bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Unphosphorylated Receiver Domain of PhoB Silences the Activity of Its Output Domain
Authors: Ellison Damon W. , McCleary William R. 
Published: July 2002

ABSTRACT

PhoB is the response regulator of the Pho regulon. It is composed of two distinct domains, an N-terminal receiver domain and a C-terminal output domain that binds DNA and interacts with ς
70
to activate transcription of the Pho regulon. Phosphorylation of the receiver domain is required for activation of the protein. The mechanism of activation by phosphorylation has not yet been determined. To better understand the function of the receiver domain in controlling the activity of the output domain, a direct comparison was made between unphosphorylated PhoB and its solitary DNA-binding domain (PhoB
DBD
) for DNA binding and transcriptional activation. Using fluorescence anisotropy, it was found that PhoB
DBD
bound to the
pho
box with an affinity seven times greater than that of unphosphorylated PhoB. It was also found that PhoB
DBD
was better able to activate transcription than the full-length, unmodified protein. We conclude that the unphosphorylated receiver domain of PhoB silences the activity of its output domain. These results suggest that upon phosphorylation of the receiver domain of PhoB, the inhibition placed upon the output domain is relieved by a conformational change that alters interactions between the unphosphorylated receiver domain and the output domain.
NorM of
Vibrio parahaemolyticus
Is an Na
+
-Driven Multidrug Efflux Pump
Authors: Morita Yuji , Kataoka Atsuko , Shiota Sumiko , Mizushima Tohru , Tsuchiya Tomofusa 
Published: July 2002

ABSTRACT

NorM of
Vibrio parahaemolyticus
apparently is a new type of multidrug efflux protein, with no significant sequence similarity to any known transport proteins. Based on the following experimental results, we conclude that NorM is an Na
+
-driven Na
+
/drug antiporter. (i) Energy-dependent ethidium efflux from cells possessing NorM was observed in the presence of Na
+
but not of K
+
. (ii) An artificially imposed, inwardly directed Na
+
gradient elicited ethidium efflux from cells. (iii) The addition of ethidium to cells loaded with Na
+
elicited Na
+
efflux. Thus, NorM is an Na
+
/drug antiporting multidrug efflux pump, the first to be found in the biological world. Judging from the similarity of the NorM sequence to those of putative proteins in sequence databases, it seems that Na
+
/drug antiporters are present not only in
V. parahaemolyticus
but also in a wide range of other organisms.
A
Xanthomonas
Alkyl Hydroperoxide Reductase Subunit C (
ahpC
) Mutant Showed an Altered Peroxide Stress Response and Complex Regulation of the Compensatory Response of Peroxide Detoxification Enzymes
Authors: Mongkolsuk Skorn , Whangsuk Wirongrong , Vattanaviboon Paiboon , Loprasert Suvit , Fuangthong Mayuree 
Published: July 2002

ABSTRACT

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for alkyl peroxide metabolism. A
Xanthomonas ahpC
mutant was constructed. The mutant had increased sensitivity to organic peroxide killing, but was unexpectedly hyperresistant to H
2
O
2
killing. Analysis of peroxide detoxification enzymes in this mutant revealed differential alteration in catalase activities in that its bifunctional catalase-peroxidase enzyme and major monofunctional catalase (Kat1) increased severalfold, while levels of its third growth-phase-regulated catalase (KatE) did not change. The increase in catalase activities was a compensatory response to lack of AhpC, and the phenotype was complemented by expression of a functional
ahpC
gene. Regulation of the catalase compensatory response was complex. The Kat1 compensatory response increase in activity was mediated by OxyR, since it was abolished in an
oxyR
mutant. In contrast, the compensatory response increase in activity for the bifunctional catalase-peroxidase enzyme was mediated by an unknown regulator, independent of OxyR. Moreover, the mutation in
ahpC
appeared to convert OxyR from a reduced form to an oxidized form that activated genes in the OxyR regulon in uninduced cells. This complex regulation of the peroxide stress response in
Xanthomonas
differed from that in other bacteria.
A Broad-Specificity Multidrug Efflux Pump Requiring a Pair of Homologous SMR-Type Proteins
Authors: Jack Donald L. , Storms Michael L. , Tchieu Jason H. , Paulsen Ian T. , Saier Milton H. 
Published: July 2002

ABSTRACT

The
Bacillus subtilis
genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in
Escherichia coli
of one such operon,
ykkCD
, but not expression of either
ykkC
or
ykkD
alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.
Response of
Bacillus subtilis
to Cerulenin and Acquisition of Resistance
Authors: Schujman Gustavo E. , Choi Keum-Hwa , Altabe Silvia , Rock Charles O. , de Mendoza Diego 
Published: July 2002

ABSTRACT

Cerulenin is a fungal mycotoxin that potently inhibits fatty acid synthesis by covalent modification of the active site thiol of the chain-elongation subtypes of β-ketoacyl-acyl carrier protein (ACP) synthases. The
Bacillus subtilis fabF
(
yjaY
) gene (

fabF
b

) encodes an enzyme that catalyzes the condensation of malonyl-ACP with acyl-ACP to extend the growing acyl chain by two carbons. There were two mechanisms by which
B. subtilis
adapted to exposure to this antibiotic. First, reporter gene analysis demonstrated that transcription of the operon containing the
fabF
gene increased eightfold in response to a cerulenin challenge. This response was selective for the inhibition of fatty acid synthesis, since triclosan, an inhibitor of enoyl-ACP reductase, triggered an increase in
fabF
reporter gene expression while nalidixic acid did not. Second, spontaneous mutants arose that exhibited a 10-fold increase in the MIC of cerulenin. The mutation mapped at the
B. subtilis fabF
locus, and sequence analysis of the mutant
fabF
allele showed that a single base change resulted in the synthesis of FabF
b
[I108F]. The purified FabF
b
and FabF
b
[I108F] proteins had similar specific activities with myristoyl-ACP as the substrate. FabF
b
exhibited a 50% inhibitory concentration (IC
50
) of cerulenin of 0.1 μM, whereas the IC
50
for FabF
b
[I108] was 50-fold higher (5 μM). These biochemical data explain the absence of an overt growth defect coupled with the cerulenin resistance phenotype of the mutant strain.
Xenorhabdus nematophilus
as a Model for Host-Bacterium Interactions:
rpoS
Is Necessary for Mutualism with Nematodes
Authors: Vivas Eugenio I. , Goodrich-Blair Heidi 
Published: July 2002

ABSTRACT

Xenorhabdus nematophilus
, a gram-negative bacterium, is a mutualist of
Steinernema carpocapsae
nematodes and a pathogen of larval-stage insects. We use this organism as a model of host-microbe interactions to identify the functions bacteria require for mutualism, pathogenesis, or both. In many gram-negative bacteria, the transcription factor ς
S
controls regulons that can mediate stress resistance, survival, or host interactions. Therefore, we examined the role of ς
S
in the ability of
X. nematophilus
to interact with its hosts. We cloned, sequenced, and disrupted the
X. nematophilus rpoS
gene that encodes ς
S
. The
X. nematophilus rpoS
mutant pathogenized insects as well as its wild-type parent. However, the
rpoS
mutant could not mutualistically colonize nematode intestines. To our knowledge, this is the first report of a specific allele that affects the ability of
X. nematophilus
to exist within nematode intestines, an important step in understanding the molecular mechanisms of this association.
Oxidation of Phenolate Siderophores by the Multicopper Oxidase Encoded by theEscherichia coli yacKGene
Authors: Kim Chulhwan , Lorenz W. Walter , Hoopes J. Todd , Dean Jeffrey F. D. 
Published: July 2002

ABSTRACTA gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned fromEscherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong opticalA610(Ε610= 10,890 M−1cm−1) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized byE. colifor iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized bySaccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron,yacKMCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, howyacKMCO moderates the sensitivity ofE. colito copper.
Properties of Spores of
Bacillus subtilis
Blocked at an Intermediate Stage in Spore Germination
Authors: Setlow Barbara , Melly Elizabeth , Setlow Peter 
Published: July 2002

ABSTRACT

Germination of mutant spores of
Bacillus subtilis
unable to degrade their cortex is accompanied by excretion of dipicolinic acid and uptake of some core water. However, compared to wild-type germinated spores in which the cortex has been degraded, the germinated mutant spores accumulated less core water, exhibited greatly reduced enzyme activity in the spore core, synthesized neither ATP nor reduced pyridine or flavin nucleotides, and had significantly higher resistance to heat and UV irradiation. We propose that the germinated spores in which the cortex has not been degraded represent an intermediate stage in spore germination, which we term stage I.
FimW Is a Negative Regulator Affecting Type 1 Fimbrial Expression inSalmonella entericaSerovar Typhimurium
Authors: Tinker Juliette K. , Hancox Lisa S. , Clegg Steven 
Published: July 2002

ABSTRACTType 1 fimbriae are proteinaceous surface appendages that carry adhesins specific for mannosylated glycoproteins. These fimbriae are found on most members of the familyEnterobacteriaceaeand are known to facilitate binding to a variety of eukaryotic cells, including those found on the mucosal surfaces of the alimentary tract. We have shown that the regulation of type 1 fimbrial expression inSalmonella entericaserovar Typhimurium is controlled, in part, by the products of four genes found within thefimgene cluster:fimZ,fimY,fimW, andfimU. To better understand the specific role of FimW in fimbrial expression, a mutation was constructed in this gene by the insertion of a kanamycin resistance DNA cassette into the chromosome. The resultingfimWmutation was characterized by mannose-sensitive hemagglutination and agglutination with fimbria-specific antiserum. Assays suggested that this mutant was more strongly fimbriate than the parental strain, exhibiting a four- to eightfold increase in fimbrial production. ThefimWmutation was introduced into a second strain ofSalmonella entericaserovar Typhimurium, and this mutant was also found to be strongly fimbriate compared to the parental strain. Consistent with the role of this protein as a negative regulator,fimA-lacZexpression in serovar Typhimurium, as well as inEscherichia coli, was increased twofold in the absence of functional FimW. Primer extension analysis determined thatfimWtranscription is initiated from its own promoter 31 bp upstream of the translation start site. Analysis using afimW-lacZreporter indicated thatfimWexpression in serovar Typhimurium was increased under conditions that select for poorly fimbriate bacteria and lowfimAexpression. FimW also appears to act as an autoregulator, since expression from thefimW-lacZreporter was increased in afimWmutant. FimW was partially purified by fusion with theE. colimaltose-binding protein. Use of this FimW protein extract, as well as others, in DNA-binding assays was unable to identify a specific binding site for FimW in thefimA,fimZ,fimY, orfimWpromoter regions. To analyze protein-protein interactions, FimW was expressed in a LexA-based two-hybrid system inE. coli. A significant interaction between FimW and the DNA-binding activator protein, FimZ, was detected using this system. These results indicate that FimW is a negative regulator of serovar Typhimurium type 1 fimbrial expression and may function by interfering with FimZ-mediated activation offimAexpression.
Starchless Mutants ofChlamydomonas reinhardtiiLack the Small Subunit of a Heterotetrameric ADP-Glucose Pyrophosphorylase
Authors: Zabawinski Christophe , Van Den Koornhuyse Nathalie , D'Hulst Christophe , Schlichting Ralf , Giersch Christoph , Delrue Brigitte , Lacroix Jean-Marie , Preiss Jack , Ball Steven 
Published: July 2002

ABSTRACTADP-glucose synthesis through ADP-glucose pyrophosphorylase defines the major rate-controlling step of storage polysaccharide synthesis in both bacteria and plants. We have isolated mutant strains defective in theSTA6locus of the monocellular green algaChlamydomonas reinhardtiithat fail to accumulate starch and lack ADP-glucose pyrophosphorylase activity. We show that this locus encodes a 514-amino-acid polypeptide corresponding to a mature 50-kDa protein with homology to vascular plant ADP-glucose pyrophosphorylase small-subunit sequences. This gene segregates independently from the previously characterizedSTA1locus that encodes the large 53-kDa subunit of the same heterotetramer enzyme. BecauseSTA1locus mutants have retained an AGPase but exhibit lower sensitivity to 3-phosphoglyceric acid activation, we suggest that the small and large subunits of the enzyme define, respectively, the catalytic and regulatory subunits of AGPase in unicellular green algae. We provide preliminary evidence that both the small-subunit mRNA abundance and enzyme activity, and therefore also starch metabolism, may be controlled by the circadian clock.
Network Identification and Flux Quantification in the Central Metabolism ofSaccharomyces cerevisiaeunder Different Conditions of Glucose Repression
Authors: Gombert Andreas Karoly , Moreira dos Santos Margarida , Christensen Bjarke , Nielsen Jens 
Published: July 2002

ABSTRACTThe network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells ofSaccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h−1or in a batch culture with a specific growth rate of 0.37 h−1. Experiments were carried out using [1-13C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism ofS. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of theMIG1gene did not cause changes in the metabolic network structure or in the flux pattern.
Five-Gene Cluster in
Clostridium thermoaceticum
Consisting of Two Divergent Operons Encoding Rubredoxin Oxidoreductase- Rubredoxin and Rubrerythrin–Type A Flavoprotein– High-Molecular-Weight Rubredoxin
Authors: Das Amaresh , Coulter Eric D. , Kurtz Donald M. , Ljungdahl Lars G. 
Published: July 2002

ABSTRACT

A five-gene cluster encoding four nonheme iron proteins and a flavoprotein from the thermophilic anaerobic bacterium
Clostridium thermoaceticum
(
Moorella thermoacetica
) was cloned and sequenced. Based on analysis of deduced amino acid sequences, the genes were identified as
rub
(rubredoxin),
rbo
(rubredoxin oxidoreductase),
rbr
(rubrerythrin),
fprA
(type A flavoprotein), and a gene referred to as
hrb
(high-molecular-weight rubredoxin). Northern blot analysis demonstrated that the five-gene cluster is organized as two subclusters, consisting of two divergently transcribed operons,
rbr-fprA-hrb
and
rbo-rub
. The
rbr, fprA
, and
rub
genes were expressed in
Escherichia coli
, and their encoded recombinant proteins were purified. The molecular masses, UV-visible absorption spectra, and cofactor contents of the recombinant rubrerythrin, rubredoxin, and type A flavoprotein were similar to those of respective homologs from other microorganisms. Antibodies raised against
Desulfovibrio vulgaris
Rbr reacted with both native and recombinant Rbr from
C. thermoaceticum
, indicating that this protein was expressed in the native organism. Since Rbr and Rbo have been recently implicated in oxidative stress protection in several anaerobic bacteria and archaea, we suggest a similar function of these proteins in oxygen tolerance of
C. thermoaceticum
.
Substitutions in Bacteriophage T4 AsiA and
Escherichia coli
ς
70
That Suppress T4
motA
Activation Mutations
Authors: Cicero Marco P. , Sharp Meghan M. , Gross Carol A. , Kreuzer Kenneth N. 
Published: July 2002

ABSTRACT

Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with
Escherichia coli
RNA polymerase holoenzyme containing the ς
70
subunit. A
motA
positive control (pc) mutant,
motA-pc1
, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς
70
mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione
S
-transferase fusions of the five mutant ς
70
proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς
70
substitutions affected the 4.2 region, which binds the −35 sequence of
E. coli
promoters. In the presence of
E. coli
RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard
E. coli
promoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support
E. coli
growth.
Genomic Analyses of Anaerobically Induced Genes in
Saccharomyces cerevisiae
: Functional Roles of Rox1 and Other Factors in Mediating the Anoxic Response
Authors: Kwast Kurt E. , Lai Liang-Chuan , Menda Nina , James David T. , Aref Susanne , Burke Patricia V. 
Published: July 2002

ABSTRACT

DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in
Saccharomyces cerevisiae
. Multiple growth conditions for wild-type and
rox1
null strains were used to identify open reading frames with a statistically robust response to this repressor. These results were compared to those obtained for a wild-type strain in response to oxygen availability. Transcripts of nearly one-sixth of the genome were differentially expressed (
P
< 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia. Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (
P
< 0.05) up-regulated in the
rox1
null strains under aerobiosis. Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in
S. cerevisiae
. Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (∼1/3), cell wall maintenance (∼1/8), carbohydrate metabolism (∼1/10), and lipid metabolism (∼1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance. Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other
trans
-acting factors in mediating the physiological response of
S. cerevisiae
to anaerobic conditions.
SitABCD Is the Alkaline Mn2+Transporter ofSalmonella entericaSerovar Typhimurium
Authors: Kehres David G. , Janakiraman Anuradha , Slauch James M. , Maguire Michael E. 
Published: July 2002

ABSTRACTMntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn2+transporter ofSalmonella entericaserovar Typhimurium andEscherichia coli. S. entericaserovar Typhimurium MntH expression is important for full virulence; however, strains carrying anmntHdeletion are only partially attenuated and display no obvious signs of Mn2+deficiency. We noted that promoter sequences formntHand for the putative Fe2+transportersitABCDappeared to have the same regulatory element responsive to Mn2+and so hypothesized thatsitABCDcould transport Mn2+with high affinity. We have now characterized transport by SitABCD inS. entericaserovar Typhimurium using54Mn2+and55Fe2+and compared its properties to those of MntH. SitABCD mediates the influx of Mn2+with an apparent affinity (Ka) identical to that of MntH, 0.1 μM. It also transports Fe2+but with aKa30 to 100 times lower, 3 to 10 μM. Inhibition of54Mn2+transport by Fe2+and of55Fe2+transport by Mn2+gave inhibition constants comparable to each cation'sKafor influx. Since micromolar concentrations of free Fe2+are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn2+transporter. The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd2+Kiof ≈1 μM and a Co2+Kiof ≈20 μM and with Ni2+, Cu2+, and Fe3+inhibiting both transporters only at concentrations of >0.1 mM. The one difference is that Zn2+exhibits potent inhibition of SitABCD (Ki= 1 to 3 μM) but inhibits MntH weakly (Ki> 50 μM). We have previously shown that MntH transports Mn2+most effectively under acidic conditions. In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn2+at slightly alkaline pHs. Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn2+uptake in different physiological environments.