bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conversion of the
Vibrio fischeri
Transcriptional Activator, LuxR, to a Repressor
Authors: Egland Kristi A. , Greenberg E. P. 
Published: July 2002

ABSTRACT

The
Vibrio fischeri
luminescence (
lux
) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal. Transcriptional activation requires the presence of a 20-base inverted repeat termed the
lux
box at a position centered 42.5 bases upstream of the transcriptional start of the
lux
operon. LuxR has proven difficult to study in vitro. A truncated form of LuxR has been purified, and together with ς
70
RNA polymerase it can activate transcription of the
lux
operon. Both the truncated LuxR and RNA polymerase are required for binding to
lux
regulatory DNA in vitro. We have constructed an artificial
lacZ
promoter with the
lux
box positioned between and partially overlapping the consensus −35 and −10 hexamers of an RNA polymerase binding site. LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant
Escherichia coli
. Furthermore, multiple
lux
boxes on an independent replicon reduced the repressor activity of LuxR. Thus, it appears that LuxR can bind to
lux
boxes independently of RNA polymerase binding to the promoter region. A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native
lux
operon. The exception was the truncated protein that had been purified and studied in vitro. This protein functioned as an activator but not as a repressor in
E. coli
. The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the
lux
promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to
lux
box-containing DNA.
Nitrate Assimilation Genes of the Marine Diazotrophic, Filamentous Cyanobacterium
Trichodesmium
sp. Strain WH9601
Authors: Wang Qingfeng , Li Hong , Post Anton F. 
Published: July 2002

ABSTRACT

A 4.0-kb DNA fragment of
Trichodesmium
sp. strain WH9601 contained gene sequences encoding the nitrate reduction enzymes,
nirA
and
narB
. A third gene positioned between
nirA
and
narB
encodes a putative membrane protein with similarity to the nitrate permeases of
Bacillus subtilis
(NasA) and
Emericella nidulans
(CrnA). The gene was shown to functionally complement a Δ
nasA
mutant of
B. subtilis
and was assigned the name
napA
(nitrate permease). NapA was involved in both nitrate and nitrite uptake by the complemented
B. subtilis
cells.
napA
is distinct from the
nrt
genes that encode the nitrate transporter of freshwater cyanobacteria.
The
Bacillus subtilis yabG
Gene Is Transcribed by SigK RNA Polymerase during Sporulation, and
yabG
Mutant Spores Have Altered Coat Protein Composition
Authors: Takamatsu Hiromu , Kodama Takeko , Imamura Atsuo , Asai Kei , Kobayashi Kazuo , Nakayama Tatsuo , Ogasawara Naotake , Watabe Kazuhito 
Published: July 2002

ABSTRACT

The expression of six novel genes located in the region from
abrB
to
spoVC
of the
Bacillus subtilis
chromosome was analyzed, and one of the genes,
yabG
, had a predicted promoter sequence conserved among SigK-dependent genes. Northern blot analysis revealed that
yabG
mRNA was first detected from 4 h after the cessation of logarithmic growth (
T
4
) in wild-type cells and in a
gerE36
(GerE
−
) mutant but not in
spoIIAC
(SigF
−
),
spoIIGAB
(SigE
−
),
spoIIIG
(SigG
−
), and
spoIVCB
(SigK
−
) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the
yabG
gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of
yabG
spores in
l
-alanine and in a mixture of
l
-asparagine,
d
-glucose,
d
-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from
yabG
spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that
yabG
encodes a sporulation-specific protein which is involved in coat protein composition in
B. subtilis
.
Kinetic Analysis of
Clostridium cellulolyticum
Carbohydrate Metabolism: Importance of Glucose 1-Phosphate and Glucose 6-Phosphate Branch Points for Distribution of Carbon Fluxes Inside and Outside Cells as Revealed by Steady-State Continuous Culture
Authors: Guedon Emmanuel , Desvaux Mickaël , Petitdemange Henri 
Published: July 2002

