bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dependence of
Mycobacterium bovis
BCG on Anaerobic Nitrate Reductase for Persistence Is Tissue Specific
Authors: Fritz Christian , Maass Silvia , Kreft Andreas , Bange Franz-Christoph 
Published: July 2002

ABSTRACT

Mycobacterium bovis
BCG, the only presently available vaccine against tuberculosis, was obtained from virulent
M. bovis
after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immune-deficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that
M. bovis
BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of
narG
, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of
M. bovis
BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.
The Cytochrome
c
Maturation Locus of
Legionella pneumophila
Promotes Iron Assimilation and Intracellular Infection and Contains a Strain-Specific Insertion Sequence Element
Authors: Viswanathan V. K. , Kurtz Sherry , Pedersen Lisa L. , Abu Kwaik Yousef , Krcmarik Kevin , Mody Sejal , Cianciotto Nicholas P. 
Published: July 2002

ABSTRACT

Previously, we obtained a
Legionella pneumophila
mutant, NU208, that is hypersensitive to iron chelators when grown on standard
Legionella
media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known
L. pneumophila
siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and
Hartmannella
and
Acanthamoeba
amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome
c
maturation gene,
ccmC
. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome
c
. Phenotypic analysis of two additional, independently derived
ccmC
mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation.
trans
-complementation analysis of NU208 confirmed that
L. pneumophila ccmC
is required for cytochrome
c
production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although
ccm
genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a
ccm
gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the
ccm
locus from strain 130b identified the genes
ccmA-H
. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between
ccmB
and
ccmC
. Southern hybridizations indicated that the open reading frame within this element (ISLp
1
) was present in multiple copies in some strains of
L. pneumophila
but was absent from others. These findings represent the first evidence for a transposable element in
Legionella
and the first identification of an
L. pneumophila
strain-specific gene.
Diversity of the Metalloprotease Toxin Produced by Enterotoxigenic
Bacteroides fragilis
Authors: Wu Shaoguang , Dreyfus Lawrence A. , Tzianabos Art O. , Hayashi Chika , Sears Cynthia L. 
Published: July 2002

ABSTRACT

Enterotoxigenic
Bacteroides fragilis
(ETBF) strains produce a 20-kDa zinc metalloprotease toxin (BFT) associated with diarrheal disease of animals, young children, and adults. BFT stimulates secretion in intestinal loops in vivo and modifies epithelial cell morphology in vitro. The
B. fragilis
toxin (
bft
) gene from ETBF strain 86-5443-2-2 (piglet;
bft
-
2
) revealed significant nucleotide and predicted amino acid differences when compared to the
bft
gene from ETBF strain VPI 13784 (lamb;
bft
-
1
). This study compares BFT-1 and BFT-2, respectively, produced by ETBF strains VPI 13784 and 86-5443-2-2 purified using the Van Tassell method (38) and a modified purification scheme described herein. Multiple differences in the protein toxins produced by these ETBF strains were identified. First, purified BFT-1 eluted from a high-resolution anion-exchange column (Mono Q) at 0.22 ± 0.005 M NaC1 versus 0.18 ± 0.001 M NaC1 for BFT-2 (
P
< 0.001). Second, BFT-1 and BFT-2 exhibited different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase fast protein liquid chromatography. Third, each BFT reacted with greater specificity to homologous rather than heterologous antisera. Fourth, BFT-2 had modest, but consistently, greater biological activity than BFT-1 when tested on HT29/C1 cells (
P
≤ 0.01). Together, these data indicate that these ETBF strains produce two distinct isotypes of BFT, termed BFT-1 (VPI 13784 BFT) and BFT-2 (86-5443-2-2 BFT) to recognize the order in which the proteins were purified and genetic sequences identified. The modified purification scheme described in this report yields about two to three times more purified BFT protein than previous protocols and is less time consuming.
Localization within a Proline-Rich Antigen (Ag2/PRA) of Protective Antigenicity against Infection withCoccidioides immitisin Mice
Authors: Peng Tao , Shubitz Lisa , Simons Julie , Perrill Robert , Orsborn Kris I. , Galgiani John N. 
Published: July 2002

