bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Cloning and Characterization of
WdPKS1
, a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in
Wangiella
(
Exophiala
)
dermatitidis
Authors: Feng Bin , Wang Xu , Hauser Melinda , Kaufmann Sarah , Jentsch Simone , Haase Gerhard , Becker Jeffery M. , Szaniszlo Paul J. 
Published: July 2002

ABSTRACT

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of
Wangiella dermatitidis
, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene,
WdPKS1
, in
W. dermatitidis
. The resulting
wdpks1
Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact
WdPKS1
gene was then successfully recovered from two plasmids. The
WdPKS1
gene was also isolated independently by complementation of the
mel3
mutation in an albino mutant of
W. dermatitidis
using a cosmid library. Sequence analysis substantiated that
WdPKS1
encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all
wdpks1
Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a
wdpks1
Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection by
Legionella pneumophila
Authors: Rossier Ombeline , Cianciotto Nicholas P. 
Published: July 2002

ABSTRACT

Previously, we had demonstrated that a
Legionella pneumophila
prepilin peptidase (
pilD
) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (

pilE
L

) mutant. To determine whether these
pilD
-dependent defects were linked to type II secretion, we have constructed two new mutants of
L. pneumophila
strain 130b. Mutations were introduced into either
lspDE
, which encodes the type II outer membrane secretin and ATPase, or
lspFGHIJK
, which encodes the pseudopilins. Unlike the wild-type and

pilE
L

strains, both
lspDE
and
lspG
mutants showed reduced secretion of six
pilD
-dependent enzymatic activities; i.e., protease, acid phosphatase,
p
-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the
pilD
mutant, suggesting that their surfaces are distinct. The
pilD, lspDE
, and
lspG
mutants were similarly and greatly impaired for growth within
Hartmannella vermiformis
, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both
lsp
mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the
pilD
mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel
pilD
-dependent mechanism for promoting
L. pneumophila
intracellular infection of human cells.
Cellular Uptake of the
Clostridium perfringens
Binary Iota-Toxin
Authors: Blöcker Dagmar , Behlke Joachim , Aktories Klaus , Barth Holger 
Published: July 2002

ABSTRACT

The binary iota-toxin is produced by
Clostridium perfringens
type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor.
Identification and Analysis of
Staphylococcus aureus
Components Expressed by a Model System of Growth in Serum
Authors: Wiltshire Michael D. , Foster Simon J. 
Published: July 2002

ABSTRACT

A model system mimicking
Staphylococcus aureus
bacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.
Escherichia coliCytotoxic Necrotizing Factor andPasteurella multocidaToxin Induce Focal Adhesion Kinase Autophosphorylation and Src Association
Authors: Thomas Warren , Pullinger Gillian D. , Lax Alistair J. , Rozengurt Enrique 
Published: July 2002

ABSTRACTCytotoxic necrotizing factor 1 andPasteurella multocidatoxin induced dose- and time-dependent increases in focal adhesion kinase (FAK) Tyr397phosphorylation in Swiss 3T3 cells. FAK autophosphorylation was sensitive to inhibitors of p160/ROCK and coincided with the formation of stable complexes between FAK and Src family members.
In Vivo Induction of Virulence and Antibiotic Resistance Transfer in
Enterococcus faecalis
Mediated by the Sex Pheromone-Sensing System of pCF10
Authors: Hirt Helmut , Schlievert Patrick M. , Dunny Gary M. 
Published: July 2002

ABSTRACT

Enterococcus faecalis
has become one of the most notable nosocomial pathogens in the last decade. Aggregation substance (AS) on the sex pheromone plasmids of
E. faecalis
has been implicated as a virulence factor in several model systems. We investigated the AS-encoding plasmid pCF10 for its ability to increase virulence in a rabbit endocarditis model. Cells containing pCF10 increased the virulence in the model significantly, as assessed by an increase in aortic valve vegetation size. The results confirmed in vivo induction of the normally tightly controlled AS. In addition to the expression of AS when
E. faecalis
cells were in contact with plasma, plasmid transfer of the tetracycline resistance-carrying plasmid was also activated in vitro and in vivo. In vivo, plasmid transfer reached remarkable frequencies of 8 × 10
−2
to 9 × 10
−2
. These values are comparable to the highest frequencies ever observed in vitro. Cells harboring pCF10 had a significant survival advantage over plasmid-free cells indicated by pCF10 present in two-thirds of the recipient population. Plasma induction was dependent on the presence of the plasmid-encoded PrgZ protein, indicating the requirement of the pheromone-sensing system in the induction process. The data suggested that the mechanism of in vivo induction may involve interference of plasma with the normal function of the pheromone peptide and its inhibitor.
Babesia bovisMerozoite Surface Antigen 1 and Rhoptry-Associated Protein 1 Are Expressed in Sporozoites, and Specific Antibodies Inhibit Sporozoite Attachment to Erythrocytes
Authors: Mosqueda Juan , McElwain Terry F. , Stiller David , Palmer Guy H. 
Published: July 2002

ABSTRACTWe examinedBabesia bovissporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed inBabesiasp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.
Growth of
Neisseria gonorrhoeae
in the Female Mouse Genital Tract Does Not Require the Gonococcal Transferrin or Hemoglobin Receptors and May Be Enhanced by Commensal Lactobacilli
Authors: Jerse Ann E. , Crow Emily T. , Bordner Amy N. , Rahman Ishrat , Cornelissen Cynthia Nau , Moench Thomas R. , Mehrazar Karim 
Published: July 2002

ABSTRACT

Neisseria gonorrhoeae
is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for
N. gonorrhoeae
in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor “on” (Hg
+
) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg
+
phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of
N. gonorrhoeae
in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.
Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of the
Escherichia coli rhaBAD
Operon
Authors: Holcroft Carolyn C. , Egan Susan M. 
Published: July 2002

ABSTRACT

The
Escherichia coli rhaBAD
operon encodes the enzymes for catabolism of the sugar
l
-rhamnose. Full
rhaBAD
activation requires the AraC family activator RhaS (bound to a site that overlaps the −35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at −92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on
rhaBAD
activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from
rhaBAD
promoter fusions. We also expressed a derivative of the α subunit of RNA polymerase deleted for the entire C-terminal domain (α-Δ235) and assayed expression from
rhaBAD
promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the α subunit (α-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on α-CTD. Overall, our results suggest that CRP interaction with α-CTD may not be required for
rhaBAD
activation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.
Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by
Pseudomonas abietaniphila
BKME-9
Authors: Martin Vincent J. J. , Mohn William W. 
Published: July 2002

ABSTRACT

We have cloned and sequenced the
dit
gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by
Pseudomonas abietaniphila
BKME-9. The
dit
gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes
ditA1A2A3
encode the α and β subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (
ditA1
::Tn
5
) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of
xylE
transcriptional fusion strains showed induction of
ditA1
and
ditA3
by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the
dit
cluster includes
ditC
, encoding an extradiol ring cleavage dioxygenase, and
ditR
, encoding an IclR-type transcriptional regulator. Although
ditR
is not strictly required for the growth of strain BKME-9 on abietanes, a
ditR
::Km
r
mutation in a
ditA3
::
xylE
reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of
ditA3
. An ORF with sequence similarity to genes encoding permeases (
ditE
) is linked with genes involved in abietane degradation.
The Redox-Sensitive Transcriptional Activator OxyR Regulates the Peroxide Response Regulon in the Obligate Anaerobe
Bacteroides fragilis
Authors: Rocha Edson R. , Owens Gary , Smith C. Jeffrey 
Published: July 2002

ABSTRACT

The peroxide response-inducible genes
ahpCF
,
dps
, and
katB
in the obligate anaerobe
Bacteroides fragilis
are controlled by the redox-sensitive transcriptional activator OxyR. This is the first functional oxidative stress regulator identified and characterized in anaerobic bacteria.
oxyR
and
dps
were found to be divergently transcribed, with an overlap in their respective promoter regulatory regions.
B. fragilis
OxyR and Dps proteins showed high identity to homologues from a closely related anaerobe,
Porphyromonas gingivalis
. Northern blot analysis revealed that
oxyR
was expressed as a monocistronic 1-kb mRNA and that
dps
mRNA was approximately 500 bases in length.
dps
mRNA was induced over 500-fold by oxidative stress in the parent strain and was constitutively induced in the peroxide-resistant mutant IB263. The constitutive peroxide response in strain IB263 was shown to have resulted from a missense mutation at codon 202 (GAT to GGT) of the
oxyR
gene [
oxyR
(Con)] with a predicted D202G substitution in the OxyR protein. Transcriptional fusion analysis revealed that deletion of
oxyR
abolished the induction of
ahpC
and
katB
following treatment with hydrogen peroxide or oxygen exposure. However,
dps
expression was induced approximately fourfold by oxygen exposure in Δ
oxyR
strains but not by hydrogen peroxide. This indicates that
dps
expression is also under the control of an oxygen-dependent OxyR-independent mechanism. Complementation of Δ
oxyR
mutant strains with wild-type
oxyR
and
oxyR
(Con) restored the inducible peroxide response and the constitutive response of the
ahpCF
,
katB
, and
dps
genes, respectively. However, overexpression of OxyR abolished the catalase activity but not
katB
expression, suggesting that higher levels of intracellular OxyR may be involved in other physiological processes. Analysis of
oxyR
expression in the parents and in Δ
oxyR
and overexpressing
oxyR
strains by Northern blotting and
oxyR
′::
xylB
fusions revealed that
B. fragilis
OxyR does not control its own expression.
The ς
70
Transcription Factor TyrR Has Zinc-Stimulated Phosphatase Activity That Is Inhibited by ATP and Tyrosine
Authors: Zhao Shimin , Zhu Qin , Somerville Ronald L. 
Published: July 2002

