bluuebunny/crossref_metadata_2025_split huggingface dataset:
MppA, a Periplasmic Binding Protein Essential for Import of the Bacterial Cell Wall Peptide
l
-Alanyl-γ-
d
-Glutamyl-
meso
-Diaminopimelate
Authors: Park James T. , Raychaudhuri Debabrata , Li Hongshan , Normark Staffan , Mengin-Lecreulx Dominique 
Published: December 2019

ABSTRACT

Mutants of a diaminopimelic acid (Dap)-requiring strain of
Escherichia coli
were isolated which failed to grow on media in which Dap was replaced by the cell wall murein tripeptide,
l
-alanyl-γ-
d
-glutamyl-
meso
-diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a new gene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, the periplasmic binding protein for Opp. A plasmid carrying the wild-type
mppA
gene allows the mutant to grow on tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity, indicating a defect in the
opp
operon. An
E. coli
strain whose entire
opp
operon was deleted but which carried the
mppA
locus was unable to grow on murein tripeptide unless it was provided with
oppBCDF
genes in
trans
. Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then transported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppA for non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys, which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, and perhaps the third OppA paralog revealed by the
E. coli
genome sequence may each bind a particular family of peptides but interact with common membrane-associated components for transport of their bound ligands into the cell. As to the physiological function of MppA, the possibility that it may be involved in signal transduction pathway(s) is discussed.
fbfB
, a Gene Encoding a Putative Galactose Oxidase, Is Involved in
Stigmatella aurantiaca
Fruiting Body Formation
Authors: Silakowski Barbara , Ehret Heidi , Schairer Hans Ulrich 
Published: December 2019

ABSTRACT

Stigmatella aurantiaca
is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn
5
-derived transposon. The gene affected (
fbfB
) in one of the mutants (AP182) was studied further. Inactivation of
fbfB
results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in
fbfB
function are mixed with those of some independent mutants defective in fruiting before starvation. The
fbfB
gene is expressed about 14 h after induction of fruiting body formation as determined by measuring β-galactosidase activity in a merodiploid strain harboring the wild-type gene and an
fbfB-Δtrp-lacZ
fusion gene or by Northern (RNA) analysis with the
Rhodobacter capsulatus pufBA
fragment fused to
fbfB
as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus
Dactylium dendroides
. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.
Molecular Characterization of the α-Glucosidase Gene (malA) from the Hyperthermophilic ArchaeonSulfolobus solfataricus
Authors: Rolfsmeier Michael , Haseltine Cynthia , Bini Elisabetta , Clark Amy , Blum Paul 
Published: December 2019

ABSTRACTAcidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences fromSulfolobus solfataricuswere cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain.malAis 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of themalAregion identified transcripts formalAand an upstream open reading frame located 5′ to the 1-kb intergenic region. ThemalAtranscription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of themalAstart codon. Gel mobility shift analysis of themalApromoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role inmalAexpression.malAhomologs were detected by Southern blot analysis in otherS. solfataricusstrains and inSulfolobus shibatae, while no homologs were evident inSulfolobus acidocaldarius, lending further support to the proposed revision of the genusSulfolobus. Phylogenetic analyses indicate that the closestS. solfataricusα-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified fromEscherichia colirevealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for theS. solfataricusα-glucosidase in carbohydrate metabolism.
Opposite Patterns of Expression of Two
Aspergillus nidulans
Xylanase Genes with Respect to Ambient pH
Authors: Maccabe A. P. , Orejas M. , Pérez-González J. A. , Ramón D. 
Published: December 2019

ABSTRACT

The
Aspergillus nidulans
xylanase genes
xlnA
and
xlnB
are subject to regulation by ambient pH via the zinc finger transcription factor PacC. In the presence of
d
-xylose,
xlnA
is expressed under conditions of alkaline ambient pH while
xlnB
is expressed at acidic ambient pH. These data have been confirmed for acidity- and alkalinity-mimicking
A. nidulans
mutants.
Contribution of the Disulfide Bond of the A Subunit to the Action of
Escherichia coli
Heat-Labile Enterotoxin
Authors: Okamoto Keinosuke , Nomura Tomohiko , Fujii Yoshio , Yamanaka Hiroyasu 
Published: December 2019

