bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chill Induction of the SigB-Dependent General Stress Response inBacillus subtilisand Its Contribution to Low-Temperature Adaptation
Authors: Brigulla Matthias , Hoffmann Tamara , Krisp Andrea , Völker Andrea , Bremer Erhard , Völker Uwe 
Published: July 2003

ABSTRACTA variety of environmental and metabolic cues trigger the transient activation of the alternative transcription factor SigB ofBacillus subtilis, which subsequently leads to the induction of more than 150 general stress genes. This general stress regulon provides nongrowing and nonsporulated cells with a multiple, nonspecific, and preemptive stress resistance. By a proteome approach we have detected the expression of the SigB regulon during continuous growth at low temperature (15°C). Using a combination of Western blot analysis and SigB-dependent reporter gene fusions, we provide evidence for high-level and persistent induction of thesigBoperon and the SigB regulon, respectively, in cells continuously exposed to low temperatures. In contrast to all SigB-activating stimuli described thus far, induction by low temperatures does not depend on the positive regulatory protein RsbV or its regulatory phosphatases RsbU and RsbP, indicating the presence of an entirely new pathway for the activation of SigB by chill stress inB. subtilis. The physiological importance of the induction of the general stress response for the adaptation ofB. subtilisto low temperatures is emphasized by the observation that growth of asigBmutant is drastically impaired at 15°C. Inclusion of the compatible solute glycine betaine in the growth medium not only improved the growth of the wild-type strain but rescued the growth defect of thesigBmutant, indicating that the induction of the general stress regulon and the accumulation of glycine betaine are independent means by whichB. subtiliscells cope with chill stress.
Membrane Binding by MinD Involves Insertion of Hydrophobic Residues within the C-Terminal Amphipathic Helix into the Bilayer
Authors: Zhou Huaijin , Lutkenhaus Joe 
Published: July 2003

ABSTRACT
MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase. Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix. This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer. To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue. Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC. In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC. Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer. We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer.
Molecular Characterization and Quantitative Analysis of Superoxide Dismutases in Virulent and Avirulent Strains of
Aeromonas salmonicida
subsp.
salmonicida
Authors: Dacanay A. , Johnson S. C. , Bjornsdottir R. , Ebanks R. O. , Ross N. W. , Reith M. , Singh R. K. , Hiu J. , Brown L. L. 
Published: July 2003

ABSTRACT

Aeromonas salmonicida
subsp.
salmonicida
is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which
A
.
salmonicida
evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs),
sodA
and
sodB
. The
sodA
gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The
sodB
gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of O
2
−
-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo- and in vitro-cultured bacteria. The regulatory sequences upstream of putative
sodA
were consistent with these differences. We could not identify other SOD isozymes such as
sodC
either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains of
A
.
salmonicida
subsp.
salmonicida
strain A449 when cultured in vitro and in vivo.
Gene Expression Analysis of Energy Metabolism Mutants of
Desulfovibrio vulgaris
Hildenborough Indicates an Important Role for Alcohol Dehydrogenase
Authors: Haveman Shelley A. , Brunelle Véronique , Voordouw Johanna K. , Voordouw Gerrit , Heidelberg John F. , Rabus Ralf 
Published: July 2003

ABSTRACT

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of
Desulfovibrio vulgaris
Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the
hyd
mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified
D. vulgaris
genes encoding proteins active in energy metabolism indicated that the
adh
gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the
adh
gene was strongly downregulated in the
hyd
mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An
adh
mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The
hyd
mutant also grew poorly on ethanol, in agreement with its low level of
adh
gene expression. The
adh
mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of
adh
in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of
D. vulgaris
under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H
2
-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.
Molecular Characterization and Expression in
Escherichia coli
of Three β-1,3-Glucanase Genes from
Lysobacter enzymogenes
Strain N4-7
Authors: Palumbo Jeffrey D. , Sullivan Raymond F. , Kobayashi Donald Y. 
Published: July 2003

