bluuebunny/crossref_metadata_2025_split huggingface dataset:
X-Prolyl Dipeptidyl Aminopeptidase Gene (
pepX
) Is Part of the
glnRA
Operon in
Lactobacillus rhamnosus
Authors: Varmanen Pekka , Savijoki Kirsi , Åvall Silja , Palva Airi , Tynkkynen Soile 
Published: April 2009

ABSTRACT

A peptidase gene expressing X-prolyl dipeptidyl aminopeptidase (PepX) activity was cloned from
Lactobacillus rhamnosus
1/6 by using the chromogenic substrate
l
-glycyl-
l
-prolyl-β-naphthylamide for screening of a genomic library in
Escherichia coli
. The nucleotide sequence of a 3.5-kb
Hin
dIII fragment expressing the peptidase activity revealed one complete open reading frame (ORF) of 2,391 nucleotides. The 797-amino-acid protein encoded by this ORF was shown to be 40, 39, and 36% identical with PepXs from
Lactobacillus helveticus
,
Lactobacillus delbrueckii
, and
Lactococcus lactis
, respectively. By Northern analysis with a
pepX
-specific probe, transcripts of 4.5 and 7.0 kb were detected, indicating that
pepX
is part of a polycistronic operon in
L. rhamnosus
. Cloning and sequencing of the upstream region of
pepX
revealed the presence of two ORFs of 360 and 1,338 bp that were shown to be able to encode proteins with high homology to GlnR and GlnA proteins, respectively. By multiple primer extension analyses, the only functional promoter in the
pepX
region was located 25 nucleotides upstream of
glnR
. Northern analysis with
glnA
- and
pepX
-specific probes indicated that transcription from
glnR
promoter results in a 2.0-kb dicistronic
glnR-glnA
transcript and also in a longer read-through polycistronic transcript of 7.0 kb that was detected with both probes in samples from cells in exponential growth phase. The
glnA
gene was disrupted by a single-crossover recombinant event using a nonreplicative plasmid carrying an internal part of
glnA
. In the disruption mutant,
glnRA
-specific transcription was derepressed 10-fold compared to the wild type, but the 7.0-kb transcript was no longer detectable with either the
glnA
- or
pepX
-specific probe, demonstrating that
pepX
is indeed part of
glnRA
operon in
L. rhamnosus
. Reverse transcription-PCR analysis further supported this operon structure. An extended stem-loop structure was identified immediately upstream of
pepX
in the
glnA-pepX
intergenic region, a sequence that showed homology to a 23S-5S intergenic spacer and to several other
L. rhamnosus
-related entries in data banks.
Genetics of
l
-Sorbose Transport and Metabolism in
Lactobacillus casei
Authors: Yebra María J. , Veyrat Ana , Santos Mario A. , Pérez-Martínez Gaspar 
Published: April 2009

ABSTRACT

Genes encoding
l
-sorbose metabolism of
Lactobacillus casei
ATCC 393 have been identified on a 6.8-kb chromosomal DNA fragment. Sequence analysis revealed seven complete genes and a partial open reading frame transcribed as two units. The deduced amino acid sequences of the first transcriptional unit (
sorRE
) showed high similarity to the transcriptional regulator and the
l
-sorbose-1-phosphate reductase of the sorbose (
sor
) operon from
Klebsiella pneumoniae
. The other genes are transcribed as one unit (
sorFABCDG
) in opposite direction to
sorRE
. The deduced peptide sequence of
sorF
showed homology with the
d
-sorbitol-6-phosphate dehydrogenase encoded in the
sor
operon from
K. pneumoniae
and
sorABCD
to components of the mannose phosphotransferase system (PTS) family but especially to domains EIIA, EIIB, EIIC and EIID of the phosphoenolpyruvate-dependent
l
-sorbose PTS from
K. pneumoniae
. Finally, the deduced amino acid sequence of a truncated gene (
sorG
) located downstream of
sorD
presented high similarity with ketose-1,6-bisphosphate aldolases. Results of studies on enzyme activities and transcriptional analysis revealed that the two gene clusters,
sorRE
and
sorFABCDG
, are induced by
l
-sorbose and subject to catabolite repression by
d
-glucose. Data indicating that the catabolite repression is mediated by components of the PTS elements and by CcpA, are presented. Results of sugar uptake assays in
L. casei
wild-type and
sorBC
mutant strains indicated that
l
-sorbose is taken up by
l
-sorbose-specific enzyme II and that
L. casei
contains an inducible
d
-fructose-specific PTS. Results of growth analysis of those strains and a
man sorBC
double mutant suggested that
l
-sorbose is probably also transported by the
d
-mannose PTS. We also present evidence, from studies on a
sorR
mutant, suggesting that the
sorR
gene encodes a positive regulator of the two
sor
operons. Sequence alignment of SorR, SorC (
K. pneumoniae
), and DeoR (
Bacillus subtilis
) revealed that they might constitute a new group of transcriptional regulators.
Straight and Curved Conformations of FtsZ Are Regulated by GTP Hydrolysis
Authors: Lu Chunlin , Reedy Mary , Erickson Harold P. 
Published: April 2009

