bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number in
Pseudomonas aeruginosa
Authors: Dasgupta Nandini , Ramphal Reuben 
Published: July 2002

ABSTRACT

Flagellar number in
Pseudomonas aeruginosa
is controlled by FleN, a putative ATP/GTP binding protein. Disruption of
fleN
results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN
24K→Q
and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.
The Default State of the Membrane-Localized Histidine Kinase PrrB of
Rhodobacter sphaeroides
2.4.1 Is in the Kinase-Positive Mode
Authors: Oh Jeong-Il , Ko In-Jeong , Kaplan Samuel 
Published: July 2002

ABSTRACT

The PrrBA two-component activation system of
Rhodobacter sphaeroides
plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the
cbb
3
terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237–4247, 2000). Therefore, the intrinsic (“default”) state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent of
prrB
gene expression is subject to the negative autoregulation of the PrrBA system.
Correlation between
Bacillus subtilis scoC
Phenotype and Gene Expression Determined Using Microarrays for Transcriptome Analysis
Authors: Caldwell Robert , Sapolsky Ron , Weyler Walter , Maile Randal R. , Causey Stuart C. , Ferrari Eugenio 
Published: July 2002

ABSTRACT

The availability of the complete sequence of the
Bacillus subtilis
chromosome (F. Kunst et al., Nature 390:249–256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of
scoC
on late-stage developmental phenomena as they relate to
aprE
expression, we studied the genome-wide effects of a
scoC
null mutant with the goal of furthering the understanding of the role of
scoC
in growth and developmental processes. In the present work we compared the expression patterns of isogenic
B. subtilis
strains, one of which carries a null mutation in the
scoC
locus (
scoC4
). The results obtained indicate that
scoC
regulates, either directly or indirectly, the expression of at least 560 genes in the
B. subtilis
genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the
scoC
mutant in light of the transcriptome changes observed.
Identification and Molecular Analysis of PcsB, a Protein Required for Cell Wall Separation of Group B Streptococcus
Authors: Reinscheid Dieter J. , Gottschalk Birgit , Schubert Axel , Eikmanns Bernhard J. , Chhatwal Gursharan S. 
Published: July 2002

ABSTRACT

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated
pcsB
, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (
M
r
, 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from
Listeria monocytogenes
. Northern blot analysis revealed a monocistronic transcriptional organization for
pcsB
in GBS. Insertional inactivation of
pcsB
in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.
Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of
Erwinia chrysanthemi
Authors: Palacios José Luis , Zaror Isabel , Martı́nez Patricio , Uribe Francisco , Opazo Patricio , Socı́as Teresa , Gidekel Manuel , Venegas Alejandro 
Published: July 2002

ABSTRACT

Erwinia chrysanthemi
exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the
E. chrysanthemi
transporter system (
prtDEF
genes) in
Escherichia coli
and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of
E. chrysanthemi
protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.
Role of the Tat Transport System in Nitrous Oxide Reductase Translocation and Cytochrome
cd
1
Biosynthesis in
Pseudomonas stutzeri
Authors: Heikkilä Mari P. , Honisch Ulrike , Wunsch Patrick , Zumft Walter G. 
Published: July 2002

ABSTRACT

By transforming N
2
O to N
2
, the multicopper enzyme nitrous oxide reductase provides a periplasmic electron sink for a respiratory chain that is part of denitrification. The signal sequence of the enzyme carries the heptameric twin-arginine consensus motif characteristic of the Tat pathway. We have identified
tat
genes of
Pseudomonas stutzeri
and functionally analyzed the unlinked
tatC
and
tatE
loci. A
tatC
mutant retained N
2
O reductase in the cytoplasm in the unprocessed form and lacking the metal cofactors. This is contrary to viewing the Tat system as specific only for fully assembled proteins. A C618V exchange in the electron transfer center Cu
A
rendered the enzyme largely incompetent for transport. The location of the mutation in the C-terminal domain of N
2
O reductase implies that the Tat system acts on a completely synthesized protein and is sensitive to a late structural variation in folding. By generating a
tatE
mutant and a reductase-overproducing strain, we show a function for TatE in N
2
O reductase translocation. Further, we have found that the Tat and Sec pathways have to cooperate to produce a functional nitrite reductase system. The cytochrome
cd
1
nitrite reductase was found in the periplasm of the
tatC
mutant, suggesting export by the Sec pathway; however, the enzyme lacked the heme D
1
macrocycle. The NirD protein as part of a complex required for heme D
1
synthesis or processing carries a putative Tat signal peptide. Since NO reduction was also inhibited in the
tatC
mutant, the Tat protein translocation system is necessary in multiple ways for establishing anaerobic nitrite denitrification.
Glucan Synthase Complex of
Aspergillus fumigatus
Authors: Beauvais A. , Bruneau J. M. , Mol P. C. , Buitrago M. J. , Legrand R. , Latgé J. P. 
Published: July 2002

ABSTRACT

The glucan synthase complex of the human pathogenic mold
Aspergillus fumigatus
has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of
A. fumigatus
were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in
Aspergillus nidulans
. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in
Escherichia coli
. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for β(1–3) glucans, showing that AfFks1p colocalized with the newly synthesized β(1–3) glucans.
AfRHO1
, the most homologous gene to
RHO1
of
Saccharomyces cerevisiae
, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H
+
-ATPase, and a 160-kDa protein which was labeled by an anti-β(1–3) glucan antibody and was homologous to ABC bacterial β(1–2) glucan transporters.
Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity
Authors: Jin Jie , Guffanti Arthur A. , Beck Catherine , Krulwich Terry A. 
Published: July 2002

ABSTRACT

A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na
+
/H
+
antiport and antiport with K
+
as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me
2+
/H
+
antiport in
Escherichia coli
vesicles.
Characterization of thed-Xylulose 5-Phosphate/d-Fructose 6-Phosphate Phosphoketolase Gene (xfp) fromBifidobacterium lactis
Authors: Meile Leo , Rohr Lukas M. , Geissmann Thomas A. , Herensperger Monique , Teuber Michael 
Published: July 2002

ABSTRACTAd-xylulose 5-phosphate/d-fructose 6-phosphate phosphoketolase (Xfp) from the probioticBifidobacterium lactiswas purified to homogeneity. The specific activity of the purified enzyme withd-fructose 6-phosphate as a substrate is 4.28 Units per mg of enzyme.Kmvalues ford-xylulose 5-phosphate andd-fructose 6-phosphate are 45 and 10 mM, respectively. The native enzyme has a molecular mass of 550,000 Da. The subunit size upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (90,000 Da) corresponds with the size (92,529 Da) calculated from the amino acid sequence of the isolated gene (namedxfp) encoding 825 amino acids. Thexfpgene was identified on the chromosome ofB. lactiswith the help of degenerated nucleotide probes deduced from the common N-terminal amino acid sequence of both the native and denatured enzyme. Comparison of the deduced amino acid sequence of the cloned gene with sequences in public databases revealed high homologies with hypothetical proteins (26 to 55% identity) in 20 microbial genomes. The amino acid sequence derived from thexfpgene contains typical thiamine diphosphate (ThDP) binding sites reported for other ThDP-dependent enzymes. Two truncated putative genes,ptaandguaA, were localized adjacent toxfpon theB. lactischromosome coding for a phosphotransacetylase and a guanosine monophosphate synthetase homologous to products of genes inMycobacterium tuberculosis. However,xfpis transcribed inB. lactisas a monocistronic operon. It is the first reported and sequenced gene of a phosphoketolase.
Sequencing of Flagellin Genes from
Natrialba magadii
Provides New Insight into Evolutionary Aspects of Archaeal Flagellins
Authors: Serganova Inna , Ksenzenko Vladimir , Serganov Alexander , Meshcheryakova Irina , Pyatibratov Michael , Vakhrusheva Olesya , Metlina Antonina , Fedorov Oleg 
Published: July 2002

