bluuebunny/crossref_metadata_2025_split huggingface dataset:
ISAfe1, an ISL3 Family Insertion Sequence fromAcidithiobacillus ferrooxidansATCC 19859
Authors: Holmes David S. , Zhao Hui-Ling , Levican Gloria , Ratouchniak Jeanine , Bonnefoy Violaine , Varela Patricia , Jedlicki Eugenia 
Published: July 2002

ABSTRACTA 1.3-kb insertion sequence, termed ISAfe1 (U66426), fromAcidithiobacillus ferrooxidansATCC 19859 is described. ISAfe1 exhibits the features of a typical bacterial insertion sequence. It has 26-bp, imperfectly matched, terminal inverted repeats and an open reading frame (ORF) that potentially encodes a transposase (TPase) of 404 amino acids (AAB07489) with significant similarity to members of the ISL3 family of insertion sequences. A potential ribosome-binding site and potential −10 and −35 promoter sites for the TPase ORF were identified, and a +1 transcriptional start site was detected experimentally. A potential outwardly directed −35 site was identified in the right inverted repeat of ISAfe1. A second ORF (ORF B), of unknown function, was found on the complementary strand with significant similarity to ORF 2 of ISAe1 fromRalstonia eutropha. Southern blot analyses demonstrated that ISAfe1-like elements can be found in multiple copies in a variety ofA. ferrooxidansstrains and that they exhibit transposition. A codon adaptation index (CAI) analysis of the TPase of ISAfe1 indicates that is has a CAI of 0.726 and can be considered well adapted to its host, suggesting that ISAfe1 might be an ancient resident ofA. ferrooxidans. Analysis of six of its target sites of insertion in the genome ofA. ferrooxidansATCC 19859 indicates a preference for 8-bp pseudopalindromic sequences, one of which resembles the termini of its inverted repeats. Evidence is presented here that is consistent with the possibility that ISAfe1 can promote both plasmid cointegrate formation and resolution inE. coli.
Novel Genes of the
sox
Gene Cluster, Mutagenesis of the Flavoprotein SoxF, and Evidence for a General Sulfur-Oxidizing System in
Paracoccus pantotrophus
GB17
Authors: Rother Dagmar , Henrich Hans-Jürgen , Quentmeier Armin , Bardischewsky Frank , Friedrich Cornelius G. 
Published: July 2002

ABSTRACT

The novel genes
soxFGH
were identified, completing the
sox
gene cluster of
Paracoccus pantotrophus
coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction.
soxF
coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome
c
sulfide dehydrogenase of
Allochromatium vinosum
. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol.
soxG
coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer.
soxH
coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476–4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome
c
reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in
soxF
and complementation analysis, it was evident that the
soxFGH
gene products were not required for lithotrophic growth with thiosulfate.
Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in
Saccharopolyspora spinosa
Authors: Madduri Krishnamurthy , Waldron Clive , Merlo Donald J. 
Published: July 2002

ABSTRACT

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in
Saccharopolyspora spinosa
are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.
Catalytic Function of an α/β Hydrolase Is Required for Energy Stress Activation of the ς
B
Transcription Factor in
Bacillus subtilis
Authors: Brody Margaret S. , Vijay Kamni , W. Price Chester 
Published: July 2002

ABSTRACT

The general stress response of
Bacillus subtilis
is controlled by the ς
B
transcription factor, which is activated in response to diverse energy and environmental stresses. These two classes of stress are transmitted by separate signaling pathways which converge on the direct regulators of ς
B
, the RsbV anti-anti-ς factor and the RsbW anti-ς factor. The energy signaling branch involves the RsbP phosphatase, which dephosphorylates RsbV in order to trigger the general stress response. The
rsbP
structural gene lies downstream from
rsbQ
in a two-gene operon. Here we identify the RsbQ protein as a required positive regulator inferred to act in concert with the RsbP phosphatase. RsbQ bound RsbP in the yeast two-hybrid system, and a large in-frame deletion in
rsbQ
had the same phenotype as a null allele of
rsbP
—an inability to activate ς
B
in response to energy stress. Genetic complementation studies indicated that this phenotype was not due to a polar effect of the
rsbQ
alteration on
rsbP
. The predicted
rsbQ
product is a hydrolase or acyltransferase of the α/β fold superfamily, members of which catalyze a wide variety of reactions. Notably, substitutions in the presumed catalytic triad of RsbQ also abolished the energy stress response but had no detectable effect on RsbQ structure, synthesis, or stability. We conclude that the catalytic activity of RsbQ is an essential constituent of the energy stress signaling pathway.
The Staphylococcal QacR Multidrug Regulator Binds a Correctly Spaced Operator as a Pair of Dimers
Authors: Grkovic Steve , Brown Melissa H. , Schumacher Maria A. , Brennan Richard G. , Skurray Ronald A. 
Published: July 2002