ABSTRACT
FtsZ assembles in vitro into protofilaments that can adopt two conformations—the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.
Involvement of Carbon Source and Acetyl Phosphate in the External-pH-Dependent Expression of Porin Genes in
Escherichia coli
Authors: Heyde Martine , Laloi Patrick , Portalier Raymond 
Published: April 2009

ABSTRACT

The porin composition of the
Escherichia coli
cell envelope was analyzed during growth at different external pHs (pHo) as a function of the acetyl phosphate (AcP) level (Δ
ackA pta
or
ackA
mutant, pyruvate or glucose as the carbon source) in the presence or absence of EnvZ. Our results indicate that the AcP level is influenced by the pHo, leading to modulation of the amount of OmpR-P and subsequent pHo-dependent expression of
ompF
and
ompC
. We also propose the existence of a specific signal, independent of EnvZ and AcP, leading to OmpR phosphorylation in response to pyruvate.
Glycine Betaine Transport in
Lactococcus lactis
Is Osmotically Regulated at the Level of Expression and Translocation Activity
Authors: van der Heide Tiemen , Poolman Bert 
Published: April 2009

ABSTRACT

Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that
Lactococcus lactis
uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438–5444, 1993), is osmotically regulated at the levels of both expression and transport activity.
Uptake of 2-Oxoglutarate in
Synechococcus
Strains Transformed with the
Escherichia coli kgtP
Gene
Authors: Vázquez-Bermúdez María Félix , Herrero Antonia , Flores Enrique 
Published: April 2009

ABSTRACT

A number of cyanobacteria from different taxonomic groups exhibited very low levels of uptake of 2-[U-
14
C]oxoglutarate.
Synechococcus
sp. strain PCC 7942 was transformed with DNA constructs carrying the
Escherichia coli kgtP
gene encoding a 2-oxoglutarate permease and a kanamycin resistance gene cassette. The
Synechococcus
sp. strains bearing the
kgtP
gene incorporated 2-oxoglutarate into the cells through an active transport process. About 75% of the radioactivity from the 2-[U-
14
C]oxoglutarate taken up that was recovered in soluble metabolites was found as glutamate and glutamine. 2-Oxoglutarate was, however, detrimental to the growth of a
Synechococcus
sp. strain bearing the
kgtP
gene.
Promoter Interference in a Bacteriophage Lambda Control Region: Effects of a Range of Interpromoter Distances
Authors: Strainic Michael G. , Sullivan Jennifer J. , Collado-Vides Julio , deHaseth Pieter L. 
Published: April 2009

ABSTRACT

The p
R
and p
RM
promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p
R
interfered with open complex formation at p
RM
and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p
RM
promoter. A model to explain these observations is proposed. A search of known
Escherichia coli
promoters identified three pairs of divergent promoters with similar separations to those investigated here.
Inorganic Polyphosphate Is Required for Motility of Bacterial Pathogens
Authors: Rashid M. Harunur , Rao Narayana N. , Kornberg Arthur 
Published: April 2009

ABSTRACT

The
ppk
gene encodes polyphosphate kinase (PPK), the principal enzyme in many bacteria responsible for the synthesis of inorganic polyphosphate (polyP) from ATP. A null mutation in the
ppk
gene of six bacterial pathogens renders them greatly impaired in motility on semisolid agar plates; this defect can be corrected by the introduction of
ppk
gene in
trans
. In view of the fact that the motility of pathogens is essential to invade and establish systemic infections in host cells, this impairment in motility suggests a crucial and essential role of PPK or polyP in bacterial pathogenesis.
Identification of SoxS-Regulated Genes in
Salmonella enterica
Serovar Typhimurium
Authors: Pomposiello Pablo J. , Demple Bruce 
Published: April 2009

