bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of T Helper Type 1 and 2 Responses to 19-Kilodalton Merozoite Surface Protein 1 in Vaccinated Healthy Volunteers and Adults Naturally Exposed to Malaria
Authors: Lee Edwin A. M. , Palmer Dupeh R. , Flanagan Katie L. , Reece William H. H. , Odhiambo Kennedy , Marsh Kevin , Pinder Margaret , Gravenor Michael B. , Keitel Wendy A. , Kester Kent E. , Diggs Carter , Kaslow David , Apostolopoulos V. , Ballou W. Ripley , Hill Adrian V. S. , Krzych Urszula , Plebanski Magdalena
Published: July 2002

Plasmodium falciparummalaria is a major cause of death in the tropics. The 19-kDa subunit ofP. falciparummerozoite surface protein 1 (MSP-119), a major blood stage vaccine candidate, is the target of cellular and humoral immune responses in animals and humans. In this phase I trial of MSP-119, immunization of nonexposed human volunteers with either of the two allelic forms of recombinant MSP-119induced high levels of antigen-specific Th1 (gamma interferon) and Th2 (interleukin 4 [IL-4] and IL-10) type lymphokines. The adjustment of the antigen dose and number of immunizations regulated the level of specificity of immune responses and Th1/Th2 bias of responses induced by vaccination. Novel conserved and allelic T-cell epitopes which induced cross-strain immune responses were identified. Importantly, responses to many of these novel epitopes were also present in adults exposed to malaria, both in east (Kenya) and west Africa (The Gambia). These data suggest that epitope-specific naturally acquired MSP-119immune responses in endemic populations can be boosted by vaccination.
Expression of Surfactant Protein D in the Human Gastric Mucosa and duringHelicobacter pyloriInfection
Authors: Murray Emma , Khamri Wafa , Walker Marjorie M. , Eggleton Paul , Moran Anthony P. , Ferris John A. , Knapp Susanne , Karim Q. Najma , Worku Mulegata , Strong Peter , Reid Kenneth B. M. , Thursz Mark R.
Published: July 2002

Helicobacterpyloriestablishes persistent infection of gastric mucosa with diverse clinical outcomes. The innate immune molecule surfactant protein D (SP-D) binds selectively to microorganisms, inducing aggregation and phagocytosis. In this study, we demonstrated the expression of SP-D in gastric mucosa by reverse transcription-PCR and immuohistochemical analysis. SP-D is present at the luminal surface and within the gastric pits, with maximal expression at the surface. Levels of expression are significantly increased inH. pylori-associated gastritis compared to those in the normal mucosa. Immunofluorescence microscopy was used to demonstrate binding and agglutination ofH. pyloriby SP-D in a lectin-specific manner. These activities resulted in a 50% reduction in the motility ofH. pylori, as judged on the basis of curvilinear velocity measured by using a Hobson BacTracker. Lipopolysaccharides extracted from threeH. pyloristrains were shown to bind SP-D in a concentration-dependent manner, and there was marked variation in the avidity of binding among the strains. SP-D may therefore play a significant role in the innate immune response toH. pyloriinfection.
Construction and Characterization of a Live, AttenuatedaroADeletion Mutant ofPseudomonas aeruginosaas a Candidate Intranasal Vaccine
Authors: Priebe Gregory P. , Brinig Mary M. , Hatano Kazue , Grout Martha , Coleman Fadie T. , Pier Gerald B. , Goldberg Joanna B.
Published: July 2002

Antibodies to the lipopolysaccharide O antigen ofPseudomonas aeruginosamediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protectiveP. aeruginosavaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarkedaroAdeletion mutant of theP. aeruginosaserogroup O2/O5 strain PAO1. The resultantaroAdeletion mutant of PAO1 is designated PAO1ΔaroA. ThearoAdeletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1ΔaroAcould be applied either intranasally or intraperitoneally at doses up to 5 × 109CFU per mouse without adverse effects. No dissemination of PAO1ΔaroAto blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypicP. aeruginosaserogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuatedP. aeruginosastrain PAO1ΔaroAappears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of theP. aeruginosaO2/O5 serogroup.
Polarized Expression of Tamm-Horsfall Protein by Renal Tubular Epithelial Cells Activates Human Granulocytes
Authors: Kreft B. , Jabs W. J. , Laskay T. , Klinger M. , Solbach W. , Kumar S. , van Zandbergen G.
Published: July 2002

