bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conserved Promoter Motif Is Required for Cell Cycle Timing of
dnaX
Transcription in
Caulobacter
Authors: Keiler Kenneth C. , Shapiro Lucy 
Published: July 2002

ABSTRACT

Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium
Caulobacter crescentus
, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including
dnaX
,
dnaA
,
dnaN
,
gyrB
, and
dnaK
, are induced at the G
1
/S-phase transition. By analyzing mutations in the
dnaX
promoter, we identified a motif between the −10 and −35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis,
Caulobacter
extracts contain an activity that binds specifically to the RRF in vitro.
Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in
Xanthomonas campestris
pv. campestris Type II Secretion Apparatus
Authors: Lee Hsien-Ming , Tyan Shiaw-Wei , Leu Wei-Ming , Chen Ling-Yun , Chen David Chanhen , Hu Nien-Tai 
Published: July 2002

ABSTRACT

The
xps
gene cluster is required for the second step of type II protein secretion in
Xanthomonas campestris
pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the
xpsM
mutant strain, and vice versa. Introduction of the wild-type
xpsM
gene carried on a plasmid into the
xpsM
mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the
xpsN
mutant strain. They were recovered either by reintroducing the wild-type
xpsN
gene or by introducing extra copies of wild-type
xpsL
or
xpsM
individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of
X. campestris
pv. campestris caused inhibition of secretion. Complementation of an
xpsL
or
xpsM
mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the
xpsN
gene on the plasmid along with the
xpsL
and the
xpsM
genes caused more severe inhibition in both cases. Furthermore, complementation of the
xpsN
mutant strain was also inhibited. In both the wild-type strain and a strain with the
xps
gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.
Formation of Intermediate Transcription Initiation Complexes at p
fliD
and p
flgM
by ς
28
RNA Polymerase
Authors: Givens Jennifer R. , McGovern Colleen L. , Dombroski Alicia J. 
Published: July 2002

ABSTRACT

The ς subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. Primary ς factors are essential for growth, while alternative ς factors are activated in response to various stimuli. Expression of class 3 genes during flagellum biosynthesis in
Salmonella enterica
serovar Typhimurium is dependent on the alternative ς factor ς
28
. Previously, a novel mechanism of transcription initiation at the
fliC
promoter by ς
28
holoenzyme was proposed. Here, we have characterized the mechanism of transcription initiation by a holoenzyme carrying ς
28
at the
fliD
and
flgM
promoters to determine if the mechanism of initiation observed at p
fliC
is a general phenomenon for all ς
28
-dependent promoters. Temperature-dependent footprinting demonstrated that promoter binding properties and low-temperature open complex formation are similar for p
fliC
, p
fliD
, and p
flgM
. However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at p
flgM
, while a sequential pattern of open complex formation occurs at p
fliD
. Open and initiated complexes formed by holoenzyme carrying ς
28
are generally unstable to heparin challenge, with the exception of initiated complexes at p
flgM
, which are stable in the presence of nucleoside triphosphates.
Regulation of rRNA Transcription Correlates with Nucleoside Triphosphate Sensing
Authors: Barker Melanie M. , Gourse Richard L. 
Published: July 2002

ABSTRACT

We have previously shown that the activity of the
Escherichia coli
rRNA promoter
rrnB
P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 different
rrnB
P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The seven
rrnB
P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the
fis
gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the
rrnB
P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which
rrnB
P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).
Molecular Characterization of Global Regulatory RNA Species That Control Pathogenicity Factors in
Erwinia amylovora
and
Erwinia herbicola
pv. gypsophilae
Authors: Ma Weilei , Cui Yaya , Liu Yang , Dumenyo C. Korsi , Mukherjee Asita , Chatterjee Arun K. 
Published: July 2002

ABSTRACT

rsmB
Ecc

specifies a nontranslatable RNA regulator that controls exoprotein production and pathogenicity in soft rot-causing
Erwinia carotovora
subsp.
carotovora
. This effect of

rsmB
Ecc

RNA is mediated mostly by neutralizing the function of RsmA
Ecc
, an RNA-binding protein of
E. carotovora
subsp.
carotovora
, which acts as a global negative regulator. To determine the occurrence of functional homologs of

rsmB
Ecc

in non-soft-rot-causing
Erwinia
species, we cloned the
rsmB
genes of
E. amylovora
(

rsmB
Ea

) and
E. herbicola
pv. gypsophilae (

rsmB
Ehg

). We show that

rsmB
Ea

in
E. amylovora
positively regulates extracellular polysaccharide (EPS) production, motility, and pathogenicity. In
E. herbicola
pv. gypsophilae,

rsmB
Ehg

elevates the levels of transcripts of a cytokinin (
etz
) gene and stimulates the production of EPS and yellow pigment as well as motility. RsmA
Ea
and RsmA
Ehg
have more than 93% identity to RsmA
Ecc
and, like the latter, function as negative regulators by affecting the transcript stability of the target gene. The
rsmB
genes reverse the negative effects of RsmA
Ea
, RsmA
Ehg
, and RsmA
Ecc
, but the extent of reversal is highest with homologous combinations of
rsm
genes. These observations and findings that