ABSTRACT

Salmonella enterica
serovar Typhimurium responds to superoxide-generating agents through
soxR
-mediated activation of the
soxS
gene, whose product, SoxS, is necessary for resistance to oxidative stress. The
S. enterica
serovar Typhimurium
soxRS
system also mediates redox-inducible resistance to diverse antibiotics, which may be relevant to clinical infections. In order to identify SoxS-regulated genes in
S. enterica
serovar Typhimurium, a
lacI
-regulated expression system for the
S. enterica
serovar Typhimurium
soxS
gene was developed. This system was used to demonstrate that
soxS
expression is sufficient for the induction of resistance to the superoxide-generating drug paraquat and for the transcriptional activation of the
sodA
and
micF
genes. In addition, a library of random
lacZ
insertions was generated and screened for clones displaying differential β-galactosidase activity in the presence or absence of SoxS. This selection yielded six independent chromosomal
lacZ
transcriptional fusions that were activated by either artificial expression of SoxS or exposure of wild-type cells to micromolar concentrations of paraquat. Moreover, disruption of the inducible genes by the insertions rendered
S. enterica
serovar Typhimurium hypersensitive to millimolar concentrations of paraquat. Nucleotide sequence determination identified the disrupted genes as
sodA
(Mn-containing superoxide dismutase),
fpr
(NADPH:ferredoxin oxidoreductase), and
ydbK
(a putative Fe-S-containing reductase).
Metabolite Repression and Inducer Exclusion in the Proline Utilization Gene Cluster of
Aspergillus nidulans
Authors: Cubero Beatriz , Gómez Dennis , Scazzocchio Claudio 
Published: April 2009

ABSTRACT

The clustered
prnB
,
prnC
, and
prnD
genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in
cis
only on the permease gene (
prnB
) while derepression of
prnD
and
prnC
is largely the result of reversal of inducer exclusion.
Reduced Flux through the Purine Biosynthetic Pathway Results in an Increased Requirement for Coenzyme A in Thiamine Synthesis in
Salmonella enterica
Serovar Typhimurium
Authors: Frodyma Michael , Rubio Aileen , Downs D. M. 
Published: April 2009

ABSTRACT

Work presented here establishes a connection between cellular coenzyme A (CoA) levels and thiamine biosynthesis in
Salmonella enterica
serovar Typhimurium. Prior work showed that
panE
mutants (
panE
encodes ketopantoate reductase) had a conditional requirement for thiamine or pantothenate. Data presented herein show that the nutritional requirement of
panE
mutants for either thiamine or pantothenate is manifest only when flux through the purine biosynthetic pathway is reduced. Further, the data show that under the above conditions it is the lack of thiamine pyrophosphate, and not decreased CoA levels, that directly prevents growth.
A
celR
Mutation Affecting Transcription of Cellulase Genes in
Thermobifida fusca
Authors: Spiridonov Nikolay A. , Wilson David B. 
Published: April 2009

ABSTRACT

Biosynthesis of extracellular cellulases in the cellulose-degrading actinomycete
Thermobifida fusca
is controlled by a transcriptional regulator, CelR, and cellobiose, which acts as an inducer interfering with the CelR-DNA interaction. We report the identification and characterization of a mutation in the
celR
gene that changes Ala
55
in the hinge helix of CelR to Thr. The wild-type and mutant
celR
genes were cloned in
Escherichia coli
, and their protein products were characterized. The CelR mutant protein bound DNA more weakly than the wild-type protein and formed a less stable complex with DNA in the presence of cellobiose. The results of Western analysis and gel retardation experiments suggest that CelR is produced constitutively and its DNA-binding activity is regulated through posttranslational modification.
Identification and Characterization of an Active Plasmid Partition Mechanism for the Novel
Lactococcus lactis
Plasmid pCI2000
Authors: Kearney Karen , Fitzgerald Gerald F. , Seegers Jos F. M. L. 
Published: April 2009

ABSTRACT

The replication region of the lactococcal plasmid pCI2000 was subcloned and analyzed. The nucleotide sequence of one 5.6-kb
Eco
RI fragment which was capable of supporting replication when cloned on a replication probe vector revealed the presence of seven putative open reading frames (ORFs). One ORF exhibited significant homology to several replication proteins from plasmids considered to replicate via a theta mode. Deletion analysis showed that this ORF, designated
repA
, is indeed required for replication. The results also suggest that the origin of replication is located outside
repA
. Upstream and divergently transcribed from
repA
, an ORF that showed significant (48 to 64%) homology to a number of proteins that are required for faithful segregation of chromosomal or plasmid DNA of gram-negative bacteria was identified. Gene interruption and transcomplementation experiments showed that this ORF, designated
parA
, is required for stable inheritance of pCI2000 and is active in
trans
. This is the first example of such a partitioning mechanism for plasmids in gram-positive bacteria.
Light-Dependent Regulation of Cyanobacterial Phytochrome Expression
Authors: García-Domínguez M. , Muro-Pastor M. I. , Reyes J. C. , Florencio F. J. 
Published: April 2009

