bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ferric Citrate Transport of
Escherichia coli
: Functional Regions of the FecR Transmembrane Regulatory Protein
Authors: Welz Dietrich , Braun Volkmar 
Published: December 2019

ABSTRACT

Transcription of the ferric citrate transport genes of
Escherichia coli
is induced by ferric citrate bound to the outer membrane receptor FecA. Additional ferric citrate-specific regulatory proteins are FecR in the cytoplasmic membrane and the FecI sigma factor in the cytoplasm. To further understand the assumed FecR-mediated signal transduction across the cytoplasmic membrane, the transmembrane topology of FecR (317 amino acids) was determined with hybrid proteins containing portions of FecR and mature BlaM β-lactamase. BlaM fused to FecR regions extending from residues 107 to 149 and residues 230 to 259 conferred high ampicillin resistance to cells, while BlaM fused to sites between residues 159 and 210 and between residues 265 and 301 conferred low resistance. Cells that synthesized FecR′-BlaM with fusion joints between residues 8 and 81 of FecR were fully sensitive to ampicillin. The ampicillin resistance of the low-resistance FecR′-BlaM hybrids was increased 2- to 10-fold by cosynthesis of plasmid-encoded GroEL GroES and SecB chaperones and in
degP
and
ompT
protease mutants, which suggested that the decreased ampicillin resistance level of these hybrids was caused by the formation of inclusion bodies and proteolytic degradation. Replacement of glycine by aspartate residues in the only hydrophobic FecR sequence (residues 85 to 100) abolished the β-lactamase activity of high-resistance FecR′-BlaM proteins, indicating that there are no other transmembrane regions in FecR that translocate BlaM into the periplasm independent of the hydrophobic sequence. All FecR′-BlaM proteins with at least 61 FecR residues complemented a
fecR
mutant such that it could grow on ferric citrate as the sole iron source and induced
fecA-lacZ
transcription independent of ferric citrate. The low resistance mediated by two FecR′-BlaM proteins in a
fecA
deletion mutant was increased 20-fold by transformation with a
fecA
-encoding plasmid. We propose that FecR spans the cytoplasmic membrane once, interacts in the periplasm with its C-terminal region with FecA occupied by ferric citrate, and transmits the information through the cytoplasmic membrane into the cytoplasm, where it converts FecI into an active sigma factor.
The Prosequence of Pro-ς
K
Promotes Membrane Association and Inhibits RNA Polymerase Core Binding
Authors: Zhang Bin , Hofmeister Antje , Kroos Lee 
Published: December 2019

ABSTRACT

Pro-ς
K
is the inactive precursor of ς
K
, a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of
Bacillus subtilis
. Upon subcellular fractionation, the majority of the pro-ς
K
was present in the membrane fraction. The rest of the pro-ς
K
was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ς
K
was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-ς
E
was analyzed. Pro-ς
K
was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-ς
K
did not require
spoIVF
gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-ς
K
processing in the mother cell. Furthermore, pro-ς
K
associated with the membrane when overproduced in vegetative cells. Overproduction of pro-ς
K
in sporulating cells resulted in more pro-ς
K
in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-ς
K
was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-ς
K
. Treatment of extracts with 0.6 M KCl appeared to free most of the pro-ς
K
and ς
K
from other cell constituents. After salt removal, ς
K
, but not pro-ς
K
, reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-ς
K
to the membrane, where it may interact with the processing machinery.
Purification, Properties, and Characterization of Recombinant
Streptomyces
sp. Strain C5 DoxA, a Cytochrome P-450 Catalyzing Multiple Steps in Doxorubicin Biosynthesis
Authors: Walczak Robbie J. , Dickens Michael L. , Priestley Nigel D. , Strohl William R. 
Published: December 2019

ABSTRACT

DoxA is a cytochrome P-450 monooxygenase involved in the late stages of daunorubicin and doxorubicin biosynthesis that has a broad substrate specificity for anthracycline glycone substrates. Recombinant DoxA was purified to homogeneity from
Streptomyces lividans
transformed with a plasmid containing the
Streptomyces
sp. strain C5
doxA
gene under the control of the strong SnpR-activated
snpA
promoter. The purified enzyme was a monomeric, soluble protein with an apparent
M
r
of 47,000. Purified DoxA catalyzed the 13-hydroxylation of 13-deoxydaunorubicin, the 13-oxidation of 13-dihydrocarminomycin and 13-dihydrodaunorubicin, and the 14-hydroxylation of daunorubicin. The pH optimum for heme activation was pH 7.5, and the temperature optimum was 30°C. The
k
cat
/