ABSTRACT

During the growth of
Clostridium cellulolyticum
in chemostat cultures with ammonia as the growth-limiting nutrient, as much as 30% of the original cellobiose consumed by
C. cellulolyticum
was converted to cellotriose, glycogen, and polysaccharides regardless of the specific growth rates. Whereas the specific consumption rate of cellobiose and of the carbon flux through glycolysis increased, the carbon flux through the phosphoglucomutase slowed. The limitation of the path through the phosphoglucomutase had a great effect on the accumulation of glucose 1-phosphate (G1P), the precursor of cellotriose, exopolysaccharides, and glycogen. The specific rates of biosynthesis of these compounds are important since as much as 16.7, 16.0, and 21.4% of the specific rate of cellobiose consumed by the cells could be converted to cellotriose, exopolysaccharides, and glycogen, respectively. With the increase of the carbon flux through glycolysis, the glucose 6-phosphate (G6P) pool decreased, whereas the G1P pool increased. Continuous culture experiments showed that glycogen biosynthesis was associated with rapid growth. The same result was obtained in batch culture, where glycogen biosynthesis reached a maximum during the exponential growth phase. Glycogen synthesis in
C. cellulolyticum
was also not subject to stimulation by nutrient limitation. Flux analyses demonstrate that G1P and G6P, connected by the phosphoglucomutase reaction, constitute important branch points for the distribution of carbon fluxes inside and outside cells. From this study it appears that the properties of the G1P-G6P branch points have been selected to control excretion of carbon surplus and to dissipate excess energy, whereas the pyruvate-acetyl coenzyme A branch points chiefly regulate the redox balance of the carbon catabolism as was shown previously (E. Guedon et al., J. Bacteriol. 181:3262–3269, 1999).
Bacillus subtilis
Locus Encoding a Killer Protein and Its Antidote
Authors: Adler Elliot , Barák Imrich , Stragier Patrick 
Published: July 2002

ABSTRACT

We have isolated mutations that block sporulation after formation of the polar septum in
Bacillus subtilis
. These mutations were mapped to the two genes of a new locus,
spoIIS
. Inactivation of the second gene,
spoIISB
, decreases sporulation efficiency by 4 orders of magnitude. Inactivation of the first gene,
spoIISA
, has no effect on sporulation but it fully restores sporulation of a
spoIISB
null mutant, indicating that SpoIISB is required only to counteract the negative effect of SpoIISA on sporulation. An internal promoter ensures the synthesis of an excess of SpoIISB over SpoIISA during exponential growth and sporulation. In the absence of SpoIISB, the sporulating cells show lethal damage of their envelope shortly after asymmetric septation, a defect that can be corrected by synthesizing SpoIISB only in the mother cell. However, forced synthesis of SpoIISA in exponentially growing cells or in the forespore leads to the same type of morphological damage and to cell death. In both cases protection against the killing effect of SpoIISA can be provided by simultaneous synthesis of SpoIISB. The
spoIIS
locus is unique to
B. subtilis
, and since it is completely dispensable for sporulation its physiological role remains elusive.
The
Agrobacterium tumefaciens rnd
Homolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid
tra
Gene Expression
Authors: Luo Zhao-Qing , Farrand Stephen K. 
Published: July 2002

ABSTRACT

Conjugal transfer of
Agrobacterium tumefaciens
Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer,
N
-(3-oxo-octanoyl)-
l
-homoserine lactone. We isolated four Tn5-induced mutants of
A. tumefaciens
C58 deficient in TraR-mediated activation of
tra
genes on pTiC58Δ
accR
. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn
5
was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by
rnd
of
Escherichia coli
, an RNase known to be involved in tRNA processing. The wild-type allele of the
rnd
homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of
cya2
, surround
A. tumefaciens rnd
, but none of these genes exerted a detectable effect on the expression of the
tra
reporter. In the mutant,
traR
was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of
tra
, but not the growth rate, was partially restored by increasing the copy number of
traR
or by disrupting
traM
, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in
rnd
also slightly reduced expression of two tested
vir
genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in
tra
gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.
Identification of a DNA Binding Region in GerE from
Bacillus subtilis
Authors: Crater Dinene L. , Moran Charles P. 
Published: July 2002

