bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and Differential Turnover of the CYS3 Regulatory Protein of
Neurospora crassa
Are Subject to Sulfur Control
Authors: Tao Ying , Marzluf George A. 
Published: December 2019

ABSTRACT

The transcription factor CYS3 of
Neurospora crassa
is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner.
cys-3
expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited.
cys-3
transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression,
cys-3
transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.
Characterization of Glucose-Specific Catabolite Repression-Resistant Mutants of
Bacillus subtilis
: Identification of a Novel Hexose:H
+
Symporter
Authors: Paulsen Ian T. , Chauvaux Sylvie , Choi Peter , Saier Milton H. 
Published: December 2019

ABSTRACT

Insertional mutagenesis was conducted on
Bacillus subtilis
cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H
+
symporter of
Escherichia coli
and the glucose/galactose:H
+
symporter of
Brucella abortus
. In a wild-type
B. subtilis
genetic background, the
glcP
::Tn
10
mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [
14
C]glucose and [
14
C]methyl α-glucoside uptake. In a Δ
pts
genetic background no phenotype was observed, suggesting that expression of the
glcP
gene required a functional phosphotransferase system. When overproduced in a Δ
pts
mutant of
E. coli
, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl α-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that
B. subtilis
possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.
Genetic Analysis, Using P22 Challenge Phage, of the Nitrogen Activator Protein DNA-Binding Site in the
Klebsiella aerogenes put
Operon
Authors: Chen Li-Mei , Goss Thomas J. , Bender Robert A. , Swift Simon , Maloy Stanley 
Published: December 2019

ABSTRACT

The
nac
gene product is a LysR regulatory protein required for nitrogen regulation of several operons from
Klebsiella aerogenes
and
Escherichia coli
. We used P22 challenge phage carrying the
put
control region from
K. aerogenes
to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the
put
operon of
Salmonella typhimurium
, which is not regulated by NAC.
Comparison of the Sensitivities of Two
Escherichia coli
Genes to In Vivo Variation of Lrp Concentration
Authors: Chen ChangFeng , Newman E. B. 
Published: December 2019

ABSTRACT

Transcription of the
Escherichia coli
genes
serA
and
gltBDF
depends on the leucine-responsive regulatory protein, Lrp, and is very much decreased in an
lrp
mutant. By the use of an Lrp-deficient host and the
lrp
gene cloned under a plasmid-borne arabinose pBAD promoter, we varied the amount of Lrp present in the cell and showed that both genes were transcribed in proportion to the amount of Lrp synthesized. The affinity of
serA
for Lrp was four to five times greater than the affinity of
gltD
. Overproduction of Lrp was lethal to the cell.
SacY, a Transcriptional Antiterminator fromBacillus subtilis, Is Regulated by Phosphorylation In Vivo
Authors: Idelson Maria , Amster-Choder Orna 
Published: December 2019

ABSTRACTSacY antiterminates transcription of thesacBgene inBacillus subtilisin response to the presence of sucrose in the growth medium. We have found that it can substitute for BglG, a homologous protein, in antiterminating transcription of thebgloperon inEscherichia coli. We therefore sought to determine whether, similarly to BglG, SacY is regulated by reversible phosphorylation in response to the availability of the inducing sugar. We show here that two forms of SacY, phosphorylated and nonphosphorylated, exist inB. subtiliscells and that the ratio between them depends on the external level of sucrose. Addition of sucrose to the growth medium after SacY phosphorylation in the cell resulted in its rapid dephosphorylation. The extent of SacY phosphorylation was found to be proportional to the cellular levels of SacX, a putative sucrose permease which was previously shown to have a negative effect on SacY activity. Thus, the mechanism by which thesacsensory system modulatessacBexpression in response to sucrose involves reversible phosphorylation of the regulator SacY, and this process appears to depend on the SacX sucrose sensor. Thesacsystem is therefore a member of the novel family of sensory systems represented bybgl.
Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the
bchI
,
-D
, and
-H
Gene Products of the Green Sulfur Bacterium
Chlorobium vibrioforme
Expressed in
Escherichia coli
Authors: Petersen Bent L. , Jensen Poul Erik , Gibson Lucien C. D. , Stummann Bjarne M. , Hunter C. Neil , Henningsen Knud W. 
Published: December 2019