ABSTRACT

Escherichia coli
heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.
Physiological and Genetic Analyses Leading to Identification of a Biochemical Role for the
moeA
(Molybdate Metabolism) Gene Product in
Escherichia coli
Authors: Hasona Adnan , Ray Ramesh M. , Shanmugam K. T. 
Published: December 2019

ABSTRACT

A unique class of chlorate-resistant mutants of
Escherichia coli
which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the
moeA
gene. Mutant strains with polar mutations in
moeA
which are also
moeB
did not produce active molybdoenzymes in any of the media tested.
moeA
mutants with a second mutation in either
cysDNCJI
or
cysH
gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of
moeA cys
double mutants suppressed the MoeA
−
phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.
Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon
Methanosarcina thermophila
Authors: Maupin-Furlow Julie A. , Aldrich Henry C. , Ferry James G. 
Published: December 2019

ABSTRACT

The 20S proteasome from the methanoarchaeon
Methanosarcina thermophila
was produced in
Escherichia coli
and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in
Escherichia coli
, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α
7
ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced
M. thermophila
20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr
1
residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys
33
with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys
33
in polarization of the Thr
1
N, which serves to strip a proton from the active-site Thr
1
Oγ nucleophile. Replacement of Asp
51
with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.
Lambda Xis Degradation In Vivo by Lon and FtsH
Authors: Leffers Gerald G. , Gottesman Susan 
Published: December 2019

ABSTRACT

Lambda Xis, which is required for site-specific excision of phage lambda from the bacterial chromosome, has a much shorter functional half-life than Int, which is required for both integration and excision (R. A. Weisberg and M. E. Gottesman, p. 489–500,
in
A. D. Hershey, ed.,
The Bacteriophage Lambda
, 1971). We found that Xis is degraded in vivo by two ATP-dependent proteases, Lon and FtsH (HflB). Xis was stabilized two- to threefold more than in the wild type in a
lon
mutant and as much as sixfold more in a
lon ftsH
double mutant at the nonpermissive temperature for the
ftsH
mutation. Integration of lambda into the bacterial chromosome was delayed in the
lon ftsH
background, suggesting that accumulation of Xis in vivo interferes with integration. Overexpression of Xis in wild-type cells from a multicopy plasmid inhibited integration of lambda and promoted curing of established lysogens, confirming that accumulation of Xis interferes with the ability of Int to establish and maintain an integrated prophage.
Identification and Characterization of a Novel Competence Gene,
comC
, Required for DNA Binding and Uptake in
Acinetobacter
sp. Strain BD413
Authors: Link Caroline , Eickernjäger Sandra , Porstendörfer Dirk , Averhoff Beate 
Published: December 2019

ABSTRACT

A gene (
comC
) essential for natural transformation was identified in
Acinetobacter
sp. strain BD413. ComC has a typical leader sequence and is similar to different type IV pilus assembly factors. A
comC
mutant (T308) is not able to bind or take up DNA but exhibits a piliation phenotype indistinguishable from the transformation wild type as revealed by electron microscopy.
Characterization of the
cvaA
and
cvi
Promoters of the Colicin V Export System: Iron-Dependent Transcription of
cvaA
Is Modulated by Downstream Sequences
Authors: Boyer Anne E. , Tai Phang C. 
Published: December 2019

