bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Fluorescence Induction Analysis of
Streptococcus pneumoniae
Identifies Genes Involved in Pathogenesis
Authors: Marra Andrea , Asundi Jyoti , Bartilson Magdalena , Lawson Stacey , Fang Flora , Christine Jillian , Wiesner Cedric , Brigham Daniel , Schneider William P. , Hromockyj Alexander E. 
Published: July 2002

ABSTRACT

Differential fluorescence induction (DFI) technology was used to identify promoters of
Streptococcus pneumoniae
induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in
S. pneumoniae
, and the entire library was screened for clones exhibiting increased
gfp
expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.
Chemokine Expression Patterns Differ within Anatomically Distinct Regions of the Genital Tract duringChlamydia trachomatisInfection
Authors: Maxion Heather K. , Kelly Kathleen A. 
Published: July 2002

ABSTRACTUntreated infections withChlamydiatrachomatiscommonly result in ascending infection to fallopian tubes and subsequent immune-mediated tubal pathology in females. The proposed immune-mediated injury may be associated with the increased recruitment of CD4 cells to the upper genital tract (GT) (oviducts) in comparison to the lower GT (cervix) during infection, as shown in animal models. To understand the mechanisms responsible for this biased recruitment of CD4 cells within the GT, we characterized chemokine expression patterns in the upper and lower GTs in mice during infection with the murine pneumonitis biovar ofChlamydiatrachomatis. Enzyme-linked immunosorbent assays of supernatants from GT homogenates revealed that the levels of the Th1-associated chemokines CXCL9 (monokine induced by gamma interferon), CXCL10 (interferon-inducible protein 10), and CCL5 (RANTES) were significantly higher in the upper GT than in the lower GT after infection, while the CCL3 (macrophage inflammatory protein 1α) level was not increased. In contrast, the level of chemokine CCL11 (eotaxin) was significantly elevated in the lower GT later in the course of infection. Increased levels of mRNA confirmed the selective differences in chemokine expression within the upper and lower GTs. The increased levels of Th1-inducible chemokines in the upper GT were not due to differences in the magnitude of infection or progesterone pretreatment. These data demonstrate that the upper and lower regions of the GT respond differently toChlamydiainfection.
Global Analysis of Outer Membrane Proteins from
Leptospira interrogans
Serovar Lai
Authors: Cullen Paul A. , Cordwell Stuart J. , Bulach Dieter M. , Haake David A. , Adler Ben 
Published: July 2002

ABSTRACT

Recombinant leptospiral outer membrane proteins (OMPs) can elicit immunity to leptospirosis in a hamster infection model. Previously characterized OMPs appear highly conserved, and thus their potential to stimulate heterologous immunity is of critical importance. In this study we undertook a global analysis of leptospiral OMPs, which were obtained by Triton X-114 extraction and phase partitioning. Outer membrane fractions were isolated from
Leptospira interrogans
serovar Lai grown at 20, 30, and 37°C with or without 10% fetal calf serum and, finally, in iron-depleted medium. The OMPs were separated by two-dimensional gel electrophoresis. Gel patterns from each of the five conditions were compared via image analysis, and 37 gel-purified proteins were tryptically digested and characterized by mass spectrometry (MS). Matrix-assisted laser desorption ionization-time-of-flight MS was used to rapidly identify leptospiral OMPs present in sequence databases. Proteins identified by this approach included the outer membrane lipoproteins LipL32, LipL36, LipL41, and LipL48. No known proteins from any cellular location other than the outer membrane were identified. Tandem electrospray MS was used to obtain peptide sequence information from eight novel proteins designated pL18, pL21, pL22, pL24, pL45, pL47/49, pL50, and pL55. The expression of LipL36 and pL50 was not apparent at temperatures above 30°C or under iron-depleted conditions. The expression of pL24 was also downregulated after iron depletion. The leptospiral major OMP LipL32 was observed to undergo substantial cleavage under all conditions except iron depletion. Additionally, significant downregulation of these mass forms was observed under iron limitation at 30°C, but not at 30°C alone, suggesting that LipL32 processing is dependent on iron-regulated extracellular proteases. However, separate cleavage products responded differently to changes in growth temperature and medium constituents, indicating that more than one process may be involved in LipL32 processing. Furthermore, under iron-depleted conditions there was no concomitant increase in the levels of the intact form of LipL32. The temperature- and iron-regulated expression of LipL36 and the iron-dependent cleavage of LipL32 were confirmed by immunoblotting with specific antisera. Global analysis of the cellular location and expression of leptospiral proteins will be useful in the annotation of genomic sequence data and in providing insight into the biology of
Leptospira
.
Proteomic Analysis of Differentially Expressed
Chlamydia pneumoniae
Genes during Persistent Infection of HEp-2 Cells
Authors: Molestina Robert E. , Klein Jon B. , Miller Richard D. , Pierce William H. , Ramirez Julio A. , Summersgill James T. 
Published: July 2002