ABSTRACT

Proteins that have a structure similar to those of LuxR and FixJ comprise a large subfamily of transcriptional activator proteins. Most members of the LuxR-FixJ family contain a similar amino-terminal receiver domain linked by a small region to a carboxy-terminal domain that contains an amino acid sequence similar to the helix-turn-helix (HTH) motif found in other DNA-binding proteins. GerE from
Bacillus subtilis
is the smallest member of the LuxR-FixJ family. Its 74-amino-acid sequence is similar over its entire length to the DNA binding region of this protein family, including the HTH motif. Therefore, GerE provides a simple model for studies of the role of this HTH domain in DNA binding. Toward this aim, we sought to identify the amino acids within this motif that are important for the specificity of binding to DNA. We examined the effects of single base pair substitutions in the high-affinity GerE binding site on the
sigK
promoter and found that nucleotides at positions +2, +3, and +4 relative to the transcription start site on the
sigK
promoter are important for a high-affinity interaction with GerE. We next examined the effects of single alanine substitutions at two positions in the HTH region of GerE on binding to wild-type or mutant target sites. We found that the substitution of an alanine for the threonine at position 42 of GerE produced a protein that binds with equal affinity to two sites that differ by 1 bp, whereas wild-type GerE binds with different affinities to these two sites. These results provide evidence that the amino acyl residues in or near the putative HTH region of GerE and potentially other members of the LuxR-FixJ family determine the specificity of DNA binding.
New Plasmid System To Select for
Saccharomyces cerevisiae
Purine-Cytosine Permease Affinity Mutants
Authors: Wagner Renaud , Straub Marie-Laure , Souciet Jean-Luc , Potier Serge , de Montigny Jacky 
Published: July 2002

ABSTRACT

The
FCY2
gene of
Saccharomyces cerevisiae
encodes a purine-cytosine permease (PCP) that mediates the active transport of purines and cytosine. A structure-function model for this PCP has been recently proposed. In this study, we developed a plasmid-based system that generated a number of affinity-mutated alleles, enabling us to define new amino acids critical for permease function.
Cellobiose Uptake in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Is Mediated by an Inducible, High-Affinity ABC Transporter
Authors: Koning Sonja M. , Elferink Marieke G. L. , Konings Wil N. , Driessen Arnold J. M. 
Published: July 2002

ABSTRACT

The hyperthermophilic archaeon
Pyrococcus furiosus
can utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (
K

m

= 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from
P. furiosus
membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The
cbtA
gene was cloned and functionally expressed in
Escherichia coli. cbtA
belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.
Characterization of PitA and PitB from
Escherichia coli
Authors: Harris Robyn M. , Webb Dianne C. , Howitt Susan M. , Cox Graeme B. 
Published: July 2002

ABSTRACT

Escherichia coli
contains two major systems for transporting inorganic phosphate (P
i
). The low-affinity P
i
transporter (
pitA
) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (
pstSCAB
) is induced at low external P
i
concentrations by the
pho
regulon and is an ABC transporter. We isolated a third putative P
i
transport gene,
pitB
, from
E. coli
K-12 and present evidence that
pitB
encodes a functional P
i
transporter that may be repressed at low P
i
levels by the
pho
regulon. While a
pitB
+
cosmid clone allowed growth on medium containing 500 μM P
i
,
E. coli
with wild-type genomic
pitB
(
pitA
Δ
pstC345
double mutant) was unable to grow under these conditions, making it indistinguishable from a
pitA pitB
Δ
pstC345
triple mutant. The mutation Δ
pstC345
constitutively activates the
pho
regulon, which is normally induced by phosphate starvation. Removal of
pho
regulation by deleting the
phoB-phoR
operon allowed the
pitB
+
pitA
Δ
pstC345
strain to utilize P
i
, with P
i
uptake rates significantly higher than background levels. In addition, the apparent
K

m

of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional
pitA
gene and lacks Pit activity when the Pst system is mutated. The
pitA
mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.
Nested Deletions of the SRL Pathogenicity Island of
Shigella flexneri
2a
Authors: Turner Sally A. , Luck Shelley N. , Sakellaris Harry , Rajakumar Kumar , Adler Ben 
Published: July 2002

ABSTRACT

In this study, we determined the boundaries of a 99-kb deletable element of
Shigella flexneri
2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the
Shigella
resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS
91
elements present at the
S. flexneri
homologs of the
Escherichia coli
genes
putA
and
mdoA
on
Not
I fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS
91
elements, resulting in a single IS
91
element spanning the two original IS
91
loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10
−5
per cell in the wild type, and mutation of a P4-like integrase gene (
int
) at the left end of the SRL PAI revealed that
int
mediates precise deletion of the PAI.
RpoS-Dependent Transcriptional Control of
sprE
: Regulatory Feedback Loop
Authors: Ruiz Natividad , Peterson Celeste N. , Silhavy Thomas J. 
Published: July 2002