ABSTRACT

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg
2+
into protoporphyrin IX. Three genes, designated
bchI
,
-D
, and
-H
, from the strictly anaerobic and obligately phototrophic green sulfur bacterium
Chlorobium vibrioforme
show a significant level of homology to the magnesium chelatase-encoding genes
bchI
,
-D
, and
-H
and
chlI
,
-D
, and
-H
of
Rhodobacter sphaeroides
and
Synechocystis
strain PCC6803, respectively. These three genes were expressed in
Escherichia coli
; the subsequent purification of overproduced BchI and -H proteins on an Ni
2+
-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase of
C. vibrioforme
is similar to the magnesium chelatases of the distantly related bacteria
R. sphaeroides
and
Synechocystis
strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the
C. vibrioforme
BchI and -D proteins and the
Synechocystis
strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the
bchI
gene product, were expressed in
E. coli
, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in
C. vibrioforme
.
luxI
- and
luxR
-Homologous Genes of
Rhizobium etli
CNPAF512 Contribute to Synthesis of Autoinducer Molecules and Nodulation of
Phaseolus vulgaris
Authors: Rosemeyer Viola , Michiels Jan , Verreth Christel , Vanderleyden Jos 
Published: December 2019

ABSTRACT

Autoinduction plays an important role in intercellular communication among symbiotic and pathogenic gram-negative bacteria. We report here that a nitrogen-fixing symbiont of
Phaseolus vulgaris
,
Rhizobium etli
CNPAF512, produces at least seven different autoinducer molecules. One of them exhibits a growth-inhibitory effect like that of the bacteriocin
small
[
N
-(3
R
-hydroxy-7-
cis
-tetradecanoyl)-
l
-homoserine lactone]. At least two of the other autoinducers are synthesized by a LuxI-homologous autoinducer synthase. The corresponding
luxI
homologous gene (
raiI
) and a
luxR
homolog (
raiR
) have been identified and characterized. Enhanced expression of
raiI
is dependent on cell density and on the presence of one or more autoinducer molecules synthesized by
R. etli
CNPAF512. A
raiI
mutant was shown to release only three different autoinducer molecules; a
raiR
mutant releases four different autoinducer molecules. Examination of different mutants for nodulation of beans showed that
raiI
is involved in the restriction of nodule number, whereas nitrogen-fixing activity in terms of acetylene reduction per nodule was not affected.
Transcriptional Analysis of the
Bordetella
Alcaligin Siderophore Biosynthesis Operon
Authors: Kang Ho Young , Armstrong Sandra K. 
Published: December 2019

ABSTRACT

The
alc
gene cluster of
Bordetella pertussis
includes three genes,
alcA
,
alcB
, and
alcC
, which are involved in alcaligin siderophore biosynthesis in response to iron starvation. The production of AlcA, AlcB, and AlcC in
Bordetella
cells and the transcriptional organization of
alcA
,
alcB
, and
alcC
were investigated by using a set of three
alc′-′lacZ
gene fusion constructs that were contiguous with the known promoter upstream of
alcA
and extended to fusion junctions within each
alc
cistron. All three
alc′-′lacZ
fusions exhibited iron-repressible reporter gene expression which was abolished by deletion of the 105-bp
alcA
promoter-operator region. In an immunoblot analysis using a monoclonal antibody specific for β-galactosidase, the AlcA-LacZ, AlcB-LacZ, and AlcC-LacZ hybrid proteins were detected in
Bordetella
cells grown under iron-depleted conditions. A
B. pertussis
mutant in which the 105-bp
alcA
promoter-operator region was deleted by allelic exchange was unable to produce detectable levels of siderophore. Hybridization analysis using gene-specific probes showed that
alc
-specific transcript levels in the mutant were negligible compared with those of the wild-type parent. These results confirm that
alcA
,
alcB
, and
alcC
are cotranscribed from an iron-regulated control region immediately upstream of
alcA
. Transcript analysis using hybridization probes representing regions downstream of
alcC
demonstrated that
alc
transcription extends approximately 3.6 kb further downstream from the
alcC
coding region, suggesting the cotranscription of additional, uncharacterized alcaligin system genes.
Both Acetate Kinase and Acetyl Coenzyme A Synthetase Are Involved in Acetate-Stimulated Change in the Direction of Flagellar Rotation in
Escherichia coli
Authors: Barak Rina , Abouhamad Walid N. , Eisenbach Michael 
Published: December 2019

