bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporal Regulation of Genes Encoding the Flagellar Proximal Rod in
Caulobacter crescentus
Authors: Boyd Charles H. , Gober James W. 
Published: July 2002

ABSTRACT

The gram-negative bacterium
Caulobacter crescentus
has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase. The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program. The flagellar regulon is arranged into a rigid
trans
-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum. The
flgBC
-
fliE
genes were identified as a result of the
C. crescentus
genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function. Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with ς
70
is responsible for regulating transcription of other early flagellar genes in
C. crescentus
. Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and
flgB
deletions do not prevent class III gene expression. This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing
fliP
, which encodes a member of the flagellum-specific protein export apparatus.
The TOL Plasmid pWW0
xylN
Gene Product from
Pseudomonas putida
Is Involved in
m-
Xylene Uptake
Authors: Kasai Yuki , Inoue Jun , Harayama Shigeaki 
Published: July 2002

ABSTRACT

The upper operon of the TOL plasmid pWW0 of
Pseudomonas putida
encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon,
xylN
, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in
Escherichia coli
. To analyze the role of the
xylN
gene product,
xylN
on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of
P. putida
harboring the wild-type and
xylN
mutant TOL plasmids were characterized. The growth of
P. putida
harboring the wild-type TOL plasmid was inhibited by a high concentration of
m
-xylene, while that of
P. putida
harboring the
xylN
mutant TOL plasmid was not. The apparent
K

s

value for the oxidation of
m
-xylene in intact cells of the
xylN
mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the
xylN
gene product is a porin involved in the transport of
m
-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.
Regulation of Gene Expression in Response to Oxygen in
Rhizobium etli
: Role of FnrN in
fixNOQP
Expression and in Symbiotic Nitrogen Fixation
Authors: Lopez Oswaldo , Morera Claudia , Miranda-Rios Juan , Girard Lourdes , Romero David , Soberón Mario 
Published: July 2002

ABSTRACT

Previously, we reported finding duplicated
fixNOQP
operons in
Rhizobium etli
CFN42. One of these duplicated operons is located in the symbiotic plasmid (
fixNOQP
d), while the other is located in a cryptic plasmid (
fixNOQP
f). Although a novel FixL-FixKf regulatory cascade participates in microaerobic expression of both
fixNOQP
duplicated operons, we found that a mutation in
fixL
eliminates
fixNOQP
f expression but has only a moderate effect on expression of
fixNOQP
d. This suggests that there are differential regulatory controls. Interestingly, only the
fixNOQP
d operon was essential for symbiotic nitrogen fixation (L. Girard, S. Brom, A. Dávalos, O. Lopez, M. Soberón, and D. Romero, Mol. Plant-Microbe Interact. 13:1283–1292, 2000). Searching for potential candidates responsible for the differential expression, we characterized two
fnrN
homologs (encoding transcriptional activators of the cyclic AMP receptor protein [CRP]-Fnr family) in
R. etli
CFN42. One of these genes (
fnrN
d) is located on the symbiotic plasmid, while the other (
fnrN
chr) is located on the chromosome. Analysis of the expression of the
fnrN
genes using transcriptional fusions with
lacZ
showed that the two
fnrN
genes are differentially regulated, since only
fnrN
d is expressed in microaerobic cultures of the wild-type strain while
fnrN
chr is negatively controlled by FixL. Mutagenesis of the two
fnrN
genes showed that both genes participate, in conjunction with FixL-FixKf, in the microaerobic induction of the
fixNOQP
d operon. Participation of these genes is also seen during the symbiotic process, in which mutations in
fnrN
d and
fnrN
chr, either singly or in combination, lead to reductions in nitrogen fixation. Therefore,
R. etli
employs a regulatory circuit for induction of the
fixNOQP
d operon that involves at least three transcriptional regulators of the CRP-Fnr family. This regulatory circuit may be important for ensuring optimal production of the
cbb
3
, terminal oxidase during symbiosis.
Regulation of the Acetoin Catabolic Pathway Is Controlled by Sigma L in
Bacillus subtilis
Authors: Ould Ali Naima , Bignon Joelle , Rapoport Georges , Debarbouille Michel 
Published: July 2002