ABSTRACT

Expression of the
Staphylococcus aureus
plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of
qacA
expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of
qacA
expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.
Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in
Escherichia coli
via Regulation of the
csgD
Gene
Authors: Prigent-Combaret Claire , Brombacher Eva , Vidal Olivier , Ambert Arnaud , Lejeune Philippe , Landini Paolo , Dorel Corinne 
Published: July 2002

ABSTRACT

The
Escherichia coli
OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the
ompR
gene, such as the
ompR234
mutation, stimulate laboratory strains of
E. coli
to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the
csgD
promoter region and stimulating its transcription. The
csgD
gene encodes the transcription regulator CsgD, which in turn activates transcription of the
csgBA
operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the
ompR
gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by
E. coli
is inhibited by increasing osmolarity in the growth medium. The
ompR234
mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the
ompR234
mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via
rpoS
-dependent transcription of the transcription regulator
cpxR
;
cpxR
-mediated repression of
csgB
and
csgD
promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.
Introduction of a Carboxyl Group in the First Transmembrane Helix ofEscherichia coliF1FoATPase Subunitcand Cytoplasmic pH Regulation
Authors: Jones Phil C. 
Published: July 2002

ABSTRACTThe multicopy subunitcof the H+-transporting F1FoATP synthase ofEscherichia colifolds across the membrane as a hairpin of two hydrophobic α helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp61inE. coli, centered in the second transmembrane helix is essential for H+transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+pumping for cytoplasmic pH regulation. In this study, Ile28was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+pumping and passive H+transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.
Tn
5
Transposase with an Altered Specificity for Transposon Ends
Authors: Naumann Todd A. , Reznikoff William S. 
Published: July 2002

ABSTRACT

Tn
5
is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn
5
contains two different specific end sequences, termed outside end (OE) and inside end (IE). In
Escherichia coli,
IE is methylated by Dam methylase (IE
ME
). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE
ME
-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE
ME
defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.
Molecular Evolution of the Intimin Gene in O111 Clones of PathogenicEscherichia coli
Authors: Tarr Cheryl L. , Whittam Thomas S. 
Published: July 2002

ABSTRACTIntimin is an important virulence factor in two groups of enteric pathogens: enteropathogenicEscherichia coli(EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagicE. coli(EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries. Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE). At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages. We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing. The sequences from both O111:H8 and O111:H9 differed from the Int-β that is typical of other clones in the same evolutionary lineage. The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-α, Int-β, or Int-γ. The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-α. The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives. Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein. An N-terminal domain that presumably functions in the periplasm may also be under positive selection.
The Active Partition GeneincCof IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts
Authors: Siddique Azeem , Figurski David H. 
Published: July 2002

ABSTRACTPlasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria. Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells. These determinants areincC, which codes for a member of the ParA family of partition ATPases;korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids. To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion ofincCin the otherwise intact 60-kb IncPα plasmid R995. R995ΔincCwas found to be highly unstable inEscherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, andAcinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts. In addition, R995ΔincCcould not be established inActinobacillus actinomycetemcomitans.trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within theincCgene, was sufficient to restore stable maintenance to wild-type levels. The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria.
The Pyrimidine Nucleotide Reductase Step in Riboflavin and F
420
Biosynthesis in
Archaea
Proceeds by the Eukaryotic Route to Riboflavin
Authors: Graupner Marion , Xu Huimin , White Robert H. 
Published: July 2002

ABSTRACT

The
Methanococcus jannaschii
gene MJ0671 was cloned and overexpressed in
Escherichia coli
, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3
H
)-pyrimidinone 5′-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3
H
)-pyrimidinone 5′-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1
H
,3
H
)-pyrimidinedione 5′-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1
H
,3
H
)-pyrimidinedione 5′-phosphate (compound 5). Only compound 3 was found to serve as a substrate for the enzyme. NADPH and NADH functioned equally well as the reductants. This specificity for the reduction of compound 3 was also confirmed by using cell extracts of
M. jannaschii
and
Methanosarcina thermophila
. Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts. The absence of the other genes in the biosynthesis of riboflavin in
Archaea
is discussed.
Effects of Pyrazinamide on Fatty Acid Synthesis by Whole Mycobacterial Cells and Purified Fatty Acid Synthase I
Authors: Boshoff Helena I. , Mizrahi Valerie , Barry Clifton E. 
Published: July 2002