K
m

values for the oxidation of anthracycline substrates by purified DoxA, incubated with appropriate electron-donating components, were as follows: for 13-deoxydaunorubicin, 22,000 M
−1
· s
−1
; for 13-dihydrodaunorubicin, 14,000 M
−1
· s
−1
; for 13-dihydrocarminomycin, 280 M
−1
· s
−1
; and for daunorubicin, 130 M
−1
· s
−1
. Our results indicate that the conversion of daunorubicin to doxorubicin by this enzyme is not a favored reaction and that the main anthracycline flux through the late steps of the daunorubicin biosynthetic pathway catalyzed by DoxA is likely directed through the 4-
O
-methyl series of anthracyclines.
( R )-Citramalate Synthase in Methanogenic Archaea
Authors: Howell David M. , Xu Huimin , White Robert H. 
Published: December 2019

ABSTRACT

The
Methanococcus jannaschii
gene MJ1392 was cloned, and its protein product was hyperexpressed in
Escherichia coli
. The resulting protein was purified and shown to catalyze the condensation of pyruvate and acetyl coenzyme A, with the formation of (
R
)-citramalate. Thus, this gene (
cimA
) encodes an (
R
)-citramalate synthase (CimA). This is the first identification of this enzyme, which is likely involved in the biosynthesis of isoleucine.
Septal Localization of Penicillin-Binding Protein 1 in
Bacillus subtilis
Authors: Pedersen Lotte B. , Angert Esther R. , Setlow Peter 
Published: December 2019

ABSTRACT

Previous studies have shown that
Bacillus subtilis
cells lacking penicillin-binding protein 1 (PBP1), encoded by
ponA
, have a reduced growth rate in a variety of growth media and are longer, thinner, and more bent than wild-type cells. It was also recently shown that cells lacking PBP1 require increased levels of divalent cations for growth and are either unable to grow or grow as filaments in media low in Mg
2+
, suggesting a possible involvement of PBP1 in septum formation under these conditions. Using epitope-tagging and immunofluorescence microscopy, we have now shown that PBP1 is localized at division sites in vegetative cells of
B. subtilis
. In addition, we have used fluorescence and electron microscopy to show that growing
ponA
mutant cells display a significant septation defect, and finally by immunofluorescence microscopy we have found that while FtsZ localizes normally in most
ponA
mutant cells, a significant proportion of
ponA
mutant cells display FtsZ rings with aberrant structure or improper localization, suggesting that lack of PBP1 affects FtsZ ring stability or assembly. These results provide strong evidence that PBP1 is localized to and has an important function in the division septum in
B. subtilis
. This is the first example of a high-molecular-weight class A PBP that is localized to the bacterial division septum.
Function of Proline Residues of MotA in Torque Generation by the Flagellar Motor of
Escherichia coli
Authors: Braun Timothy F. , Poulson Susan , Gully Jonathan B. , Empey J. Courtney , Van Way Susan , Putnam Angélica , Blair David F. 
Published: December 2019

ABSTRACT

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from
Escherichia coli
) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D
2
O versus H
2
O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.
Membrane Topology of MntB, the Transmembrane Protein Component of an ABC Transporter System for Manganese in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Bartsevich Victor V. , Pakrasi Himadri B. 
Published: December 2019

ABSTRACT

The structure of the membrane protein MntB, a component of a manganese transporter system in
Synechocystis
sp. strain PCC 6803, was examined with a series of fusions to the reporter proteins alkaline phosphatase and β-galactosidase. The results support a topological model for MntB consisting of nine transmembrane segments, with the amino terminus of the protein being in the periplasm and the carboxyl terminus being in the cytoplasm.
Increased
rrn
Gene Dosage Causes Intermittent Transcription of rRNA in
Escherichia coli
Authors: Voulgaris Justina , French Sarah , Gourse Richard L. , Squires Craig , Squires Catherine L. 
Published: December 2019