ABSTRACT

Escherichia coli
strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.
Control of
ftsZ
Expression, Cell Division, and Glutamine Metabolism in Luria-Bertani Medium by the Alarmone ppGpp in
Escherichia coli
Authors: Powell Bradford S. , Court Donald L. 
Published: December 2019

ABSTRACT

Inactivation of transcription factor ς
54
, encoded by
rpoN
(
glnF
), restores high-temperature growth in Luria-Bertani (LB) medium to strains containing the heat-sensitive cell division mutation
ftsZ84
. Mutational defects in three other genes involved in general nitrogen control (
glnD
,
glnG
, and
glnL
) also suppress lethal filamentation. Since addition of glutamine to LB medium fully blocks suppression by each mutation, the underlying cause of suppression likely derives from a stringent response to the limitation of glutamine. This model is supported by several observations. The
glnL
mutation requires RelA-directed synthesis of the nutrient alarmone ppGpp to suppress filamentation. Artificially elevated levels of ppGpp suppress
ftsZ84
, as do RNA polymerase mutations that reproduce global effects of the ppGpp-induced state. Both the
glnF
null mutation and an elevated copy number of the
relA
gene similarly affect transcription from the upstream (pQ) promoters of the
ftsQAZ
operon, and both of these genetic conditions increase the steady-state level of the FtsZ84 protein. Physiological suppression of
ftsZ84
by a high salt concentration was also shown to involve RelA. Additionally, we found that the growth of a
glnF
or
glnD
strain on LB medium depends on RelA or supplemental glutamine in the absence of RelA function. These data expand the roles for ppGpp in the regulation of glutamine metabolism and the expression of FtsZ during cell division.
Variation in Flagellin Genes and Proteins of
Burkholderia cepacia
Authors: Hales Barbara A. , Morgan J. Alun W. , Hart C. Anthony , Winstanley Craig 
Published: December 2019

ABSTRACT

The majority of isolates of
Burkholderia cepacia
, an important opportunistic pathogen associated with cystic fibrosis, can be classified into two types on the basis of flagellin protein size. Electron microscopic analysis indicates that the flagella of strains with the larger flagellin type (type I) are wider in diameter. Flagellin genes representative of both types were cloned and sequenced to design oligonucleotide primers for PCR amplification of the central variable domain of
B. cepacia
flagellin genes. PCR-restriction fragment length polymorphism analysis of amplified
B. cepacia
flagellin gene products from 16 strains enabled flagellin type classification on the basis of product size and revealed considerable differences in sequence, indicating that the flagellin gene is a useful biomarker for epidemiological and phylogenetic studies of this organism.
The
narA
Locus of
Synechococcus
sp. Strain PCC 7942 Consists of a Cluster of Molybdopterin Biosynthesis Genes
Authors: Rubio Luis M. , Flores Enrique , Herrero Antonia 
Published: December 2019