ABSTRACT

We have determined the nucleotide sequence of a flagellin gene locus from the haloalkaliphilic archaeon
Natrialba magadii
, identified the gene products among proteins forming flagella, and demonstrated cotranscription of the genes. Based on the sequence analysis we suggest that different regions of the genes might have distinct evolutionary histories including possible genetic exchange with bacterial flagellin genes.
Regulation of themetC-cysKOperon, Involved in Sulfur Metabolism inLactococcus lactis
Authors: Fernández María , Kleerebezem Michiel , Kuipers Oscar P. , Siezen Roland J. , van Kranenburg Richard 
Published: July 2002

ABSTRACTSulfur metabolism in gram-positive bacteria is poorly characterized. Information on the molecular mechanisms of regulation of genes involved in sulfur metabolism is limited, and no regulator genes have been identified. Here we describe the regulation of the lactococcalmetC-cysKoperon, encoding a cystathionine β-lyase (metC) and cysteine synthase (cysK). Its expression was shown to be negatively affected by high concentrations of cysteine, methionine, and glutathione in the culture medium, while sulfur limitation resulted in a high level of expression. Other sulfur sources tested showed no significant effect onmetC-cysKgene expression. In addition we found thatO-acetyl-l-serine, the substrate of cysteine synthase, was an inducer of themetC-cysKoperon. Using a random mutagenesis approach, we identified two genes,cmbRandcmbT, involved in regulation ofmetC-cysKexpression. ThecmbTgene is predicted to encode a transport protein, but its precise role in regulation remains unclear. Disruption ofcmbTresulted in a two- to threefold reduction ofmetC-cysKtranscription. A 5.7-kb region containing thecmbRgene was cloned and sequenced. The encoded CmbR protein is homologous to the LysR family of regulator proteins and is an activator of themetC-cysKoperon. In analogy to CysB fromEscherichia coli, we propose that CmbR requires acetylserine to be able to bind the activation sites and subsequently activate transcription of themetC-cysKoperon.
Differential Regulation of Twitching Motility and Elastase Production by Vfr in
Pseudomonas aeruginosa
Authors: Beatson Scott A. , Whitchurch Cynthia B. , Sargent Jennifer L. , Levesque Roger C. , Mattick John S. 
Published: July 2002

ABSTRACT

Vfr, a homolog of
Escherichia coli
cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and
regA
transcription in
Pseudomonas aeruginosa
. We identified a twitching motility-defective mutant that carries a transposon insertion in
vfr
and confirmed that
vfr
is required for twitching motility by construction of an independent allelic deletion-replacement mutant of
vfr
that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type
vfr
. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK
lasI
::Tc) with a spontaneous deletion in
vfr
(S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in
vfr
-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to
E. coli
CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.
Penicillin-Binding Proteins 1a and 1b Form Independent Dimers in
Escherichia coli
Authors: Charpentier Xavier , Chalut Christian , Rémy Marie-Hélène , Masson Jean-Michel 
Published: July 2002

ABSTRACT

We report here that PBP1a can dimerize but does not interact with PBP1b to form PBP1a/PBP1b heterodimers in
Escherichia coli
. These findings support the idea of a relevant involvement of dimerization of both PBP1a and PBP1b during murein synthesis and suggest the existence of different peptidoglycan synthesis complexes.
Genome Sequence and Analysis of the Oral Bacterium
Fusobacterium nucleatum
Strain ATCC 25586
Authors: Kapatral Vinayak , Anderson Iain , Ivanova Natalia , Reznik Gary , Los Tamara , Lykidis Athanasios , Bhattacharyya Anamitra , Bartman Allen , Gardner Warren , Grechkin Galina , Zhu Lihua , Vasieva Olga , Chu Lien , Kogan Yakov , Chaga Oleg , Goltsman Eugene , Bernal Axel , Larsen Niels , D'Souza Mark , Walunas Theresa , Pusch Gordon , Haselkorn Robert , Fonstein Michael , Kyrpides Nikos , Overbeek Ross 
Published: July 2002

