bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of a Two-Component Sensor-Kinase and Response-Regulator System (DcuS-DcuR) Controlling Gene Expression in Response to C
4
-Dicarboxylates in
Escherichia coli
Authors: Golby Paul , Davies Suzanne , Kelly David J. , Guest John R. , Andrews Simon C. 
Published: December 2019

ABSTRACT

The
dcuB
gene of
Escherichia coli
encodes an anaerobic C
4
-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition,
dcuB
expression is strongly induced by C
4
-dicarboxylates, suggesting the presence of a novel C
4
-dicarboxylate-responsive regulator in
E. coli
. This paper describes the isolation of a Tn
10
mutant in which the 160-fold induction of
dcuB
expression by C
4
-dicarboxylates is absent. The corresponding Tn
10
mutation resides in the
yjdH
gene, which is adjacent to the
yjdG
gene and close to the
dcuB
gene at ∼93.5 min in the
E. coli
chromosome. The
yjdHG
genes (redesignated
dcuSR
) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the
dcuSR
operon restored the C
4
-dicarboxylate inducibility of
dcuB
expression in the
dcuS
mutant to levels exceeding those of the
dcuS
+
strain by approximately 1.8-fold. The
dcuS
mutation affected the expression of other genes with roles in C
4
-dicarboxylate transport or metabolism. Expression of the fumarate reductase (
frdABCD
) operon and the aerobic C
4
-dicarboxylate transporter (
dctA
) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C
4
-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the
dcuS
mutant was normal. However, under aerobic conditions with C
4
-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a
dctA
mutant. Studies employing a
dcuA dcuB dcuC
triple mutant unable to transport C
4
-dicarboxylates anaerobically revealed that C
4
-dicarboxylate transport is not required for C
4
-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a ∼140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C
4
-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of
Klebsiella pneumoniae
. DcuS is not closely related to the C
4
-dicarboxylate-sensing DctS or DctB protein of
Rhodobacter capsulatus
or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.
Analysis of the Type 1 Pilin Gene Cluster
fim
in
Salmonella
: Its Distinct Evolutionary Histories in the 5′ and 3′ Regions
Authors: Boyd E. Fidelma , Hartl Daniel L. 
Published: December 2019

ABSTRACT

The type 1 pilin encoded by
fim
is present in both
Escherichia coli
and
Salmonella
natural isolates, but several lines of evidence indicate that similarities at the
fim
locus may be an example of independent acquisition rather than common ancestry. For example, the
fim
gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the
fim
gene cluster of
Salmonella
, the genes of which show high nucleotide sequence divergence from their
E. coli
counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the
fim
gene cluster is present in all isolates of
S. enterica
but is absent from
S. bongori
. Molecular phylogenetic analyses of the
fimA
and
fimI
genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the
fimZ
,
fimY
, and
fimW
genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the
fimZ
,
fimY
, and
fimW
intergenic regions in the 3′ region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the
fimZ
,
fimY
, and
fimW
genes are less than 46%, which is considerably lower than those of the other six genes of the
fim
cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.
Transcription of the Stationary-Phase-Associated
hspX
Gene of
Mycobacterium tuberculosis
Is Inversely Related to Synthesis of the 16-Kilodalton Protein
Authors: Hu Yanmin , Coates Anthony R. M. 
Published: December 2019