ABSTRACT

The effects of low extracellular pH and intracellular accumulation of weak organic acids were compared with respect to fatty acid synthesis by whole cells of
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
. The profile of fatty acids synthesized during exposure to benzoic, nicotinic, or pyrazinoic acids, as well as that observed during intracellular hydrolysis of the corresponding amides, was not a direct consequence of modulation of fatty acid synthesis by these compounds but reflected the response to inorganic acid stress. Analysis of fatty acid synthesis in crude mycobacterial cell extracts demonstrated that pyrazinoic acid failed to directly modulate the fatty acid synthase activity catalyzed by fatty acid synthase I (FAS-I). However, fatty acid synthesis was irreversibly inhibited by 5-chloro-pyrazinamide in a time-dependent fashion. Moreover, we demonstrate that pyrazinoic acid does not inhibit purified mycobacterial FAS-I, suggesting that this enzyme is not the immediate target of pyrazinamide.
Expression, Autoregulation, and DNA Binding Properties of the
Mycobacterium tuberculosis
TrcR Response Regulator
Authors: Haydel Shelley E. , Benjamin William H. , Dunlap Nancy E. , Clark-Curtiss Josephine E. 
Published: July 2002

ABSTRACT

The TrcRS two-component system of
Mycobacterium tuberculosis
is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the
trcR
and
trcS
two-component system genes are transcribed in broth-grown
M. tuberculosis
. Analysis of the
trcR
and
trcS
genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of
M. tuberculosis
growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a
trcR-lacZ
fusion plasmid and a TrcR expression plasmid were cotransformed into
Escherichia coli
. Upon induction of the TrcR protein, there was a >500-fold increase in β-galactosidase activity from the
trcR-lacZ
fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the
trcR
promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping
trcR
PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the
trcR
promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the
trcR
promoter was mutated and analyzed in
lacZ
transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the
trcR
promoter resulted in the loss of
trcR
transcriptional activation in the presence of TrcR. This report indicates that the
M. tuberculosis
TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the
trcR
promoter.
Identification of [2Fe-2S] Clusters in Microbial Ferrochelatases
Authors: Dailey Tamara A. , Dailey Harry A. 
Published: July 2002

ABSTRACT

The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria
Caulobacter crescentus
and
Mycobacterium tuberculosis
possess [2Fe-2S] clusters. The enzyme from
C. crescentus
is a homodimeric, membrane-associated protein while the enzyme from
M. tuberculosis
is monomeric and soluble. The clusters of the
C. crescentus
and
M. tuberculosis
ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast
Schizosaccharomyces pombe
. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.
Comparison of Fluorescent In Situ Hybridization and Conventional Culturing for Detection of
Helicobacter pylori
in Gastric Biopsy Specimens
Authors: Rüssmann Holger , Kempf Volkhard A. J. , Koletzko Sibylle , Heesemann Jürgen , Autenrieth Ingo B. 
Published: July 2002

ABSTRACT

In this study, we have investigated 201 gastric biopsy specimens obtained from dyspeptic patients for the presence of
Helicobacter pylori
. By means of fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescence-labeled oligonucleotide probes specific for
H. pylori
, this pathogen was detected in 63 biopsy specimens. By using conventional culturing,
H. pylori
was isolated from 49 of these 63 gastric biopsy specimens. In contrast, FISH failed to identify
H. pylori
in four samples from which the pathogen was cultured. The lowest sensitivity was obtained by using the urease test.
H. pylori
was detected indirectly by this method in 43 of 67 biopsy specimens, which were positive for the pathogen as determined by FISH and/or culturing. All 49
H. pylori
isolates that were detected by FISH and culturing underwent antimicrobial susceptibility testing for clarithromycin, a macrolide drug that is a key component in the therapy of peptic ulcer disease caused by this pathogen. Clarithromycin susceptibility testing of cultured isolates was carried out by the E-test, whereas FISH was used on biopsy specimens to detect clarithromycin-resistant mutant strains. No discrepancies were found between these two methods. Thirty-seven strains were clarithromycin sensitive, and eight
H. pylori
isolates were resistant to the macrolide. From another four biopsy specimens, a mixture of clarithromycin-sensitive and -resistant strains was identified by both methods. Thus, FISH is a reliable technique for determining the clarithromycin susceptibility of this pathogen. Taken together, FISH is a more sensitive and rapid technique than culturing for detection of
H. pylori
in gastric biopsy specimens. However, in the microbiology routine diagnostic laboratory, the combination of both FISH and conventional culturing significantly increases the sensitivity in detection of
H. pylori
.