ABSTRACT

Bacillus subtilis
grown in media containing amino acids or glucose secretes acetate, pyruvate, and large quantities of acetoin into the growth medium. Acetoin can be reused by the bacteria during stationary phase when other carbon sources have been depleted. The
acoABCL
operon encodes the E1α, E1β, E2, and E3 subunits of the acetoin dehydrogenase complex in
B. subtilis
. Expression of this operon is induced by acetoin and repressed by glucose in the growth medium. The
acoR
gene is located downstream from the
acoABCL
operon and encodes a positive regulator which stimulates the transcription of the operon. The product of
acoR
has similarities to transcriptional activators of sigma 54-dependent promoters. The four genes of the operon are transcribed from a −12, −24 promoter, and transcription is abolished in
acoR
and
sigL
mutants. Deletion analysis showed that DNA sequences more than 85 bp upstream from the transcriptional start site are necessary for full induction of the operon. These upstream activating sequences are probably the targets of AcoR. Analysis of an
acoR
′-′
lacZ
strain of
B. subtilis
showed that the expression of
acoR
is not induced by acetoin and is repressed by the presence of glucose in the growth medium. Transcription of
acoR
is also negatively controlled by CcpA, a global regulator of carbon catabolite repression. A specific interaction of CcpA in the upstream region of
acoR
was demonstrated by DNase I footprinting experiments, suggesting that repression of transcription of
acoR
is mediated by the binding of CcpA to the promoter region of
acoR
.
Genetic Isolation of Meningococci of the Electrophoretic Type 37 Complex
Authors: Claus H. , Stoevesandt J. , Frosch M. , Vogel U. 
Published: July 2002

ABSTRACT

Neisseria meningitidis
(the meningococcus) is a naturally competent bacterial species in which intra- and interspecific horizontal gene transfer is a major source of genetic diversity. In strains of the electrophoretic type 37 (ET-37) complex and of the A4 cluster, we identified genomic DNA coding for a novel restriction-modification system and for the tail of a previously unidentified prophage. Furthermore, a novel 7.2-kb DNA segment restricted to clones of the ET-37 complex and the A4 cluster was isolated and shown to occur both as a plasmid (pJS-B) and as a chromosomal integration. Neither the genomic loci nor pJS-B was present in ET-5 complex, lineage 3, or serogroup A meningococci. The differential distribution of the DNA segments described herein, as well as of
opcA, porB, nmeAI, nmeBI
, and
nmeDI
described previously, supports the concept of genetic isolation of hypervirulent lineages responsible for most cases of serogroup C disease worldwide.
Transcriptional Regulation of the
orf19
Gene and the
tir-cesT-eae
Operon of Enteropathogenic
Escherichia coli
Authors: Sánchez-SanMartı́n Claudia , Bustamante Vı́ctor H. , Calva Edmundo , Puente José Luis 
Published: July 2002

ABSTRACT

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic
Escherichia coli
(EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The
tir
gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by
orf19
and downstream by the
cesT
and
eae
genes. With the use of a series of
cat
transcriptional fusions and primer extension analysis, we confirmed that
tir, cesT
, and
eae
form the
LEE5
operon, which is under the control of a promoter located upstream from
tir
, and found that the
orf19
gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the
tir
and the
orf19
promoters and that a sequence motif located between positions −204 and −157 was needed for the Ler-dependent activation of the
tir
operon. Sequence elements located between positions −204 and −97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the
tir
promoter was fully active even in the absence of Ler, indicating that
tir
expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25°C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of
orf19
, but not of the
tir
operon, became Ler independent in an
hns
mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene.
Biochemical, Molecular, and Genetic Analyses of the Acetone Carboxylases from
Xanthobacter autotrophicus
Strain Py2 and
Rhodobacter capsulatus
Strain B10
Authors: Sluis Miriam K. , Larsen Rachel A. , Krum Jonathan G. , Anderson Ruth , Metcalf William W. , Ensign Scott A. 
Published: July 2002