ABSTRACT

The 16-kDa protein, an α-crystallin homologue, is one of the most abundant proteins in stationary-phase
Mycobacterium tuberculosis
. Here, transcription and translation of the
hspX
gene, which encodes the 16-kDa protein, have been investigated by Northern blotting analysis, primer extension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a microaerophilic stationary-phase model. Two transcripts of about 2.5 and 1.1 kb were demonstrated by Northern blot analysis and hybridized to the
hspX
gene probe. Primer extension analysis revealed that the transcription start site is located 33 nucleotides upstream of the
hspX
gene start codon. The cellular level of the
hspX
mRNA was maximum in log-phase bacilli and was markedly reduced after 20 days in unagitated culture, when the organisms had entered the stationary phase. A third transcript of 0.5 kb was detected 0.6 kb downstream of the
hspX
gene; this transcript has a transcriptional pattern completely different from that of the 1.1- and 2.5-kb products, suggesting that there may be another gene in this region. In contrast to the high level of
hspX
mRNA in log-phase bacilli, 16-kDa protein synthesis was low in log-phase bacteria and rose to its maximum after 20 days. In both log-phase and stationary-phase bacteria the mRNA was unstable, with a half-life of 2 min, which indicated that the transcript stability was growth rate independent and not a general means for controlling the gene expression. However, the cellular content of 16-kDa protein, while low in log-phase bacteria, rose to a maximum at 10 days and remained at this high level for up to 50 days, which indicates that this protein is a stable molecule with a low turnover rate. These data suggest that the regulation of
hspX
expression during entry into and maintenance of stationary phase involves translation initiation efficiency and protein stability as potential mechanisms.
Analysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from
Ralstonia eutropha
: Generation of β-Alanine Auxotrophic Tn
5
Mutants and Cloning of the
panD
Gene Region
Authors: Hoppensack Astrid , Rehm Bernd H. A. , Steinbüchel Alexander 
Published: December 2019

ABSTRACT

The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from
Ralstonia eutropha
by 4-phosphopantetheine was investigated. Four β-alanine auxotrophic Tn
5
-induced mutants of
R. eutropha
HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the
panD
gene from
Escherichia coli
, encoding
l
-aspartate-1-decarboxylase (EC
4.1.1.15
), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)
+
transhydrogenase (EC
1.6.1.1
) alpha 1 subunit, encoded by the
pntAA
gene from
Escherichia coli
. The
panD
gene was cloned by complementation of the
panD
mutant of
R. eutropha
Q20. DNA sequencing of the
panD
gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from
E. coli
and
Bacillus subtilis
, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS
−
led to complementation of the
panD
mutants of
R. eutropha
and
E. coli
SJ16, respectively.
panD
-encoded
l
-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of
panD
, an ORF with strong similarity to
pntAA
from
E. coli
, encoding NAD(P)
+
transhydrogenase subunit alpha 1 was found; downstream of
panD
, two ORFs with strong similarity to
pntAB
and
pntB
, encoding subunits alpha 2 and beta of the NAD(P)
+
transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of
pan
and
pnt
genes was found in
R. eutropha
. Labeling experiments using one of the
R. eutropha panD
mutants and [2-
14
C]β-alanine provided no evidence that
R. eutropha
PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the
E. coli panD
mutant exhibit detectable labeling of functional PHA synthase from
R. eutropha
.
Identification and Characterization of SirA, an Iron-Regulated Protein from
Staphylococcus aureus
Authors: Heinrichs Jon H. , Gatlin LaVette E. , Kunsch Charles , Choi Gil H. , Hanson Mark S. 
Published: December 2019

ABSTRACT

The acquisition of iron by pathogenic bacteria is often a crucial step in establishing infection. To accomplish this, many bacteria, including
Staphylococcus aureus
, produce low-molecular-weight iron-chelating siderophores. However, the secretion and transport of these molecules in gram-positive organisms are poorly understood. The sequence, organization, and regulation of genes involved in siderophore transport are conserved among gram-negative bacteria. We used this information to identify a putative siderophore transport locus from an
S. aureus
genomic sequence database. This locus contains three predicted open reading frames with a high degree of homology to genes involved in siderophore uptake in several bacterial species, in particular the
cbr
locus of the plant pathogen
Erwinia chrysanthemi
. The first gene in the locus, which we have designated
sir
for staphylococcal iron regulated, encodes a putative lipoprotein with a molecular mass of 37 kDa. The open reading frame is preceded by a 19-bp region of dyad symmetry with homology for operator sequences controlling iron-regulated expression of genes in other bacteria. Fur titration experiments indicate that this region of dyad symmetry is sufficient for Fur-dependent regulation in
Escherichia coli
. The expression of this gene was repressed, in a dose-dependent manner, by the addition of iron to the
S. aureus
culture medium.
sir
-encoded proteins may be involved in iron acquisition in vivo and therefore may be targets for antimicrobial agents.
A Novel Reduced Flavin Mononucleotide-Dependent Methanesulfonate Sulfonatase Encoded by the Sulfur-RegulatedmsuOperon ofPseudomonas aeruginosa
Authors: Kertesz Michael A. , Schmidt-Larbig Karen , Wüest Thomas 
Published: December 2019