ABSTRACT

Lysobacter enzymogenes
strain N4-7 produces multiple biochemically distinct extracellular β-1,3-glucanase activities. The
gluA
,
gluB
, and
gluC
genes, encoding enzymes with β-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of β-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of
gluA
and
gluC
gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the
gluC
product. Analysis of the
gluB
gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in
Escherichia coli
resulted in the production of proteins with β-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45°C and that GluB is most active between pH 4.5 and 5.0 at 41°C. Activity of recombinant proteins against various β-1,3 glucan substrates indicates that GluA and GluC are most active against linear β-1,3 glucans, while GluB is most active against the insoluble β-1,3 glucan substrate zymosan A. These data suggest that the contribution of β-1,3-glucanases to the biocontrol activity of
L. enzymogenes
may be due to complementary activities of these enzymes in the hydrolysis of β-1,3 glucans from fungal cell walls.
TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388)
Authors: Schröder Gunnar , Lanka Erich 
Published: July 2003

ABSTRACTTraG-like proteins are essential components of type IV secretion systems. During secretion, TraG is thought to translocate defined substrates through the inner cell membrane. The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity. TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA. TrwB (R388) is a hexameric TraG-like protein that binds ATP. Both proteins, however, lack NTPase activity under in vitro conditions. We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraGΔ2 and TrwBΔ1) and/or containing a point mutation at the putative nucleotide-binding site (TraGΔ2K187T and TraGK187T). Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates. Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase. TraGΔ2 and TrwBΔ1 bound ATP and, with similar affinity, ADP. Binding of ATP and ADP was strongly inhibited by the presence of Mg2+or single-stranded DNA and was competed for by other nucleotides. Compared to the activity of TraGΔ2, the ATP- and ADP-binding activity of the point mutation derivative TraGΔ2K187T was significantly reduced. Each TraG derivative bound DNA with an affinity similar to that of the native protein. DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg2+. Thus, both nucleotide binding and DNA binding were sensitive to Mg2+and were competitive with respect to each other.
YjdE (AdiC) Is the Arginine:Agmatine Antiporter Essential for Arginine-Dependent Acid Resistance in
Escherichia coli
Authors: Gong Shimei , Richard Hope , Foster John W. 
Published: July 2003

ABSTRACT

To survive in extremely acidic conditions,
Escherichia coli
has evolved three adaptive acid resistance strategies thought to maintain internal pH. While the mechanism behind acid resistance system 1 remains enigmatic, systems 2 and 3 are known to require external glutamate (system 2) and arginine (system 3) to function. These latter systems employ specific amino acid decarboxylases and putative antiporters that exchange the extracellular amino acid substrate for the intracellular by-product of decarboxylation. Although GadC is the predicted antiporter for system 2, the antiporter specific for arginine/agmatine exchange has not been identified. A computer-based homology search revealed that the
yjdE
(now called
adiC
) gene product shared an overall amino acid identity of 22% with GadC. A series of
adiC
mutants isolated by random mutagenesis and by targeted deletion were shown to be defective in arginine-dependent acid resistance. This defect was restored upon introduction of an

adiC
+

-containing plasmid. An
adiC
mutant proved incapable of exchanging extracellular arginine for intracellular agmatine but maintained wild-type levels of arginine decarboxylase protein and activity. Western blot analysis indicated AdiC is an integral membrane protein. These data indicate that the arginine-to-agmatine conversion defect of
adiC
mutants was at the level of transport. The
adi
gene region was shown to be organized into two transcriptional units,
adiAY
and
adiC
, which are coordinately regulated but independently transcribed. The data also illustrate that the AdiA decarboxylase:AdiC antiporter system is designed to function only at acid levels sufficient to harm the cell.
Characterization of
Staphylococcus aureus
SarA Binding Sites
Authors: Sterba Kristen M. , Mackintosh Samuel G. , Blevins Jon S. , Hurlburt Barry K. , Smeltzer Mark S. 
Published: July 2003