ABSTRACT

When the number of rRNA (
rrn
) operons in an
Escherichia coli
cells is increased by adding an
rrn
operon on a multicopy plasmid, the rate of rRNA expression per operon is reduced to maintain a constant concentration of rRNA in the cell. We have used electron microscopy to examine rRNA transcription in cells containing a multicopy plasmid carrying
rrnB
. We found that there were fewer RNA polymerase molecules transcribing the
rrn
genes, as predicted from previous gene dosage studies. Furthermore, RNA polymerase molecules were arranged in irregularly spaced groups along the operon. No apparent pause or transcription termination sites that would account for the irregular spacing of the groups of polymerase molecules were observed. We also found that the overall transcription elongation rate was unchanged when the
rrn
gene dosage was increased. Our data suggest that when
rrn
gene dosage is increased, initiation events, or promoter-proximal elongation events, are interrupted at irregular time intervals.
The
Yersinia enterocolitica
pYV Virulence Plasmid Contains Multiple Intrinsic DNA Bends Which Melt at 37°C
Authors: Rohde John R. , Luan Xing-she , Rohde Harold , Fox James M. , Minnich S. A. 
Published: December 2019

ABSTRACT

Temperature has a pleiotropic effect on
Yersinia enterocolitica
gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (≤30°C) and virulence plasmid-encoded Yop secretion (37°C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the
Y. enterocolitica
virulence plasmid, pYV, undergoes a conformational transition between 30 and 37°C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including
virF
, the positive activator of virulence genes. These bends are detectable at 30°C but melt at 37°C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from
Y. enterocolitica
were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the
Y. enterocolitica
pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in
Y. enterocolitica
and other enteric pathogens.
Glucose Transport in the Extremely Thermoacidophilic
Sulfolobus solfataricus
Involves a High-Affinity Membrane-Integrated Binding Protein
Authors: Albers Sonja-V. , Elferink Marieke G. L. , Charlebois Robert L. , Sensen Christoph W. , Driessen Arnold J. M. , Konings Wil N. 
Published: December 2019

ABSTRACT

The archaeon
Sulfolobus solfataricus
grows optimally at 80°C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars. Cells rapidly accumulate glucose. This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (

K
d

of 0.43 μM) and retains high glucose affinity at very low pH values (as low as pH 0.6). The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein. The gene coding for the binding protein was identified in the
S. solfataricus
P2 genome by means of the amino-terminal amino acid sequence of the purified protein. Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment. Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in
Pyrococcus horikoshii
OT3 was found to be organized in an operon. These data indicate that
S. solfataricus
utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose.
Regulation of Gene Expression by Glucose inSaccharomyces cerevisiae: a Role for ADA2and ADA3/NGG1
Authors: Wu Mei , Newcomb Laura , Heideman Warren 
Published: December 2019

ABSTRACTWhenSaccharomyces cerevisiaecells are transferred from poor medium to fresh medium containing glucose, they rapidly increase the transcription of a large group of genes as they resume rapid growth and accelerate progress through the cell cycle. Among those genes induced by glucose isCLN3, encoding a G1cyclin that is thought to play a pivotal role in progression through Start. Deletion ofCLN3delays the increase in proliferation normally observed in response to glucose medium.ADA2andADA3/NGG1are necessary for the rapid induction ofCLN3message levels in response to glucose. Loss of eitherADA2orADA3/NGG1also affects a large number of genes and inhibits the rapid global increase in transcription that occurs in response to glucose. Surprisingly, these effects are transitory, and expression ofCLN3and total poly(A)+RNA appear normal whenADA2orADA3/NGG1deletion mutants are examined in log-phase growth. These results indicate a role forADA2andADA3/NGG1in allowing rapid transcriptional responses to environmental signals. Consistent with the role of the Ada proteins in positive regulation ofCLN3, deletion ofRPD3, encoding a histone deacetylase, prevented the down regulation ofCLN3mRNA in the absence of glucose.
Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange
Authors: Pavelka Martin S. , Jacobs William R. 
Published: December 2019

ABSTRACT
Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organismsMycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains ofM. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organismMycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.
Moraxella (Branhamella)catarrhalis BRO β-Lactamase: a Lipoprotein of Gram-Positive Origin?
Authors: Bootsma Hester J. , Aerts Piet C. , Posthuma George , Harmsen Theo , Verhoef Jan , van Dijk Hans , Mooi Frits R. 
Published: December 2019

ABSTRACTIn the past 20 years, BRO β-lactamase-producingMoraxella catarrhalisstrains have emerged. We show that BRO is expressed as a 33-kDa lipoprotein associated with the inner leaflet of the outer membrane. To our knowledge, this is the first description of a lipidated β-lactamase in a gram-negative species.
Escherichia coli Gene ydeA Encodes a Major Facilitator Pump Which Exports l-Arabinose and Isopropyl-β-d-Thiogalactopyranoside
Authors: Carolé Sandra , Pichoff Sébastien , Bouché Jean-Pierre 
Published: December 2019

ABSTRACT
Inactivation of the Escherichia coli geneydeA, which encodes a member of the major facilitator superfamily, decreased the efflux of l-arabinose, thereby affecting the expression of AraC-regulated genes. In addition, overexpression of ydeA decreased the expression of genes regulated by isopropyl-β-d-thiogalactopyranoside.
An Autoregulatory Circuit Affecting Peptide Signaling in
Bacillus subtilis
Authors: Lazazzera Beth A. , Kurtser Iren G. , McQuade Ryan S. , Grossman Alan D. 
Published: December 2019

ABSTRACT

The competence and sporulation factor (CSF) of
Bacillus subtilis
is an extracellular pentapeptide produced from the product of
phrC
. CSF has at least three activities: (i) at low concentrations, it stimulates expression of genes activated by the transcription factor ComA; at higher concentrations, it (ii) inhibits expression of those same genes and (iii) stimulates sporulation. Because the activities of CSF are concentration dependent, we measured the amount of extracellular CSF produced by cells. We found that by mid-exponential phase, CSF accumulated to concentrations (1 to 5 nM) that stimulate ComA-dependent gene expression. Upon entry into stationary phase, CSF reached 50 to 100 nM, concentrations that stimulate sporulation and inhibit ComA-dependent gene expression. Transcription of
phrC
was found to be controlled by two promoters: P1, which precedes
rapC
, the gene upstream of
phrC
; and P2, which directs transcription of
phrC
only. Both RapC and CSF were found to be part of autoregulatory loops that affect transcription from P1, which we show is activated by ComA∼P. RapC negatively regulates its own expression, presumably due to its ability to inhibit accumulation of ComA∼P. CSF positively regulates its own expression, presumably due to its ability to inhibit RapC activity. Transcription from P2, which is controlled by the alternate sigma factor ς
H
, increased as cells entered stationary phase, contributing to the increase in extracellular CSF at this time. In addition to controlling transcription of
phrC
, ς
H
appears to control expression of at least one other gene required for production of CSF.
Salmonella typhimurium
LT2 Catabolizes Propionate via the 2-Methylcitric Acid Cycle
Authors: Horswill Alexander R. , Escalante-Semerena Jorge C. 
Published: December 2019

ABSTRACT

We previously identified the
prpBCDE
operon, which encodes catabolic functions required for propionate catabolism in
Salmonella typhimurium
. Results from
13
C-labeling experiments have identified the route of propionate breakdown and determined the biochemical role of each Prp enzyme in this pathway. The identification of catabolites accumulating in wild-type and mutant strains was consistent with propionate breakdown through the 2-methylcitric acid cycle. Our experiments demonstrate that the α-carbon of propionate is oxidized to yield pyruvate. The reactions are catalyzed by propionyl coenzyme A (propionyl-CoA) synthetase (PrpE), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (probably PrpD), 2-methylisocitrate hydratase (probably PrpD), and 2-methylisocitrate lyase (PrpB). In support of this conclusion, the PrpC enzyme was purified to homogeneity and shown to have 2-methylcitrate synthase activity in vitro.
1
H nuclear magnetic resonance spectroscopy and negative-ion electrospray ionization mass spectrometry identified 2-methylcitrate as the product of the PrpC reaction. Although PrpC could use acetyl-CoA as a substrate to synthesize citrate, kinetic analysis demonstrated that propionyl-CoA is the preferred substrate.
A Single-Ring Mitochondrial Chaperonin (Hsp60-Hsp10) Can Substitute for GroEL-GroES In Vivo
Authors: Nielsen Kåre L. , McLennan Neil , Masters Millicent , Cowan Nicholas J. 
Published: December 2019

ABSTRACT

Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an
Escherichia coli
strain engineered so that the
groE
operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.