ABSTRACTWhenPseudomonas aeruginosais grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library ofP. aeruginosaPAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have namedmsuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of themsuoperon was analyzed with a transcriptionalmsuD::xylEfusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intactcysBgene, and themsuoperon is therefore part of thecysregulon, since sulfite utilization was found to be CysB independent in this species. Measurements ofmsuD::xylEexpression incysNandcysIgenetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulatingmsuexpression, and sulfonate utilization therefore appears to be tightly regulated.
Molecular Characterization of the Nitrite-Reducing System of
Staphylococcus carnosus
Authors: Neubauer H. , Pantel I. , Götz F. 
Published: December 2019

ABSTRACT

Characterization of a nitrite reductase-negative
Staphylococcus carnosus
Tn
917
mutant led to the identification of the
nir
operon, which encodes NirBD, the dissimilatory NADH-dependent nitrite reductase; SirA, the putative oxidase and chelatase, and SirB, the uroporphyrinogen III methylase, both of which are necessary for biosynthesis of the siroheme prosthetic group; and NirR, which revealed no convincing similarity to proteins with known functions. We suggest that NirR is essential for
nir
promoter activity. In the absence of NirR, a weak promoter upstream of
sirA
seems to drive transcription of
sirA
,
nirB
,
nirD
, and
sirB
in the stationary-growth phase. In primer extension experiments one predominant and several weaker transcription start sites were identified in the
nir
promoter region. Northern blot analyses indicated that anaerobiosis and nitrite are induction factors of the
nir
operon: cells grown aerobically with nitrite revealed small amounts of full-length transcript whereas cells grown anaerobically with or without nitrite showed large amounts of full-length transcript. Although a transcript is detectable, no nitrite reduction occurs in cells grown aerobically with nitrite, indicating an additional oxygen-controlled step at the level of translation, enzyme folding, assembly, or insertion of prosthetic groups. The nitrite-reducing activity expressed during anaerobiosis is switched off reversibly when the oxygen tension increases, most likely due to competition for electrons with the aerobic respiratory chain. Another gene,
nirC
, is located upstream of the
nir
operon.
nirC
encodes a putative integral membrane-spanning protein of unknown function. A
nirC
mutant showed no distinct phenotype.
Environmental Signals Modulate ToxT-Dependent Virulence Factor Expression in
Vibrio cholerae
Authors: Schuhmacher Darren A. , Klose Karl E. 
Published: December 2019

ABSTRACT

The regulatory protein ToxT directly activates the transcription of virulence factors in
Vibrio cholerae
, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of
toxT
. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in
V. cholerae
at 30°C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37°C. Also, expression of six ToxT-dependent Tn
phoA
fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by
ctxAp
- and
tcpAp
-luciferase fusions confirmed that negative regulation by 37°C or bile occurs at the transcriptional level in
V. cholerae
. Interestingly, ToxT-dependent transcription of these same promoters in
Salmonella typhimurium
was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in
V. cholerae
and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.
Protein ProQ Influences Osmotic Activation of Compatible Solute Transporter ProP in
Escherichia coli
K-12
Authors: Kunte H. Jörg , Crane Rebecca A. , Culham Doreen E. , Richmond Deborah , Wood Janet M. 
Published: December 2019