ABSTRACT

Recent data have shown that the respiratory pathogen
Chlamydia pneumoniae
expresses an altered gene transcription profile during gamma interferon (IFN-γ)-induced persistent infection in vitro. In the present study, we examined, by proteomics, expression of
C. pneumoniae
proteins labeled intracellularly with [
35
S]methionine/cysteine under normal conditions or IFN-γ-mediated persistence. The identity of differentially expressed proteins during persistent infection was determined by matching spots to those of proteins identified in
C. pneumoniae
elementary bodies by matrix-assisted laser desorption ionization mass spectrometry. Upon treatment with 50 U of IFN-γ per ml, a marked upregulation of major outer membrane protein (MOMP), heat shock protein 60 (Hsp-60/GroEL), and proteins with functions in DNA replication (GyrA), transcription (RpoA, PnP), translation (Rrf), glycolysis (PgK, GlgP), and type III secretion (SctN) was observed at 24 h of infection. In contrast, no significant decreases in bacterial protein expression were found in
C. pneumoniae
-infected cells due to IFN-γ treatment. Upregulation of
C. pneumoniae
proteins involved in diverse functions during persistent infection may allow the organism to resist the inhibitory effects of IFN-γ while retaining basic functions. Future studies should examine the differential expression of chlamydial proteins during the developmental cycle under IFN-γ pressure to obtain a finer representation of the gene products involved in establishing persistence.
Identification of a Quorum-Sensing Signal Molecule in the Facultative Intracellular PathogenBrucella melitensis
Authors: Taminiau Bernard , Daykin Mavis , Swift Simon , Boschiroli Maria-Laura , Tibor Anne , Lestrate Pascal , De Bolle Xavier , O'Callaghan David , Williams Paul , Letesson Jean-Jacques 
Published: July 2002

ABSTRACTBrucella melitensisis a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant fromB. melitensisinduces bioluminescence in anEscherichia coliacyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein ofPseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule asN-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of eitherB. melitensisorBrucella suis1330 reduces the transcription of thevirBoperon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence inBrucella.
Sigma B Contributes to PrfA-Mediated Virulence inListeria monocytogenes
Authors: Nadon Celine A. , Bowen Barbara M. , Wiedmann Martin , Boor Kathryn J. 
Published: July 2002

ABSTRACTTranscription of theListeria monocytogenespositive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream ofprfA(prfAp1andprfAp2) or from the upstreamplcApromoter. We demonstrate thatprfAp2is a functional σB-dependent promoter and that asigBdeletion mutation affects the virulence phenotype ofL. monocytogenes. Thus, the alternative sigma factor σBcontributes to virulence inL. monocytogenes.
Deletion Analysis of the
Escherichia coli
Taurine and Alkanesulfonate Transport Systems
Authors: Eichhorn Eric , van der Ploeg Jan R. , Leisinger Thomas 
Published: July 2002

