bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteomic Analysis of Wild-Type
Sinorhizobium meliloti
Responses to
N
-Acyl Homoserine Lactone Quorum-Sensing Signals and the Transition to Stationary Phase
Authors: Chen Hancai , Teplitski Max , Robinson Jayne B. , Rolfe Barry G. , Bauer Wolfgang D. 
Published: August 2003

ABSTRACT

Proteome analysis revealed that two long-chain
N
-acyl homoserine lactones (AHLs) produced by
Sinorhizobium meliloti
1021 induced significant differences in the accumulation of more than 100 polypeptides in early-log-phase cultures of the wild type. Fifty-six of the corresponding proteins have been identified by peptide mass fingerprinting. The proteins affected by addition of these two AHLs had diverse functions in carbon and nitrogen metabolism, energy cycles, metabolite transport, DNA synthesis, and protein turnover. Two hours of exposure to 3-oxo-C
16:1
-homoserine lactone (3-oxo-C
16:1
-HL) affected the accumulation of 40 of the 56 identified proteins, whereas comparable exposure to C
14
-HL affected 13 of the 56 proteins. Levels of four proteins were affected by both AHLs. Exposure to 3-oxo-C
16:1
-HL for 8 h affected the accumulation of 17 proteins, 12 of which had reduced accumulation. Of the 80 proteins identified as differing in accumulation between early-log- and early-stationary-phase cultures, only 13 were affected by exposure to 3-oxo-C
16:1
-HL or C
14
-HL. These results provide a foundation for future studies of the functions regulated by AHL quorum sensing in
S. meliloti
and help to establish proteomic analysis as a powerful global approach to the identification of quorum-sensing regulatory patterns in wild-type bacteria.
Open Reading Frame
all0601
from
Anabaena
sp. Strain PCC 7120 Represents a Novel Gene,
cnaT
, Required for Expression of the Nitrate Assimilation
nir
Operon
Authors: Frías José E. , Herrero Antonia , Flores Enrique 
Published: August 2003

ABSTRACT

Expression of the nitrate assimilation
nir
operon in the filamentous, heterocyst-forming cyanobacterium
Anabaena
sp. strain PCC 7120 requires the action of both the global nitrogen control transcription factor NtcA and the pathway-specific transcriptional regulator NtcB. In the genome of this cyanobacterium, the
ntcB
gene is found in a cluster of genes located in the complementary strand, upstream from the
nir
operon. Just downstream of
ntcB
, there is an open reading frame,
all0601
(previously designated
orf356
and now designated the
cnaT
gene), that putatively encodes a protein similar to proteins with glycosyl transferase activity and that is also present clustered together with
ntcB
homologues or nitrate assimilation structural genes in other cyanobacterial genomes. An insertional mutant of
cnaT
was generated and found to be unable to assimilate nitrate, although it could use ammonium or dinitrogen as a source of nitrogen for growth. In the mutant, under derepression conditions,
nir
operon mRNA (as determined by RNA-DNA hybridization and primer extension analysis) and enzymes of the nitrate reduction system (i.e., nitrate reductase and nitrite reductase) were expressed at low or undetectable levels. Inactivation of
cnaT
did not impair expression of
ntcB
, and expression of
cnaT
itself was constitutive and regulated by neither NtcA nor NtcB. Regulation of expression of the
nir
operon in
Anabaena
sp. strain PCC 7120 by CnaT and the previously described regulatory elements, NtcA and NtcB, is discussed.
Control of SXT Integration and Excision
Authors: Burrus Vincent , Waldor Matthew K. 
Published: August 2003