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.
Increased Virulence of a Fibronectin-Binding Protein Mutant of
Staphylococcus aureus
in a Rat Model of Pneumonia
Authors: McElroy Mary C. , Cain David J. , Tyrrell Christine , Foster Timothy J. , Haslett Christopher
Published: July 2002

Fibronectin-binding proteins mediate
Staphylococcus aureus
internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using
S. aureus
strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate
S. aureus
internalization into alveolar epithelial cells in vitro and that
S. aureus
internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of
S. aureus
-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation.
S. aureus
growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that
S. aureus
was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of
S. aureus
in pneumonia.
Purification and Characterization of a Membrane-Bound Hydrogenase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Sapra Rajat , Verhagen Marc F. J. M. , Adams Michael W. W.
Published: July 2002

Highly washed membrane preparations from cells of the hyperthermophilic archaeon
Pyrococcus furiosus
contain high hydrogenase activity (9.4 μmol of H
2
evolved/mg at 80°C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with
n
-dodecyl-β-
d
-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (α and β) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained ∼1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or
P. furiosus
ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H
2
as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the α and β subunits were located in the genome database within a putative 14-gene operon (termed
mbh
). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of
P. furiosus
and show that the α subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the α and β subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from
Methanosarcina barkeri
,
Escherichia coli
, and
Rhodospirillum rubrum
. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that
P. furiosus
, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H
2
metabolism is coupled to energy conservation.
Identification of Iron-Responsive, Differential Gene Expression in the CyanobacteriumSynechocystissp. Strain PCC 6803 with a Customized Amplification Library
Authors: Singh Abhay K. , Sherman Louis A.
Published: July 2002

We describe the use of a method called differential expression using customized amplification library (DECAL) to study the global changes in gene expression in iron-deficient versus iron-reconstituting cells ofSynechocystissp. strain PCC 6803. We identified a number of genes, such asisiA,idiA,psbA,cpcG, andslr0374, whose expression either increased or decreased in response to iron availability. Further analysis led to the identification of additional genes related to those identified by DECAL (e.g.,psbC,psbO,psaA,apcABC,cpcBAC1C2D, andnblA) that were differentially regulated by iron availability. Expression ofcpcG,psbC,psbO,psaA,apcABC, andcpcBAC1C2Dincreased, whereas that ofisiA,idiA,nblA,psbA, andslr0374 decreased, in iron-reconstituting cells. S1 nuclease protection studies showed that increased transcript levels ofpsbAin iron-deficient cells was due to the increased expression of bothpsbA2andpsbA3genes, although the steady-state level ofpsbA2remained higher than that ofpsbA3.
A Novel Bacterial ATP-Binding Cassette Transporter System That Allows Uptake of Macromolecules
Authors: Momma Keiko , Okamoto Masako , Mishima Yumiko , Mori Shigetarou , Hashimoto Wataru , Murata Kousaku
Published: July 2002

A gram-negative bacterium,
Sphingomonas
sp. strain A1, isolated as a producer of alginate lyase, has a characteristic cell envelope structure and forms a mouth-like pit on its surface. The pit is produced only when the cells have to incorporate and assimilate alginate. An alginate uptake-deficient mutant was derived from cells of strain A1. One open reading frame,
algS
(1,089 bp), exhibiting homology to the bacterial ATP-binding domain of an ABC transporter, was cloned as a fragment complementing the mutation.
algS
was followed by two open reading frames,
algM1
(972 bp) and
algM2
(879 bp), which exhibit homology with the transmembrane permeases of ABC transporters. Disruption of
algS
of strain A1 resulted in the failure to incorporate alginate and to form a pit. Hexahistidine-tagged AlgS protein (AlgS
His6
) overexpressed in
Escherichia coli
and purified by Ni
2+
affinity column chromatography showed ATPase activity. Based on these results, we propose the occurrence of a novel pit-dependent ABC transporter system that allows the uptake of macromolecules.
Identification of the
Escherichia coli
Nicotinic Acid Mononucleotide Adenylyltransferase Gene
Authors: Mehl Ryan A. , Kinsland Cynthia , Begley Tadhg P.
Published: July 2002

