bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence that the Enterococcal Polysaccharide Antigen Gene (
epa
) Cluster Is Widespread in
Enterococcus faecalis
and Influences Resistance to Phagocytic Killing of
E. faecalis
Authors: Teng Fang , Jacques-Palaz Karen D. , Weinstock George M. , Murray Barbara E. 
Published: July 2002

ABSTRACT

In previous studies, we cloned a cluster of genes involved in polysaccharide biosynthesis (
epa
) from
Enterococcus faecalis
strain OG1RF and showed that this gene cluster mediated synthesis of a polysaccharide in
Escherichia coli
. Disruption of two open reading frames in the
epa
gene cluster of OG1RF generated two mutants, TX5179 and TX5180, which were attenuated in a mouse peritonitis model. In the current study, Western blotting was performed with serum from a patient with
E. faecalis
endocarditis and polysaccharide extracts from OG1RF and the mutants TX5179 and TX5180. OG1RF showed a smear in the high-molecular-weight region and discrete bands in the low-molecular-weight region, which were missing from the mutants; periodate treatment and carbohydrate staining confirmed the polysaccharide nature of this material. In a neutrophil killing assay using OG1RF-absorbed normal human serum, the mutants TX5179 and TX5180, respectively, were 50 and 2.4 times more susceptible to killing than wild-type OG1RF (
P
≤ 0.01). With a fluorescence phagocytosis assay, 2.5 to 3 times more of the mutants were taken up by neutrophils than OG1RF (
P
≤ 0.001). Finally, with restriction digestion and hybridization under high-stringency conditions, the
epa
gene cluster of OG1RF (which is also present in the sequenced
E. faecalis
strain V583) was detected in 12 of 12 other clonally distinct
E. faecalis
strains tested: a similar polysaccharide pattern was detected for the 12 strains on Western blots using an
E. faecalis
endocarditis patient serum, and sera from four other patients with
E. faecalis
endocarditis all reacted with polysaccharide extracts of OG1RF. These results indicate that the
epa
gene cluster is widespread among
E. faecalis
and confers some protection against human host defenses.
Haemophilus ducreyi
Requires the
flp
Gene Cluster for Microcolony Formation In Vitro
Authors: Nika Joseph R. , Latimer Jo L. , Ward Christine K. , Blick Robert J. , Wagner Nikki J. , Cope Leslie D. , Mahairas Gregory G. , Munson Robert S. , Hansen Eric J. 
Published: July 2002

ABSTRACT

Haemophilus ducreyi
, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in
H. ducreyi
that encoded predicted protein products with significant homology to those encoded by the
tad
(for tight adhesion) locus in
Actinobacillus actinomycetemcomitans
that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this
H. ducreyi
gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the
tad
locus; this 15-gene cluster in
H. ducreyi
was designated
flp
. RT-PCR analysis indicated that the
H. ducreyi flp
gene cluster was likely to be a polycistronic operon. Mutations within the
flp
gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An
H. ducreyi
mutant with an inactivated
tadA
gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another
H. ducreyi
mutant with inactivated
flp-1
and
flp-2
genes was as virulent as the wild-type parent strain. These results indicate that the
flp
gene cluster is essential for microcolony formation by
H. ducreyi
, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.
Expression of the Biofilm-Associated Protein Interferes with Host Protein Receptors of
Staphylococcus aureus
and Alters the Infective Process
Authors: Cucarella Carme , Tormo M. Ángeles , Knecht Erwin , Amorena Beatriz , Lasa Íñigo , Foster Timothy J. , Penadés José R. 
Published: July 2002

ABSTRACT

The adherence of
Staphylococcus aureus
to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections.
S. aureus
presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on
S. aureus
adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.
Sulfolobicins, Specific Proteinaceous Toxins Produced by Strains of the Extremely Thermophilic Archaeal Genus
Sulfolobus
Authors: Prangishvili David , Holz Ingelore , Stieger Evelyn , Nickell Stephan , Kristjansson Jakob K. , Zillig Wolfram 
Published: July 2002