ABSTRACT

Acetone carboxylase is the key enzyme of bacterial acetone metabolism, catalyzing the condensation of acetone and CO
2
to form acetoacetate. In this study, the acetone carboxylase of the purple nonsulfur photosynthetic bacterium
Rhodobacter capsulatus
was purified to homogeneity and compared to that of
Xanthobacter autotrophicus
strain Py2, the only other organism from which an acetone carboxylase has been purified. The biochemical properties of the enzymes were virtually indistinguishable, with identical subunit compositions (α
2
β
2
γ
2
multimers of 85-, 78-, and 20-kDa subunits), reaction stoichiometries (CH
3
COCH
3
+ CO
2
+ ATP→CH
3
COCH
2
COO
−
+ H
+
+ AMP + 2P
i
), and kinetic properties (

K
m

for acetone, 8 μM;
k
cat
= 45 min
−1
). Both enzymes were expressed to high levels (17 to 25% of soluble protein) in cells grown with acetone as the carbon source but were not present at detectable levels in cells grown with other carbon sources. The genes encoding the acetone carboxylase subunits were identified by transposon mutagenesis of
X. autotrophicus
and sequence analysis of the
R. capsulatus
genome and were found to be clustered in similar operons consisting of the genes
acxA
(β subunit),
acxB
(α subunit), and
acxC
(γ subunit). Transposon mutagenesis of
X. autotrophicus
revealed a requirement of σ
54
and a σ
54
-dependent transcriptional activator (AcxR) for acetone-dependent growth and acetone carboxylase gene expression. A potential σ
54
-dependent promoter 122 bp upstream of
X. autotrophicus acxABC
was identified. An AcxR gene homolog was identified 127 bp upstream of
acxA
in
R. capsulatus
, but this activator lacked key features of σ
54
-dependent activators, and the associated
acxABC
lacked an apparent σ
54
-dependent promoter, suggesting that σ
54
is not required for expression of
acxABC
in
R. capsulatus.
These studies reveal a conserved strategy of ATP-dependent acetone carboxylation and the involvement of transcriptional enhancers in acetone carboxylase gene expression in gram-negative acetone-utilizing bacteria.
Characterization and Regulation of the
gbuA
Gene, Encoding Guanidinobutyrase in the Arginine Dehydrogenase Pathway of
Pseudomonas aeruginosa
PAO1
Authors: Nakada Yuji , Itoh Yoshifumi 
Published: July 2002

ABSTRACT

The arginine dehydrogenase (or oxidase) pathway catabolically converts arginine to succinate via 2-ketoglutarate and 4-guanidinobutyrate (4-GB) with the concomitant formation of CO
2
and urea. Guanidinobutyrase (GBase; EC 3.5.3.7) catalyzes the conversion of 4-guanidinobutyrate to 4-aminobutyrate and urea in this pathway. We investigated the structure and regulation of the gene for GBase (designated
gbuA
) of
Pseudomonas aeruginosa
PAO1 and characterized the
gbuA
product. The
gbuA
and the adjacent
gbuR
genes were cloned by functional complementation of a
gbuA9005
mutant of strain PAO1 defective in 4-GB utilization. The deduced amino acid sequence of GbuA (319 amino acids;
M
r
34,695) assigned GBase to the arginase/agmatinase family of C-N hydrolases. Purified GbuA was a homotetramer of 140 kDa that catalyzed the specific hydrolysis of 4-GB with

