bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthetic
lac
Operator Substitutions for Studying the Nitrate- and Nitrite-Responsive NarX-NarL and NarQ-NarP Two-Component Regulatory Systems of
Escherichia coli
K-12
Authors: Stewart Valley , Bledsoe Peggy J. 
Published: March 2003

ABSTRACT

The NarX and NarQ sensor-histidine kinases control phosphorylation of the NarL and NarP response regulators in response to the respiratory oxidants nitrate and nitrite. Target operon transcription is activated by the Fnr protein in response to anaerobiosis, and it is further activated and/or repressed by the phospho-NarL and phospho-NarP proteins, which bind to heptamer DNA sequences. The location and arrangement of heptamers vary widely among different target operon control regions. We have constructed a series of monocopy
lac
operon control region constructs in which the primary operator O1-
lac
has been replaced by 7-2-7 heptamer pairs from the
nrfA
,
nirB
,
napF
, and
fdnG
operon control regions. These constructs provide tools for dissecting various aspects of ligand interactions with sensor-kinases, sensor interactions with response regulators, and phospho-response regulator interactions with DNA targets. Expression of the
lacZ
gene from these constructs was repressed to various degrees by nitrate and nitrite. In response to nitrate, the
nrfA
and
nirB
operon 7-2-7 heptamer pairs at operator O1 each mediated greater than 100-fold repression of
lacZ
gene expression, whereas the
napF
operon 7-2-7 heptamer pair mediated approximately tenfold repression. Introduction of
narL
,
narP
,
narX
, and
narQ
null alleles in various combinations allowed the in vivo interactions between different sensor-regulator pairs to be evaluated and compared.
Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon
Sulfolobus solfataricus
Authors: Potters M. Ben , Solow Barbara T. , Bischoff Kenneth M. , Graham David E. , Lower Brian H. , Helm Richard , Kennelly Peter J. 
Published: March 2003

ABSTRACT

When soluble extracts of the extreme acidothermophilic archaeon
Sulfolobus solfataricus
were incubated with [γ-
32
P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ∼46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame
sso0417
, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame
sso0417
was therefore cloned, and its protein product was expressed in
Escherichia coli
. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co
2+
or Mn
2+
. The recombinant protein underwent autophosphorylation when incubated with either [γ-
32
P]ATP or [γ-
32
P]GTP. The site of phosphorylation was identified as Ser
59
, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the
32
P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of
32
P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.
Identification of ZipA, a Signal Recognition Particle-Dependent Protein from
Neisseria gonorrhoeae
Authors: Du Ying , Arvidson Cindy Grove 
Published: March 2003

ABSTRACT

A genetic screen designed to identify proteins that utilize the signal recognition particle (SRP) for targeting in
Escherichia coli
was used to screen a
Neisseria gonorrhoeae
plasmid library. Six plasmids were identified in this screen, and each is predicted to encode one or more putative cytoplasmic membrane (CM) proteins. One of these, pSLO7, has three open reading frames (ORFs), two of which have no similarity to known proteins in GenBank other than sequences from the closely related
N. meningitidis
. Further analyses showed that one of these, SLO7ORF3, encodes a protein that is dependent on the SRP for localization. This gene also appears to be essential in
N. gonorrhoeae
since it was not possible to generate null mutations in the gene. Although appearing unique to
Neisseria
at the DNA sequence level, SLO7ORF3 was found to share some features with the cell division gene
zipA
of
E. coli
. These features included similar chromosomal locations (with respect to linked genes) as well as similarities in the predicted protein domain structures. Here, we show that SLO7ORF3 can complement an
E. coli
conditional
zipA
mutant and therefore encodes a functional ZipA homolog in
N. gonorrhoeae
. This observation is significant in that it is the first ZipA homolog identified in a non-rod-shaped organism. Also interesting is that this is the fourth cell division protein (the others are FtsE, FtsX, and FtsQ) shown to utilize the SRP for localization, which may in part explain why the genes encoding the three SRP components are essential in bacteria.
Genomic Diversification among Archival Strains of
Salmonella enterica
Serovar Typhimurium LT7
Authors: Liu Gui-Rong , Edwards Kelly , Eisenstark Abraham , Fu Ying-Mei , Liu Wei-Qiao , Sanderson Kenneth E. , Johnston Randal N. , Liu Shu-Lin 
Published: March 2003

ABSTRACT

To document genomic changes during long periods of storage, we analyzed
Salmonella enterica
serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at −70°C or in serovar Typhimurium LT2 stocked either at −70°C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at −70°C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.
A Single Gene on the Staphylococcal Multiresistance Plasmid pSK1 Encodes a Novel Partitioning System
Authors: Simpson Alice E. , Skurray Ronald A. , Firth Neville 
Published: March 2003

