) suggests. EstV is also the first lipase from an epsilon-proteobacterium to be described. Furthermore, this enzyme is a new member of family V, probably the least-known family of bacterial lipases, and the first lipase of this family for which kinetic behavior, inhibition by natural substances, and other key biochemical features are reported.
Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Different Lifestyles
Authors: Dicksved Johan , Flöistrup Helen , Bergström Anna , Rosenquist Magnus , Pershagen Göran , Scheynius Annika , Roos Stefan , Alm Johan S. , Engstrand Lars , Braun-Fahrländer Charlotte , von Mutius Erika , Jansson Janet K.
Published: February 2007

In this population-based study, 90 children from three European countries were examined to determine the impact of lifestyle on the fecal microbiota. The study was designed to assess the impact of two extreme lifestyles that we hypothesized could impact the microbial composition in the gut: i.e., an anthroposophic lifestyle (restricted use of antibiotics, greater consumption of fermented vegetables, etc.) versus living on a farm (greater consumption of farm milk, contact with animals, etc.). In previous studies, these lifestyles correlated with lower prevalence of allergies. Terminal restriction fragment length polymorphism (T-RFLP) was used to assess the bacterial composition in fecal samples since recent studies have shown that the majority of this community cannot be cultivated. The T-RFLP data were used to calculate richness and evenness of the fecal microbiota. Children that were attending Steiner schools (anthroposophic children) had a significantly higher diversity of microbes in their feces than farm children, who in turn also had lower diversity than the control groups. Specific primers were also used to focus on the
Lactobacillus
-like community (lactic acid bacteria [LAB]). Large differences were found in the LAB subpopulations in the sampled groups. In some children, the LAB subpopulation was dominated by a species that has not yet been cultivated.
Widespread Occurrence and Genomic Context of Unusually Small Polyketide Synthase Genes in Microbial Consortia Associated with Marine Sponges
Authors: Fieseler Lars , Hentschel Ute , Grozdanov Lubomir , Schirmer Andreas , Wen Gaiping , Platzer Matthias , Hrvatin Siniša , Butzke Daniel , Zimmermann Katrin , Piel Jörn
Published: February 2007

Numerous
marine sponges harbor enormous amounts of as-yet-uncultivated bacteria
in their tissues. There is increasing evidence that these symbionts
play an important role in the synthesis of protective metabolites, many
of which are of great pharmacological interest. In this study, genes
for the biosynthesis of polyketides, one of the most important classes
of bioactive natural products, were systematically investigated in 20
demosponge species from different oceans. Unexpectedly, the sponge
metagenomes were dominated by a ubiquitously present, evolutionarily
distinct, and highly sponge-specific group of polyketide synthases
(PKSs). Open reading frames resembling animal fatty acid genes were
found on three corresponding DNA regions isolated from the metagenomes
of
Theonella swinhoei
and
Aplysina aerophoba
. Their
architecture suggests that methyl-branched fatty acids are the
metabolic product. According to a phylogenetic analysis of housekeeping
genes, at least one of the PKSs belongs to a bacterium of the
Deinococcus-Thermus
phylum. The results provide new insights
into the chemistry of sponge symbionts and allow inference of a
detailed phylogeny of the diverse functional PKS types present in
sponge metagenomes. Based on these qualitative and quantitative data,
we propose a significantly simplified strategy for the targeted
isolation of biomedically relevant PKS genes from complex
sponge-symbiont
associations.
Expression Profiles of Bovine Genes in the Rectoanal Junction Mucosa during Colonization with
Escherichia coli
O157:H7
Authors: Li Jie , Hovde Carolyn J.
Published: February 2007

A bovine-specific cDNA microarray was used to characterize gene expression in the bovine rectoanal junction mucosa in response to
Escherichia coli
O157:H7 colonization, and results were confirmed using quantitative real-time PCR. The results showed involvement of cell processes including immune response, cell structure/dynamics, signal transduction, intercellular communication, and metabolism.
A Derepression System Based on the
Bacillus subtilis
Sporulation Pathway Offers Dynamic Control of Heterologous Gene Expression
Authors: Nijland Reindert , Veening Jan-Willem , Kuipers Oscar P.
Published: February 2007

By rewiring the sporulation gene-regulatory network of
Bacillus subtilis
, we generated a novel expression system relying on derepression. The gene of interest is placed under the control of the
abrB
promoter, which is active only when Spo0A is absent, and Spo0A is controlled via an IPTG (isopropyl-β-
d
-thiogalactopyranoside)-inducible promoter.
Detection and Identification by PCR of a Highly Virulent Phylogenetic Subgroup among Extraintestinal Pathogenic
Escherichia coli
B2 Strains
Authors: Bidet Philippe , Metais Arnaud , Mahjoub-Messai Farah , Durand Lionel , Dehem Marie , Aujard Yannick , Bingen Edouard , Nassif Xavier , Bonacorsi Stéphane
Published: February 2007

Closely related
Escherichia coli
B2 strains O1:K1, O2:K1, O18:K1, and O45:K1 constitute a major subgroup causing extraintestinal infections. A DNA pathoarray analysis was used to develop a PCR specific for this subgroup that was included in the multiplex phylogenetic-grouping PCR method. Our PCR may serve to identify this virulent subgroup among different ecological niches.
Clonal Population Structure and Specific Genotypes of Multidrug-Resistant
Campylobacter coli
from Turkeys
Authors: D'lima C. B. , Miller W. G. , Mandrell R. E. , Wright S. L. , Siletzky R. M. , Carver D. K. , Kathariou S.
Published: February 2007

Commercial turkey flocks in North Carolina have been found to be colonized frequently with
Campylobacter coli
strains that are resistant to several antimicrobials (tetracycline, streptomycin, erythromycin, kanamycin, and ciprofloxacin/nalidixic acid). Such strains have been designated multidrug resistant (MDR). However, the population structure of MDR
C. coli
from turkeys remains poorly characterized. In this study, an analysis of multilocus sequence typing (MLST)-based sequence types (STs) of 59 MDR strains from turkeys revealed that the majority of these strains corresponded to one of 14 different STs, with three STs accounting for 41 (69%) of the strains. The major STs were turkey specific, and most (87%) of the strains with these STs were resistant to the entire panel of antibiotics mentioned above. Some (13%) of the strains with these STs were susceptible to just one or two of the antibiotics in this panel. Further subtyping using
fla
typing and pulsed-field gel electrophoresis with SmaI and KpnI revealed that the major MDR STs corresponded to strains of related but distinct subtypes, providing evidence for genomic diversification within these STs. These findings suggest that MDR strains of
C. coli
from turkeys have a clonal population structure characterized by the presence of a relatively small number of clonal groups that appear to be disseminated in the turkey production system. In addition, the observed correlation between STs and the MDR profiles of the microbes indicates that MLST-based typing holds potential for source-tracking applications specific to the animal source (turkeys) and the antimicrobial resistance profile (MDR status) of
C. coli
.
Enhancement of Biodesulfurization in Two-Liquid Systems by Heterogeneous Expression of
Vitreoscilla
Hemoglobin
Authors: Xiong Xiaochao , Xing Jianmin , Li Xin , Bai Xuejing , Li Wangliang , Li Yuguang , Liu Huizhou
Published: February 2007

The
vgb
gene, encoding
Vitreoscilla
hemoglobin (VHb), was introduced into a specific desulfurization bacterium,
Rhodococcus erythropolis
LSSE8-1. The VHb-specific spectrum was observed for the recombinant. Compared to the wild type, the strain bearing
vgb
showed a higher biomass yield and desulfurizing activity.
Weissellicin 110, a Newly Discovered Bacteriocin from
Weissella cibaria
110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand
Authors: Srionnual Sirinat , Yanagida Fujitoshi , Lin Li-Hsiu , Hsiao Kuang-Nan , Chen Yi-sheng
Published: February 2007

Weissella cibaria
110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from
W. cibaria
110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.
The SAR92 Clade: an Abundant Coastal Clade of Culturable Marine Bacteria Possessing Proteorhodopsin
Authors: Stingl Ulrich , Desiderio Russell A. , Cho Jang-Cheon , Vergin Kevin L. , Giovannoni Stephen J.
Published: February 2007

Proteorhodopsin (PR) is a protein that is abundant in marine bacterioplankton. PR is hypothesized to be a light-dependent proton pump, thus creating a proton gradient that can be used for energy production without electron transport. Currently, the only culture that has been reported to possesses PR is the highly abundant alphaproteobacterium “
Candidatus
Pelagibacter ubique” (SAR11 clade), but surprisingly, its growth in batch culture was not enhanced by light. Here, we present the first cultured gammaproteobacterium that possesses a PR gene. Genome sequencing and analysis of HTCC2207 showed that the PR gene is present as a lone transcriptional unit directly followed by an operon containing genes that are presumably involved in the synthesis of retinal, the chromophore of PR. Half-time decay times of different PR intermediates in native HTCC2207 cells ranged between 2 and 15 ms, and the absorbance maximum of PR was determined to be 528 nm. Proteorhodopsin was identified in three additional strains, using a specific PCR assay on other cultured members of the SAR92 clade. Phylogenetic analyses of the PR genes determined that they form a deeply rooting cluster not closely related to any PR genes recovered so far. Fluorescence in situ hybridization and RNA blots showed that the SAR92 clade reaches up to 10% of the total bacterial population in surface waters close to the Oregon coast and decreases over depth and distance from the shore. Although the growth of HTCC2207 is limited by the amount of available carbon that is present in the medium applied, these cultures do not grow at higher rates nor do they have higher growth yields when incubated under light.
Simultaneous Detection of
Pseudomonas fragi
,
P. lundensis
, and
P. putida
from Meat by Use of a Multiplex PCR Assay Targeting the
carA
Gene
Authors: Ercolini Danilo , Russo Federica , Blaiotta Giuseppe , Pepe Olimpia , Mauriello Gianluigi , Villani Francesco
Published: February 2007

Species-specific primers and a multiplex PCR assay were developed for the simultaneous identification and differentiation of
Pseudomonas fragi
,
P. lundensis
, and
P. putida
based on the coamplification of different portions of the small subunit of the carbamoyl phosphate synthase gene (
carA
). The
carA
multiplex PCR was used to detect the presence of the three
Pseudomonas
species from beef, chicken, and pork samples and proved to be effective in showing their evolution during the storage of meat.
Assessing Adhesion Forces of Type I and Type IV Pili of
Xylella fastidiosa
Bacteria by Use of a Microfluidic Flow Chamber
Authors: De La Fuente Leonardo , Montanes Emilie , Meng Yizhi , Li Yaxin , Burr Thomas J. , Hoch H. C. , Wu Mingming
Published: February 2007

Xylella fastidiosa
, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type
X. fastidiosa
cells was 147 ± 11 pN. Mutant cells possessing only type I pili required a force of 204 ± 22 pN for removal, whereas cells possessing only type IV pili required 119 ± 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.
An NAD(P)H-Nicotine Blue Oxidoreductase Is Part of the Nicotine Regulon and May Protect
Arthrobacter nicotinovorans
from Oxidative Stress during Nicotine Catabolism
Authors: Mihasan Marius , Chiribau Calin-Bogdan , Friedrich Thorsten , Artenie Vlad , Brandsch Roderich
Published: February 2007

An NAD(P)H-nicotine blue (quinone) oxidoreductase was discovered as a member of the nicotine catabolic pathway of
Arthrobacter nicotinovorans
. Transcriptional analysis and electromobility shift assays showed that the enzyme gene was expressed in a nicotine-dependent manner under the control of the transcriptional activator PmfR and thus was part of the nicotine regulon of
A. nicotinovorans
. The flavin mononucleotide-containing enzyme uses NADH and, with lower efficiency, NADPH to reduce, by a two-electron transfer, nicotine blue to the nicotine blue leuco form (hydroquinone). Besides nicotine blue, several other quinones were reduced by the enzyme. The NAD(P)H-nicotine blue oxidoreductase may prevent intracellular one-electron reductions of nicotine blue which may lead to semiquinone radicals and potentially toxic reactive oxygen species.
Characterization of the Sporulation Control Protein SsgA by Use of an Efficient Method To Create and Screen Random Mutant Libraries in Streptomycetes
Authors: Traag Bjørn A. , Seghezzi Nicolas , Vijgenboom Erik , van Wezel Gilles P.
Published: February 2007

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of
Streptomyces coelicolor ssgA
. The variants were amplified directly from deep-frozen biomass suspensions. Around 800
ssgA
variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an
ssgA
mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.
Induction of Glutathione
S
-Transferase in Biofilms and Germinating Spores of
Mucor hiemalis
Strain EH5 from Cold Sulfidic Spring Waters
Authors: Hoque Enamul , Pflugmacher Stephan , Fritscher Johannes , Wolf Manfred
Published: February 2007

The occurrence and activation of glutathione
S
-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of
Mucor hiemalis
isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (∼27.8- and ∼25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.
In the Yeast Heat Shock Response, Hsf1-Directed Induction of Hsp90 Facilitates the Activation of the Slt2 (Mpk1) Mitogen-Activated Protein Kinase Required for Cell Integrity
Authors: Truman Andrew W. , Millson Stefan H. , Nuttall James M. , Mollapour Mehdi , Prodromou Chrisostomos , Piper Peter W.
Published: February 2007

Yeast is rendered temperature sensitive with loss of the C-terminal (CT) domain of heat shock transcription factor (Hsf1). This domain loss was found to abrogate heat stimulation of Slt2 (Mpk1), the mitogen-activated protein kinase that directs the reinforced cell integrity gene expression needed for high-temperature growth. In Hsf1 CT domain-deficient cells, Slt2 still undergoes Mkk1/2-directed dual-Thr/Tyr phosphorylation in response to the heat stimulation of cell integrity pathway signaling, but the low Hsp90 expression level suppresses any corresponding increase in Slt2 kinase activity due to Slt2 being a “client” of the Hsp90 chaperone. A non-Hsf1-directed Hsp90 overexpression restored the heat induction of Slt2 activity in these cells, as well as both Slt2-dependent (Rlm1, Swi4) and Slt2-independent (MBF) transcriptional activities. Their high-temperature growth was also rescued, not just by this Hsp90 overexpression but by osmotic stabilization, by the expression of a Slt2-independent form of the Rlm1 transcriptional regulator of cell integrity genes, and by a multicopy
SLT2
gene vector. In providing the elevated Hsp90 needed for an efficient activation of Slt2, heat activation of Hsf1 indirectly facilitates (Slt2-directed) heat activation of yet another transcription factor (Rlm1). This provides an explanation as to why, in earlier transcript analysis compared to chromatin immunoprecipitation studies, many more genes of yeast displayed an Hsf1-dependent transcriptional activation by heat than bound Hsf1 directly. The levels of Hsp90 expression affecting transcription factor regulation by Hsp90 client protein kinases also provides a mechanistic model for how heat shock factor can influence the expression of several non-
hsp
genes in higher organisms.
Molecular and Biochemical Characterization of a Novel Intracellular Invertase fromAspergillus nigerwith Transfructosylating Activity
Authors: Goosen Coenie , Yuan Xiao-Lian , van Munster Jolanda M. , Ram Arthur F. J. , van der Maarel Marc J. E. C. , Dijkhuizen Lubbert
Published: February 2007

A novel subfamily of putative intracellular invertase enzymes (glycoside hydrolase family 32) has previously been identified in fungal genomes. Here, we report phylogenetic, molecular, and biochemical characteristics of SucB, one of two novel intracellular invertases identified inAspergillus niger. ThesucBgene was expressed inEscherichia coliand an invertase-negative strain ofSaccharomyces cerevisiae. Enzyme purified fromE. colilysate displayed a molecular mass of 75 kDa, judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Its optimum pH and temperature for sucrose hydrolysis were determined to be 5.0 and 37 to 40°C, respectively. In addition to sucrose, the enzyme hydrolyzed 1-kestose, nystose, and raffinose but not inulin and levan. SucB produced 1-kestose and nystose from sucrose and 1-kestose, respectively. With nystose as a substrate, products up to a degree of polymerization of 4 were observed. SucB displayed typical Michaelis-Menten kinetics with substrate inhibition on sucrose (apparentKm,Ki, andVmaxof 2.0 ± 0.2 mM, 268.1 ± 18.1 mM, and 6.6 ± 0.2 μmol min−1mg−1of protein [total activity], respectively). At sucrose concentrations up to 400 mM, transfructosylation (FTF) activity contributed approximately 20 to 30% to total activity. At higher sucrose concentrations, FTF activity increased to up to 50% of total activity. Disruption ofsucBinA. nigerresulted in an earlier onset of sporulation on solid medium containing various carbon sources, whereas no alteration of growth in liquid culture medium was observed. SucB thus does not play an essential role in inulin or sucrose catabolism inA. nigerbut may be needed for the intracellular conversion of sucrose to fructose, glucose, and small oligosaccharides.
Retinal Biosynthesis in Fungi: Characterization of the Carotenoid Oxygenase CarX fromFusarium fujikuroi
Authors: Prado-Cabrero Alfonso , Scherzinger Daniel , Avalos Javier , Al-Babili Salim
Published: February 2007

Thecargene cluster of the ascomyceteFusarium fujikuroiencodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid inF. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion ofcarXdid not impede neurosporaxanthin biosynthesis. On the contrary, ΔcarXmutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in β-carotene-accumulatingEscherichia colicells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its β-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as γ-carotene, torulene, and β-apo-8′-carotenal. Our data indicate that the occurrence of at least one β-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the β-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.
Role of DNA Repair by Nonhomologous-End Joining in
Bacillus subtilis
Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation
Authors: Moeller Ralf , Stackebrandt Erko , Reitz Günther , Berger Thomas , Rettberg Petra , Doherty Aidan J. , Horneck Gerda , Nicholson Wayne L.
Published: February 2007

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (
recA
,
splB
,
ykoU
,
ykoV
, and
ykoU ykoV
mutants) of
Bacillus subtilis
. NHEJ-defective spores with mutations in
ykoU
,
ykoV
, and
ykoU ykoV
were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.
Catalytic Properties of
Staphylococcus aureus
and
Bacillus
Members of the Secondary Cation/Proton Antiporter-3 (Mrp) Family Are Revealed by an Optimized Assay in an
Escherichia coli
Host
Authors: Swartz Talia H. , Ito Masahiro , Ohira Takayuki , Natsui Shinsuke , Hicks David B. , Krulwich Terry A.
Published: February 2007

Monovalent cation proton antiporter-3 (Mrp) family antiporters are widely distributed and physiologically important in prokaryotes. Unlike other antiporters, they require six or seven hydrophobic gene products for full activity. Standard fluorescence-based assays of Mrp antiport in membrane vesicles from
Escherichia coli
transformants have not yielded strong enough signals for characterization of antiport kinetics. Here, an optimized assay protocol for vesicles of antiporter-deficient
E. coli
EP432 transformants produced higher levels of secondary Na
+
(Li
+
)/H
+
antiport than previously reported. Assays were conducted on Mrps from alkaliphilic
Bacillus pseudofirmus
OF4 and
Bacillus subtilis
and the homologous antiporter of
Staphylococcus aureus
(Mnh), all of which exhibited Na
+
(Li
+
)/H
+
antiport. A second paralogue of
S. aureus
(Mnh2) did not. K
+
, Ca
2+
, and Mg
2+
did not support significant antiport by any of the test antiporters. All three Na
+
(Li
+
)/H
+
Mrp antiporters had alkaline pH optima and apparent

K
m

values for Na
+
that are among the lowest reported for bacterial Na
+
/H
+
antiporters. Using a fluorescent probe of the transmembrane electrical potential (ΔΨ), Mrp Na
+
/H
+
antiport was shown to be ΔΨ consuming, from which it is inferred to be electrogenic. These assays also showed that membranes from
E. coli
EP432 expressing Mrp antiporters generated higher ΔΨ levels than control membranes, as did membranes from
E. coli
EP432 expressing plasmid-borne NhaA, the well-characterized electrogenic
E. coli
antiporter. Assays of respiratory chain components in membranes from Mrp and control
E. coli
transformants led to a hypothesis explaining how activity of secondary, ΔΨ-consuming antiporters can elicit increased capacity for ΔΨ generation in a bacterial host.
Isolation of the
rstA
Gene as a Multicopy Suppressor of YjeE, an Essential ATPase of Unknown Function in
Escherichia coli
Authors: Campbell Tracey L. , Ederer Claudia S. , Allali-Hassani Abdellah , Brown Eric D.
Published: February 2007

YjeE is an essential ATPase in
Escherichia coli
whose cellular function remains uncharacterized. Using a genomic library, we have identified
rstA
as a multicopy suppressor of a conditional
yjeE
deletion strain. High-copy
rstA
is the first recorded suppressor for a lesion in
yjeE
, and this newly charted genetic interaction has the potential to be informative about the function, with further study of the interacting partners.