ABSTRACT

A histidine kinase protein (Cph1) with sequence homology and spectral characteristics very similar to those of the plant phytochrome has been recently identified in the cyanobacterium
Synechocystis
sp. strain PCC 6803. Cph1 together with Rcp1 (a protein homologue to the response regulator CheY) forms a light-regulated two-component system whose function is presently unknown. Levels of
cph1 rcp1
mRNA increase in the dark and decrease upon reillumination. A dark-mediated increase in
cph1 rcp1
mRNA levels was inhibited by the presence of glucose, but not by inhibition of the photosynthetic electron flow. The half-life of
cph1 rcp1
transcript in the light was about fourfold shorter than in the dark, indicating that control of
cph1 rcp1
transcript stability is one of the mechanisms by which light regulates expression of the cyanobacterial phytochrome. After 15 min of darkness, 3-min pulses of red, blue, green, and far-red light were equally efficient in decreasing the
cph1 rcp1
mRNA levels. Red light downregulation was not reversed by far-red light, suggesting that
cph1 rcp1
mRNA levels are not controlled by a phytochrome-like photoreceptor. Furthermore, a
Synechocystis
strain containing an H538R Cph1 point mutation, unable to phosphorylate Rcp1, shows normal light-dark regulation of the
cph1 rcp1
transcript levels. Our data suggest a role of cyanobacterial phytochrome in the control of processes required for adaptation in light-dark and dark-light transitions.
The VirR Response Regulator from
Clostridium perfringens
Binds Independently to Two Imperfect Direct Repeats Located Upstream of the
pfoA
Promoter
Authors: Cheung Jackie K. , Rood Julian I. 
Published: April 2009

ABSTRACT

Regulation of toxin production in the gram-positive anaerobe
Clostridium perfringens
occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the
virS
gene, while its cognate response regulator is encoded by the
virR
gene. We have constructed a VirR expression plasmid in
Escherichia coli
and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the
pfoA
gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated
plc
,
colA
, and
pfoR
genes, which encode alpha-toxin, collagenase, and a putative
pfoA
regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the
pfoA
promoter. When this region was deleted, VirR was no longer able to bind to the
pfoA
promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the
pfoA
promoter and activating transcription.
Role of the Tetraheme Cytochrome CymA in Anaerobic Electron Transport in Cells of
Shewanella putrefaciens
MR-1 with Normal Levels of Menaquinone
Authors: Myers Judith M. , Myers Charles R. 
Published: April 2009

ABSTRACT

Shewanella putrefaciens
MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome
c
. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the
cymA
gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a
cymA
knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the
cymA
transcript and CymA protein was also restored, by complementation of MR1-CYMA with the
cymA
gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.
Copper/Zinc-Superoxide Dismutase Is Required for Oxytetracycline Resistance of
Saccharomyces cerevisiae
Authors: Avery Simon V. , Malkapuram Srividya , Mateus Carolina , Babb Kimberly S. 
Published: April 2009

ABSTRACT

Saccharomyces cerevisiae
, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of
SOD1
(encoding Cu/Zn superoxide dismutase) rendered
S. cerevisiae
hypersensitive to oxytetracycline (OTC): a
sod1Δ
mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 μg ml
−1
, whereas concentrations of up to 1,000 μg ml
−1
had no effect on the growth of the wild type. OTC resistance was restored in the
sod1
Δ mutant by complementation with wild-type
SOD1
. The effect of OTC appeared to be cytotoxic and was not evident in a
ctt1
Δ (cytosolic catalase) mutant or in the presence of tetracycline.
SOD1
transcription was not induced by OTC, suggesting that constitutive
SOD1
expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and
sod1
Δ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the
sod1
Δ mutant, but not the wild type, to OTC. We propose that Sod1p protects
S. cerevisiae
against a mode of OTC action that is dependent on oxidative damage.
Characterization of the Endogenous Plasmid from
Pseudomonas alcaligenes
NCIB 9867: DNA Sequence and Mechanism of Transfer
Authors: Kwong Stephen M. , Yeo Chew Chieng , Suwanto Antonius , Poh Chit Laa 
Published: April 2009

ABSTRACT

The endogenous plasmid pRA2 from
Pseudomonas alcaligenes
NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The
Pac
25I restriction-modification system and two mobile elements, Tn
5563
and IS
1633
, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the ΩStr
r
/Spc
r
gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of
P. alcaligenes
NCIB 9867 were observed at high frequencies (2.4 × 10
−4
per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into
Pseudomonas putida
RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in
trans
. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host,
P. alcaligenes
NCIB 9867, as well as in
P. putida
RA713 after 100 generations of nonselective growth. Disruption of the pRA2
pac25I
restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.