bluuebunny/crossref_metadata_2025 huggingface dataset:
Adherence Properties of
Staphylococcus aureus
under Static and Flow Conditions: Roles of
agr
and
sar
Loci, Platelets, and Plasma Ligands
Authors: Shenkman Boris , Rubinstein Ethan , Cheung Ambrose L. , Brill Grigory E. , Dardik Rima , Tamarin Ilya , Savion Naphtali , Varon David
Published: July 2002

Global regulatory genes in
Staphylococcus aureus
, including
agr
and
sar
, are known to regulate the expression of multiple virulence factors, including cell wall adhesins. In the present study, the adherence of
S. aureus
RN6390 (wild type), RN6911 (
agr
), ALC136 (
sar
), and ALC135 (
agr sar
) to immobilized fibrinogen, fibronectin, von Willebrand factor (vWF), extracellular matrix (ECM), and human endothelial cells (EC) EAhy.926 was studied. Bacteria grown to postexponential phase were subjected to light oscillation (static condition) or to shear stress at 200 s
−1
(flow condition) on tissue culture polystyrene plates coated with either protein ligands, ECM, or EC. Adherence of nonlabeled bacteria to immobilized ligands was measured by an image analysis system, while adherence of [
3
H]thymidine-labeled
S. aureus
to ECM and EC was measured by a β-scintillation counter. The results showed increased adherence of
agr
and
agr sar
mutants to immobilized fibrinogen and higher potential of these mutants to induce platelet aggregation in suspension, decreased adherence of
sar
and
agr sar
mutants to immobilized fibronectin and vWF as well as to ECM and EC, increased adherence of both
S. aureus
wild type and
sar
mutant to EC treated with platelet-rich plasma (PRP) compared to platelet-poor plasma (PPP) and to EC treated with PPP compared to the control, and increased adherence of
S. aureus
wild type to EC coated with PRP in which platelets were activated with phorbol 12-myristate 13-acetate compared to intact PRP. This finding paralleled the increased adherence to EC of activated compared to intact platelets. It is suggested that platelet-mediated
S. aureus
adherence to EC depends on platelet activation and the number of adherent platelets and available receptors on the platelet membrane. In conclusion, the
agr
locus downregulates
S. aureus
adherence to fibrinogen, while the
sar
locus upregulates
S. aureus
adherence to fibronectin, vWF, ECM, and EC. The effect of both
agr
and
sar
on
S. aureus
adherence properties develops primarily under flow conditions, which suggests different adhesion mechanisms in static and flow conditions.
Protection against Anthrax Lethal Toxin Challenge by Genetic Immunization with a Plasmid Encoding the Lethal Factor Protein
Authors: Price Brian M. , Liner Adriane L. , Park Sukjoon , Leppla Stephen H. , Mateczun Alfred , Galloway Darrell R.
Published: July 2002

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) ofBacillus anthracisLF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) ofB. anthracisPA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 μg of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.
Proteomics Reveals Open Reading Frames in
Mycobacterium tuberculosis
H37Rv Not Predicted by Genomics
Authors: Jungblut Peter R. , Müller Eva-Christina , Mattow Jens , Kaufmann Stefan H. E.
Published: July 2002

Genomics revealed the sequence of 3924 genes of the H37Rv strain of
Mycobacterium tuberculosis
. Proteomics complements genomics in showing which genes are really expressed, and here we show the expression of six genes not predicted by genomics, as proved by two-dimensional electrophoresis and matrix-assisted laser desorption ionization and nano-electrospray mass spectrometry.
Induction of Gamma Interferon and Nitric Oxide by Truncated Pneumolysin That Lacks Pore-Forming Activity
Authors: Baba Hisashi , Kawamura Ikuo , Kohda Chikara , Nomura Takamasa , Ito Yutaka , Kimoto Terumi , Watanabe Isao , Ichiyama Satoshi , Mitsuyama Masao
Published: July 2002