ABSTRACT

Secretion of the
Escherichia coli
toxin colicin V was previously determined to be iron regulated via the Fur (ferric uptake regulator) protein, based on studies in
fur
mutants. The iron dependence of transcription and expression of
cvaA
, which encodes a transporter accessory protein, and
cvi
, encoding the colicin V immunity protein, was assessed under conditions of iron excess or depletion. Immunoblots showed that production of both Cvi and CvaA is iron dependent. The iron-dependent transcriptional start for
cvaA
identified by primer extension and S1 nuclease analysis, P1, lies 320 bp upstream of the translational start and is associated with a newly identified Fur binding site. β-Galactosidase activity in transcriptional
lacZ
fusions with the P1 promoter alone is higher than with downstream sequences present and is induced 10-fold by iron depletion. Including immediate downstream regions with P1 enhances activity from P1 even more but reduces the induction by iron depletion fivefold. Including subsequent downstream sequences, however, down-modulates overall transcription from P1 almost fourfold. Deletion of a long stem-loop structure in this region alleviates the down-modulation by increasing transcription, indicating that the sequences or structure of this element may contribute to this down-regulation. Characterization of the
cvi
promoter by primer extension showed that it resides where predicted, about 50 bp upstream of
cvi
associated with a previously identified Fur binding site. The
cvi
promoter is also inducible by iron depletion. The modulating sequences from
cvaA
were placed downstream of the
cvi
promoter to test their effects in transcriptional fusions of the
cvi
promoter to
lacZ
. The fusion results showed that these sequences also modulate transcription of the
cvi
promoter in a manner similar to that of the
cvaA
promoter. The potential for up- and down-regulation within the long untranslated region downstream of the
cvaA
promoter suggests a novel mechanism that fine-tunes expression of the colicin V secretion genes.
Characterization of thebvgRLocus ofBordetella pertussis
Authors: Merkel Tod J. , Barros Cassia , Stibitz Scott 
Published: December 2019

ABSTRACTBordetella pertussis, the causative agent of whooping cough, produces a wide array of factors that are associated with its ability to cause disease. The expression and regulation of these virulence factors is dependent upon thebvglocus (originally designated thevirlocus), which encodes two proteins: BvgA, a 23-kDa cytoplasmic protein, and BvgS, a 135-kDa transmembrane protein. It is proposed that BvgS responds to environmental signals and interacts with BvgA, a transcriptional regulator which upon modification by BvgS binds to specific promoters and activates transcription. An additional class of genes is repressed by thebvglocus. Expression of this class, thebvg-repressed genes (vrgs [forvir-repressed genes]), is reduced under conditions in which expression of the aforementionedbvg-activated virulence factors is maximal; this repression is dependent upon the presence of an intactbvgASlocus. We have previously identified a locus required for regulation of all of the knownbvg-repressed genes inB. pertussis. This locus, designatedbvgR, maps to a location immediately downstream ofbvgAS. We have undertaken deletion and complementation studies, as well as sequence analysis, in order to identify thebvgRopen reading frame and identify thecis-acting sequences required for regulated expression ofbvgR. Studies utilizing transcriptional fusions ofbvgRto the gene encoding alkaline phosphatase have demonstrated thatbvgRis activated at the level of transcription and that this activation is dependent upon an intactbvgASlocus.
Mutation of a Gene Encoding a Putative Glycoprotease Leads to Reduced Salt Tolerance, Altered Pigmentation, and Cyanophycin Accumulation in the CyanobacteriumSynechocystissp. Strain PCC 6803
Authors: Zuther Ellen , Schubert Hendrik , Hagemann Martin 
Published: December 2019

ABSTRACTThe salt-sensitive mutant 549 of the cyanobacteriumSynechocystissp. strain PCC 6803 was genetically and physiologically characterized. The mutated site and corresponding wild-type site were cloned and partially sequenced. The genetic analysis revealed that during the mutation about 1.8 kb was deleted from the chromosome of mutant 549. This deletion affected four open reading frames: agcpgene homolog, thepsaFJgenes, and an unknown gene. After construction of mutants with single mutations, only thegcpmutant showed a reduction in salt tolerance comparable to that of the initial mutant, indicating that the deletion of this gene was responsible for the salt sensitivity and that the other genes were of minor importance. Besides the reduced salt tolerance, a remarkable change in pigmentation was observed that became more pronounced in salt-stressed cells. The phycobilipigment content decreased, and that of carotenoids increased. Investigations of changes in the ultrastructure revealed an increase in the amount of characteristic inclusion bodies containing the high-molecular-weight nitrogen storage polymer cyanophycin (polyaspartate and arginine). The salt-induced accumulation of cyanophycin was confirmed by chemical estimations. The putative glycoprotease encoded by thegcpgene might be responsible for the degradation of cyanophycin inSynechocystis. Mutation of this gene leads to nitrogen starvation of the cells, accompanied by characteristic changes in pigmentation, ultrastructure, and salt tolerance level.
Characterization of the Starvation-Survival Response of
Staphylococcus aureus
Authors: Watson Sean P. , Clements Mark O. , Foster Simon J. 
Published: December 2019

ABSTRACT

The starvation-survival response of
Staphylococcus aureus
as a result of glucose, amino acid, phosphate, or multiple-nutrient limitation was investigated. Glucose and multiple-nutrient limitation resulted in the loss of viability of about 99 to 99.9% of the population within 2 days. The remaining surviving cells developed increased survival potential, remaining viable for months. Amino acid or phosphate limitation did not lead to the development of a stable starvation-survival state, and cells became nonculturable within 7 days. For multiple-nutrient limitation, the development of the starvation-survival state was cell density dependent. Starvation survival was associated with a decrease in cell size and increase in resistance to acid shock and oxidative stress. There was no evidence for the formation of a viable but nonculturable state during starvation as demonstrated by flow cytometry. Long-term survival of cells was dependent on cell wall and protein biosynthesis. Analysis of [
35
S]methionine incorporation and labelled proteins demonstrated that differential protein synthesis occurred deep into starvation.
The TATA-Binding Protein (TBP) from the Human Fungal PathogenCandida albicansCan Complement Defects in Human and Yeast TBPs
Authors: Leng Ping , Carter Philip E. , Brown Alistair J. P. 
Published: December 2019

ABSTRACTCandida albicansis the major fungal pathogen in humans, yet little is known about transcriptional regulation in this organism. Therefore, we have isolated, characterized, and expressed theC. albicansTATA-binding protein (TBP) gene (TBP1), because this general transcription initiation factor plays a key role in the activation and regulation of eukaryotic promoters. Southern and Northern blot analyses suggest that a singleC. albicans TBP1locus is expressed at similar levels in the yeast and hyphal forms of this fungus. TheTBP1open reading frame is 716 bp long and encodes a functional TBP of 27 kDa.C. albicansTBP is capable of binding specifically to a TATA box in vitro, substituting for the human TBP to activate basal transcription in vitro, and suppressing the lethal Δspt15mutation inSaccharomyces cerevisiae. The predicted amino acid sequences of TBPs fromC. albicansand other organisms reveal a striking pattern of C-terminal conservation and N-terminal variability: the C-terminal DNA-binding domain displays at least 80% amino acid sequence identity to TBPs from fungi, flies, nematodes, slime molds, plants, and humans. Sequence differences between human and fungal TPBs in the DNA-binding domain may represent potential targets for antifungal therapy.
Positive and Negative Transcriptional Regulation of the
Escherichia coli
Gluconate Regulon Gene
gntT
by GntR and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Authors: Peekhaus Norbert , Conway T. 
Published: December 2019

ABSTRACT

The
gntT
gene of
Escherichia coli
is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor of
gntT
expression since gluconate is a catabolite-repressing sugar. In a
gntR
deletion mutant, the expression of a chromosomal
gntT
::
lacZ
fusion is both high and constitutive, confirming that GntR is the negative regulator of
gntT
. Indeed, GntR binds to two consensus
gnt
operator sites; one overlaps the −10 region of the
gntT
promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when
gntR
deletion strains are grown in the presence of gluconate, there is a twofold decrease in
gntT
expression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of
gntR
mutants to the inducer is termed ultrarepression. Transcription of
gntT
is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the
gntT
transcription start site.