ABSTRACT

Several novel strains of “
Sulfolobus islandicus
” produced proteinaceous toxins, termed sulfolobicins, which killed cells of other strains of the same species, as well as of
Sulfolobus solfataricus
P1 and
Sulfolobus shibatae
B12, but not of the producer strains and of
Sulfolobus acidocaldarius
DSM639. The sulfolobicin purified from the strain HEN2/2 had a molecular mass of about 20 kDa. It was found to be associated with the producer cells as well as with cell-derived S-layer-coated spherical membrane vesicles 90 to 180 nm in diameter and was not released from the cells in soluble form.
Yersinia enterocolitica
TyeA, an Intracellular Regulator of the Type III Machinery, Is Required for Specific Targeting of YopE, YopH, YopM, and YopN into the Cytosol of Eukaryotic Cells
Authors: Cheng Luisa W. , Schneewind Olaf 
Published: July 2002

ABSTRACT

Pathogenic
Yersinia
species employ type III machines to target effector Yops into the cytosol of eukaryotic cells.
Yersinia tyeA
mutants are thought to be defective in the targeting of YopE and YopH without affecting the injection of YopM, YopN, YopO, YopP, and YopT into the cytosol of eukaryotic cells. One model suggests that TyeA may form a tether between YopN (LcrE) and YopD on the bacterial surface, a structure that may translocate YopE and YopH across the plasma membrane of eukaryotic cells (M. Iriarte, M. P. Sory, A. Boland, A. P. Boyd, S. D. Mills, I. Lambermont, and G. R. Cornelis, EMBO J. 17:1907–1918, 1998). We have examined the injection of Yop proteins by
tyeA
mutant yersiniae with the digitonin fractionation technique. We find that
tyeA
mutant yersiniae not only secreted YopE, YopH, YopM, and YopN into the extracellular medium but also targeted these polypeptides into the cytosol of HeLa cells. Protease protection, cell fractionation, and affinity purification experiments suggest that TyeA is located intracellularly and binds to YopN or YopD. We propose a model whereby TyeA functions as a negative regulator of the type III targeting pathway in the cytoplasm of yersiniae, presumably by preventing the export of YopN.
Evidence for a Signaling System in
Helicobacter pylori
: Detection of a
luxS
-Encoded Autoinducer
Authors: Joyce Elizabeth A. , Bassler Bonnie L. , Wright Andrew 
Published: July 2002

ABSTRACT

Helicobacter pylori
possesses a homolog of the
luxS
gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in
Vibrio harveyi
. The genomes of several other species of bacteria, notably
Escherichia coli
,
Salmonella enterica
serovar Typhimurium, and
Vibrio cholerae
, also include
luxS
homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in
V. harveyi
. In this report, we demonstrate that
H. pylori
also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in
V. harveyi
. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in
H. pylori
and that a
luxS
null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in
H. pylori
and suggest possible roles that it may play in
H. pylori
infection.
Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in
Klebsiella pneumoniae
Strains Bearing the
Rhodospirillum rubrum dra
Operon
Authors: Halbleib Cale M. , Zhang Yaoping , Roberts Gary P. , Ludden Paul W. 
Published: July 2002

ABSTRACT

The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in
Klebsiella pneumoniae
strains transformed by plasmids carrying
dra
genes from
Rhodospirillum rubrum
. The
dra
operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in
R. rubrum
. In bacteria containing the
dra
operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. The
dra
gene products, expressed at a low level in
K. pneumoniae
, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in
R. rubrum
. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion of
nifD
, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.
Characterization of a Second
tfd
Gene Cluster for Chlorophenol and Chlorocatechol Metabolism on Plasmid pJP4 in
Ralstonia eutropha
JMP134(pJP4)
Authors: Laemmli Caroline M. , Leveau Johan H. J. , Zehnder Alexander J. B. , van der Meer Jan Roelof 
Published: July 2002