Pneumolysin (PLY), an important virulence factor ofStreptococcus pneumoniae, is known to exert various effects on the host immune cells, including cytokine induction, in addition to its known cytolytic activity as a member of the thiol-activated cytolysins. It is of interest to determine whether cytolytic activity is involved in triggering the cytokine production. In this study, we constructed full-length recombinant PLY and noncytolytic truncated PLYs with C-terminal deletions to examine the response of spleen cells to these PLY preparations. When cytolytic activity was blocked by treatment with cholesterol, full-length PLY was capable of inducing gamma interferon (IFN-γ) production. Truncated PLYs that originally exhibited no cytolytic activity were also active in IFN-γ induction. Therefore, the IFN-γ-inducing ability of PLY appeared to be independent of the cytolytic activity. Furthermore, IFN-γ-inducing preparations were also capable of inducing nitric oxide synthase expression and nitric oxide (NO) production, and the addition of neutralizing antibody to IFN-γ abolished the NO production. These results clearly demonstrated that PLY is capable of inducing IFN-γ production in spleen cells by a mechanism different from pore formation and that the induced IFN-γ stimulates NO production. These findings were discussed with reference to the contribution of PLY to the virulence ofS. pneumoniaein vivo.
Type III Secretion of
Salmonella enterica
Serovar Typhimurium Translocated Effectors and SseFG
Authors: Hansen-Wester Imke , Stecher Bärbel , Hensel Michael
Published: July 2002

The type III secretion system (TTSS) encoded by
Salmonella
enterica
serovar Typhimurium pathogenicity island 2 (SPI2) is employed by
Salmonella enterica
for interaction with host cells during the intracellular phase of pathogenesis. This TTSS secretes a set of SPI2-encoded proteins in vitro and translocates
Salmonella
serovar Typhimurium translocated effectors (STE) that are encoded by genes outside of SPI2 into host cells. Using an epitope-tagging approach, we analyzed secretion of proteins by the TTSS of SPI2 and identified SseF and SseG as further secreted substrate proteins. Three members of the STE family, SifA, SifB, and SseJ, were secreted under conditions that also induce secretion of SPI2-encoded substrate proteins.
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

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

Untreated 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

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

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

Brucella 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

Transcription 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

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

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

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

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

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

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

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

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

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

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.
Secreted Euryarchaeal Microhalocins Kill Hyperthermophilic Crenarchaea
Authors: Haseltine Cynthia , Hill Tiffany , Montalvo-Rodriguez Rafael , Kemper Samantha K. , Shand Richard F. , Blum Paul
Published: July 2002

Few antibiotics targeting members of the archaeal domain are currently available for genetic studies. Since bacterial antibiotics are frequently directed against competing and related organisms, archaea by analogy might produce effective antiarchaeal antibiotics. Peptide antibiotic (halocin) preparations from euryarchaeal halophilic strains S8a, GN101, and TuA4 were found to be toxic for members of the hyperthermophilic crenarchaeal genus
Sulfolobus
. No toxicity was evident against representative bacteria or eukarya. Halocin S8 (strain S8a) and halocin R1 (strain GN101) preparations were cytostatic, while halocin A4 (strain TuA4) preparations were cytocidal. Subsequent studies focused on the use of halocin A4 preparations and
Sulfolobus solfataricus
. Strain TuA4 cell lysates were not toxic for
S. solfataricus
, and protease (but not nuclease) treatment of the halocin A4 preparation inactivated toxicity, indicating that the A4 toxic factor must be a secreted protein. Potassium chloride supplementation of the
Sulfolobus
assay medium potentiated toxicity, implicating use of a salt-dependent mechanism. The utility of halocin A4 preparations for genetic manipulation of
S. solfataricus
was assessed through the isolation of UV-induced resistant mutants. The mutants exhibited stable phenotypes and were placed into distinct classes based on their levels of resistance.
The SHI-3 Iron Transport Island of
Shigella boydii
0-1392 Carries the Genes for Aerobactin Synthesis and Transport
Authors: Purdy Georgiana E. , Payne Shelley M.
Published: July 2002