ABSTRACTSubunits of a proline-rich coccidioidal antigen (Ag2/PRA) ofCoccidioides immitiswere analyzed by comparison as vaccines in mice. The optimal dose of plasmid vaccine encoding full-length Ag2/PRA was determined to be between 10 and 100 μg. Mice vaccinated with plasmids encoding amino acids (aa) 1 to 106 were as protective as full-length Ag2/PRA (aa 1 to 194). The subunit from aa 27 to 106 was significantly but less protective. Plasmids encoding aa 90 to 151 or aa 90 to 194 were not protective. Analogous results were obtained with recombinant vaccines of the same amino acid sequences. In addition, mixtures of aa 90 to 194 with either aa 1 to 106 or aa 27 to 106 did not enhance protection compared to the active single-recombinant subunits alone. Humoral response of total immunoglobulin G (IgG) and subclasses IgG1 and IgG2a were detectable in subunit vaccinations but at significantly (100-fold) lower concentrations than after vaccination with plasmids encoding full-length Ag2/PRA. Since virtually all protection by vaccination with full-length Ag2/PRA can be accounted for in the first half of the protein (aa 1 to 106), this subunit could make a multicomponent vaccine more feasible by reducing the quantity of protein per dose and the possibility of an untoward reactions to a foreign protein.
Assessment of Virulence of Uropathogenic
Escherichia coli
Type 1 Fimbrial Mutants in Which the Invertible Element Is Phase-Locked On or Off
Authors: Gunther IV Nereus W. , Snyder Jennifer A. , Lockatell Virginia , Blomfield Ian , Johnson David E. , Mobley Harry L. T. 
Published: July 2002

ABSTRACT

Type 1 fimbria is a proven virulence factor of uropathogenic
Escherichia coli
(UPEC), causing urinary tract infections. Expression of the fimbria is regulated at the transcriptional level by a promoter situated on an invertible element, which can exist in one of two different orientations. The orientation of the invertible element that allows the expression of type 1 fimbriae is defined as “on,” and the opposite orientation, in which no transcription occurs, is defined as “off.” During the course of a urinary tract infection, we have observed that the infecting
E. coli
population alternates between fimbriated and nonfimbriated states, with the fimbriated on orientation peaking at 24 h. We propose that the ability of the invertible element to switch orientations during infection is itself a virulence trait. To test this hypothesis, nucleotide sequence changes were introduced in the left inverted repeat of the invertible element of UPEC pyelonephritis strain CFT073 that locked the invertible elements permanently in either the on or the off orientation. The virulence of these mutants was assessed in the CBA mouse model of ascending urinary tract infection at 4, 24, 48, and 72 h postinoculation (hpi). We conducted independent challenges, in which bladders of mice were inoculated with either a single mutant or the wild type, and cochallenges, in which a mutant and the wild type were inoculated together to allow direct competition in the urinary tract. In both sets of experimental infections, the locked-off mutant was recovered from the urine, bladder, and kidneys in significantly lower numbers than the wild type at 24 hpi (
P
≤ 0.05), demonstrating its attenuation. Conversely, the locked-on mutant was recovered in higher numbers than the wild type at 24 hpi (
P
≤ 0.05), showing enhanced virulence of this mutant. No significant differences were seen between the mutants and wild type in the urine or the bladder at 48 or 72 hpi. However, the wild type outcompeted the locked-off mutant in the kidneys during the cochallenge experiment at 72 hpi (
P
= 0.009). Overall, these data suggest that the ability of the invertible element controlling type 1 fimbria expression to phase vary contributes significantly to virulence early (24 hpi) in the course of a urinary tract infection by UPEC and most profoundly influences colonization of the bladder.
Neisseria lactamica
Protects against Experimental Meningococcal Infection
Authors: Oliver Kerry J. , Reddin Karen M. , Bracegirdle Philippa , Hudson Michael J. , Borrow Ray , Feavers Ian M. , Robinson Andrew , Cartwright Keith , Gorringe Andrew R. 
Published: July 2002

ABSTRACT

Immunological and epidemiological evidence suggests that the development of natural immunity to meningococcal disease results from colonization of the nasopharynx by commensal
Neisseria
spp., particularly with
N. lactamica
. We report here that immunization with
N. lactamica
killed whole cells, outer membrane vesicles, or outer membrane protein (OMP) pools and protected mice against lethal challenge by a number of diverse serogroup B and C meningococcal isolates in a model of bacteremic infection. Sera raised to
N. lactamica
killed whole cells, OMPs, or protein pools were found to cross-react with meningococcal isolates of a diverse range of genotypes and phenotypes. The results confirm the potential of
N. lactamica
to form the basis of a vaccine against meningococcal disease.
opdA
, a
Salmonella enterica
Serovar Typhimurium Gene Encoding a Protease, Is Part of an Operon Regulated by Heat Shock
Authors: Conlin Christopher A. , Miller Charles G. 
Published: July 2002

ABSTRACT

The
opdA
(
prlC
) gene of
Salmonella enterica
serovar Typhimurium and
Escherichia coli
encodes the metalloprotease oligopeptidase A (OpdA). We report that
opdA
is cotranscribed with a downstream open reading frame,
yhiQ
. Transcription of this operon is induced after a temperature shift (30 to 42°C), and this induction depends on the heat shock sigma factor encoded by the
rpoH
(
htpR
) gene.
Arginine Catabolism in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803 Involves the Urea Cycle and Arginase Pathway
Authors: Quintero María José , Muro-Pastor Alicia María , Herrero Antonia , Flores Enrique 
Published: July 2002

ABSTRACT

Cells of the unicellular cyanobacterium
Synechocystis
sp. strain PCC 6803 supplemented with micromolar concentrations of
l
-[
14
C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of
l
-[
14
C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [
14
C]ornithine preceded that of [
14
C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with
l
-[
14
C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Δ
1
pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an
argF
(ornithine carbamoyltransferase) insertional mutant was impaired in the production of [
14
C]citrulline from [
14
C]arginine; (ii) a
proC
(Δ
1
pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [
14
C]proline, [
14
C]glutamate, and [
14
C]glutamine from [
14
C]arginine or [
14
C]ornithine; and (iii) a
putA
(proline oxidase) insertional mutant did not produce [
14
C]glutamate from
l
-[
14
C]arginine,
l
-[
14
C]ornithine, or
l
-[
14
C]proline. Mutation of two open reading frames (
sll0228
and
sll1077
) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of
argD
(
N
-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Δ
1
pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [
14
C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-
l
-arginyl-poly(
l
-aspartic acid)], a reserve material unique to cyanobacteria.
Penicillin Binding Protein 5 Affects Cell Diameter, Contour, and Morphology of
Escherichia coli
Authors: Nelson David E. , Young Kevin D. 
Published: July 2002

ABSTRACT

Although general physiological functions have been ascribed to the high-molecular-weight penicillin binding proteins (PBPs) of
Escherichia coli
, the low-molecular-weight PBPs have no well-defined biological roles. When we examined the morphology of a set of
E. coli
mutants lacking multiple PBPs, we observed that strains expressing active PBP 5 produced cells of normal shape, while mutants lacking PBP 5 produced cells with altered diameters, contours, and topological features. These morphological effects were visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After filamentation, cellular diameter varied erratically along the length of individual filaments and many filaments exhibited extensive branching. Also, in general, the mean diameter of cells lacking PBP 5 was significantly increased compared to that of cells from isogenic strains expressing active PBP 5. Expression of cloned PBP 5 reversed the effects observed in Δ
dacA
mutants. Although deletion of PBP 5 was required for these phenotypes, the absence of additional PBPs magnified the effects. The greatest morphological alterations required that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were aberrant. In no case did mutation of another PBP produce the same drastic morphological effects. We conclude that among the low-molecular-weight PBPs, PBP 5 plays a principle role in determining cell diameter, surface uniformity, and overall topology of the peptidoglycan sacculus.
Purification and Properties of Ornithine Racemase from
Clostridium sticklandii
Authors: Chen Hao-Ping , Lin Chin-Fen , Lee Ya-Jung , Tsay San-San , Wu Shih-Hsiung 
Published: July 2002