ABSTRACT

The
Escherichia coli tauABCD
and
ssuEADCB
gene clusters are required for the utilization of taurine and alkanesulfonates as sulfur sources and are expressed only under conditions of sulfate or cysteine starvation.
tauD
and
ssuD
encode an α-ketoglutarate-dependent taurine dioxygenase and a reduced flavin mononucleotide-dependent alkanesulfonate monooxygenase, respectively. These enzymes are responsible for the desulfonation of taurine and alkanesulfonates. The amino acid sequences of SsuABC and TauABC exhibit similarity to those of components of the ATP-binding cassette transporter superfamily, suggesting that two uptake systems for alkanesulfonates are present in
E. coli
. Chromosomally located in-frame deletions of the
tauABC
and
ssuABC
genes were constructed in
E. coli
strain EC1250, and the growth properties of the mutants were studied to investigate the requirement for the TauABC and SsuABC proteins for growth on alkanesulfonates as sulfur sources. Complementation analysis of in-frame deletion mutants confirmed that the growth phenotypes obtained were the result of the in-frame deletions constructed. The range of substrates transported by these two uptake systems was largely reflected in the substrate specificities of the TauD and SsuD desulfonation systems. However, certain known substrates of TauD were transported exclusively by the SsuABC system. Mutants in which only formation of hybrid transporters was possible were unable to grow with sulfonates, indicating that the individual components of the two transport systems were not functionally exchangeable. The TauABCD and SsuEADCB systems involved in alkanesulfonate uptake and desulfonation thus are complementary to each other at the levels of both transport and desulfonation.
Novel Genes Coding for Lithotrophic Sulfur Oxidation of
Paracoccus pantotrophus
GB17
Authors: Friedrich Cornelius G. , Quentmeier Armin , Bardischewsky Frank , Rother Dagmar , Kraft Regine , Kostka Susanne , Prinz Heino 
Published: July 2002

ABSTRACT

The gene region coding for lithotrophic sulfur oxidation of
Paracoccus pantotrophus
GB17 is located on a 13-kb insert of plasmid pEG12. Upstream of the previously described six open reading frames (ORFs)
soxABCDEF
with a partial sequence of
soxA
and
soxF
(C. Wodara, F. Bardischewsky, and C. G. Friedrich, J. Bacteriol. 179:5014–5023, 1997), 4,350 bp were sequenced. The sequence completed
soxA
, and uncovered six new ORFs upstream of
soxA
, designated ORF1, ORF2, and ORF3, and
soxXYZ
. ORF1 could encode a 275-amino-acid polypeptide of 29,332 Da with a 61 to 63% similarity to LysR transcriptional regulators. ORF2 could encode a 245-amino-acid polypeptide of 26,022 Da with the potential to form six transmembrane helices and with a 48 to 51% similarity to proteins involved in redox transport in cytochrome
c
biogenesis. ORF3 could encode a periplasmic polypeptide of 186 amino acids of 20,638 Da with a similarity to thioredoxin-like proteins and with a putative signal peptide of 21 amino acids. Purified SoxXA, SoxYZ, and SoxB are essential for thiosulfate or sulfite-dependent cytochrome
c
reduction in vitro. N-terminal and internal amino acid sequences identified SoxX, SoxY, SoxZ, and SoxA to be coded by the respective genes. The molecular masses of the mature proteins determined by electrospray ionization spectroscopy (SoxX, 14,834 Da; SoxY, 11,094 Da; SoxZ, 11,717 Da; and SoxA, 30,452 Da) were identical or close to those deduced from the nucleotide sequence with differences for the covalent heme moieties. SoxXA represents a novel type of periplasmic
c
-type cytochromes, with SoxX as a monoheme and SoxA as a hybrid diheme cytochrome
c
. SoxYZ is an as-yet-unprecedented soluble protein. SoxY has a putative signal peptide with a twin arginine motif and possibly cotransports SoxZ to the periplasm. SoxYZ neither contains a metal nor a complex redox center, as proposed for proteins likely to be transported via the Tat system.
Purification and Characterization of
glpX
-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of
Escherichia coli
Authors: Donahue Janet L. , Bownas Jennifer L. , Niehaus Walter G. , Larson Timothy J. 
Published: July 2002

ABSTRACT

In
Escherichia coli
, gene products of the
glp
regulon mediate utilization of glycerol and
sn
-glycerol 3-phosphate. The
glpFKX
operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described
fbp
-encoded enzyme. The purified enzyme was dimeric, dependent on Mn
2+
for activity, and exhibited an apparent

K
m

of 35 μM for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.
The
rfaE
Gene from
Escherichia coli
Encodes a Bifunctional Protein Involved in Biosynthesis of the Lipopolysaccharide Core Precursor ADP-
l
-
glycero
-
d
-
manno
-Heptose
Authors: Valvano Miguel A. , Marolda Cristina L. , Bittner Mauricio , Glaskin-Clay Mike , Simon Tania L. , Klena John D. 
Published: July 2002

ABSTRACT

The intermediate steps in the biosynthesis of the ADP-
l
-
glycero
-
d
-
manno
-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn
10
insertion that confers a heptoseless LPS phenotype in the chromosome of
Escherichia coli
K-12. The mutation was in a gene homologous to the previously reported
rfaE
gene from
Haemophilus influenzae
. The
E. coli rfaE
gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the
aut
gene product of
Ralstonia eutrophus
. Each domain was expressed individually, demonstrating that only Domain I could complement the
rfaE
::Tn
10
mutation in
E. coli
, as well as the
rfaE543
mutation of
Salmonella enterica
SL1102. DNA sequencing of the
rfaE543
gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the
aut
::Tn
5
mutation in
R. eutrophus
is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the
E. coli
Domain II. Thus, both domains of
rfaE
are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of
d
-
glycero
-
d
-
manno
-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-
d
-
glycero
-
d
-
manno
-heptose.
Novel Toluene Elimination System in a Toluene-Tolerant Microorganism
Authors: Kobayashi Hideki , Uematsu Katsuyuki , Hirayama Hisako , Horikoshi Koki 
Published: July 2002

ABSTRACT

In studies of
Pseudomonas putida
IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.
Biochemical and Genetic Evidence that
Enterococcus faecium
L50 Produces Enterocins L50A and L50B, the
sec
-Dependent Enterocin P, and a Novel Bacteriocin Secreted without an N-Terminal Extension Termed Enterocin Q
Authors: Cintas Luis M. , Casaus Pilar , Herranz Carmen , Håvarstein Leiv Sigve , Holo Helge , Hernández Pablo E. , Nes Ingolf F. 
Published: July 2002

ABSTRACT

Enterococcus faecium
L50 grown at 16 to 32°C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47°C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the
sec
-dependent pediocin-like enterocin P produced by
E. faecium
P13 (L. M. Cintas, P. Casaus, L. S. Håvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321–4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of
E. faecium
L50 containing the enterocin P structural gene (
entP
) and the putative immunity protein gene (
entiP
) reveals a genetic organization identical to that previously found in
E. faecium
P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed
entQ
, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed
orf2
, was located immediately upstream of and in opposite orientation to
entQ
and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by
E. faecium
L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47°C and maximally detected at 47 and 37 to 47°C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25°C and are not detected at 37°C or above.
Increased Sensitivity to Oxidative Challenges Associated with
topA
Deletion in
Escherichia coli
Authors: Tse-Dinh Yuk-Ching 
Published: July 2002

ABSTRACT

Deletion of
topA
in
Escherichia coli
was found to result in a higher level of killing after treatment with either hydrogen peroxide or
N
-ethylmaleimide. This effect on oxidative challenge response represents a new role for
E. coli
DNA topoisomerase I in addition to prevention of excessive negative supercoiling of DNA.
Visualization of Phospholipid Domains in
Escherichia coli
by Using the Cardiolipin-Specific Fluorescent Dye 10-
N
-Nonyl Acridine Orange
Authors: Mileykovskaya Eugenia , Dowhan William 
Published: July 2002

ABSTRACT

Cardiolipin (CL)-specific fluorescent dye 10-
N
-nonyl-acridine orange (NAO) was used to visualize CL distribution in
Escherichia coli
cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.
Membrane Association of the
Escherichia coli
Enterobactin Synthase Proteins EntB/G, EntE, and EntF
Authors: Hantash Feras M. , Earhart Charles F. 
Published: July 2002

ABSTRACT

The cytosolic proteins EntE, EntF, and EntB/G, which are
Escherichia coli
enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.
Role of Motility in Adherence to and Invasion of a Fish Cell Line by
Vibrio anguillarum
Authors: Ormonde Patricia , Hörstedt Per , O'Toole Ronan , Milton Debra L. 
Published: July 2002

ABSTRACT

To understand further the role of the flagellum of
Vibrio anguillarum
in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by
V. anguillarum
, both in vivo and in vitro.