ABSTRACT

The stationary-phase response exhibited by
Escherichia coli
upon nutrient starvation is mainly induced by a decrease of the ClpXP-dependent degradation of the alternate primary ς factor RpoS. Although it is known that the specific regulation of this proteolysis is exercised by the orphan response regulator SprE, it remains unclear how SprE's activity is regulated in vivo. Previous studies have demonstrated that the cellular content of SprE itself is paradoxically increased in stationary-phase cells in an RpoS-dependent fashion. We show here that this RpoS-dependent upregulation of SprE levels is due to increased transcription. Furthermore, we demonstrate that
sprE
is part of the two-gene
rssA-sprE
operon, but it can also be transcribed from an additional RpoS-dependent promoter located in the
rssA-sprE
intergenic region. In addition, by using an in-frame deletion in
rssA
we found that RssA does not regulate either SprE or RpoS under the conditions tested.
Structures of Bacterial Flagellar Motors from Two FliF-FliG Gene Fusion Mutants
Authors: Thomas Dennis , Morgan David Gene , DeRosier David J. 
Published: July 2002

ABSTRACT

Flagella purified from
Salmonella enterica
serovar Typhimurium contain FliG, FliM, and FliN, cytoplasmic proteins that are important in torque generation and switching, and FliF, a transmembrane structural protein. The motor portion of the flagellum (the basal body complex) has a cytoplasmic C ring and a transmembrane M ring. Incubation of purified basal bodies at pH 4.5 removed FliM and FliN but not FliG or FliF. These basal bodies lacked C rings but had intact M rings, suggesting that FliM and FliN are part of the C ring but not a detectable part of the M ring. Incubation of basal bodies at pH 2.5 removed FliG, FliM, and FliN but not FliF. These basal bodies lacked the C ring, and the cytoplasmic face of the M ring was altered, suggesting that FliG makes up at least part of the cytoplasmic face of the M ring. Further insights into FliG were obtained from cells expressing a fusion protein of FliF and FliG. Flagella from these mutants still rotated but cells were not chemotactic. One mutant is a full-length fusion of FliF and FliG; the second mutant has a deletion lacking the last 56 residues of FliF and the first 94 residues of FliG. In the former, C rings appeared complete, but a portion of the M ring was shifted to higher radius. The C-ring–M-ring interaction appeared to be altered. In basal bodies with the fusion-deletion protein, the C ring was smaller in diameter, and one of its domains occupied space vacated by missing portions of FliF and FliG.
Quantification of Expression of
Staphylococcus epidermidis
Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions
Authors: Vandecasteele S. J. , Peetermans W. E. , Merckx R. , Van Eldere J. 
Published: July 2002

ABSTRACT

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (
gmk
, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (
tpi
, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene;
hsp-60
, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the
hsp-60
gene was induced by exposure to a 10°C heat shock (37 to 47°C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for
Escherichia coli
that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).
Complex I and Its Involvement in Redox Homeostasis and Carbon and Nitrogen Metabolism in
Rhodobacter capsulatus
Authors: Tichi Mary A. , Meijer Wim G. , Tabita F. Robert 
Published: July 2002

ABSTRACT

A transposon mutant of
Rhodobacter capsulatus
, strain Mal7, that was incapable of photoautotrophic and chemoautotrophic growth and could not grow photoheterotrophically in the absence of an exogenous electron acceptor was isolated. The phenotype of strain Mal7 suggested that the mutation was in some gene(s) not previously shown to be involved in CO
2
fixation control. The site of transposition in strain Mal7 was identified and shown to be in the gene
nuoF
, which encodes one of the 14 subunits for NADH ubiquinone-oxidoreductase, or complex I. To confirm the role of complex I and
nuoF
for CO
2
-dependent growth, a site-directed
nuoF
mutant was constructed (strain SBC1) in wild-type strain SB1003. The complex I-deficient strains Mal7 and SBC1 exhibited identical phenotypes, and the pattern of CO
2
fixation control through the Calvin-Benson-Bassham pathway was the same for both strains. It addition, it was shown that electron transport through complex I led to differential control of the two major
cbb
operons of this organism. Complex I was further shown to be linked to the control of nitrogen metabolism during anaerobic photosynthetic growth of
R. capsulatus
.
Unexpected Presence of Defective
glpR
Alleles in Various Strains of
Escherichia coli
Authors: Holtman C. Kay , Thurlkill Richard , Pettigrew Donald W. 
Published: July 2002

ABSTRACT

Alleles of
glpR
associated with the same GlpR
−
phenotype produce substitutions in different conserved portions of the glycerol 3-phosphate repressor which are not part of the helix-turn-helix motif. Analysis of the effects on growth and enzyme expression show that glucose repression of glycerol utilization is not dependent on a functional repressor.