bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunocytochemical Localization of a Calmodulinlike Protein in
Bacillus subtilis
Cells
Authors: Dominguez Delfina C. , Adams Hank , Hageman James H. 
Published: December 2019

ABSTRACT

To determine possible functions of the calmodulinlike protein of
Bacillus subtilis
, the time course of its expression during sporulation and its cellular localization were studied. The protein was expressed in a constitutive manner from the end of logarithmic growth through 8 h of sporulation as determined by antibody cross-reactivity immunoblots and enzyme-linked immunosorbent assays (ELISAs). In partially purified extracts, the immunopositive protein comigrated upon electrophoresis with a protein which selectively bound [
45
Ca]CaCl
2
, ruthenium red, and Stains-all. Previous studies showed increased extractability of the calmodulinlike protein from
B. subtilis
cells when urea and 2-mercaptoethanol were used in breakage buffers, implying that the protein might be partially associated with the membrane fraction. This was confirmed by demonstrating that isolated membrane vesicles of
B. subtilis
also gave positive immunological tests with Western blotting and ELISAs. To more precisely locate the protein in cells, thin sections of late-log-phase cells, sporulating cells, and free spores were reacted first with bovine brain anticalmodulin specific antibodies and then with gold-conjugated secondary antibodies; the thin sections were examined by transmission electron microscopy. The calmodulinlike protein was found almost exclusively associated with the cell envelope of these fixed, sectioned cells. A possible function of the calmodulinlike protein in sensing calcium ions or regulating calcium ion transport is suggested.
Alteration of the Repressor Activity of MarR, the Negative Regulator of the
Escherichia coli marRAB
Locus, by Multiple Chemicals In Vitro
Authors: Alekshun Michael N. , Levy Stuart B. 
Published: December 2019

ABSTRACT

MarR negatively regulates expression of the multiple antibiotic resistance operon (
marRAB
) in
Escherichia coli
. In this study, it was demonstrated that sodium salicylate, plumbagin, 2,4-dinitrophenol, and menadione–inducers of the
marRAB
operon in whole cells–all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.
Growth and Glucose Repression Are Controlled by Glucose Transport in
Saccharomyces cerevisiae
Cells Containing Only One Glucose Transporter
Authors: Ye Ling , Kruckeberg Arthur L. , Berden Jan A. , van Dam Karel 
Published: December 2019

ABSTRACT

A set of
Saccharomyces cerevisiae
strains with variable expression of only the high-affinity Hxt7 glucose transporter was constructed by partial deletion of the
HXT7
promoter in vitro and integration of the gene at various copy numbers into the genome of an
hxt1-7 gal2
deletion strain. The glucose transport capacity increased in strains with higher levels of
HXT7
expression. The consequences for various physiological properties of varying the glucose transport capacity were examined. The control coefficient of glucose transport with respect to growth rate was 0.54. At high extracellular glucose concentrations, both invertase activity and the rate of oxidative glucose metabolism increased manyfold with decreasing glucose transport capacity, which is indicative of release from glucose repression. These results suggest that the intracellular glucose concentration produces the signal for glucose repression.
Succinate Dehydrogenase (Sdh) from
Bradyrhizobium japonicum
Is Closely Related to Mitochondrial Sdh
Authors: Westenberg David J. , Guerinot Mary Lou 
Published: December 2019

ABSTRACT

The
sdhCDAB
operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont
Bradyrhizobium japonicum
. Sdh from
B. japonicum
is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in
Escherichia coli
.
The
Streptomyces peucetius dpsC
Gene Determines the Choice of Starter Unit in Biosynthesis of the Daunorubicin Polyketide
Authors: Bao Wuli , Sheldon Paul J. , Wendt-Pienkowski Evelyn , Hutchinson C. Richard 
Published: December 2019

ABSTRACT

The starter unit used in the biosynthesis of daunorubicin is propionyl coenzyme A (CoA) rather than acetyl-CoA, which is used in the production of most of the bacterial aromatic polyketides studied to date. In the daunorubicin biosynthesis gene cluster of
Streptomyces peucetius
, directly downstream of the genes encoding the β-ketoacyl:acyl carrier protein synthase subunits, are two genes,
dpsC
and
dpsD
, encoding proteins that are believed to function as the starter unit-specifying enzymes. Recombinant strains containing plasmids carrying
dpsC
and
dpsD
, in addition to other daunorubicin polyketide synthase (PKS) genes, incorporate the correct starter unit into polyketides made by these genes, suggesting that, contrary to earlier reports, the enzymes encoded by
dpsC
and
dpsD
play a crucial role in starter unit specification. Additionally, the results of a cell-free synthesis of 21-carbon polyketides from propionyl-CoA and malonyl-CoA that used the protein extracts of recombinant strains carrying other daunorubicin PKS genes to which purified DpsC was added suggest that this enzyme has the primary role in starter unit discrimination for daunorubicin biosynthesis.
Glucose Metabolism in
gcr
Mutants of
Saccharomyces cerevisiae
Authors: Uemura H. , Fraenkel D. G. 
Published: December 2019

ABSTRACT

A
gcr2
null mutant of
Saccharomyces cerevisiae
grows well on glucose in spite of its lower level of glycolytic enzymes between triose phosphates and pyruvate. A quantitative analysis shows that these levels are adequate to the flux but glycerate phosphates are elevated.
Staphylococcus aureus cap5P Encodes a UDP- N -Acetylglucosamine 2-Epimerase with Functional Redundancy
Authors: Kiser Kevin B. , Bhasin Navneet , Deng Lingyi , Lee Jean C. 
Published: December 2019

ABSTRACTThe serotype 5 capsule gene cluster ofStaphylococcus aureuscomprises 16 genes (cap5Athroughcap5P), but little is known about how the putative gene products function in capsule biosynthesis. We propose that theN-acetylmannosaminuronic acid (ManNAcA) component of theS. aureusserotype 5 capsular polysaccharide (CP5) is synthesized from a UDP-N-acetylglucosamine (UDP-GlcNAc) precursor that is epimerized to UDP-N-acetylmannosamine (UDP-ManNAc) and then oxidized to UDP-ManNAcA. We report the purification and biochemical characterization of a recombinant UDP-GlcNAc 2-epimerase encoded byS. aureus cap5P. Purified Cap5P converted ∼10% of UDP-GlcNAc to UDP-ManNAc as detected by gas chromatography-mass spectrometry. The epimerization of UDP-GlcNAc to UDP-ManNAc occurred over a wide pH range and was unaffected by divalent cations. Surprisingly, CP5 expression inS. aureuswas unaffected by insertional inactivation ofcap5P. Sequence homology searches of the publicS. aureusgenomic databases revealed the presence of another putative UDP-GlcNAc 2-epimerase on theS. aureuschromosome that showed 61% identity to Cap5P. Redundancy of UDP-GlcNAc 2-epimerase function inS. aureuswas demonstrated by cloning thecap5Phomologue from strain Newman and complementing anEscherichia coli rffEmutant defective in UDP-GlcNAc 2-epimerase activity. Our results confirm the putative function of theS. aureus cap5Pgene product and demonstrate the presence of a second gene on the staphylococcal chromosome with a similar function.
Complementary and Overlapping Selectivity of the Two-Peptide Bacteriocins Plantaricin EF and JK
Authors: Moll Gert N. , van den Akker Emile , Hauge Håvard H. , Nissen-Meyer Jon , Nes Ingolf F. , Konings Wil N. , Driessen Arnold J. M. 
Published: December 2019

ABSTRACTPlantaricin EF and JK are both two-peptide bacteriocins produced byLactobacillus plantarumC11. The mechanism of plantaricin EF and JK action was studied onL. plantarum965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Δψ) and pH gradient (ΔpH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate thatL. plantarumC11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.
The Adherence-Associated Lipoprotein P100, Encoded by an opp Operon Structure, Functions as the Oligopeptide-Binding Domain OppA of a Putative Oligopeptide Transport System in Mycoplasma hominis
Authors: Henrich Birgit , Hopfe Miriam , Kitzerow Annette , Hadding Ulrich 
Published: December 2019

ABSTRACTMycoplasma hominis, a cell-wall-less prokaryote, was shown to be cytoadherent by the participation of a 100-kDa membrane protein (P100). To identify the gene encoding P100, peptides of P100 were partially sequenced to enable the synthesis of P100-specific oligonucleotides suitable as probes for the detection of the P100 gene. With this strategy, we identified a genomic region of about 10.4 kb inM. hominisFBG carrying the P100 gene. Analysis of the complete deduced protein sequence suggests that P100 is expressed as a pre-lipoprotein with a structure in the N-terminal region common to peptide-binding proteins and an ATP- or GTP-binding P-loop structure in the C-terminal region. Downstream of the P100 gene, an additional four open reading frames putatively encoding the four core domains of an active transport system, OppBCDF, were localized. The organization of the P100 gene andoppBCDFin a transcriptionally active operon structure was demonstrated in Northern blot and reverse transcription-PCR analyses, as all gene-specific probes detected a common RNA of 9.5 kb. Primer extension analysis revealed that the transcriptional initiation site was localized 323 nucleotides upstream of the methionine-encoding ATG of the P100 gene. The peptide-binding character of the P100 protein was confirmed by fluorescence spectroscopy and strongly suggests that the cytoadherence-mediating lipoprotein P100 represents OppA, the substrate-binding domain of a peptide transport system inM. hominis.
Transcript Cleavage, Attenuation, and an Internal Promoter in the Rhodobacter capsulatus puc Operon
Authors: LeBlanc H. , Lang A. S. , Beatty J. T. 
Published: December 2019

ABSTRACT
The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in thepuf operon. Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII. We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies. Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur. A putative RNA stem-loop structure appears to attenuate transcription initiated at the pucoperon major promoter. We also found that a minorpucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa γ (PucE) protein.
Genome Scanning in Haemophilus influenzae for Identification of Essential Genes
Authors: Reich Karl A. , Chovan Linda , Hessler Paul 
Published: December 2019

ABSTRACTWe have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames ofHaemophilus influenzaeby PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.
Characterization of the yrbA Gene ofBacillus subtilis, Involved in Resistance and Germination of Spores
Authors: Takamatsu Hiromu , Kodama Takeko , Nakayama Tatsuo , Watabe Kazuhito 
Published: December 2019

ABSTRACTInsertional inactivation of theyrbAgene ofBacillus subtilisreduced the resistance of the mutant spores to lysozyme. TheyrbAmutant spores lost their optical density at the same rate as the wild-type spores upon incubation withl-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this mediumyrbAspores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis ofyrbAtranscripts in varioussigmutants indicated thatyrbAwas transcribed by RNA polymerase with ςEbeginning at 2 h after the start of sporulation. TheyrbApromoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intactyrbAmutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination ofyrbAspores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of theyrbAmutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.
Identification and Characterization ofhupT, a Gene Involved in Negative Regulation of Hydrogen Oxidation in Bradyrhizobium japonicum
Authors: Van Soom C. , Lerouge I. , Vanderleyden J. , Ruiz-Argüeso T. , Palacios J. M. 
Published: December 2019

ABSTRACTTheBradyrhizobium japonicum hupTgene was sequenced, and its gene product was found to be homologous to NtrB-like histidine kinases. AhupTmutant expresses higher levels of hydrogenase activity than the wild-type strain under hydrogenase-inducing conditions (i.e., microaerobiosis plus hydrogen, or symbiosis), whereas in noninducedhupTcells,hupSLexpression is derepressed but does not lead to hydrogenase activity. We conclude that HupT is involved in the repression of HupSL synthesis at the transcriptional level but that enzymatic activation requires inducing conditions.
Combined Genetic and Physical Map of the Complex Genome of
Agrobacterium tumefaciens
Authors: Goodner Brad W. , Markelz Brian P. , Flanagan M. Casey , Crowell Chris B. , Racette Jodi L. , Schilling Brittany A. , Halfon Leah M. , Mellors J. Scott , Grabowski Gregory 
Published: December 2019

ABSTRACT

A combined genetic and physical map of the
Agrobacterium tumefaciens
A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.
Timing of FtsZ Assembly in
Escherichia coli
Authors: Den Blaauwen Tanneke , Buddelmeijer Nienke , Aarsman Mirjam E. G. , Hameete Cor M. , Nanninga Nanne 
Published: December 2019

ABSTRACT

The timing of the appearance of the FtsZ ring at the future site of division in
Escherichia coli
was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.
Amino Acid-DNA Contacts by RhaS: an AraC Family Transcription Activator
Authors: Bhende Prasanna M. , Egan Susan M. 
Published: December 2019

ABSTRACT

RhaS, an AraC family protein, activates
rhaBAD
transcription by binding to
rhaI
, a site consisting of two 17-bp inverted repeat half-sites. In this work, amino acids in RhaS that make base-specific contacts with
rhaI
were identified. Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix. Assays of
rhaB-lacZ
activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact
rhaI
. The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites. We first made targeted, random mutations and then made alanine substitutions within this region of RhaS. Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding. A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts. In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp −65 and −67 in
rhaI
1
, and that Arg202 contacts −46 and Arg206 contacts −48 in
rhaI
2
. In motif 2, we found that Asp250 and Asn252 both contact the bp −79 in
rhaI
1
. Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.
Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of
Escherichia coli
: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite
Authors: Lee Angela I. , Delgado Asunción , Gunsalus Robert P. 
Published: December 2019

ABSTRACT

The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using
Escherichia coli
membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 μM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 μM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 μM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.
Identification of a Conserved N-Terminal Sequence Involved in Transmembrane Signal Transduction in EnvZ
Authors: Waukau Jill , Forst Steven 
Published: December 2019

ABSTRACT

To determine whether N-terminal sequences are involved in the transmembrane signaling mechanism of EnvZ, the nucleotide sequences of
envZ
genes from several enteric bacteria were determined. Comparative analysis revealed that the amino acid sequence between Pro41 and Glu53 was highly conserved. To further analyze the role of the conserved sequence,
envZ
of
Escherichia coli
was subjected to random PCR mutagenesis and mutant alleles that produced a high-osmolarity phenotype, in which
ompF
was repressed, were isolated. The mutations identified clustered within, as well as adjacent to, the Pro41-to-Glu53 sequence. These findings suggest that the conserved Pro41-to-Glu53 sequence is involved in the signal transduction mechanism of EnvZ.
Bacterial Conjugation Protein MobA Mediates Integration of Complex DNA Structures into Plant Cells
Authors: Bravo-Angel Ana María , Gloeckler Véronique , Hohn Barbara , Tinland Bruno 
Published: December 2019

ABSTRACT

Agrobacterium tumefaciens
transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by
Agrobacterium
to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5′ end. Isolation and analysis of the most conserved 5′ ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins.
Identification of a Functional
fur
Gene in
Bradyrhizobium japonicum
Authors: Hamza Iqbal , Hassett Richard , O’Brian Mark R. 
Published: December 2019

ABSTRACT

The recent identification of the iron response regulator (Irr) in
Bradyrhizobium japonicum
raised the question of whether the global regulator Fur is present in that organism. A
fur
gene homolog was isolated by the functional complementation of an
Escherichia coli fur
mutant. The
B. japonicum
Fur bound to a Fur box DNA element in vitro, and a
fur
mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus,
B. japonicum
expresses at least two regulators of iron metabolism.