rsmB
Ea

and

rsmB
Ehg

RNA bind RsmA
Ecc
indicate that the
rsmB
effect is channeled via RsmA. Additional support for this conclusion comes from the observation that the
rsmB
genes are much more effective as positive regulators in a RsmA
+
strain of
E. carotovora
subsp.
carotovora
than in its RsmA
−
derivative.
E. herbicola
pv. gypsophilae produces a 290-base
rsmB
transcript that is not subject to processing. By contrast,
E. amylovora
produces 430- and 300-base
rsmB
transcripts, the latter presumably derived by processing of the primary transcript as previously noted with the transcripts of

rsmB
Ecc

. Southern blot hybridizations revealed the presence of
rsmB
homologs in
E. carotovora, E. chrysanthemi, E. amylovora, E. herbicola, E. stewartii
and
E. rhapontici
, as well as in other enterobacteria such as
Escherichia coli, Salmonella enterica
serovar Typhimurium,
Serratia marcescens, Shigella flexneri, Enterobacter aerogenes, Klebsiella pneumoniae, Yersinia enterocolitica
, and
Y. pseudotuberculosis
. A comparison of
rsmB
sequences from several of these enterobacterial species revealed a highly conserved 34-mer region which is predicted to play a role in positive regulation by
rsmB
RNA.
Genetic Evidence that the α5 Helix of the Receiver Domain of PhoB Is Involved in Interdomain Interactions
Authors: Allen Mindy P. , Zumbrennen Kimberly B. , McCleary William R. 
Published: July 2002

ABSTRACT

Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from
Escherichia coli
, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the
phoB
gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the
phoA
gene. However, another deletion mutant that contained the C-terminal α-helix of the receiver domain (α5) in addition to the entire output domain was unable to activate transcription of
phoA
. This result suggests that the α5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal ∼85% of CheY's receiver domain to the β5-α5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the α5 helix of its unphosphorylated receiver domain and its output domain.
Engineered Fatty Acid Biosynthesis in
Streptomyces
by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III
Authors: Smirnova Natalya , Reynolds Kevin A. 
Published: July 2002

ABSTRACT

The
Streptomyces glaucescens
β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in
S. glaucescens
resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D
3
acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in
Escherichia coli
and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within
S. glaucescens
.
Two Regions of GerE Required for Promoter Activation in
Bacillus subtilis
Authors: Crater Dinene L. , Moran Charles P. 
Published: July 2002

ABSTRACT

GerE from
Bacillus subtilis
is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent
cotC
promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent
cotX
promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the
cotX
promoter, but not from the
cotC
promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the
cotC
and
cotX
promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.
The Membrane-Associated Protein-Serine/Threonine Kinase fromSulfolobus solfataricusIs a Glycoprotein
Authors: Lower Brian H. , Kennelly Peter J. 
Published: July 2002

ABSTRACTTreatment of a sodium dodecyl sulfate-polyacrylamide gel with periodic acid-Schiff (PAS) stain or blotting withGalanthus nivalisagglutinin revealed the presence of several glycosylated polypeptides in a partially purified detergent extract of the membrane fraction ofSulfolobus solfataricus.One of the glycoproteins comigrated with the membrane-associated protein-serine/threonine kinase fromS. solfataricus, which had been radiolabeled by autophosphorylation with [32P]ATP in vitro. Treatment with a chemical deglycosylating agent, trifluoromethanesulfonic acid, abolished PAS staining and reduced theMrof the protein kinase from ∼67,000 to ∼62,000. Protein kinase activity also adhered to, and could be eluted from, agarose beads containing boundG. nivalisagglutinin. Glycosylation of the protein kinase implies that at least a portion of this integral membrane protein resides on the external surface of the cell membrane.
Assembly of Colicin A in the Outer Membrane of Producing
Escherichia coli
Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion
Authors: Cavard Daniele 
Published: July 2002

ABSTRACT

Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of
Escherichia coli
. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the
cal
gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.
PII T-Loop Mutations Affecting Signal Transduction to NtrB Also Abolish Yeast Two-Hybrid Interactions
Authors: Martínez-Argudo Isabel , Contreras Asunción 
Published: July 2002

ABSTRACT

Mutations A49P and Δ47-53 at the T loop of the
Escherichia coli
GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schepke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment. The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.
Roles of the C-Terminal End of SecY in Protein Translocation and Viability of
Escherichia coli
Authors: Chiba Kazuhiko , Mori Hiroyuki , Ito Koreaki 
Published: July 2002

ABSTRACT
SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation.
Genetic Organization of the
Vibrio harveyi dnaA
Gene Region and Analysis of the Function of the
V. harveyi
DnaA Protein in
Escherichia coli
Authors: Berenstein Dvora , Olesen Kirsten , Speck Christian , Skovgaard Ole 
Published: July 2002

ABSTRACT

The
Vibrionaceae
family is distantly related to
Enterobacteriaceae
within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the
dnaA
gene region of
Vibrio harveyi
and found that although the organization of the
V. harveyi dnaA
region differs from that of
Escherichia coli
, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the
dnaA
promoter region. The DnaA proteins of
V. harveyi
and
E. coli
are interchangeable and function nearly identically in controlling
dnaA
transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.
Direct Quantification of Human Cytomegalovirus Immediate-Early and Late mRNA Levels in Blood of Lung Transplant Recipients by Competitive Nucleic Acid Sequence-Based Amplification
Authors: Greijer Astrid E. , Verschuuren Erik A. M. , Harmsen Martin C. , Dekkers Chantal A. J. , Adriaanse Henriëtte M. A. , The T. Hauw , Middeldorp Jaap M. 
Published: July 2002

ABSTRACT

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10
4
copies per 100 μl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.