The gene (
ybeN
) coding for nicotinate mononucleotide adenylyltransferase, an NAD(P) biosynthetic enzyme, has been identified and overexpressed in
Escherichia coli
. This enzyme catalyzes the reversible adenylation of nicotinate mononucleotide and shows product inhibition. The rate of adenylation of nicotinate mononucleotide is at least 20 times faster than the rate of adenylation of nicotinamide mononucleotide.
Identification and Characterization of a Membrane Permease Involved in Iron-Hydroxamate Transport in
Staphylococcus aureus
Authors: Sebulsky M. Tom , Hohnstein Dan , Hunter Meredith D. , Heinrichs David E.
Published: July 2002

Staphylococcus aureus
was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An
S. aureus
mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn
917
-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn
917
-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of
Bacillus subtilis
. DNA sequence information was then used to clone a larger fragment of DNA surrounding the
fhuG
gene, and this resulted in the identification of an operon of three genes,
fhuCBG
, all of which show significant similarities to ferric hydroxamate uptake (
fhu
) genes in
B. subtilis
. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the
S. aureus
FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in
S. aureus
. This regulation is attributable to Fur in
S. aureus
because a strain containing an insertionally inactivated
fur
gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of
fhuCBG
are recognized by the
Escherichia coli
Fur protein.
Identification of the
Pseudomonas aeruginosa glmM
Gene, Encoding Phosphoglucosamine Mutase
Authors: Tavares Isabel M. , Jolly Laure , Pompeo Frédérique , Leitão Jorge H. , Fialho Arsénio M. , Sá-Correia Isabel , Mengin-Lecreulx Dominique
Published: July 2002

A search for a potential
algC
homologue within the
Pseudomonas aeruginosa
PAO1 genome database has revealed an open reading frame (ORF) of unknown function, ORF540 in contig 54 (July 1999
Pseudomonas
genome release), that theoretically coded for a 445-amino-acid-residue polypeptide (I. M. Tavares, J. H. Leitão, A. M. Fialho, and I. Sá-Correia, Res. Microbiol. 150:105–116, 1999). The product of this gene is here identified as the phosphoglucosamine mutase (GlmM) which catalyzes the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, an essential step in the formation of the cell wall precursor UDP-
N
-acetylglucosamine. The
P. aeruginosa
gene has been cloned into expression vectors and shown to restore normal peptidoglycan biosynthesis and cell growth of a
glmM Escherichia coli
mutant strain. The GlmM enzyme from
P. aeruginosa
has been overproduced to high levels and purified to homogeneity in a six-histidine-tagged form. Beside its phosphoglucosamine mutase activity, the
P. aeruginosa
enzyme is shown to exhibit phosphomannomutase and phosphoglucomutase activities, which represent about 20 and 2% of its GlmM activity, respectively.
Relation of Capsular Polysaccharide Production and Colonial Cell Organization to Colony Morphology in
Vibrio parahaemolyticus
Authors: Enos-Berlage Jodi L. , McCarter Linda L.
Published: July 2002

Vibrio parahaemolyticus
is a ubiquitous, gram-negative marine bacterium that undergoes phase variation between opaque and translucent colony morphologies. The purpose of this study was to determine the factor(s) responsible for the opaque and translucent phenotypes and to examine cell organization within both colony types. Examination of thin sections of ruthenium red-stained bacterial cells by electron microscopy revealed a thick, electron-dense layer surrounding the opaque cells that was absent in preparations from translucent strains. Extracellular polysaccharide (EPS) material was extracted from both opaque and translucent strains, and the opaque strain was shown to produce abundant levels of polysaccharide, in contrast to the translucent strain. Compositional analysis of the EPS identified four major sugars: glucose, galactose, fucose, and
N
-acetylglucosamine. Confocal scanning laser microscopy was used to investigate cell organization within opaque and translucent colonies. Cells within both types of colonies exhibited striking organization; rod-shaped cells were aligned parallel to one another and perpendicular to the agar surface throughout the depth of the colony. Cells within translucent colonies appeared more tightly packed than cells in opaque colonies. In addition, a dramatic difference in the structural integrity of these two colony types was observed. When colonies were perturbed, the cell organization of the translucent colonies was completely disrupted while the organization of the opaque colonies was maintained. To our knowledge, this study represents the first description of how cells are organized in the interior of a viable bacterial colony. We propose that the copious amount of EPS produced by the opaque strain fills the intercellular space within the colony, resulting in increased structural integrity and the opaque phenotype.
Trk1 and Trk2 Define the Major K
+
Transport System in Fission Yeast
Authors: Calero Fernando , Gómez Néstor , Ariño Joaquín , Ramos José
Published: July 2002

The
trk1
+
gene has been proposed as a component of the K
+
influx system in the fission yeast
Schizosaccharomyces pombe
. Previous work from our laboratories revealed that
trk1
mutants do not show significantly altered content or influx of K
+
, although they are more sensitive to Na
+
. Genome database searches revealed that
S. pombe
encodes a putative gene (designated here
trk2
+
) that shows significant identity to
trk1
+
. We have analyzed the characteristics of potassium influx in
S. pombe
by using
trk1 trk2
mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics.
trk2
mutants do not show altered K
+
transport and exhibit only a slightly reduced Na
+
tolerance. However,
trk1 trk2
double mutants fail to grow at low K
+
concentrations and show a dramatic decrease in Rb
+
influx, as a result of loss of the high-affinity transport component. Furthermore,
trk1 trk2
cells are very sensitive to Na
+
, as would be expected for a strain showing defective potassium transport. When
trk1 trk2
cells are maintained in K
+
-free medium, the potassium content remains higher than that of the wild type or
trk
single mutants. In addition, the
trk1 trk2
strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K
+
transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.
The C-Terminal Portion of the Tail Fiber Protein of Bacteriophage Lambda Is Responsible for Binding to LamB, Its Receptor at the Surface of
Escherichia coli
K-12
Authors: Wang Jiang , Hofnung Maurice , Charbit Alain
Published: July 2002

Bacteriophage λ adsorbs to its
Escherichia coli
K-12 host by interacting with LamB, its cell-surface receptor. We fused C-terminal portions of J, the tail fiber protein of λ, to maltose-binding protein. Solid-phase binding assays demonstrated that a purified fusion protein comprising only the last 249 residues of J could bind to LamB trimers and inhibited recognition by anti-LamB antibodies. Electron microscopy further demonstrated that the fusion protein could also bind to LamB at the surface of intact cells. This interaction prevented λ adsorption but affected only partially maltose uptake.
The
Streptococcus pneumoniae
Beta-Galactosidase Is a Surface Protein
Authors: Zähner Dorothea , Hakenbeck Regine
Published: July 2002

The β-galactosidase gene of
Streptococcus pneumoniae
,
bgaA
, encodes a putative 2,235-amino-acid protein with the two amino acid motifs characteristic of the glycosyl hydrolase family of proteins. In addition, an N-terminal signal sequence and a C-terminal LPXTG motif typical of surface-associated proteins of gram-positive bacteria are present. Trypsin treatment of cells resulted in solubilization of the enzyme, documenting that it is associated with the cell envelope. In order to obtain defined mutants suitable for
lacZ
reporter experiments, the
bgaA
gene was disrupted, resulting in a complete absence of endogenous β-galactosidase activity. The results are consistent with β-galactosidase being a surface protein that seems not to be involved in lactose metabolism but that may play a role during pathogenesis.
A Sheep in Wolf's Clothing:
Listeria innocua
Strains with Teichoic Acid-Associated Surface Antigens and Genes Characteristic of
Listeria monocytogenes
Serogroup 4
Authors: Lan Zheng , Fiedler Franz , Kathariou Sophia
Published: July 2002

Listeria monocytogenes
serotype 4b has been implicated in numerous food-borne epidemics and in a substantial fraction of sporadic listeriosis. A unique lineage of the nonpathogenic species
Listeria innocua
was found to express teichoic acid-associated surface antigens that were otherwise expressed only by
L. monocytogenes
of serotype 4b and the rare serotypes 4d and 4e. These
L. innocua
strains were also found to harbor sequences homologous to the gene
gtcA
, which has been shown to be essential for teichoic acid glycosylation in
L. monocytogenes
serotype 4b. Transposon mutagenesis and genetic studies revealed that the
gtcA
gene identified in this lineage of
L. innocua
was functional in serotype 4b-like glycosylation of the teichoic acids of these organisms. The genomic organization of the
gtcA
region was conserved between this lineage of
L. innocua
and
L. monocytogenes
serotype 4b. Our data are in agreement with the hypothesis that, in this lineage of
L. innocua
,
gtcA
was acquired by lateral transfer from
L. monocytogenes
serogroup 4. The high degree of nucleotide sequence conservation in the
gtcA
sequences suggests that such transfer was relatively recent. Transfer events of this type may alter the surface antigenic properties of
L. innocua
and may eventually lead to evolution of novel pathogenic lineages through additional acquisition of genes from virulent listeriae.
Role of thedinBGene Product in Spontaneous Mutation inEscherichia coliwith an Impaired Replicative Polymerase
Authors: Strauss B. S. , Roberts R. , Francis L. , Pouryazdanparast P.
Published: July 2002

We isolated several new mutator mutations of theEscherichia colireplicative polymerasednaEsubunit alpha and used them and a previously reporteddnaEmutation to study spontaneous frameshift and base substitution mutations. Two of thesednaEstrains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium. A differential effect of the medium was not observed when thesednaEmutations were combined with a mismatch repair mutation. The selection scheme for thednaEmutations required that they be able to complement a temperature-sensitive strain. However, the ability to complement is not related to the mutator effect for at least one of the mutants. Comparison of the mutation rates for frameshift and base substitution mutations inmutSanddnaE mutSstrains suggests that the mismatch repair proteins respond differently to the two types of change. Deletion ofdinBfrom both chromosome and plasmid resulted in a four- to fivefold decrease in the rate of frameshift and base substitution mutations in adnaE mutSdouble mutant background. This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in thisdnaEmutant. Deletion ofdinBfrom strains carrying a wild-typednaEhad a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result ofdinBpolymerase action even in cells with a normal replicative polymerase.
Increase of External Osmolarity Reduces Morphogenetic Defects and Accumulation of Chitin in a
gas1
Mutant of
Saccharomyces cerevisiae
Authors: Turchini Anna , Ferrario Laura , Popolo Laura
Published: July 2002

We have performed a physiological analysis of the effects of high-osmolarity media on
gas1
Δ cells. The reductions in the duplication time, number of pluribudded cells, hypersensitivity to Calcofluor and sodium dodecyl sulfate, and chitin level indicate a partial suppression of the mutant phenotype.
GAS1
deletion was found to be lethal in the absence of the Bck1 and Slt2 (Mpk1) proteins of the cell integrity pathway.
pTAR-Encoded Proteins in Plasmid Partitioning
Authors: Kalnin Kirill , Stegalkina Svetlana , Yarmolinsky Michael
Published: July 2002

Partition cassettes, essential for the segregational stability of low-copy-number bacterial plasmids, typically encode two autoregulated proteins and an adjacent
cis
-acting centromere analog to which one or perhaps both proteins bind. The diminutive partition region of pTAR of
Agrobacterium
spp. was reported to be exceptional, encoding only a single protein, ParA (D. R. Gallie and C. I. Kado, J. Mol. Biol. 193:465–478, 1987). However, resequencing of the region revealed two small downstream genes,
parB
and
orf-84
, of which only
parB
was found to be essential for partitioning in
A. tumefaciens
. Purified ParA exhibited a weak ATPase activity that was modestly increased by nonspecific DNA. ParB bound in vitro to repeated sequences present in a region,
parS
, that possesses centromere and operator functions and within which we identified the primary transcription start site by primer extension. In certain respects the Par proteins behave normally in the foreign host
Escherichia coli
. In
E. coli
, as in
A. tumefaciens
, ParB repressed the partition operon; ParA, inactive alone, augmented this repression. Functional similarities between the partition system of pTAR and those of other plasmids and bacteria are prominent, despite differences in size, organization, and amino acid sequence.
Characterization of the Enzymatic Component of
Clostridium perfringens
Iota-Toxin
Authors: Nagahama Masahiro , Sakaguchi Yoshihiko , Kobayashi Keiko , Ochi Sadayuki , Sakurai Jun
Published: July 2002

The iota
a
component (i
a
) of
Clostridium perfringens
ADP ribosylates nonmuscle β/γ actin and skeletal muscle α-actin. Replacement of Arg-295 in i
a
with alanine led to a complete loss of NAD
+
-glycohydrolase (NADase) and ADP-ribosyltransferase (ARTase); that of the residue with lysine caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them. Substitution of alanine for Glu-378 and Glu-380 caused a complete loss of NADase and ARTase. However, exchange of Glu-378 to aspartic acid or glutamine resulted in little effect on NADase activity but a drastic reduction in ARTase activity (<0.1% of the wild-type activity). Exchange of Glu-380 to aspartic acid caused a drastic reduction in NADase and ARTase activities (<0.1% of the wild-type activities) but did not completely diminish them; that of the residue to glutamine caused a complete loss of ARTase activity. Replacement of Ser-338 with alanine resulted in 0.7 to 2.3% wild-type activities, and that of Ser-340 and Thr-339 caused a reduction in these activities of 5 to 30% wild-type activities. The kinetic analysis showed that Arg-295 and Ser-338 also play an important role in the binding of NAD
+
to i
a
, that Arg-295, Glu-380, and Ser-338 play a crucial role in the catalytic rate of NADase activity, and that these three amino acid residues and Glu-378 are essential for ARTase activity. The effect of amino acid replacement in i
a
on ARTase activity was similar to that on lethal and cytotoxic activities, suggesting that lethal and cytotoxic activities in i
a
are dependent on ARTase activity.
1-Deoxy-
d
-Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in
Rhodobacter capsulatus
Authors: Hahn Frederick M. , Eubanks Lisa M. , Testa Charles A. , Blagg Brian S. J. , Baker Jonathan A. , Poulter C. Dale
Published: July 2002

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and
d
-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-
d
-xylulose 5-phosphate synthase. In
Rhodobacter capsulatus
, two open reading frames (ORFs) carry the genes that encode 1-deoxy-
d
-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-
d
-xylulose 5-phosphate synthase A and B, containing a His
6
tag, were synthesized in
Escherichia coli
and purified to greater than 95% homogeneity in two steps. 1-Deoxy-
d
-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-
d
-xylulose 5-phosphate synthase A and B:

K
m

pyruvate
= 0.61 and 3.0 mM,

K
m

d
-glyceraldehyde 3-phosphate

= 150 and 120 μM, and
V
max
= 1.9 and 1.4 μmol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-
d
-xylulose 5-phosphate synthase B complemented the disrupted essential
dxs
gene in
E. coli
strain FH11.
Cloning and Analysis of a
Candida albicans
Gene That Affects Cell Surface Hydrophobicity
Authors: Singleton David R. , Masuoka James , Hazen Kevin C.
Published: July 2002

The opportunistic pathogenic yeast
Candida albicans
exhibits growth phase-dependent changes in cell surface hydrophobicity, which has been correlated with adhesion to host tissues. Cell wall proteins that might contribute to the cell surface hydrophobicity phenotype were released by limited glucanase digestion. These proteins were initially characterized by their rates of retention during hydrophobic interaction chromatography–high-performance liquid chromatography and used as immunogens for monoclonal antibody production. The present work describes the cloning and functional analysis of a
C. albicans
gene encoding a 38-kDa protein recognized by the monoclonal antibody 6C5-H4CA. The 6C5-H4CA antigen was resolved by two-dimensional electrophoresis, and a partial protein sequence was determined by mass spectrometry analysis of tryptic fragments. The obtained peptides were used to identify the gene sequence from the unannotated
C. albicans
DNA database. The antibody epitope was provisionally mapped by peptide display panning, and a peptide sequence matching the epitope was identified in the gene sequence. The gene sequence encodes a novel open reading frame (ORF) of unknown function that is highly similar to several other
C. albicans
ORFs and to a single
Saccharomyces cerevisiae
ORF. Knockout of the gene resulted in a decrease in measurable cell surface hydrophobicity and in adhesion of
C. albicans
to fibronectin. The results suggest that the 38-kDa protein is a hydrophobic surface protein that meditates binding to host target proteins.
Rebinding of Extracellular Adherence Protein Eap toStaphylococcus aureusCan Occur through a Surface-Bound Neutral Phosphatase
Authors: Flock Margareta , Flock Jan-Ingmar
Published: July 2002