ABSTRACT

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium
Fusobacterium nucleatum
. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens
Porphyromonas gingivalis
and
Bacteroides forsythus
. The
F. nucleatum
strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (
http://www.integratedgenomics.com
). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive
Clostridium
spp.,
Enterococcus
spp., and
Lactococcus
spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H
2
S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of
F. nucleatum
revealed by its genome are therefore consistent with its specialized niche in the mouth.
Properties of the Glucose-6-Phosphate Transporter from
Chlamydia pneumoniae
(HPTcp) and the Glucose-6-Phosphate Sensor from
Escherichia coli
(UhpC)
Authors: Schwöppe Christian , Winkler Herbert H. , Neuhaus H. Ekkehard 
Published: July 2002

ABSTRACT

The amino acid sequence of the proposed glucose-6-phosphate (Glc6P) transporter from
Chlamydia pneumoniae
(HPTcp; hexose phosphate transporter [
Chlamydia pneumoniae
]) exhibits a higher degree of similarity to the
Escherichia coli
Glc6P sensor (UhpC) than to the
E. coli
Glc6P transporter (UhpT). Overexpression of His-UhpC in a UhpT-deficient
E. coli
strain revealed that the sensor protein is also able to transport Glc6P and exhibits an apparent

K
m

(Glc6P)
of 25 μM, whereas His-HPTcp exhibits an apparent

K
m

(Glc6P)
of 98 μM. His-HPTcp showed a four-times-lower specific activity than His-UhpT but a 56-times-higher specific activity than His-UhpC. Like His-UhpT and His-UhpC, the carrier His-HPTcp performs a sugar-phosphate/inorganic-phosphate antiporter mode of transport. Surprisingly, while physiological concentrations of inorganic phosphate competitively inhibited transport mediated by the
E. coli
proteins His-UhpT and His-UhpC, transport mediated by His-HPTcp was not inhibited. Interestingly, C
3
-organophosphates stimulated His-HPTcp activity but not His-UhpT- or His-UhpC-catalyzed Glc6P transport. In contrast to His-UhpC, the His-HPTcp protein does not act as a Glc6P sensor in the
uhp
regulon.
Characterization of the Interaction of
Bacillus subtilis
PyrR with
pyr
mRNA by Site-Directed Mutagenesis of the Protein
Authors: Savacool Heather K. , Switzer Robert L. 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
PyrR protein regulates transcriptional attenuation of the pyrimidine nucleotide (
pyr
) operon by binding in a uridine nucleotide-dependent manner to specific sites on
pyr
mRNA and stabilizing a secondary structure of the downstream RNA that favors termination of transcription. The high-resolution structure of unliganded PyrR was used to guide site-directed mutagenesis of 12 amino acid residues that were thought likely to be involved in the binding of RNA. Missense mutations were constructed and evaluated for their effects on regulation of
pyr
genes in vivo and their uracil phosphoribosyltransferase activity, which is catalyzed by wild-type PyrR. A substantial fraction of the mutant PyrR proteins did not have native structures, but eight PyrR mutants were purified and characterized physically, for their uracil phosphoribosyltransferase activity and for their ability to bind
pyr
RNA in vitro. On the basis of these studies Thr-18, His-22, Arg-141, and Arg-146 were implicated in RNA binding. Arg-27 and Lys-152 were also likely to be involved in RNA binding, but Gln substitution mutations in these residues may have altered their subunit-subunit interactions slightly. Arg-19 was implicated in
pyr
regulation, but a specific role in RNA binding could not be demonstrated because the R19Q mutant protein could not be purified in native form. The results confirm a role in RNA binding of a positively charged face of PyrR previously identified from the crystallographic structure. The RNA binding residues lie in two sequence segments that are conserved in PyrR proteins from many species.