In
Shigella boydii
0-1392, genes encoding the synthesis and transport of the hydroxamate siderophore aerobactin are located within a 21-kb iron transport island between
lysU
and the
pheU
tRNA gene. DNA sequence analysis of the
S. boydii
0-1392 island, designated SHI-3 for
Shigella
island 3, revealed a conserved aerobactin operon associated with a P4 prophage-like integrase gene and numerous insertion sequences (IS). SHI-3 is present at the
pheU
tRNA locus in some
S. boydii
isolates but not in others. The map locations of the aerobactin genes vary among closely related species. The association of the aerobactin operon with phage genes and mobile elements and its presence at different locations within the genomes of enteric pathogens suggest that these virulence-enhancing genes may have been acquired by bacteriophage integration or IS element-mediated transposition. An
S. boydii
aerobactin synthesis mutant, 0-1392
iucB
, was constructed and was similar to the wild type in tissue culture assays of invasion and intercellular spread.
Analysis of Functional Domains of the
Enterococcus faecalis
Pheromone-Induced Surface Protein Aggregation Substance
Authors: Waters C. M. , Dunny G. M.
Published: July 2002

Pheromone-inducible aggregation substance (AS) proteins of
Enterococcus faecalis
are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons Tn
lacZ
/in and Tn
phoA
/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.
nag
Genes of
Ralstonia
(Formerly
Pseudomonas
) sp. Strain U2 Encoding Enzymes for Gentisate Catabolism
Authors: Zhou Ning-Yi , Fuenmayor Sergio L. , Williams Peter A.
Published: July 2002

Ralstonia
sp. strain U2 metabolizes naphthalene via gentisate to central metabolites. We have cloned and sequenced a 21.6-kb region spanning the
nag
genes. Upstream of the pathway genes are
nagY,
homologous to chemotaxis proteins, and
nagR,
a regulatory gene of the LysR family. Divergently transcribed from
nagR
are the genes for conversion of naphthalene to gentisate (
nagAaGHAbAcAdBFCQED
) (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522–2530, 1998), which except for the insertion of
nagGH
, encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of
Pseudomonas putida
PpG7. Downstream of
nahD
is a cluster of genes (
nagJIKLMN
) which are probably cotranscribed with
nagAaGHAbAcAdBFCQED
as a single large operon. By cloning into expression vectors and by biochemical assays, three of these genes (
nagIKL
) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate. NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates. NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The three other genes (
nagJMN
) have also been cloned and overexpressed, but no biochemical activities have been attributed to them. NagJ is homologous to a glutathione
S
-transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function. Downstream of the operon is a partial sequence with homology to a transposase.
Regulation of
osmC
Gene Expression by the Two-Component System
rcsB-rcsC
in
Escherichia coli
Authors: Davalos-Garcia Marcela , Conter Annie , Toesca Isabelle , Gutierrez Claude , Cam Kaymeuang
Published: July 2002

The
Escherichia coli osmC
gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters,
osmCp
1
and
osmCp
2
. Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses
osmCp
1
and activates
osmCp
2
, the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor ς
s
specifically recognizes
osmCp
2
. This work reports the identification of an additional regulatory element, the two-component system
rcsB
-
rcsC
, affecting positively the distal promoter
osmCp
1
. The response regulator of the system, RcsB, does not affect expression of the proximal promoter
osmCp
2
. Deletion analysis located the site necessary for RcsB activation just upstream of
osmCp
1
. In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at
osmCp
1
.
Anaerobic Metabolism of 3-Hydroxybenzoate by the Denitrifying Bacterium
Thauera aromatica
Authors: Laempe Diana , Jahn Martina , Breese Klaus , Schägger Hermann , Fuchs Georg
Published: July 2002

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium
Thauera aromatica
. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate–CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate–CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of β-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.
SigE Is a Chaperone for the
Salmonella enterica
Serovar Typhimurium Invasion Protein SigD
Authors: Darwin K. Heran , Robinson Lloyd S. , Miller Virginia L.
Published: July 2002

SigD is translocated into eucaryotic cells by a type III secretion system. In this work, evidence that the putative chaperone SigE directly interacts with SigD is presented. A bacterial two-hybrid system demonstrated that SigE can interact with itself and SigD. In addition, SigD was specifically copurified with SigE-His
6
on a nickel column.
The Wide-Domain Carbon Catabolite Repressor CreA Indirectly Controls Expression of the
Aspergillus nidulans xlnB
Gene, