bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in
Streptococcus pneumoniae
Authors: Luo Ping , Morrison Donald A. 
Published: December 2002

ABSTRACT

Natural transformation in
Streptococcus pneumoniae
is regulated by a quorum-sensing system that acts through accumulation and sensing of a peptide pheromone (competence-stimulating peptide [CSP]) to control many competence-specific genes acting in DNA uptake, processing, and integration. The period of competence induced by CSP lasts only 15 min (quarter-height peak width). The recently identified regulator ComX is required for the CSP-dependent expression of many competence-specific genes that share an unusual consensus sequence (TACGAATA) at their promoter regions. To test the hypothesis that this regulator acts as a transient alternative sigma factor, ComX was purified from an
Escherichia coli
overexpression strain and core RNA polymerase was purified from a
comX
-deficient
S. pneumoniae
strain. The reconstituted ComX-polymerase holoenzyme produced transcripts for the competence-specific genes
ssbB
,
cinA
,
cglA
,
celA
, and
dalA
and was inhibited by anti-ComX antibody, but not by anti-σ
70
antibody. Western blotting using antibodies specific for ComX, σ
70
, and poly-His revealed a transient presence of ComX for a period of 15 to 20 min after CSP treatment, while RNA polymerase remained at a constant level and σ
A
remained between 60 and 125% of its normal level. ComX reached a molar ratio to RNA polymerase of at least 1.5. We conclude that ComX is unstable and acts as a competence-specific sigma factor.
Binding of σAand σBto Core RNA Polymerase after Environmental Stress inBacillus subtilis
Authors: Rollenhagen Claudia , Antelmann Haike , Kirstein Janine , Delumeau Olivier , Hecker Michael , Yudkin Michael D. 
Published: December 2002

ABSTRACTInBacillus subtilis, the alternative sigma factor σBis activated in response to environmental stress or energy depletion. The general stress regulon under the control of σBprovides the cell with multiple stress resistance. Experiments were designed to determine how activated σBreplaces σAas a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the σA-dependent stress geneclpEunder σB-inducing conditions showed that expression was higher in asigBmutant background than in the wild type. The relative affinities of σAand σBfor binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of σBfor E was 60-fold lower than that of σA. Western blot analyses with antibodies against σA, σB, and E showed that, after exposure to ethanol stress, the concentration of σBwas only twofold higher than those of σAand E. Thus, the concentration of σBafter stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of σBto E after stress.
Transcriptional Regulation and Signature Patterns Revealed by Microarray Analyses of
Streptococcus pneumoniae
R6 Challenged with Sublethal Concentrations of Translation Inhibitors
Authors: Ng Wai-Leung , Kazmierczak Krystyna M. , Robertson Gregory T. , Gilmour Raymond , Winkler Malcolm E. 
Published: December 2002

ABSTRACT

The effects of sublethal concentrations of four different classes of translation inhibitors (puromycin, tetracycline, chloramphenicol, and erythromycin) on global transcription patterns of
Streptococcus pneumoniae
R6 were determined by microarray analyses. Consistent with the general mode of action of these inhibitors, relative transcript levels of genes that encode ribosomal proteins and translation factors or that mediate tRNA charging and amino acid biosynthesis increased or decreased, respectively. Transcription of the heat shock regulon was induced only by puromycin or streptomycin treatment, which lead to truncation or mistranslation, respectively, but not by other antibiotics that block translation, transcription, or amino acid charging of tRNA. In contrast, relative transcript amounts of certain genes involved in transport, cellular processes, energy metabolism, and purine nucleotide (
pur
) biosynthesis were changed by different translation inhibitors. In particular, transcript amounts from a
pur
gene cluster and from purine uptake and salvage genes were significantly elevated by several translation inhibitors, but not by antibiotics that target other cellular processes. Northern blotting confirmed increased transcript amounts from part of the
pur
gene cluster in cells challenged by translation inhibitors and revealed the presence of a 10-kb transcript. Purine metabolism genes were negatively regulated by a homologue of the PurR regulatory protein, and full derepression in a Δ
purR
mutant depended on optimal translation. Unexpectedly, hierarchical clustering of the microarray data distinguished among the global transcription patterns caused by antibiotics that inhibit different steps in the translation cycle. Together, these results show that there is extensive control of transcript amounts by translation in
S. pneumoniae
, especially for de novo purine nucleotide biosynthesis. In addition, these global transcription patterns form a signature that can be used to classify the mode of action and potential mechanism of new translation inhibitors.
Choline Starvation Induces the Gene
licD2
in
Streptococcus pneumoniae
Authors: Desai Bhushan V. , Reiter Harvard , Morrison Donald A. 
Published: December 2002

ABSTRACT

Mutant strains of
Streptococcus pneumoniae
were constructed to monitor the regulation of three dispersed genes known or predicted to act in choline metabolism. One gene (
licD2
) was regulated in response to choline deprivation over a 30-fold range. The other two (SP1860 and
licC
) responded little if at all to the same challenge.
Membrane Topology of the ZntB Efflux System of
Salmonella enterica
Serovar Typhimurium
Authors: Caldwell Andreia M. , Smith Ronald L. 
Published: December 2002

ABSTRACT

The membrane topology of the ZntB Zn
2+
transport protein of
Salmonella enterica
serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to
blaM
or
lacZ
cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.
The
Pseudomonas aeruginosa rhlAB
Operon Is Not Expressed during the Logarithmic Phase of Growth Even in the Presence of Its Activator RhlR and the Autoinducer
N
-Butyryl-Homoserine Lactone
Authors: Medina Gerardo , Juárez Katy , Soberón-Chávez Gloria 
Published: December 2002

ABSTRACT

The
Pseudomonas aeruginosa rhlAB
operon encodes the enzyme rhamnosyltransferase 1, which produces the biosurfactant mono-rhamnolipid;
rhlAB
induction is dependent on the quorum-sensing transcription activator RhlR complexed with the autoinducer
N
-butyryl-homoserine lactone (C
4
-HSL). In this work we studied
rhlAB
induction in a
P. aeruginosa
and
Escherichia coli
background. We found that, in both bacteria, its expression is not induced during the logarithmic phase of growth even in the presence of RhlR and C
4
-HSL. Additionally, we found that
rhlAB
expression is partially σ
s
dependent.
Identification of a Helix-Turn-Helix Motif of
Bacillus thermoglucosidasius
HrcA Essential for Binding to the CIRCE Element and Thermostability of the HrcA-CIRCE Complex, Indicating a Role as a Thermosensor
Authors: Hitomi Masafumi , Nishimura Hiroshi , Tsujimoto Yoshiyuki , Matsui Hiroshi , Watanabe Kunihiko 
Published: December 2002

ABSTRACT

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the
groE
and
dnaK
operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile
Bacillus thermoglucosidasius
KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in
B. thermoglucosidasius
HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from
Bacillus subtilis
and
B. thermoglucosidasius
, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of
B. thermoglucosidasius
and
B. subtilis
and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.
Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in
Sphingomonas paucimobilis
SYK-6
Authors: Hara Hirofumi , Masai Eiji , Miyauchi Keisuke , Katayama Yoshihiro , Fukuda Masao 
Published: December 2002

ABSTRACT

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by
Sphingomonas paucimobilis
SYK-6. In the 10.5-kb
Eco
RI fragment carrying the genes for PCA 4,5-dioxygenase (
ligAB
), 2-pyrone-4,6-dicarboxylate hydrolase (
ligI
), 4-oxalomesaconate hydratase (
ligJ
), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (
ligC
), we found the
ligK
gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The
ligK
gene was located 1,183 bp upstream of
ligI
and transcribed in the same direction as
ligI
. We also found the
ligR
gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of
ligK
. The
ligK
gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of
ligK
showed 57 to 88% identity with those of the corresponding genes recently reported in
Sphingomonas
sp. strain LB126,
Comamonas testosteroni
BR6020,
Arthrobacter keyseri
12B, and
Pseudomonas ochraceae
NGJ1. The
ligK
gene was expressed in
Escherichia coli
, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO
2
. LigK is a hexamer, and its isoelectric point is 5.1. The

K
m

for CHA and oxaloacetate are 11.2 and 136 μM, respectively. Inactivation of
ligK
in
S. paucimobilis
SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that
ligK
encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of
S. paucimobilis
SYK-6 consisted of four transcriptional units, including the
ligK-orf1-ligI-lsdA
cluster, the
ligJAB
cluster, and the monocistronic
ligR
and
ligC
genes.
A Bacitracin-Resistant
Bacillus subtilis
Gene Encodes a Homologue of the Membrane-Spanning Subunit of the
Bacillus licheniformis
ABC Transporter
Authors: Ohki Reiko , Tateno Kozue , Okada Youji , Okajima Haruo , Asai Kei , Sadaie Yoshito , Murata Makiko , Aiso Toshiko 
Published: December 2002

ABSTRACT

Bacitracin is a peptide antibiotic nonribosomally produced by
Bacillus licheniformis
. The
bcrABC
genes which confer bacitracin resistance to the bacitracin producer encode ATP binding cassette (ABC) transporter proteins, which are hypothesized to pump out bacitracin from the cells.
Bacillus subtilis
168, which has no bacitracin synthesizing operon, has several genes homologous to
bcrABC
. It was found that the disruption of
ywoA
, a gene homologous to
bcrC
, resulted in hypersensitivity to bacitracin. Resistance to other drugs such as surfactin, iturin A, vancomycin, tunicamycin, gramicidin D, valinomycin and several cationic dyes were not changed in the
ywoA
disruptant. Spontaneous bacitracin-resistant mutants (Bcr-1 and -2) isolated in the presence of bacitracin have a single base substitution from A to G in the ribosome binding region. Northern hybridization analysis and determination of the expression of
ywoA-LacZ
transcriptional fusion gene revealed that the transcription of the
ywoA
gene was dependent on extracytoplasmic function (ECF) σ factors σ
M
and σ
X
. Preincubation of wild-type cells in the presence of a low concentration of bacitracin induced increased resistance to bacitracin about two- to threefold, although the mechanism of this induction has not yet been elucidated. It has been reported that a commercially available bacitracin is a mixture of several components and also contains impurity. Bacitracin A was purified by reverse phase high-performance liquid chromatography (HPLC). Similar results were obtained with bacitracin A as those with crude bacitracin, indicating that contaminating substances were not responsible for the results obtained in this study.
TheStreptococcus pneumoniae ciaRegulon: CiaR Target Sites and Transcription Profile Analysis
Authors: Mascher Thorsten , Zähner Dorothea , Merai Michelle , Balmelle Nadège , de Saizieu Antoine B. , Hakenbeck Regine 
Published: December 2002

ABSTRACTTheciaR-ciaHsystem is one of 13 two-component signal-transducing systems of the human pathogenStreptococcus pneumoniae. Mutations in the histidine protein kinase CiaH confer increased resistance to beta-lactam antibiotics and interfere with the development of genetic competence. In order to identify the genes controlled by theciasystem, theciaregulon, DNA fragments targeted by the response regulator CiaR were isolated from restricted chromosomal DNA using the solid-phase DNA binding assay and analyzed by hybridization to an oligonucleotide microarray representing theS. pneumoniaegenome. A set of 18 chromosomal regions containing 26 CiaR target sites were detected and proposed to represent the minimalciaregulon. The putative CiaR target loci included genes important for the synthesis and modification of cell wall polymers, peptide pheromone and bacteriocin production, and thehtrA-spo0Jregion. In addition, the transcription profile ofcialoss-of-function mutants and those with an apparent activatedciasystem representing the off and on states of the regulatory system were analyzed. The transcript analysis confirmed thecia-dependent expression of seven putative target loci and revealed three additionalcia-regulated loci. Five putative target regions were silent under all conditions, and for the remaining three regions, nocia-dependent expression could be detected. Furthermore, the competence regulon, including thecomCDEoperon required for induction of competence, was completely repressed by theciasystem.
Function of Oxygen Resistance Proteins in the Anaerobic, Sulfate-Reducing Bacterium
Desulfovibrio vulgaris
Hildenborough
Authors: Fournier Marjorie , Zhang Yi , Wildschut Janine D. , Dolla Alain , Voordouw Johanna K. , Schriemer David C. , Voordouw Gerrit 
Published: December 2002

ABSTRACT

Two mutant strains of
Desulfovibrio vulgaris
Hildenborough lacking either the
sod
gene for periplasmic superoxide dismutase or the
rbr
gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H
2
O
2
) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a
sor
mutant lacking the gene for the cytoplasmic superoxide reductase. The
sor
mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the
sod
mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the
rbr
mutant, indicating that H
2
O
2
resistance may also be conferred by two other
rbr
genes in the
D. vulgaris
genome. Inhibition of Sod activity by azide and H
2
O
2
, but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of
D. vulgaris
. Thus, Sor plays a key role in oxygen defense of
D. vulgaris
under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.
The SecB Chaperone Is Bifunctional in
Serratia marcescens
: SecB Is Involved in the Sec Pathway and Required for HasA Secretion by the ABC Transporter
Authors: Sapriel Guillaume , Wandersman Cécile , Delepelaire Philippe 
Published: December 2002

ABSTRACT

HasA is the secreted hemophore of the heme acquisition system (Has) of
Serratia marcescens
. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in
Escherichia coli
, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the
secB
gene from
S. marcescens
. We show that
S. marcescens
SecB is 93% identical to
E. coli
SecB and complements the secretion defects of a
secB
mutant of
E. coli
for both the Sec and ABC pathways of HasA secretion. In
S. marcescens
, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that
S. marcescens
SecB is the genuine chaperone for HasA secretion in
S. marcescens
. These results also demonstrate that
S. marcescens
SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of
secB
point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.
Mutational Analysis of a Conserved Signal-Transducing Element: the HAMP Linker of theEscherichia coliNitrate Sensor NarX
Authors: Appleman J. Alex , Stewart Valley 
Published: December 2002

ABSTRACTThe HAMP linker, a predicted structural element observed in sensor proteins from all domains of life, is proposed to transmit signals between extracellular sensory input domains and cytoplasmic output domains. HAMP (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase) linkers are located just inside the cytoplasmic membrane and are projected to form two short amphipathic α-helices (AS-1 and AS-2) joined by an unstructured connector. The presumed helices are comprised of hydrophobic residues in heptad repeats, with only three positions exhibiting strong conservation. We generated missense mutations at these three positions and throughout the HAMP linker in theEscherichia colinitrate sensor kinase NarX and screened the resulting mutants for defective responses to nitrate. Most missense mutations in this region resulted in a constitutive phenotype mimicking the ligand-bound state, and only one residue (a conserved Glu before AS-2) was essential for HAMP linker function. We also scanned thenarXHAMP linker with an overlapping set of seven-residue deletions. Deletions in AS-1 and the connector resulted in constitutive phenotypes. Two deletions in AS-2 resulted in a novel reversed response phenotype in which the response to ligand was the opposite of that seen for thenarX+strain. These observations are consistent with the proposed HAMP linker structure, show that the HAMP linker plays an active role in transmembrane signal transduction, and indicate that the two amphipathic α-helices have different roles in signal transduction.
Lack of the ApbC or ApbE Protein Results in a Defect in Fe-S Cluster Metabolism in
Salmonella enterica
Serovar Typhimurium
Authors: Skovran Elizabeth , Downs Diana M. 
Published: December 2002

ABSTRACT

The
isc
genes function in the assembly of Fe-S clusters and are conserved in many prokaryotic and eukaryotic organisms. In most bacteria studied, the
isc
operon can be deleted without loss of cell viability, indicating that additional systems for Fe-S cluster assembly must exist. Several laboratories have described nutritional and biochemical defects resulting from mutations in the
isc
operon. Here we demonstrate that null mutations in two genes of unknown function,
apbC
and
apbE
, result in similar cellular deficiencies. Exogenous ferric chloride suppressed these deficiencies in the
apbC
and
apbE
mutants, distinguishing them from previously described
isc
mutants. The deficiencies caused by the
apbC
and
isc
mutations were additive, which is consistent with Isc and ApbC's having redundant functions or with Isc and ApbC's functioning in different areas of Fe-S cluster metabolism (e.g., Fe-S cluster assembly and Fe-S cluster repair). Both the ApbC and ApbE proteins are similar in sequence to proteins that function in metal cofactor assembly. Like the enzymes with sequence similarity to ApbC, purified ApbC protein was able to hydrolyze ATP. The data herein are consistent with the hypothesis that the ApbC and ApbE proteins function in Fe-S cluster metabolism in vivo.
Improving the utility of prostate specific antigen (PSA) in the diagnosis of prostate cancer: the use of PSA derivatives and novel markers
Authors: Jain S , Bhojwani A G , Mellon J K 
Published: December 2002

Abstract
Prostate specific antigen (PSA) testing is now a routine part of the investigation of men with suspected prostate cancer. While a very useful test it still has its problems, in particular its lack of specificity means abnormal results are often caused by benign disease. This review describes the current problems with PSA testing in prostate cancer diagnosis and highlights potential ways in which these may be reduced.
Epidemiology of perforated colonic diverticular disease
Authors: Morris C R , Harvey I M , Stebbings W S L , Speakman C T M , Kennedy H J , Hart A R 
Published: December 2002

Abstract
Perforated colonic diverticular disease results in considerable mortality and morbidity. This review appraises existing evidence on the epidemiology and mechanisms of perforation, highlights areas of further study, and suggests an epidemiological approach towards preventing the condition. Computerised searches were used to identify published articles relating to the epidemiology, pathophysiology, and clinical features of perforated colonic diverticular disease. Several drug and dietary exposures have potential biological mechanisms for causing perforation. Of these only non-steroidal anti-inflammatory drugs have been consistently identified as risk factors in aetiological studies. The causes of perforated colonic diverticular disease remain largely unknown. Further aetiological studies, looking specifically at perforation, are required to investigate whether cause-effect relationships exist for both drug and dietary exposures. The identification of risk factors for perforation would allow primary public health prevention, secondary risk factor modification, and early prophylactic surgery to be aimed at people at high risk.