ABSTRACT

The staphylococcal accessory regulator locus (
sarA
) encodes a DNA-binding protein (SarA) that modulates expression of over 100 genes. Whether this occurs via a direct interaction between SarA and
cis
elements associated with its target genes is unclear, partly because the definitive characteristics of a SarA binding site have not been identified. In this work, electrophoretic mobility shift assays (EMSAs) were used to identify a SarA binding site(s) upstream of the SarA-regulated gene
cna
. The results suggest the existence of multiple high-affinity binding sites within the
cna
promoter region. Using a SELEX (systematic evolution of ligands by exponential enrichment) procedure and purified, recombinant SarA, we also selected DNA targets that contain a high-affinity SarA binding site from a random pool of DNA fragments. These fragments were subsequently cloned and sequenced. Randomly chosen clones were also examined by EMSA. These DNA fragments bound SarA with affinities comparable to those of recognized SarA-regulated genes, including
cna
,
fnbA
, and
sspA
. The composition of SarA-selected DNAs was AT rich, which is consistent with the nucleotide composition of the
Staphylococcus aureus
genome. Alignment of selected DNAs revealed a 7-bp consensus (ATTTTAT) that was present with no more than one mismatch in 46 of 56 sequenced clones. By using the same criteria, consensus binding sites were also identified upstream of the
S. aureus
genes
spa
,
fnbA
,
sspA
,
agr
,
hla
, and
cna
. With the exception of
cna
, which has not been previously examined, this 7-bp motif was within the putative SarA binding site previously associated with each gene.
The Core Dimerization Domains of Histidine Kinases Contain Recognition Specificity for the Cognate Response Regulator
Authors: Ohta Noriko , Newton Austin 
Published: July 2003

ABSTRACT

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the
Caulobacter crescentus
cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.
Genetic Variation at thevlsELocus ofBorrelia burgdorferiwithin Ticks and Mice over the Course of a Single Transmission Cycle
Authors: Ohnishi Jun , Schneider Brad , Messer William B. , Piesman Joseph , de Silva Aravinda M. 
Published: July 2003

ABSTRACTThe Lyme disease spirochete,Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete. One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens. ThevlsElocus represents the best-studied example of antigenic variation inB. burgdorferi. During vertebrate host infection, recombination between the activevlsElocus and silent, partialvlsEcopies leads to gene conversion events and the generation of novel alleles at the expression site. In the present study, we followed a population ofB. burgdorferiorganisms moving through vertebrate host and tick stages to complete one transmission cycle. The major goal of the study was to determine if thevlsElocus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle. We report here that thevlsEgenetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages. Therefore, naturally infected ticks are likely to transmit spirochete populations with multiplevlsEalleles into naive vertebrate hosts. AlthoughvlsEgenetic diversity in mice was maintained through tick stages, the dominantvlsEalleles were different between tick stages as well as between individual ticks. We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominantvlsEalleles. AlthoughvlsEgenetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector.
Purification and Biochemical Characterization of the F
1
F
o
-ATP Synthase from Thermoalkaliphilic
Bacillus
sp. Strain TA2.A1
Authors: Cook Gregory M. , Keis Stefanie , Morgan Hugh W. , von Ballmoos Christoph , Matthey Ulrich , Kaim Georg , Dimroth Peter 
Published: July 2003

ABSTRACT

We describe here purification and biochemical characterization of the F
1
F
o
-ATP synthase from the thermoalkaliphilic organism
Bacillus
sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F
1
F
o
-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F
1
subunits α, β, γ, δ, and ε and F
o
subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F
1
F
o
holoenzyme or the isolated F
1
moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F
1
. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide
m
-chlorophenylhydrazone, while the electroneutral Na
+
/H
+
antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na
+
ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic
Bacillus
species. The ATP synthase was specifically modified at its c subunits by
N
,
N
′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.
Novel Psychrophilic and Thermolabile
l
-Threonine Dehydrogenase from Psychrophilic
Cytophaga
sp. Strain KUC-1
Authors: Kazuoka Takayuki , Takigawa Shouhei , Arakawa Noriaki , Hizukuri Yoshiyuki , Muraoka Ikuo , Oikawa Tadao , Soda Kenji 
Published: July 2003

ABSTRACT

A psychrophilic bacterium,
Cytophaga
sp. strain KUC-1, that abundantly produces a NAD
+
-dependent
l
-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the
Cytophaga
enzyme is distinct from other
l
-threonine dehydrogenases that have thus far been studied.
l
-Threonine and
dl
-
threo
-3-hydroxynorvaline were the substrates, and NAD
+
and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The

K
m

for
l
-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD
+
binds to an enzyme prior to
l
-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into
Escherichia coli
, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from
Staphylococcus aureus
and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast,
l
-threonine dehydrogenase from
E. coli
belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the
Cytophaga
enzyme.
l
-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the
E. coli
and human UDP-glucose 4-epimerases are highly conserved in the
Cytophaga
enzyme, except for the residues participating in the substrate binding.