bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of Signature-Tagged Mutagenesis in
Burkholderia pseudomallei
To Identify Genes Important in Survival and Pathogenesis
Authors: Cuccui J. , Easton A. , Chu K. K. , Bancroft G. J. , Oyston P. C. F. , Titball R. W. , Wren B. W. 
Published: December 2006

ABSTRACT

Burkholderia pseudomallei
, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in
recA
involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine,
p
-aminobenzoic acid, and a mutant with an insertion in
aroB
causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the
aroB
mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.
Functional Analysis of Nine Putative Chemoreceptor Proteins inSinorhizobium meliloti
Authors: Meier Veronika M. , Muschler Paul , Scharf Birgit E. 
Published: December 2006

ABSTRACTThe genome of the symbiotic soil bacteriumSinorhizobium meliloticontains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression withlacZfusions, we have identifiedmcpUandmcpXas being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins inS. melilotican sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lackingmcpUwere swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state ofS. meliloti.
Weak Rolling Adhesion Enhances Bacterial Surface Colonization
Authors: Anderson Brett N. , Ding Albert M. , Nilsson Lina M. , Kusuma Kaoru , Tchesnokova Veronika , Vogel Viola , Sokurenko Evgeni V. , Thomas Wendy E. 
Published: December 2006

ABSTRACT

Bacterial adhesion to and subsequent colonization of surfaces are the first steps toward forming biofilms, which are a major concern for implanted medical devices and in many diseases. It has generally been assumed that strong irreversible adhesion is a necessary step for biofilm formation. However, some bacteria, such as
Escherichia coli
when binding to mannosylated surfaces via the adhesive protein FimH, adhere weakly in a mode that allows them to roll across the surface. Since single-point mutations or even increased shear stress can switch this FimH-mediated adhesion to a strong stationary mode, the FimH system offers a unique opportunity to investigate the role of the strength of adhesion independently from the many other factors that may affect surface colonization. Here we compare levels of surface colonization by
E. coli
strains that differ in the strength of adhesion as a result of flow conditions or point mutations in FimH. We show that the weak rolling mode of surface adhesion can allow a more rapid spreading during growth on a surface in the presence of fluid flow. Indeed, an attempt to inhibit the adhesion of strongly adherent bacteria by blocking mannose receptors with a soluble inhibitor actually increased the rate of surface colonization by allowing the bacteria to roll. This work suggests that (i) a physiological advantage to the weak adhesion demonstrated by commensal variants of FimH bacteria may be to allow rapid surface colonization and (ii) antiadhesive therapies intended to prevent biofilm formation can have the unintended effect of enhancing the rate of surface colonization.
Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in
Escherichia coli
Authors: Macomber Lee , Rensing Christopher , Imlay James A. 
Published: December 2006

ABSTRACT

Because copper catalyzes the conversion of H
2
O
2
to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes,
copA
,
cueO
, and
cusCFBA
, rendered
Escherichia coli
sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H
2
O
2
than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H
2
O
2
damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H
2
O
2
resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H
2
O
2
-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while
E. coli
responds to H
2
O
2
stress by inducing iron sequestration proteins, H
2
O
2
-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism.
Bacteroides fragilis
Synthesizes a DNA Invertase Affecting both a Local and a Distant Region
Authors: Roche-Hakansson Hazeline , Chatzidaki-Livanis Maria , Coyne Michael J. , Comstock Laurie E. 
Published: December 2006

ABSTRACT

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of
Bacteroides fragilis
was characterized. Its gene,
tsr19
, is adjacent to
mpi
, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of
B. fragilis
, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.
Characterization of Colony Morphology Variants Isolated fromStreptococcus pneumoniaeBiofilms
Authors: Allegrucci Magee , Sauer Karin 
Published: December 2006

