bluuebunny/crossref_metadata_2025 huggingface dataset:
The
Pseudomonas aeruginosa devB/SOL
Homolog,
pgl
, Is a Member of the
hex
Regulon and Encodes 6-Phosphogluconolactonase
Authors: Hager Paul W. , Calfee M. Worth , Phibbs Paul V.
Published: July 2002

A cyclic version of the Entner-Doudoroff pathway is used by
Pseudomonas aeruginosa
to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the
hex
regulon. Within the
hex
cluster is an open reading frame (ORF) with homology to the
devB/SOL
family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5′ end of
zwf
(encodes glucose-6-phosphate dehydrogenase) and is followed immediately by
eda
(encodes the Entner-Doudoroff aldolase). The
devB/SOL
homolog was inactivated in
P. aeruginosa
PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned
devB/SOL
homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as
pgl
and propose that the
devB/SOL
family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and
Plasmodium falciparum
, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in
P. aeruginosa
PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in
P. aeruginosa
PAO8026 (
hexR
). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by
pgl
, a structural gene of the
hex
regulon.
Vibrio cholerae
H-NS Silences Virulence Gene Expression at Multiple Steps in the ToxR Regulatory Cascade
Authors: Nye Melinda B. , Pfau James D. , Skorupski Karen , Taylor Ronald K.
Published: July 2002

H-NS is an abundant nucleoid-associated protein involved in the maintenance of chromosomal architecture in bacteria. H-NS also has a role in silencing the expression of a variety of environmentally regulated genes during growth under nonpermissive conditions. In this study we demonstrate a role for H-NS in the negative modulation of expression of several genes within the ToxR virulence regulon of
Vibrio cholerae
. Deletion of
hns
resulted in high, nearly constitutive levels of expression of the genes encoding cholera toxin, toxin-coregulated pilus, and the ToxT virulence gene regulatory protein. For the cholera toxin- and ToxT-encoding genes, elevated expression in an
hns
mutant was found to occur in the absence of the cognate activator proteins, suggesting that H-NS functions directly at these promoters to decrease gene expression. Deletion analysis of the region upstream of
toxT
suggests that an extensive region located far upstream of the transcriptional start site is required for complete H-NS-mediated repression of gene expression. These data indicate that H-NS negatively influences multiple levels of gene expression within the
V. cholerae
virulence cascade and raise the possibility that the transcriptional activator proteins in the ToxR regulon function to counteract the repressive effects of H-NS at the various promoters as well as to recruit RNA polymerase.
Mutations in Each of thetolGenes ofPseudomonas putidaReveal that They Are Critical for Maintenance of Outer Membrane Stability
Authors: Llamas María A. , Ramos Juan L. , Rodríguez-Herva José J.
Published: July 2002

The outer membrane of gram-negative bacteria functions as a permeability barrier that protects cells against a large number of antibacterial agents. OprL protein ofPseudomonas putidahas been shown to be crucial to maintain the stability of this cell component (J. J. Rodrı́guez-Herva, M.-I. Ramos-González, and J. L. Ramos. J. Bacteriol. 178:1699–1706, 1996). In the present study we cloned and mutagenized theorf1,tolQ,tolR,tolA, andtolBgenes fromP. putidaKT2440, which were located upstream of theoprLgene. Polar and nonpolar mutations of theP. putida tolQ,tolR,tolA, andtolBgenes were generated in vitro by using the Ω-Kmrinterposon, which carries two transcriptional stop signals, or a promoterlessxylEcassette, lacking any transcriptional stop signal, respectively. The mutant constructs were used to inactivate, by reverse genetics procedures, the corresponding chromosomal copies of the genes. The phenotype of each mutant strain was analyzed and compared with those of the wild-type strain and the previously characterizedP. putida oprL::xylEmutant. All mutant strains exhibited a similar phenotype: altered cell morphology, bleb formation at the cell surface, release of periplasmic and outer membrane proteins to the extracellular medium, increased sensitivity to a variety of compounds (i.e., EDTA, sodium dodecyl sulfate, deoxycholate, and some antibiotics), filament formation, and severely reduced cell motility. Altogether, these results demonstrate the importance of the Tol-OprL system for the maintenance of outer membrane integrity inP. putidaand suggest a possible role of these proteins in assembling outer membrane components.
Role of aCandida albicansP1-Type ATPase in Resistance to Copper and Silver Ion Toxicity
Authors: Riggle Perry J. , Kumamoto Carol A.
Published: July 2002

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identifyCandida albicansgenes that are involved in resistance to copper ion toxicity, a library containing inserts ofC. albicansgenomic DNA was used to complement the copper sensitivity phenotype of aSaccharomyces cerevisiae cup1Δ strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termedCRD1(for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termedCRD2. Transcription of theCRD1gene was found to increase upon growth with increasing copper levels, while theCRD2mRNA was expressed at a constant level. Strains with theCRD1gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 μM CuSO4. Thesecrd1strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with theCRD2gene disrupted showed reduced growth rate with increasing copper concentration, thecrd2mutants eventually attained wild-type levels of growth, demonstrating thatCRD2is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence ofC. albicansas a nosocomial pathogen.
The Amino Terminus of
Pseudomonas aeruginosa
Outer Membrane Protein OprF Forms Channels in Lipid Bilayer Membranes: Correlation with a Three-Dimensional Model
Authors: Brinkman Fiona S. L. , Bains Manjeet , Hancock Robert E. W.
Published: July 2002

Pseudomonas aeruginosa
OprF forms 0.36-nS channels and, rarely, 2- to 5-nS channels in lipid bilayer membranes. We show that a protein comprising only the N-terminal 162-amino-acid domain of OprF formed the smaller, but not the larger, channels in lipid bilayers. Circular dichroism spectroscopy indicated that this protein folds into a β-sheet-rich structure, and three-dimensional comparative modeling revealed that it shares significant structural similarity with the amino terminus of the orthologous protein
Escherichia coli
OmpA, which has been shown to form a β-barrel. OprF and OmpA share only 15% identity in this domain, yet these results support the utility of modeling such widely divergent β-barrel domains in three dimensions in order to reveal similarities not readily apparent through primary sequence comparisons. The model is used to further hypothesize why porin activity differs for the N-terminal domains of OprF and OmpA.
Yersinia enterocolitica
ClpB Affects Levels of Invasin and Motility
Authors: Badger Julie L. , Young Briana M. , Darwin Andrew J. , Miller Virginia L.
Published: July 2002

Expression of the
Yersinia enterocolitica inv
gene is dependent on growth phase and temperature.
inv
is maximally expressed at 23°C in late-exponential- to early-stationary-phase cultures. We previously reported the isolation of a
Y. enterocolitica
mutant (JB1A8v) that shows a decrease in invasin levels yet is hypermotile when grown at 23°C. JB1A8v has a transposon insertion within
uvrC
. Described here is the isolation and characterization of a clone that suppresses these mutant phenotypes of the
uvrC
mutant JB1A8v. This suppressing clone encodes ClpB (a Clp ATPase homologue). The
Y. enterocolitica
ClpB homologue is 30 to 40% identical to the ClpB proteins from various bacteria but is 80% identical to one of the two ClpB homologues of
Yersinia pestis
. A
clpB
::Tn
Max2
insertion mutant (JB69Qv) was constructed and determined to be deficient in invasin production and nonmotile when grown at 23°C. Analysis of
inv
and
fleB
(flagellin gene) transcript levels in JB69Qv suggested that ClpB has both transcriptional and posttranscriptional effects. In contrast, a
clpB
null mutant, BY1v, had no effect on invasin levels or motility. A model accounting for these observations is presented.
Maltose and Maltodextrin Transport in the Thermoacidophilic Gram-Positive Bacterium
Alicyclobacillus acidocaldarius
Is Mediated by a High-Affinity Transport System That Includes a Maltose Binding Protein Tolerant to Low pH
Authors: Hülsmann Anja , Lurz Rudi , Scheffel Frank , Schneider Erwin
Published: July 2002

We have studied the uptake of maltose in the thermoacidophilic gram-positive bacterium
Alicyclobacillus acidocaldarius
, which grows best at 57°C and pH 3.5. Under these conditions, accumulation of [
14
C]maltose was observed in cells grown with maltose but not in those grown with glucose. At lower temperatures or higher pH values, the transport rates substantially decreased. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The kinetic parameters (

K
m

of 0.91 ± 0.06 μM and
V
max
ranging from 0.6 to 3.7 nmol/min/mg of protein) are consistent with a binding protein-dependent ATP binding cassette (ABC) transporter. A corresponding binding protein (MalE) that interacts with maltose with high affinity (

K
d

of 1.5 μM) was purified from the culture supernatant of maltose-grown cells. Immunoelectron microscopy revealed distribution of the protein throughout the cell wall. The
malE
gene was cloned and sequenced. Five additional open reading frames, encoding components of a maltose transport system (MalF and MalG), a putative transcriptional regulator (MalR), a cyclodextrinase (CdaA), and an α-glucosidase (GlcA), were identified downstream of
malE
. The
malE
gene lacking the DNA sequence that encodes the signal sequence was expressed in
Escherichia coli
. The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80°C. Recombinant MalE cross-reacted with an antiserum raised against the wild-type protein, thereby indicating that the latter is the product of the
malE
gene. The MalE protein might be well suited as a model to study tolerance of proteins to low pH.
The Unphosphorylated Receiver Domain of PhoB Silences the Activity of Its Output Domain
Authors: Ellison Damon W. , McCleary William R.
Published: July 2002

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

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

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

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

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

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

A 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

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

Type 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

ADP-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

The 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

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

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

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

MntH, 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.
Determinants of the C-Terminal Domain of the
Escherichia coli
RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters
Authors: Savery Nigel J. , Lloyd Georgina S. , Busby Stephen J. W. , Thomas Mark S. , Ebright Richard H. , Gourse Richard L.
Published: July 2002

Alanine scanning of the
Escherichia coli
RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the
CC(−61.5)
promoter and/or the
lacP1
promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.
Pathogenesis of Primary Respiratory Disease Induced by Isolates from a New Genetic Cluster of Bovine Viral Diarrhea Virus Type I
Authors: Baule C. , Kulcsár G. , Belák K. , Albert M. , Mittelholzer C. , Soós T. , Kucsera L. , Belák S.
Published: July 2002

The pathogenesis of infection induced by cytopathogenic isolates from the newly identified genetic cluster Id of bovine viral diarrhea virus (BVDV) type I was studied in two experimental infections of previously seronegative, immunocompetent calves. Experiment 1 focused on the evaluation of clinical patterns, viremia, and serological responses. All infected calves in this experiment developed respiratory symptoms and seroconverted to BVDV positivity. Contact calves also contracted a respiratory tract infection following exposure to infected animals. Viremia was demonstrated between postinfection days 2 and 17, and the virus was detected in organ specimens of all but one each of the infected and contact calves. In experiment 2, the distribution of BVDV in various tissues of calves euthanized at defined days postinfection was studied. In two of these calves recurrent shedding of BVDV in nasal secretions was shown. BVDV was detected in various tissues of all infected calves throughout the experiment and also following seroconversion and the clearance of BVDV from the circulatory system. Despite the widespread distribution of the virus in various organs, significant tissue damage was found mainly in respiratory tract and lymphoid tissues. These experiments revealed that viruses from cluster Id of BVDV are able to induce primary respiratory disease in previously seronegative, immunocompetent calves. Contact transmission and virus recurrence, contrary to observations from acute experimental infections with noncytopathogenic BVDV, are likely to reflect differences in biological features of these cytopathogenic isolates. Virus shedding and its presence in tissues following peripheral clearance and in the presence of antibodies may have implications in the diagnosis, pathogenesis, and epidemiology of BVDV-induced syndromes in cattle.
Simple, Sensitive, and Specific Detection of Human Immunodeficiency Virus Type 1 Subtype B DNA in Dried Blood Samples for Diagnosis in Infants in the Field
Authors: Beck Ingrid A. , Drennan Kathryn D. , Melvin Ann J. , Mohan Kathey M. , Herz Arnd M. , Alarcón Jorge , Piscoya Julia , Velázquez Carlos , Frenkel Lisa M.
Published: July 2002

The detection of virus is used to diagnose human immunodeficiency virus type 1 (HIV-1) infection in infants due to the persistence of maternal antibodies for a year or more. An HIV-1 DNA PCR assay with simple specimen collection and processing was developed and evaluated. Whole blood was collected on filter paper that lysed cells and bound the DNA, eliminating specimen centrifugation and extraction procedures. The DNA remained bound to the filter paper during PCR amplification. Assays of copy number standards showed reproducible detection of 5 to 10 copies of HIV-1 in 5 μl of whole blood. The sensitivity of the assay did not decrease after storage of the standards on filter paper for 3 months at room temperature or after incubation at 37 or 45°C for 20 h. The primers used for nested PCR of the HIV-1
pol
gene amplified templates from a reference panel of multiple HIV-1 subtypes but did not amplify a subtype A or a subtype C virus from children living in Seattle. The assay had a sensitivity of 98.4% and a specificity of 98.3% for testing of 122 specimens from 35 HIV-1-infected and 16 uninfected children and 43 seronegative adults living in Washington. The assay had a sensitivity of 99% and a specificity of 100% for testing of 102 HIV-1-positive (as determined by enzyme immunoassay) Peruvian women and 6 seropositive and 34 seronegative infants. This assay, with adsorption of whole blood to filter paper and no specimen processing, provides a practical, economical, sensitive, and specific method for the diagnosis of HIV-1 subtype B infection in infants.
Investigation of Optimal Specimen Type and Sampling Time for Detection of Measles Virus RNA during a Measles Epidemic
Authors: Riddell Michaela A. , Chibo Doris , Kelly Heath A. , Catton Michael G. , Birch Christopher J.
Published: July 2002

At various times postonset of rash, 74 patients positive for measles virus-specific immunoglobulin M provided samples for detection of measles virus RNA by a reverse transcriptase PCR. Of lymphocytes, urine, throat swab, and serum specimens, throat swab specimens were optimal for detection of measles virus RNA during the first 2 weeks after the rash.
Coinfection with ThreeEhrlichiaSpecies in Dogs from Thailand and Venezuela with Emphasis on Consideration of 16S Ribosomal DNA Secondary Structure
Authors: Suksawat Jiraporn , Pitulle Christian , Arraga-Alvarado Cruz , Madrigal Karina , Hancock Susan I. , Breitschwerdt Edward B.
Published: July 2002

As part of a larger study to investigate tick-borne infections in dogs from Thailand and Venezuela, documentation of coinfection with threeEhrlichiaspecies in two dogs, one from each country, became the focus of the present study. Although neither dog had clinical signs attributable to ehrlichiosis, both dogs were anemic and neutropenic and the Thai dog was thrombocytopenic. Genus- and species-specific PCR targeting the 16S rRNA genes indicated that both dogs were coinfected withEhrlichia canis,E. platys, andE. equi. To our knowledge, these results provide the first molecular documentation for the presence ofE. equiin dogs from these countries. Using universal bacterial PCR primers, one nearly full-length 16S rRNA gene could be amplified from each dog. The sequences were identical to each other and almost identical to that ofE. platys(AF156784), providing the firstE. platys16S ribosomal DNA (rDNA) sequences reported from these two geographically divergent countries. To determine whether these sequence differences allow differentiation between these two strains and other published 16S rDNAE. platyssequences, we performed a phylogenetic analysis of the rRNA, incorporating the consideration of secondary structure.
Methicillin-Resistant
Staphylococcus aureus
: Comparison of Susceptibility Testing Methods and Analysis of
mecA
-Positive Susceptible Strains
Authors: Sakoulas George , Gold Howard S. , Venkataraman Lata , DeGirolami Paola C. , Eliopoulos George M. , Qian Qinfang
Published: July 2002

Methicillin-resistant
Staphylococcus aureus
(MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the
mecA
gene used as the “gold standard” assay. Analysis of 107 methicillin-susceptible
S. aureus
(MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked
mecA
was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the
mecA
gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that
mecA
- and PBP 2a-positive
S. aureus
isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.
In Vitro Culture, Ultrastructure, Antigenic, and Molecular Characterization of
Encephalitozoon cuniculi
Isolated from Urine and Sputum Samples from a Spanish Patient with AIDS
Authors: del Aguila Carmen , Moura Hercules , Fenoy Soledad , Navajas Raquel , Lopez-Velez Rogelio , Li Lixia , Xiao Lihua , Leitch Gordon J. , da Silva Alexandre , Pieniazek Norman J. , Lal Altaf A. , Visvesvara Govinda S.
Published: July 2002

In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as
Encephalitozoon cuniculi
, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.
Rapid Typing of
Borrelia burgdorferi
Sensu Lato Species in Specimens from Patients with Different Manifestations of Lyme Borreliosis
Authors: Lünemann Jan D. , Zarmas Silvia , Priem Susanne , Franz Juliane , Zschenderlein Rolf , Aberer Elisabeth , Klein Rolf , Schouls Leo , Burmester Gerd R. , Krause Andreas
Published: July 2002

To further investigate the pathogenic potential of different
Borrelia burgdorferi
genospecies, specimens from 27 patients with different manifestations of Lyme borreliosis were analyzed by PCR and reverse line blotting (RLB). In samples from Lyme arthritis patients,
B. burgdorferi
sensu stricto was predominantly identified, while in patients with neuroborreliosis or acrodermatitis,
Borrelia garinii
and
Borrelia afzelii
, respectively, were exclusively detected. The results demonstrate that PCR-RLB is a valuable tool for epidemiological and pathogenetic studies of Lyme borreliosis.
Particular Biochemical Profiles for EnterohemorrhagicEscherichia coliO157:H7 Isolates on the ID 32E System
Authors: Leclercq Alexandre , Lambert Bernard , Pierard Denis , Mahillon Jacques
Published: July 2002

The ability of the ID 32E system to identify and discriminate 74Escherichia coliO157 isolates among 106E. colinon-O157 isolates was evaluated. The results showed atypical biochemical reactions but accurate identification at the species level and no unique biochemical profile numbers forE. coliO157, although these numbers were distinct from those of other serotypes.