ABSTRACT

Ornithine racemase has been purified to homogeneity from
Clostridium sticklandii
, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This is the first racemase known to be highly specific to ornithine. This PLP-dependent enzyme has an
M
r
of 92,000, with a

K
m

for
l
-ornithine of 0.77 ± 0.05 mM and a

k
cat

of 980 ± 20 s
−1
.
Autophagy and the cvt Pathway Both Depend on
AUT9
Authors: Lang Thomas , Reiche Steffen , Straub Michael , Bredschneider Monika , Thumm Michael 
Published: July 2002

ABSTRACT

In growing cells of the yeast
Saccharomyces cerevisiae
, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen for
aut
−
mutants and isolated
aut9-1
cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid
aut9-1
mutant cells with a genomic library, in this study we identified and characterized the
AUT9
gene, which is allelic with
CVT7. aut9
-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.
Roles of Aconitase in Growth, Metabolism, and Morphological Differentiation of
Streptomyces coelicolor
Authors: Viollier P. H. , Nguyen K. T. , Minas W. , Folcher M. , Dale G. E. , Thompson C. J. 
Published: July 2002

ABSTRACT

The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184–3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in
Streptomyces coelicolor
development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned
acoA
gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an
acoA
disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an
acoA citA
mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription of
acoA
was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.
Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria
Authors: Karlin Samuel , Mrázek Jan , Campbell Allan , Kaiser Dale 
Published: July 2002

ABSTRACT

Predicted highly expressed (PHX) genes are characterized for the completely sequenced genomes of the four fast-growing bacteria
Escherichia coli,
Haemophilus influenzae, Vibrio cholerae,
and
Bacillus subtilis
. Our approach to ascertaining gene expression levels relates to codon usage differences among certain gene classes: the collection of all genes (average gene), the ensemble of ribosomal protein genes, major translation/transcription processing factors, and genes for polypeptides of chaperone/degradation complexes. A gene is predicted highly expressed (PHX) if its codon frequencies are close to those of the ribosomal proteins, major translation/transcription processing factor, and chaperone/degradation standards but strongly deviant from the average gene codon frequencies. PHX genes identified by their codon usage frequencies among prokaryotic genomes commonly include those for ribosomal proteins, major transcription/translation processing factors (several occurring in multiple copies), and major chaperone/degradation proteins. Also PHX genes generally include those encoding enzymes of essential energy metabolism pathways of glycolysis, pyruvate oxidation, and respiration (aerobic and anaerobic), genes of fatty acid biosynthesis, and the principal genes of amino acid and nucleotide biosyntheses. Gene classes generally not PHX include most repair protein genes, virtually all vitamin biosynthesis genes, genes of two-component sensor systems, most regulatory genes, and most genes expressed in stationary phase or during starvation. Members of the set of PHX aminoacyl-tRNA synthetase genes contrast sharply between genomes. There are also subtle differences among the PHX energy metabolism genes between
E. coli
and
B. subtilis
, particularly with respect to genes of the tricarboxylic acid cycle. The good agreement of PHX genes of
E. coli
and
B. subtilis
with high protein abundances, as assessed by two-dimensional gel determination, is verified. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are also examined. The spatial distribution of PHX genes within each genome reveals clusters and significantly long regions without PHX genes.
Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate
Authors: Miyazaki Junichi , Kobashi Nobuyuki , Nishiyama Makoto , Yamane Hisakazu 
Published: July 2002

ABSTRACT

Our previous studies revealed that lysine is synthesized through α-aminoadipate in an extremely thermophilic bacterium,
Thermus thermophilus
HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from α-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an
argD
homolog of
T. thermophilus
HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the
argD
-like gene, now termed
lysJ
, in
T. thermophilus
HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The
lysJ
gene was cloned into a plasmid and overexpressed in
Escherichia coli
, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the ɛ-amino group of
N
2
-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When
N
2
-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the δ-amino group of
N
2
-acetylornithine to 2-oxoglutarate 16 times more efficiently than when
N
2
-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in
T. thermophilus
HB27 is functionally and evolutionarily related to arginine biosynthesis.