ABSTRACT

The TyrR protein of
Escherichia coli
(513 amino acid residues) is the chief transcriptional regulator of a group of genes that are essential for aromatic amino acid biosynthesis and transport. The TyrR protein can function either as a repressor or as an activator. The central region of the TyrR protein (residues 207 to 425) is similar to corresponding polypeptide segments of the NtrC protein superfamily. Like the NtrC protein, TyrR has intrinsic ATPase activity. Here, we report that TyrR possesses phosphatase activity. This activity is subject to inhibition by
l
-tyrosine and its analogues and by ATP and ATP analogues. Zinc ion (2 mM) stimulated the phosphatase activity of the TyrR protein by a factor of 57. The phosphatase-active site of TyrR was localized to a 31-kDa domain (residues 191 to 467) of the protein. However, mutational alteration of distant amino acid residues at both the N terminus and the C terminus of TyrR altered the phosphatase activity.
Haemophilus influenzae
TyrR (318 amino acid residues), a protein with a high degree of sequence similarity to the C terminus of the
E. coli
TyrR protein, exhibited a phosphatase activity similar to that of
E. coli
TyrR.
Characterization and Role of
tbuX
in Utilization of Toluene by
Ralstonia pickettii
PKO1
Authors: Kahng Hyung-Yeel , Byrne Armando M. , Olsen Ronald H. , Kukor Jerome J. 
Published: July 2002

ABSTRACT

The
tbu
regulon of
Ralstonia pickettii
PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the
tbu
pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of
tbuT
, which is located immediately downstream of
tbuA1UBVA2C
, and the associated promoter (
PtbuA1
) is unique in that it results in a cascade type of up-regulation of
tbuT
in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of
tbuT
, 63 bp downstream of the
tbuT
stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated
tbuX
. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in
Escherichia coli
. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in
R. pickettii
PKO1. In addition, the expression of
tbuX
appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.
FabF Is Required for Piezoregulation of
cis
-Vaccenic Acid Levels and Piezophilic Growth of the Deep-Sea Bacterium
Photobacterium profundum
Strain SS9
Authors: Allen Eric E. , Bartlett Douglas H. 
Published: July 2002

ABSTRACT

To more fully explore the role of unsaturated fatty acids in high-pressure, low-temperature growth, the
fabF
gene from the psychrotolerant, piezophilic deep-sea bacterium
Photobacterium profundum
strain SS9 was characterized and its role and regulation were examined. An SS9 strain harboring a disruption in the
fabF
gene (strain EA40) displayed growth impairment at elevated hydrostatic pressure concomitant with diminished
cis
-vaccenic acid (18:1) production. However, growth ability at elevated pressure could be restored to wild-type levels by the addition of exogenous 18:1 to the growth medium. Transcript analysis did not indicate that the SS9
fabF
gene is transcriptionally regulated, suggesting that the elevated 18:1 levels produced in response to pressure increase result from posttranscriptional changes. Unlike many pressure-adapted bacterial species such as SS9, the mesophile
Escherichia coli
did not regulate its fatty acid composition in an adaptive manner in response to changes in hydrostatic pressure. Moreover, an
E. coli fabF
strain was as susceptible to elevated pressure as wild-type cells. It is proposed that the SS9
fabF
product, β-ketoacyl–acyl carrier protein synthase II has evolved novel pressure-responsive characteristics which facilitate SS9 growth at high pressure.
Cloning, Expression, and Characterization of
cis
-Polyprenyl Diphosphate Synthase from the Thermoacidophilic Archaeon
Sulfolobus acidocaldarius
Authors: Hemmi Hisashi , Yamashita Satoshi , Shimoyama Takefumi , Nakayama Toru , Nishino Tokuzo 
Published: July 2002

ABSTRACT

cis
-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of
cis
-polyprenyl diphosphate synthase from a thermoacidophilic archaeon,
Sulfolobus acidocaldarius
, and characterized the recombinant enzyme.