ABSTRACT

Within the 5.9-kb DNA region between the
tfdR
and
tfdK
genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from
Ralstonia eutropha
JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (
tfdCDEF
and
tfdB
) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows:
tfdD
II
C
II
E
II
F
II
and
tfdB
II
(in short, the
tfd
II
cluster), by analogy to
tfdCDEF
and
tfdB
(the
tfd
I
cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown
R. eutropha
JMP134 identified a single transcription start site in front of the first gene of the cluster,
tfdD
II
, suggesting an operon-like organization for the
tfd
II
genes. By expressing each ORF in
Escherichia coli
, we confirmed that
tfdD
II
coded for a chloromuconate cycloisomerase,
tfdC
II
coded for a chlorocatechol 1,2-dioxygenase,
tfdE
II
coded for a dienelactone hydrolase,
tfdF
II
coded for a maleylacetate reductase, and
tfdB
II
coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from
R. eutropha
JMP134 showed that both
tfd
I
and
tfd
II
genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the
tfd
II
genes seem to be redundant with respect to those of the
tfd
I
cluster. One reason why the
tfd
II
genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression
tfdR
and
tfdS
.
rpoS
Mutants in Archival Cultures of
Salmonella enterica
Serovar Typhimurium
Authors: Sutton Amy , Buencamino Raphael , Eisenstark Abraham 
Published: July 2002

ABSTRACT

Long-term survival under limited growth conditions presents bacterial populations with unique environmental challenges. The existence of
Salmonella enterica
serovar Typhimurium cultures undisturbed in sealed nutrient agar stab vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures. We chose to start with examination of the
rpoS
gene since, among gram-negative bacteria, many genes needed for survival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the
rpoS
start codon UUG instead of the expected AUG of
Salmonella
and
Escherichia coli
strains recorded in GenBank. Ten of the 27 had additional mutations in the
rpoS
gene compared with the
X77752
wild-type strain currently recorded in GenBank. The
rpoS
mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially. Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it is assumed that the 10
rpoS
mutations occurred during storage. Since the remaining 17 sequences were wild type (other than in the start codon), it is obvious that
rpoS
remained relatively stable during decades of sealed storage.
Competition between the Yops of
Yersinia enterocolitica
for Delivery into Eukaryotic Cells: Role of the SycE Chaperone Binding Domain of YopE
Authors: Boyd Aoife P. , Lambermont Isabelle , Cornelis Guy R. 
Published: July 2002

ABSTRACT

A type III secretion-translocation system allows
Yersinia
adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca
2+
-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant
Yersinia
devoid of most of the Yop effectors (ΔHOPEM and ΔTHE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE
15
-Cya as efficiently as YopE
130
-Cya. They were also able to deliver YopE
Δ17–77
. SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type
Yersinia
suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca
2+
chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.
Understanding the Growth Phenotype of the Yeast
gcr1
Mutant in Terms of Global Genomic Expression Patterns
Authors: López M. Cecilia , Baker Henry V. 
Published: July 2002

ABSTRACT

The phenotype of an organism is the manifestation of its expressed genome. The
gcr1
mutant of yeast grows at near wild-type rates on nonfermentable carbon sources but exhibits a severe growth defect when grown in the presence of glucose, even when nonfermentable carbon sources are available. Using DNA microarrays, the genomic expression patterns of wild-type and
gcr1
mutant yeast growing on various media, with and without glucose, were compared. A total of 53 open reading frames (ORFs) were identified as
GCR1
dependent based on the criterion that their expression was reduced twofold or greater in mutant versus wild-type cultures grown in permissive medium consisting of YP supplemented with glycerol and lactate. The
GCR1
-dependent genes, so defined, fell into three classes: (i) glycolytic enzyme genes, (ii) ORFs carried by Ty elements, and (iii) genes not previously known to be
GCR1
dependent. In wild-type cultures,
GCR1
-dependent genes accounted for 27% of the total hybridization signal, whereas in mutant cultures, they accounted for 6% of the total. Glucose addition to the growth medium resulted in a reprogramming of gene expression in both wild-type and mutant yeasts. In both strains, glycolytic enzyme gene expression was induced by the addition of glucose, although the expression of these genes was still impaired in the mutant compared to the wild type. By contrast, glucose resulted in a strong induction of Ty-borne genes in the mutant background but did not greatly affect their already high expression in the wild-type background. Both strains responded to glucose by repressing the expression of genes involved in respiration and the metabolism of alternative carbon sources. Thus, the severe growth inhibition observed in
gcr1
mutants in the presence of glucose is the result of normal signal transduction pathways and glucose repression mechanisms operating without sufficient glycolytic enzyme gene expression to support growth via glycolysis alone.
A Single-Transformation Gene Function Test in Diploid
Candida albicans
Authors: Enloe Brian , Diamond Aviva , Mitchell Aaron P. 
Published: July 2002

ABSTRACT

The fungal pathogen
Candida albicans
is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (
UAU1
) for which two copies may be selected. Insertion of
UAU1
into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (
ADE2, RIM20
, and
YGR189
), while only allelic triplications were found at two essential loci (
SNF1
and
CDC28
). We have unexpectedly isolated homozygous mutants with mutations at
CDC25
; they are viable but defective in filamentation on serum-containing medium. The
UAU1
cassette is thus useful to assess rapidly the essentiality of
C. albicans
genes.
The
torYZ
(
yecK bisZ
) Operon Encodes a Third Respiratory Trimethylamine
N
-Oxide Reductase in
Escherichia coli
Authors: Gon Stéphanie , Patte Jean-Claude , Méjean Vincent , Iobbi-Nivol Chantal 
Published: July 2002

ABSTRACT

The
bisZ
gene of
Escherichia coli
was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study,
bisZ
has been renamed
torZ
based on the findings that (i) the
torZ
gene product, TorZ, is able to reduce trimethylamine
N
-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii)
torZ
belongs to the
torYZ
operon, and the first gene,
torY
(formerly
yecK
), encodes a pentahemic
c
-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the
torYZ
operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The
torYZ
and the
torCAD
operons may have diverged from a common ancestor, but, surprisingly, no
torD
homologue is found in the sequences around
torYZ
. Moreover, the
torYZ
operon is expressed at very low levels under the conditions tested, and, in contrast to
torCAD
, it is not induced by TMAO or dimethyl sulfoxide.
Acquisition of the
rfb-gnd
Cluster in Evolution of
Escherichia coli
O55 and O157
Authors: Tarr Phillip I. , Schoening Laura M. , Yea Yoo-Lee , Ward Teresa R. , Jelacic Srdjan , Whittam Thomas S. 
Published: July 2002

ABSTRACT

The
rfb
region specifies the structure of lipopolysaccharide side chains that comprise the diverse gram-negative bacterial somatic (O) antigens. The
rfb
locus is adjacent to
gnd
, which is a polymorphic gene encoding 6-phosphogluconate dehydrogenase. To determine if
rfb
and
gnd
cotransfer, we sequenced
gnd
in five O55 and 13 O157 strains of
Escherichia coli. E. coli
O157:H7 has a
gnd
allele (allele
A
) that is only 82% identical to the
gnd
allele (allele
D
) of closely related
E. coli
O55:H7. In contrast,
gnd
alleles of
E. coli
O55 in distant lineages are >99.9% identical to
gnd
allele
D
. Though
gnd
alleles
B
and
C
in
E. coli
O157 that are distantly related to
E. coli
O157:H7 are more similar to allele
A
than to allele
D
, there are nucleotide differences at 4 to 6% of their sites. Alleles
B
and
C
can be found in
E. coli
O157 in different lineages, but we have found allele
A
only in
E. coli
O157 belonging to the DEC5 lineage. DNA 3′ to the O55
gnd
allele in diverse
E. coli
lineages has sequences homologous to
tnpA
of the
Salmonella enterica
serovar Typhimurium IS
200
element,
E. coli Rhs
elements (including an H-rpt gene), and portions of the O111 and O157
rfb
regions. We conclude that
rfb
and
gnd
cotransferred into
E. coli
O55 and O157 in widely separated lineages and that recombination was responsible for recent antigenic shifts in the emergence of pathogenic
E. coli
O55 and O157.