ABSTRACTTheorf245gene is located immediately upstream of, and divergently transcribed from, the replication initiation gene,rep, of theStaphylococcus aureusmultiresistance plasmid pSK1, and related genes have been found in association with a range of evolutionarily distinct replication genes on plasmids from various gram-positive genera.orf245has been shown previously to extend the segregational stability of a pSK1 minireplicon. Here we describe an investigation into the basis oforf245-mediated stabilization.orf245was not found to influence transcription of pSK1rep, indicating that it is not directly involved in plasmid replication. This was confirmed by demonstrating thatorf245is able to enhance the segregational stability of heterologous theta- and rolling-circle-replicating replicons, suggesting that it encodes a plasmid maintenance function. Evidence inconsistent with postsegregational killing and multimer resolution mechanisms was obtained; however, the intergenic region upstream oforf245was found to mediateorf245-dependent incompatibility, as would be expected if it encodes acis-acting centromere-like site. Taken together, these findings implicate active partitioning as the probable basis of the activity oforf245, which is therefore redesignatedpar. Since it is unrelated to any gene known to play a role in plasmid segregation, it seems likely that pSK1parpotentially represents the prototype of a novel class of active partitioning systems that are distinguished by their capacity to enhance plasmid segregational stability via a single protein-encoding gene.
Roles of YqjH and YqjW, Homologs of the
Escherichia
coli
UmuC/DinB or Y Superfamily of DNA Polymerases, in Stationary-Phase Mutagenesis and UV-Induced Mutagenesis of
Bacillus
subtilis
Authors: Sung Huang-Mo , Yeamans Gabriel , Ross Christian A. , Yasbin Ronald E. 
Published: March 2003

ABSTRACT

YqjH and YqjW are
Bacillus
subtilis
homologs of the UmuC/DinB or Y superfamily of DNA polymerases that are involved in SOS-induced mutagenesis in
Escherichia
coli
. While the functions of YqjH and YqjW in
B
.
subtilis
are still unclear, the comparisons of protein structures demonstrate that YqjH has 36% identity to
E
.
coli
DNA polymerase IV (DinB protein), and YqjW has 26% identity to
E
.
coli
DNA polymerase V (UmuC protein). In this report, we demonstrate that both YqjH and the products of the
yqjW
operon are involved in UV-induced mutagenesis in this bacterium. Furthermore, resistance to UV-induced damage is significantly reduced in cells lacking a functional YqjH protein. Analysis of stationary-phase mutagenesis indicates that absences of YqjH, but not that of YqjW, decreases the ability of
B
.
subtilis
to generate revertants at the
hisC952
allele via this system. These data suggest a role for YqjH in the generation of at least some types of stationary-phase-induced mutagenesis.
Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from
Moritella
abyssi
Authors: Xu Ying , Feller Georges , Gerday Charles , Glansdorff Nicolas 
Published: March 2003

ABSTRACT

The enzyme ornithine carbamoyltransferase (OTCase) of
Moritella
abyssi
(OTCase
Mab
), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase
Mab
displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from
Escherichia
coli
or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric
Pyrococcus
furiosus
OTCase and for a thermolabile trimeric mutant thereof. OTCase
Mab
displays higher

K
m

values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-
N
-phosphonoacetyl-
l
-ornithine (PALO). OTCase
Mab
differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high

K
m

values and the lower sensitivity to PALO. The

K
m

for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (
k
cat
/

K
m

) of OTCases adapted to different temperatures showed that OTCase
Mab
activity remains suboptimal at low temperature despite the 4.5-fold decrease in the

K
m

value for ornithine observed when the temperature is brought from 20 to 5°C. OTCase
Mab
adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.
Enterococcus faecalisPlasmid pAD1-Encoded Fst Toxin Affects Membrane Permeability and Alters Cellular Responses to Lantibiotics
Authors: Weaver Keith E. , Weaver Dariel M. , Wells Carol L. , Waters Christopher M. , Gardner Marshall E. , Ehli Erik A. 
Published: March 2003

ABSTRACTFst is a peptide toxin encoded by thepartoxin-antitoxin stability determinant ofEnterococcus faecalisplasmid pAD1. Intracellular overproduction of Fst resulted in simultaneous inhibition of all cellular macromolecular synthesis concomitant with cell growth inhibition and compromised the integrity of the cell membrane. Cells did not lyse or noticeably leak intracellular contents but had specific defects in chromosome partitioning and cell division. Extracellular addition of synthetic Fst had no effect on cell growth. Spontaneous Fst-resistant mutants had a phenotype consistent with changes in membrane composition. Interestingly, overproduction of Fst sensitized cells to the lantibiotic nisin, and Fst-resistant mutants were cross-resistant to nisin and the pAD1-encoded cytolysin.
Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of
Escherichia coli
Authors: Ghosh Anindya S. , Young Kevin D. 
Published: March 2003

ABSTRACT

Penicillin binding protein (PBP) 5, a
dd
-carboxypeptidase that removes the terminal
d
-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of
Escherichia coli
. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant,
E. coli
CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp
218
and Lys
219
residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of
E. coli
PBP 5.
The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from theMyxococcus xanthusChromosome
Authors: Tojo Nobuki , Komano Teruya 
Published: March 2003

ABSTRACTDuring lysogenization of myxophage Mx8, phage DNA can be integrated into theattBsite of theMyxococcus xanthuschromosome through site-specific recombination. We previously demonstrated that the Mx8attPsite is located within the coding sequence of the Mx8intPgene. Hence, the integration of Mx8 into theM. xanthuschromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of theintP-attPorintR-attRregions as well as thelacZgene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designatedxiswas shown to be required in addition tointR.
Architecture of a Fur Binding Site: a Comparative Analysis
Authors: Lavrrar Jennifer L. , McIntosh Mark A. 
Published: March 2003

ABSTRACT

Fur is an iron-binding transcriptional repressor that recognizes a 19-bp consensus site of the sequence 5′-GATAATGATAATCATTATC-3′. This site can be defined as three adjacent hexamers of the sequence 5′-GATAAT-3′, with the third being slightly imperfect (an F-F-F configuration), or as two hexamers in the forward orientation separated by one base pair from a third hexamer in the reverse orientation (an F-F-x-R configuration). Although Fur can bind synthetic DNA sequences containing the F-F-F arrangement, most natural binding sites are variations of the F-F-x-R arrangement. The studies presented here compared the ability of Fur to recognize synthetic DNA sequences containing two to four adjacent hexamers with binding to sequences containing variations of the F-F-x-R arrangement (including natural operator sequences from the
entS
and
fepB
promoter regions of
Escherichia coli
). Gel retardation assays showed that the F-F-x-R architecture was necessary for high-affinity Fur-DNA interactions and that contiguous hexamers were not recognized as effectively. In addition, the stoichiometry of Fur at each binding site was determined, showing that Fur interacted with its minimal 19-bp binding site as two overlapping dimers. These data confirm the proposed overlapping-dimer binding model, where the unit of interaction with a single Fur dimer is two inverted hexamers separated by a C:G base pair, with two overlapping units comprising the 19-bp consensus binding site required for the high-affinity interaction with two Fur dimers.
Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR
Authors: Wallecha Anu , Correnti Jason , Munster Vincent , van der Woude Marjan 
Published: March 2003

ABSTRACT

OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of
agn43
from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of
agn43
. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses
agn43
transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of
agn43.
OxyR(ox) repressed in vitro transcription but only from an unmethylated
agn43
template. The −10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the
agn43
regulatory region DNA with an affinity similar to that for the regulatory regions of
katG
and
oxyS
, which are activated by OxyR(ox), indicating that binding at
agn43
can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at
agn43
is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.
Interaction of the Initiator Protein of an IncB Plasmid with Its Origin of DNA Replication
Authors: Betteridge T. , Yang J. , Pittard A. J. , Praszkier J. 
Published: March 2003

ABSTRACT

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5′AANCNGCAA3′. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to
ori
B
interact with one another.
Involvement of the Multidomain Regulatory Protein XynR in Positive Control of Xylanase Gene Expression in the Ruminal Anaerobe
Prevotella bryantii
B
1
4
Authors: Miyazaki Kohji , Miyamoto Hiroyuki , Mercer Derry K. , Hirase Tatsuaki , Martin Jennifer C. , Kojima Yoichi , Flint Harry J. 
Published: March 2003

ABSTRACT

The xylanase gene cluster from the rumen anaerobe
Prevotella bryantii
B
1
4 was found to include a gene (
xynR
) that encodes a multidomain regulatory protein and is downstream from the xylanase and β-xylosidase genes
xynA
and
xynB.
Additional genes identified upstream of
xynA
and
xynB
include
xynD
, which encodes an integral membrane protein that has homology with Na:solute symporters;
xynE
, which is related to the genes encoding acylhydrolases and arylesterases; and
xynF
, which has homology with the genes encoding α-glucuronidases. XynR includes, in a single 833-amino-acid polypeptide, a putative input domain unrelated to other database sequences, a likely transmembrane domain, histidine kinase motifs, response regulator sequences, and a C-terminal AraC-type helix-turn-helix DNA binding domain. Two transcripts (3.7 and 5.8 kb) were detected with a
xynA
probe, and the start site of the 3.7-kb transcript encoding
xynABD
was mapped to a position upstream of
xynD
. The DNA binding domain of XynR was purified after amplification and overexpression in
Escherichia coli
and was found to bind to a 141-bp DNA fragment from the region immediately upstream of
xynD.
In vitro transcription assays demonstrated that XynR stimulates transcription of the 3.7-kb transcript. We concluded that XynR acts as a positive regulator that activates expression of
xynABD
in
P. bryantii
B
1
4. This is the first regulatory protein that demonstrates significant homology with the two-component regulatory protein superfamily and has been shown to be involved in the regulation of polysaccharidase gene expression.