Extracellular adherence protein Eap secreted fromStaphylococcus aureuswas previously found to enhance the adherence ofS. aureusto eukaryotic cells. This enhancement effect is due to the ability of Eap to rebind toS. aureusand to bind to eukaryotic cells and several plasma and matrix proteins. In this study we defined one potential binding target for Eap on the surface ofS. aureus, a surface-located neutral phosphatase. This phosphatase lacks an LPXTG region, but around 80% is retained on the cell surface. The soluble phosphatase can form a complex with Eap at a nonrandom molar ratio, and phosphatase activity is retained. The phosphatase can also bind to fibronectin. The cell surface-located portion presumably contributes to adherence ofS. aureusto fibronectin.
Overexpression of
yccL
(
gnsA
) and
ydfY
(
gnsB
) Increases Levels of Unsaturated Fatty Acids and Suppresses both the Temperature-Sensitive
fabA6
Mutation and Cold-Sensitive
secG
Null Mutation of
Escherichia coli
Authors: Sugai Rie , Shimizu Hisayo , Nishiyama Ken-ichi , Tokuda Hajime
Published: July 2002

A multicopy suppressor of the cold-sensitive
secG
null mutation was isolated. The suppressor contained
sfa
and
yccL
, the former of which has been reported to be a multicopy suppressor of the
fabA6
mutation carried by a temperature-sensitive unsaturated fatty acid auxotroph. Subcloning of the suppressor gene revealed that
yccL
, renamed
gnsA
(
secG
null mutant suppressor), was responsible for the suppression of both the
secG
null mutation and the
fabA6
mutation. In contrast, the
sfa
gene did not suppress the
fabA6
mutation. The
ydfY
(
gnsB
) gene, encoding a protein which is highly similar to GnsA, also suppressed both the
secG
null mutation and the
fabA6
mutation. Although both
gnsA
and
gnsB
are linked to cold shock genes, the levels of GnsA and GnsB did not exhibit a cold shock response. A
gnsA-gnsB
double null mutant grew normally under all conditions examined; thus, the in vivo functions of
gnsA
and
gnsB
remain unresolved. However, overexpression of
gnsA
and
gnsB
stimulated proOmpA translocation of the
secG
null mutant at low temperature and caused a significant increase in the unsaturated fatty acid content of phospholipids. Taken together, these results suggest that an increase in membrane fluidity due to the increase in unsaturated fatty acids compensates for the absence of the SecG function, especially at low temperature.
Salmonella enterica
Serovar Typhimurium Swarming Mutants with Altered Biofilm-Forming Abilities: Surfactin Inhibits Biofilm Formation
Authors: Mireles Joe Robert , Toguchi Adam , Harshey Rasika M.
Published: July 2002

Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the isolation of swarming-defective transposon mutants of
Salmonella enterica
serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation. We report that surfactin inhibits biofilm formation of wild-type
S. enterica
grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested had similar effects.
RecA-Mediated Rescue ofEscherichia coliStrains with Replication Forks Arrested at the Terminus
Authors: Maisnier-Patin Sophie , Nordström Kurt , Dasgupta Santanu
Published: July 2002

The recombinational rescue of chromosome replication was investigated inEscherichia colistrains with the unidirectional originoriR1, from the plasmid R1, integrated withinoriCin clockwise (intR1CW) or counterclockwise (intR1CC) orientations. Only theintR1CCstrain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, theintR1CCstrain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar inoriCandintR1CCstrains. In theintR1CCstrain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of theintR1CCstrain by inactivating Tus, the protein needed to block replication forks atTersites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.
Molecular Cloning and Functional Analysis of the MutY Homolog of
Deinococcus radiodurans
Authors: Li Xianghong , Lu A-Lien
Published: July 2002

The
mutY
homolog gene (
mutY
Dr
) from
Deinococcus radiodurans
encodes a 39.4-kDa protein consisting of 363 amino acids that displays 35% identity to the
Escherichia coli
MutY (MutY
Ec
) protein. Expressed MutY
Dr
is able to complement
E. coli mutY
mutants but not
mutM
mutants to reduce the mutation frequency. The glycosylase and binding activities of MutY
Dr
with an A/G-containing substrate are more sensitive to high salt and EDTA concentrations than the activities with an A/7,8-dihydro-8-oxoguanine (GO)-containing substrate are. Like the MutY
Ec
protein, purified recombinant MutY
Dr
expressed in
E. coli
has adenine glycosylase activity with A/G, A/C, and A/GO mismatches and weak guanine glycosylase activity with a G/GO mismatch. However, MutY
Dr
exhibits limited apurinic/apyrimidinic lyase activity and can form only weak covalent protein-DNA complexes in the presence of sodium borohydride. This may be due to an arginine residue that is present in MutY
Dr
at the position corresponding to the position of MutY
Ec
Lys142, which forms the Schiff base with DNA. The kinetic parameters of MutY
Dr
are similar to those of MutY
Ec
. Although MutY
Dr
has similar substrate specificity and a binding preference for an A/GO mismatch over an A/G mismatch, as MutY
Ec
does, the binding affinities for both mismatches are slightly lower for MutY
Dr
than for MutY
Ec
. Thus, MutY
Dr
can protect the cell from GO mutational effects caused by ionizing radiation and oxidative stress.
Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number in
Pseudomonas aeruginosa
Authors: Dasgupta Nandini , Ramphal Reuben
Published: July 2002

Flagellar number in
Pseudomonas aeruginosa
is controlled by FleN, a putative ATP/GTP binding protein. Disruption of
fleN
results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN
24K→Q
and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.
The Default State of the Membrane-Localized Histidine Kinase PrrB of
Rhodobacter sphaeroides
2.4.1 Is in the Kinase-Positive Mode
Authors: Oh Jeong-Il , Ko In-Jeong , Kaplan Samuel
Published: July 2002

The PrrBA two-component activation system of
Rhodobacter sphaeroides
plays a major role in the induction of photosynthesis gene expression under oxygen-limiting or anaerobic conditions. The PrrB histidine kinase is composed of two structurally identifiable regions, the conserved C-terminal kinase/phosphatase domain and the N-terminal membrane-spanning domain with six transmembrane helices framing three periplasmic and two cytoplasmic loops. Using a set of PrrB mutants with lesions in the transmembrane domain, we demonstrate that the central portion of the PrrB transmembrane domain including the second periplasmic loop plays an important role in both sensing and signal transduction. Signal transduction via the transmembrane domain is ultimately manifested by controlling the activity of the C-terminal kinase/phosphatase domain. The extent of signal transduction is determined by the ability of the transmembrane domain to sense the strength of the inhibitory signal received from the
cbb
3
terminal oxidase (J.-I Oh, and S. Kaplan, EMBO J. 19:4237–4247, 2000). Therefore, the intrinsic (“default”) state of PrrB is in the kinase-dominant mode. It is also demonstrated that the extent of
prrB
gene expression is subject to the negative autoregulation of the PrrBA system.
Correlation between
Bacillus subtilis scoC
Phenotype and Gene Expression Determined Using Microarrays for Transcriptome Analysis
Authors: Caldwell Robert , Sapolsky Ron , Weyler Walter , Maile Randal R. , Causey Stuart C. , Ferrari Eugenio
Published: July 2002

The availability of the complete sequence of the
Bacillus subtilis
chromosome (F. Kunst et al., Nature 390:249–256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of
scoC
on late-stage developmental phenomena as they relate to
aprE
expression, we studied the genome-wide effects of a
scoC
null mutant with the goal of furthering the understanding of the role of
scoC
in growth and developmental processes. In the present work we compared the expression patterns of isogenic
B. subtilis
strains, one of which carries a null mutation in the
scoC
locus (
scoC4
). The results obtained indicate that
scoC
regulates, either directly or indirectly, the expression of at least 560 genes in the
B. subtilis
genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the
scoC
mutant in light of the transcriptome changes observed.
Identification and Molecular Analysis of PcsB, a Protein Required for Cell Wall Separation of Group B Streptococcus
Authors: Reinscheid Dieter J. , Gottschalk Birgit , Schubert Axel , Eikmanns Bernhard J. , Chhatwal Gursharan S.
Published: July 2002

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated
pcsB
, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (
M
r
, 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from
Listeria monocytogenes
. Northern blot analysis revealed a monocistronic transcriptional organization for
pcsB
in GBS. Insertional inactivation of
pcsB
in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.
Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of
Erwinia chrysanthemi
Authors: Palacios José Luis , Zaror Isabel , Martı́nez Patricio , Uribe Francisco , Opazo Patricio , Socı́as Teresa , Gidekel Manuel , Venegas Alejandro
Published: July 2002

Erwinia chrysanthemi
exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the
E. chrysanthemi
transporter system (
prtDEF
genes) in
Escherichia coli
and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of
E. chrysanthemi
protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.