K
m

and
K
cat
values of 49 mM and 1,012 s
−1,
respectively. The divergent
gbuR
gene, which shared the intergenic promoter region of 206 bp with
gbuA
, encoded a putative regulatory protein (297 amino acids;
M
r
33,385) homologous to the LysR family of proteins. Insertional inactivation of
gbuR
by a gentamicin resistance cassette caused a defect in 4-GB utilization. GBase and
gbuA
′::′
lacZ
fusion assays demonstrated that this
gbuR
mutation abolishes the inducible expression of
gbuA
by exogenous 4-GB, indicating that GbuR participates in the regulation of this gene. Northern blotting located an inducible promoter for
gbuA
in the intergenic region, and primer extension localized the transcription start site of this promoter at 40 bp upstream from the initiation codon of
gbuA
. The
gbuRA
genes at the genomic map position of 1547000 are unlinked to the 2-ketoarginine utilization gene
kauB
at 5983000, indicative of at least two separate genetic units involved in the arginine dehydrogenase pathway.
Identification of Target Genes Regulated by the Two-Component System HP166-HP165 of
Helicobacter pylori
Authors: Dietz Patricia , Gerlach Gabriele , Beier Dagmar 
Published: July 2002

ABSTRACT

Two-component systems are signal transduction systems which enable bacteria to regulate cellular functions in response to changing environmental conditions. In most cases regulation is accomplished on the transcriptional level by a response regulator protein, which, according to the phosphorylation state of its receiver domain, displays different affinities for its target promoters. Here we describe identification of genes regulated by the two-component system HP166-HP165 of
Helicobacter pylori
and characterization of the corresponding target promoters. We demonstrated that expression of the HP166-HP165 two-component system is negatively autoregulated under conditions favoring autophosphorylation of the histidine kinase. Furthermore, we found that response regulator HP166 activates transcription of genes encoding a protein family with an unknown function present in
H. pylori
26695, as well as an operon composed of five
H. pylori-
specific genes. While open reading frame HP166 is an essential gene, the target genes of the response regulator are not required for growth under in vitro culture conditions.
Luteolin and GroESL Modulate In Vitro Activity of NodD
Authors: Yeh Kuo-Chen , Peck Melicent C. , Long Sharon R. 
Published: July 2002

ABSTRACT

In the early stages of symbiosis between the soil bacterium
Sinorhizobium meliloti
and its leguminous host plant, alfalfa, bacterial nodulation (
nod
) genes are controlled by NodD1, NodD2, and NodD3, members of the LysR family of transcriptional regulators, in response to flavonoid and other inducers released by alfalfa. To gain an understanding of the biochemical aspects of this action, epitope-tagged recombinant NodD1 and NodD3 were overexpressed in
Escherichia coli
. The DNA binding properties of the purified recombinant NodD proteins were indistinguishable from those of NodD isolated from
S. meliloti
. In addition, the
E. coli
GroEL chaperonin copurified with the recombinant NodD proteins. In this study, we showed that NodD proteins are in vitro substrates of the GroESL chaperonin system and that their DNA binding activity is modulated by GroESL. This confirmed the earlier genetic implication that the GroESL chaperonin system is essential for the function of these regulators. Increased DNA binding activity by NodD1 in the presence of luteolin confirmed that NodD1 is involved in recognizing the plant signal during the early stages of symbiosis.
Nonenzymatic Turnover of anErwinia carotovoraQuorum-Sensing Signaling Molecule
Authors: Byers Joseph T. , Lucas Claire , Salmond George P. C. , Welch Martin 
Published: July 2002

ABSTRACTThe production of virulence factors and carbapenem antibiotic in the phytopathogenErwinia carotovorais under the control of quorum sensing. The quorum-sensing signaling molecule,N-(3-oxohexanoyl)-l-homoserine lactone (OHHL), accumulates in log-phase culture supernatants ofE. carotovorabut diminishes in concentration during the stationary phase. In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur. Rather, it was caused by degradation of the molecule. The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing. The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to ∼8.5. OHHL became unstable over a narrow pH range (pH 7 to 8). Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30°C) by buffering the samples at pH 6.8. These results may provide a rationale for the observation that an early response of plants which are under attack byErwiniais to activate a proton pump which alkalizes the site of infection to a pH of >8.2.
act
Operon Control of Developmental Gene Expression in
Myxococcus xanthus
Authors: Gronewold Thomas M. A. , Kaiser Dale 
Published: July 2002

ABSTRACT

Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the
csgA
gene, which encodes the C-signal, is directed by four genes of the
act
operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the
act
genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in
actA
,
actB
,
actC
, or
actD
mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null
act
mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The
actA
and
actB
null mutations substantially decreased the expression of all these reporters. The other
act
null mutations caused either premature expression to wild-type levels (
actC
) or delayed expression (
actD
), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.
Identification of Two
myo-
Inositol Transporter Genes of
Bacillus subtilis
Authors: Yoshida Ken-Ichi , Yamamoto Yoshiyuki , Omae Kaoru , Yamamoto Mami , Fujita Yasutaro 
Published: July 2002

ABSTRACT

Among hundreds of mutants constructed systematically by the Japanese groups participating in the functional analysis of the
Bacillus subtilis
genome project, we found that a mutant with inactivation of
iolT
(
ydjK
) exhibited a growth defect on
myo
-inositol as the sole carbon source. The putative product of
iolT
exhibits significant similarity with many bacterial sugar transporters in the databases. In
B. subtilis
, the
iolABCDEFGHIJ
and
iolRS
operons are known to be involved in inositol utilization, and its transcription is regulated by the IolR repressor and induced by inositol. Among the
iol
genes,
iolF
was predicted to encode an inositol transporter. Inactivation of
iolF
alone did not cause such an obvious growth defect on inositol as the
iolT
inactivation, while simultaneous inactivation of the two genes led to a more severe defect than the single
iolT
inactivation. Determination of inositol uptake by the mutants revealed that
iolT
inactivation almost completely abolished uptake, but uptake by IolF itself was slightly detectable. These results, as well as the

K
m

and
V
max
values for the IolT and IolF inositol transporters, indicated that
iolT
and
iolF
encode major and minor inositol transporters, respectively. Northern and primer extension analyses of
iolT
transcription revealed that the gene is monocistronically transcribed from a promoter likely recognized by ςsgr;
A
RNA polymerase and negatively regulated by IolR as well. The interaction between IolR and the
iolT
promoter region was analyzed by means of gel retardation and DNase I footprinting experiments, it being suggested that the mode of interaction is quite similar to that found for the promoter regions of the
iol
divergon.
Rubredoxins Involved in Alkane Oxidation
Authors: van Beilen Jan B. , Neuenschwander Martin , Smits Theo H. M. , Roth Christian , Balada Stefanie B. , Witholt Bernard 
Published: July 2002

ABSTRACT

Rubredoxins (Rds) are essential electron transfer components of bacterial membrane-bound alkane hydroxylase systems. Several Rd genes associated with alkane hydroxylase or Rd reductase genes were cloned from gram-positive and gram-negative organisms able to grow on
n-
alkanes (Alk-Rds). Complementation tests in an
Escherichia coli
recombinant containing all
Pseudomonas putida
GPo1 genes necessary for growth on alkanes except Rd 2 (AlkG) and sequence comparisons showed that the Alk-Rds can be divided in AlkG1- and AlkG2-type Rds. All alkane-degrading strains contain AlkG2-type Rds, which are able to replace the GPo1 Rd 2 in
n-
octane hydroxylation. Most strains also contain AlkG1-type Rds, which do not complement the deletion mutant but are highly conserved among gram-positive and gram-negative bacteria. Common to most Rds are the two iron-binding CXXCG motifs. All Alk-Rds possess four negatively charged residues that are not conserved in other Rds. The AlkG1-type Rds can be distinguished from the AlkG2-type Rds by the insertion of an arginine downstream of the second CXXCG motif. In addition, the glycines in the two CXXCG motifs are usually replaced by other amino acids. Mutagenesis of residues conserved in either the AlkG1- or the AlkG2-type Rds, but not between both types, shows that AlkG1 is unable to transfer electrons to the alkane hydroxylase mainly due to the insertion of the arginine, whereas the exchange of the glycines in the two CXXCG motifs only has a limited effect.