ABSTRACT

ProP is an osmoregulatory compatible solute transporter in
Escherichia coli
K-12. Mutation
proQ220
::Tn
5
decreased the rate constant for and the extent of ProP activation by an osmotic upshift but did not alter
proP
transcription or the ProP protein level. Allele
proQ220
::Tn
5
was isolated, and the
proQ
sequence was determined. Locus
proQ
is upstream from
prc
(
tsp
) at 41.2 centisomes on the genetic map. The
proQ220
::Tn
5
and
prc
phenotypes were different, however. Gene
proQ
is predicted to encode a 232-amino-acid, basic, hydrophilic protein (molecular mass, 25,876 Da; calculated isoelectric point, 9.66; 32% D, E, R, or K; 54.5% polar amino acids). The insertion of PCR-amplified
proQ
into vector pBAD24 produced a plasmid containing the wild-type
proQ
open reading frame, the expression of which yielded a soluble protein with an apparent molecular mass of 30 kDa. Antibodies raised against the overexpressed ProQ protein detected cross-reactive material in
proQ
+
bacteria but not in
proQ220
::Tn
5
bacteria. ProQ may be a structural element that influences the osmotic activation of ProP at a posttranslational level.
Correlation of Activity Regulation and Substrate Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase Activity in
Rhodospirillum rubrum
Authors: Kim Kitai , Zhang Yaoping , Roberts Gary P. 
Published: December 2019

ABSTRACT

In
Rhodospirillum rubrum
, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.
Role of the Alternative Sigma Factor ς
S
in Expression of the AlkS Regulator of the
Pseudomonas oleovorans
Alkane Degradation Pathway
Authors: Canosa Inés , Yuste Luis , Rojo Fernando 
Published: December 2019

ABSTRACT

The AlkS protein activates transcription from the
PalkB
promoter, allowing the expression of a number of genes required for the assimilation of alkanes in
Pseudomonas oleovorans
. We have identified the promoter from which the
alkS
gene is transcribed,
PalkS
, and analyzed its expression under different conditions and genetic backgrounds. Transcription from
PalkS
was very low during the exponential phase of growth and increased considerably when cells reached the stationary phase. The
PalkS
−10 region was similar to the consensus described for promoters recognized by
Escherichia coli
RNA polymerase bound to the alternative sigma factor ς
S
, which directs the expression of many stationary-phase genes. Reporter strains containing
PalkS-lacZ
transcriptional fusions showed that
PalkS
promoter is very weakly expressed in a
Pseudomonas putida
strain bearing an inactivated allele of the gene coding for ς
S
,
rpoS
. When
PalkS
was transferred to
E. coli
, transcription started at the same site and expression was higher in stationary phase only if ς
S
-RNA polymerase was present. The low levels of AlkS protein generated in the absence of ς
S
were enough to support a partial induction of the
PalkB
promoter. The −10 and −35 regions of
PalkS
promoter also show some similarity to the consensus recognized by ς
D
-RNA polymerase, the primary form of RNA polymerase. We propose that in exponential phase
PalkS
is probably recognized both by ς
D
-RNA polymerase (inefficiently) and by ς
S
-RNA polymerase (present at low levels), leading to low-level expression of the
alkS
gene. ς
S
-RNA polymerase would be responsible for the high level of activity of
PalkS
observed in stationary phase.
Uracil-Induced Down-Regulation of the Yeast Uracil Permease
Authors: Séron Karin , Blondel Marie-Odile , Haguenauer-Tsapis Rosine , Volland Christiane 
Published: December 2019

ABSTRACT

In
Saccharomyces cerevisiae
the
FUR4
-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the
FUR4
mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.
Differential Dependence of Levansucrase and α-Amylase Secretion on SecA (Div) during the Exponential Phase of Growth of
Bacillus subtilis
Authors: Leloup Laurence , Driessen Arnold J. M. , Freudl Roland , Chambert Régis , Petit-Glatron Marie-Françoise 
Published: December 2019

ABSTRACT

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a
degU32
(Hy)
Bacillus subtilis
strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and α-amylase overexpressed under the control of the
sacB
leader region. The level of SecA was reduced in the presence of sodium azide and in the
div341
thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing
secA
. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, α-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the α-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.