ABSTRACT

The
narA
locus required for nitrate reduction in
Synechococcus
sp. strain PCC 7942 is shown to consist of a cluster of genes, namely,
moeA
,
moaC
,
moaD
,
moaE
, and
moaA
, involved in molybdenum cofactor biosynthesis. The product of the
moaC
gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from
Escherichia coli
and in its C-terminal half to MoaB or Mog. Overexpression of the
Synechococcus moaC
gene in
E. coli
resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of
E. coli
. Insertional inactivation of the
moeA
,
moaC
,
moaE
, and
moaA
genes showed that the
moeA-moa
gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The
moaCDEA
genes constitute an operon which is transcribed divergently from the
moeA
gene. Expression of the
moeA
gene and the
moa
operon was little affected by the nitrogen source present in the culture medium.
The
Yersinia
Yop Virulon: LcrV Is Required for Extrusion of the Translocators YopB and YopD
Authors: Sarker Mahfuzur R. , Neyt Cécile , Stainier Isabelle , Cornelis Guy R. 
Published: December 2019

ABSTRACT

LcrV, an essential piece of the Yop virulon, is encoded by the large
lcrGVsycDyopBD
operon. In spite of repeated efforts, the role of LcrV in the Yop virulon remains elusive. In an attempt to clarify this, we engineered a complete deletion of
lcrV
in the pYV plasmid of
Yersinia enterocolitica
E40 and characterized the phenotype of the mutant. Complementation experiments showed that the mutation was not polar with regard to
yopB
and
yopD
. Nevertheless the mutation abolished secretion of YopB and YopD, while secretion of the other Yops was unaffected or even increased. Northern blot analysis showed that transcription of
yopD
was not affected. YopD could be detected inside the bacteria, showing that the lack of its secretion was not due to a lack of translation or to proteolysis. This indicated that LcrV is specifically involved in the process of release of YopB and YopD. We then investigated the possible interactions between LcrV and YopB or YopD. We constructed a glutathione
S
-transferase–LcrV hybrid protein, and we observed that either YopB or YopD could be copurified with it. The same approach showed that LcrV also interacts with LcrG but not with the chaperone SycD. Using deletants of
lcrV
, we then identified a definite LcrG-binding domain in the C terminus of LcrV.
Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required for
pap
Phase Variation
Authors: Kaltenbach Linda , Braaten Bruce , Tucker Julie , Krabbe Margareta , Low David 
Published: December 2019

ABSTRACT

The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as
ilvIH
, activation of
papBA
transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate
ilvIH
transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on
ilvIH
transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for
pap
and
ilvIH
DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity for
pap
Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-locked
pap
derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on
pap
transcription state.
When an ATPase Is Not an ATPase: at Low Temperatures the C-Terminal Domain of the ABC Transporter CvaB Is a GTPase
Authors: Zhong Xiaotian , Tai Phang C. 
Published: December 2019

ABSTRACT

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from
Escherichia coli
is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg
2+
and Mn
2+
dependent. However, BCTD possesses similar GTPase and ATPase activities at 37°C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10°C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.
An
rne-1 pnp-7
Double Mutation Suppresses the Temperature-Sensitive Defect of
lacZ
Gene Expression in a
divE
Mutant
Authors: Aiso Toshiko , Ohki Reiko 
Published: December 2019

ABSTRACT

A
divE
mutant, which has a temperature-sensitive mutation in the tRNA
1
Ser
gene, exhibits differential loss of the synthesis of certain proteins, such as β-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In
Escherichia coli
, the UCA codon is recognized only by tRNA
1
Ser
. Several genes containing UCA codons are normally expressed after a temperature shift to 42°C in the
divE
mutant. Therefore, it is unlikely that the defect in protein synthesis at 42°C is simply caused by a defect in the decoding function of the mutant tRNA
1
Ser
. In this study, we sought to determine the cause of the defect in
lacZ
gene expression in the
divE
mutant. It has also been shown that the defect in
lacZ
gene expression is accompanied by a decrease in the amount of
lacZ
mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in
lacZ
gene expression, we constructed
divE
mutants containing
rne-1
,
rnb-500
, and
pnp-7
mutations in various combinations. We found that the defect was almost completely restored by introducing an
rne-1 pnp-7
double mutation into the
divE
mutant. Northern hybridization analysis showed that the
rne-1
mutation stabilized
lacZ
mRNA, whereas the
pnp-7
mutation stabilized mutant tRNA
1
Ser
, at 44°C. We present a mechanism that may explain these results.