ABSTRACT

The
Vibrio cholerae
SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics. SXT integrates in a site-specific fashion at
prfC
and excises from the chromosome to form a circular but nonreplicative extrachromosomal form. Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int. Here we found that Int is necessary and sufficient for SXT integration and that
int
expression in recipient cells requires the SXT activators SetC and SetD. Although no
xis
-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision. We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration. Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs. Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors. In the absence of
prfC
, SXT integrated into several secondary attachment sites but preferentially into the 5′ end of
pntB
. SXT excision and transfer from a donor containing
pntB
::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility.
Composition, Acquisition, and Distribution of the Vi Exopolysaccharide-Encoding
Salmonella enterica
Pathogenicity Island SPI-7
Authors: Pickard Derek , Wain John , Baker Stephen , Line Alexandra , Chohan Sonia , Fookes Maria , Barron Andrew , Ó Gaora Peadar , Chabalgoity José A. , Thanky Niren , Scholes Christoph , Thomson Nicholas , Quail Michael , Parkhill Julian , Dougan Gordon 
Published: August 2003

ABSTRACT

Vi capsular polysaccharide production is encoded by the
viaB
locus, which has a limited distribution in
Salmonella enterica
serovars. In
S. enterica
serovar Typhi,
viaB
is encoded on a 134-kb pathogenicity island known as SPI-7 that is located between partially duplicated tRNA

pheU

sites. Functional and bioinformatic analysis suggests that SPI-7 has a mosaic structure and may have evolved as a consequence of several independent insertion events. Analysis of
viaB
-associated DNA in Vi-positive
S. enterica
serovar Paratyphi C and
S. enterica
serovar Dublin isolates revealed the presence of similar SPI-7 islands. In
S. enterica
serovars Paratyphi C and Dublin, the SopE bacteriophage and a 15-kb fragment adjacent to the intact tRNA

pheU

site were absent. In
S. enterica
serovar Paratyphi C only, a region encoding a type IV pilus involved in the adherence of
S. enterica
serovar Typhi to host cells was missing. The remainder of the SPI-7 islands investigated exhibited over 99% DNA sequence identity in the three serovars. Of 30 other
Salmonella
serovars examined, 24 contained no insertions at the equivalent tRNA

pheU

site, 2 had a 3.7-kb insertion, and 4 showed sequence variation at the tRNA

pheU

-
phoN
junction, which was not analyzed further. Sequence analysis of the SPI-7 region from
S. enterica
serovar Typhi strain CT18 revealed significant synteny with clusters of genes from a variety of saprophytic bacteria and phytobacteria, including
Pseudomonas aeruginosa
and
Xanthomonas axonopodis
pv. citri. This analysis suggested that SPI-7 may be a mobile element, such as a conjugative transposon or an integrated plasmid remnant.
Membrane Localization of Motility, Signaling, and Polyketide Synthetase Proteins in
Myxococcus xanthus
Authors: Simunovic Vesna , Gherardini Frank C. , Shimkets Lawrence J. 
Published: August 2003

ABSTRACT

Myxococcus xanthus
cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm
−3
). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm
−3
) and low buoyant density (1.169 to 1.171 g cm
−3
). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm
−3
) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.
Coordinate Regulation of the
Escherichia coli
Formate Dehydrogenase
fdnGHI
and
fdhF
Genes in Response to Nitrate, Nitrite, and Formate: Roles for NarL and NarP
Authors: Wang Henian , Gunsalus Robert P. 
Published: August 2003

ABSTRACT

Escherichia coli
possesses three distinct formate dehydrogenase enzymes encoded by the
fdnGHI
,
fdhF
, and
fdoGHI
operons. To examine how two of the formate dehyrogenase operons (
fdnGHI
and
fdhF
) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with
fdnG-lacZ
and
fdhF-lacZ
reporter fusions. Complementary patterns of gene expression were seen. Optimal
fdhF-lacZ
expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast,
fdnG-lacZ
expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce
fdnG-lacZ
expression. However, NarP could not induce expression; rather, it functioned as an antagonist of
fdnG-lacZ
expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.
Protein Content of Polyhedral Organelles Involved in Coenzyme B
12
-Dependent Degradation of 1,2-Propanediol in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Havemann Gregory D. , Bobik Thomas A. 
Published: August 2003

ABSTRACT

Salmonella enterica
forms polyhedral organelles during coenzyme B
12
-dependent growth on 1,2-propanediol (1,2-PD). Previously, these organelles were shown to consist of a protein shell partly composed of the PduA protein, the majority of the cell's B
12
-dependent diol dehydratase, and additional unidentified proteins. In this report, the polyhedral organelles involved in B
12
-dependent 1,2-PD degradation by
S. enterica
were purified by a combination of detergent extraction and differential and density gradient centrifugation. The course of the purification was monitored by electron microscopy and gel electrophoresis, as well as enzymatic assay of B
12
-dependent diol dehydratase. Following one- and two-dimensional gel electrophoresis of purified organelles, the identities and relative abundance of their constituent proteins were determined by N-terminal sequencing, protein mass fingerprinting, Western blotting, and densitometry. These analyses indicated that the organelles consisted of at least 15 proteins, including PduABB′CDEGHJKOPTU and one unidentified protein. Seven of the proteins identified (PduABB′JKTU) have some sequence similarity to the shell proteins of carboxysomes (a polyhedral organelle involved in autotrophic CO
2
fixation), suggesting that the
S. enterica
organelles and carboxysomes have a related multiprotein shell. In addition,
S. enterica
organelles contained four enzymes: B
12
-dependent diol dehydratase, its putative reactivating factor, aldehyde dehydrogenase, and ATP cob(I)alamin adenosyltransferase. This complement of enzymes indicates that the primary catalytic function of the
S. enterica
organelles is the conversion of 1,2-PD to propionyl coenzyme A (which is consistent with our prior proposal that the
S. enterica
organelles function to minimize aldehyde toxicity during growth on 1,2-PD). The possibility that similar protein-bound organelles may be more widespread in nature than currently recognized is discussed.
RtsA and RtsB Coordinately Regulate Expression of the Invasion and Flagellar Genes inSalmonella entericaSerovar Typhimurium
Authors: Ellermeier Craig D. , Slauch James M. 
Published: August 2003

ABSTRACTSalmonella entericaserovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process. The bacterium responds by tightly regulating the expression of virulence genes. We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNAPheUinS. entericaserovar Typhimurium. RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein. In a random screen, we identified five RtsA-regulated fusions, all belonging to theSalmonellapathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells. We show that RtsA increases expression of the invasion genes by inducinghilAexpression. RtsA also induces expression ofhilD,hilC, and theinvFoperon. However, induction ofhilAis independent of HilC and HilD and is mediated by direct binding of RtsA to thehilApromoter. The phenotype of anrtsAnull mutation is similar to the phenotype of ahilCmutation, both of which decrease expression of SPI1 genes approximately twofold. We also show that RtsA can induce expression of a SPI1 TTSS effector,slrP, independent of any SPI1 regulatory protein. RtsB represses expression of the flagellar genes by binding to theflhDCpromoter region. Repression of the positive activatorsflhDCdecreases expression of the entire flagellar regulon. We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine.
Regulation of Expression of Scaffoldin-Related Genes in
Clostridium thermocellum
Authors: Dror Tali W. , Rolider Adi , Bayer Edward A. , Lamed Raphael , Shoham Yuval 
Published: August 2003

ABSTRACT

Clostridium thermocellum
produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose. The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound. The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin. The transcriptional level of the
olpB
,
orf2
,
sdbA
, and
cipA
genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation. The mRNA level of
olpB
,
orf2
, and
cipA
varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h
−1
and 2 to 10 transcripts per cell at a growth rate of 0.35 h
−1
in batch culture. The mRNA level of
sdbA
was about three transcripts per cell and was not influenced by growth rate. Primer extension analysis revealed two major transcriptional start sites, at −81 and −50 bp, upstream of the translational start site of the
cipA
gene. The potential promoters exhibited homology to the known sigma factors σ
A
and σ
L
(σ
54
) of
Bacillus subtilis
. Transcription from the σ
L
-like promoter was found under all growth conditions, whereas transcription from the σ
A
-like promoter was significant only under carbon limitation. The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.
ClpE from
Lactococcus lactis
Promotes Repression of CtsR-Dependent Gene Expression
Authors: Varmanen Pekka , Vogensen Finn K. , Hammer Karin , Palva Airi , Ingmer Hanne 
Published: August 2003

ABSTRACT

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in
Lactococcus lactis
is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of
clpP
encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in
Bacillus subtilis
. Thus, our data point to a regulatory role of ClpE in turning off
clpP
gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.
Anthranilate Synthase Can Generate Sufficient Phosphoribosyl Amine for Thiamine Synthesis in
Salmonella enterica
Authors: Ramos I. , Downs Diana M. 
Published: August 2003

ABSTRACT

In bacteria, the biosynthetic pathway for the hydroxymethyl pyrimidine moiety of thiamine shares metabolic intermediates with purine biosynthesis. The two pathways branch after the compound aminoimidazole ribotide. Past work has shown that the first common metabolite, phosphoribosyl amine (PRA), can be generated in the absence of the first enzyme in purine biosynthesis, PurF. PurF-independent PRA synthesis is dependent on both strain background and growth conditions. Standard genetic approaches have not identified a gene product singly responsible for PurF-independent PRA formation. This result has led to the hypothesis that multiple enzymes contribute to PRA synthesis, possibly as the result of side products from their dedicated reaction. A mutation that was able to restore PRA synthesis in a
purF gnd
mutant strain was identified and found to map in the gene coding for the TrpD subunit of the anthranilate synthase (AS)-phosphoribosyl transferase (PRT) complex. Genetic analyses indicated that wild-type AS-PRT was able to generate PRA in vivo and that the P362L mutant of TrpD facilitated this synthesis. In vitro activity assays showed that the mutant AS was able to generate PRA from ammonia and phosphoribosyl pyrophosphate. This work identifies a new reaction catalyzed by AS-PRT and considers it in the context of cellular thiamine synthesis and metabolic flexibility.
Mutational Loss of a K
+
and NH
4
+
Transporter Affects the Growth and Endospore Formation of Alkaliphilic
Bacillus pseudofirmus
OF4
Authors: Wei Yi , Southworth Thomas W. , Kloster Hilde , Ito Masahiro , Guffanti Arthur A. , Moir Anne , Krulwich Terry A. 
Published: August 2003

ABSTRACT

A putative transport protein (Orf9) of alkaliphilic
Bacillus pseudofirmus
OF4 belongs to a transporter family (CPA-2) of diverse K
+
efflux proteins and cation antiporters. Orf9 greatly increased the concentration of K
+
required for growth of a K
+
uptake mutant of
Escherichia coli
. The cytoplasmic K
+
content of the cells was reduced, consistent with an efflux mechanism. Orf9-dependent translocation of K
+
in
E. coli
is apparently bidirectional, since ammonium-sensitive uptake of K
+
could be shown in K
+
-depleted cells. The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9. No Orf9-dependent K
+
(Na
+
)/H
+
antiport activity was found in membrane vesicles. Nonpolar deletion mutants in the
orf9
locus of the alkaliphile chromosome exhibited no K
+
-related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen. Their patterns of growth and ammonium content suggested a physiological role for the
orf9
locus in bidirectional ammonium transport. Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an
orf8
deletion. Uptake was proton motive force dependent and was inhibited by K
+
. Orf9 is proposed to be designated AmhT (ammonium homeostasis). Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation. Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the
orf9
and
orf7
-
orf10
deletion mutants.
Cyclic AMP Receptor Protein-Dependent Activation of the
Escherichia coli acs
P2 Promoter by a Synergistic Class III Mechanism
Authors: Beatty Christine M. , Browning Douglas F. , Busby Stephen J. W. , Wolfe Alan J. 
Published: August 2003

ABSTRACT

The cyclic AMP receptor protein (CRP) activates transcription of the
Escherichia coli acs
gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of
acs
, the distal
acs
P1 and the proximal
acs
P2. In this study, we demonstrated that
acs
P2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to
acs
P2. We proposed that CRP activates transcription from
acs
P2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of
acs
expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that
acs
transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to
acs
transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.