ABSTRACTIn this study, we report the isolation of colony morphology variants fromStreptococcus pneumoniaeserotype 3 biofilms. The colony variants differed in colony size (large, medium, and small) and their mucoid appearance on blood agar. The small nonmucoid variant (SCV) emerged during the initial attachment stage ofS. pneumoniaebiofilm formation and dominated over the course of biofilm growth. Mucoid variants appeared at later biofilm developmental stages. The reduction in colony size/mucoidy correlated with a decrease in capsule production and an increase in initial attachment. The large mucoid variant formed flat unstructured biofilms, failed to aggregate in liquid culture, and adhered poorly to solid surfaces. In contrast, SCVs autoaggregated in liquid culture, hyperadhered to solid surfaces, and formed biofilms with significant three-dimensional structure, mainly in the form of microcolonies. The variants showed similar antibiotic resistance/susceptibility based on a modified Kirby-Bauer test and when grown as biofilms. However, antimicrobial treatment ofS. pneumoniaebiofilms altered the colony variant's distribution and mainly affected the most interior areas of biofilm microcolonies. To further explore the nature of the variants, the capsule biosynthetic operon (cps3DSUM) was explored in greater detail. The genetic analysis indicated that the emergence of nonmucoid variants was due to a deletion comprisingcps3DSUas well as additional genes upstream of thecps3operon. Overall, our findings suggest that in vitro biofilm formation ofS. pneumoniaeserotype 3 coincides with the emergence of colony variants with distinct genotypic and phenotypic characteristics.
Specific Binding of D1 Protein Degradation Products to the
psbAI
Promoter in
Synechococcus
sp. Strain PCC 7942
Authors: Stelljes Christian , Koenig Friederike 
Published: December 2006

ABSTRACT

The D1:1 protein and its potentially occurring degradation products were overexpressed in
Escherichia coli
. Protein-DNA interaction is shown for the promoter region of
psbAI
. The D1:1 degradation products may be involved in transcription regulation of
psbAI
by binding in the promoter region. Additionally, C-terminal fragments of the D1 protein bind to a sequence with similarity to
isiB
, a gene which encodes a flavodoxin-like protein.
An Actin Homolog of the Archaeon
Thermoplasma acidophilum
That Retains the Ancient Characteristics of Eukaryotic Actin
Authors: Hara Futoshi , Yamashiro Kan , Nemoto Naoki , Ohta Yoshinori , Yokobori Shin-ichi , Yasunaga Takuo , Hisanaga Shin-ichi , Yamagishi Akihiko 
Published: December 2006

ABSTRACT

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon
Thermoplasma acidophilum
, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol.
358:
145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg
2+
, critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.
On Torque and Tumbling in Swimming
Escherichia coli
Authors: Darnton Nicholas C. , Turner Linda , Rojevsky Svetlana , Berg Howard C. 
Published: December 2006

ABSTRACT

Bacteria swim by rotating long thin helical filaments, each driven at its base by a reversible rotary motor. When the motors of peritrichous cells turn counterclockwise (CCW), their filaments form bundles that drive the cells forward. We imaged fluorescently labeled cells of
Escherichia coli
with a high-speed charge-coupled-device camera (500 frames/s) and measured swimming speeds, rotation rates of cell bodies, and rotation rates of flagellar bundles. Using cells stuck to glass, we studied individual filaments, stopping their rotation by exposing the cells to high-intensity light. From these measurements we calculated approximate values for bundle torque and thrust and body torque and drag, and we estimated the filament stiffness. For both immobilized and swimming cells, the motor torque, as estimated using resistive force theory, was significantly lower than the motor torque reported previously. Also, a bundle of several flagella produced little more torque than a single flagellum produced. Motors driving individual filaments frequently changed directions of rotation. Usually, but not always, this led to a change in the handedness of the filament, which went through a sequence of polymorphic transformations, from normal to semicoiled to curly 1 and then, when the motor again spun CCW, back to normal. Motor reversals were necessary, although not always sufficient, to cause changes in filament chirality. Polymorphic transformations among helices having the same handedness occurred without changes in the sign of the applied torque.
Role of mRNA Stability in Growth Phase Regulation of Gene Expression in the Group A Streptococcus
Authors: Barnett Timothy C. , Bugrysheva Julia V. , Scott June R. 
Published: December 2006

ABSTRACTThe impressive disease spectrum ofStreptococcus pyogenes(the group A streptococcus [GAS]) is believed to be determined by its ability to modify gene expression in response to environmental stimuli. Virulence gene expression is controlled tightly by several different transcriptional regulators in this organism. In addition, expression of most, if not all, GAS genes is determined by a global mechanism dependent on growth phase. To begin an analysis of growth-phase regulation, we compared the transcriptome 2 h into stationary phase to that in late exponential phase of a serotype M3 GAS strain. We identified thearctranscript as more abundant in stationary phase in addition to thesagandsdatranscripts that had been previously identified. We found that in stationary phase, the stability ofsagA,sda, andarcTtranscripts increased dramatically. We found that polynucleotide phosphorylase (PNPase [encoded bypnpA]) is rate limiting for decay ofsagAandsdatranscripts in late exponential phase, since the stability of these mRNAs was greater in apnpAmutant, while stability of control mRNAs was unaffected by this mutation. Complementation restored the wild-type decay rate. Furthermore, in apnpAmutant, thesagAmRNA appeared to be full length, as determined by Northern hybridization. It seems likely that mRNAs abundant in stationary phase are insensitive to the normal decay enzyme(s) and instead require PNPase for this process. It is possible that PNPase activity is limited in stationary phase, allowing persistence of these important virulence factor transcripts at this phase of growth.
Repression of the Inner Membrane Lipoprotein NlpA by Rns in EnterotoxigenicEscherichia coli
Authors: Bodero Maria D. , Pilonieta M. Carolina , Munson George P. 
Published: December 2006

ABSTRACTThe expression of the inner membrane protein NlpA is repressed by the enterotoxigenicEscherichia coli(ETEC) virulence regulator Rns, a member of the AraC/XylS family. The Rns homologs CfaD from ETEC and AggR from enteroaggregativeE. colialso repress expression ofnlpA. In vitro DNase I and potassium permanganate footprinting revealed that Rns binds to a site overlapping the start codon ofnlpA, preventing RNA polymerase from forming an open complex atnlpAp. A second Rns binding site between positions −152 and −195 relative to thenlpAtranscription start site is not required for repression. NlpA is not essential for growth ofE. coliunder laboratory conditions, but it does contribute to the biogenesis of outer membrane vesicles. As outer membrane vesicles have been shown to contain ETEC heat-labile toxin, the repression ofnlpAmay be an indirect mechanism through which the virulence regulators Rns and CfaD limit the release of toxin.
Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression
Authors: Grote Jens , König Simone , Ackermann Doreen , Sopalla Claudia , Benedyk Malgorzata , Los Marek , Kerkhoff Claus 
Published: December 2006

Abstract

Background
S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887).

Results
In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ).

Conclusion
The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.
On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg
Authors: Castillo Betzaida , Bansal Vibha , Ganesan Ashok , Halling Peter , Secundo Francesco , Ferrer Amaris , Griebenow Kai , Barletta Gabriel 
Published: December 2006

Abstract

Background
Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results
As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-β-cyclodextrin (MβCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion
These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.
CESAR: conventional ventilatory support vs extracorporeal membrane oxygenation for severe adult respiratory failure
Authors: Peek Giles J , Clemens Felicity , Elbourne Diana , Firmin Richard , Hardy Pollyanna , Hibbert Clare , Killer Hilliary , Mugford Miranda , Thalanany Mariamma , Tiruvoipati Ravin , Truesdale Ann , Wilson Andrew 
Published: December 2006

Abstract
Background
An estimated 350 adults develop severe, but potentially reversible respiratory failure in the UK annually. Current management uses intermittent positive pressure ventilation, but barotrauma, volutrauma and oxygen toxicity can prevent lung recovery. An alternative treatment, extracorporeal membrane oxygenation, uses cardio-pulmonary bypass technology to temporarily provide gas exchange, allowing ventilator settings to be reduced. While extracorporeal membrane oxygenation is proven to result in improved outcome when compared to conventional ventilation in neonates with severe respiratory failure, there is currently no good evidence from randomised controlled trials to compare these managements for important clinical outcomes in adults, although evidence from case series is promising.

Methods/Design
The aim of the randomised controlled trial of Conventional ventilatory support vs extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR) is to assess whether, for patients with severe, but potentially reversible, respiratory failure, extracorporeal membrane oxygenation will increase the rate of survival without severe disability ('confined to bed' and 'unable to wash or dress') by six months post-randomisation, and be cost effective from the viewpoints of the NHS and society, compared to conventional ventilatory support. Following assent from a relative, adults (18–65 years) with severe, but potentially reversible, respiratory failure (Murray score ≥ 3.0 or hypercapnea with pH < 7.2) will be randomised for consideration of extracorporeal membrane oxygenation at Glenfield Hospital, Leicester or continuing conventional care in a centre providing a high standard of conventional treatment. The central randomisation service will minimise by type of conventional treatment centre, age, duration of high pressure ventilation, hypoxia/hypercapnea, diagnosis and number of organs failed, to ensure balance in key prognostic variables. Extracorporeal membrane oxygenation will not be available for patients meeting entry criteria outside the trial. 180 patients will be recruited to have 80% power to be able to detect a one third reduction in the primary outcome from 65% at 5% level of statistical significance (2-sided test). Secondary outcomes include patient morbidity and health status at 6 months.

Discussion
Analysis will be based on intention to treat. A concurrent economic evaluation will also be performed to compare the costs and outcomes of both treatments.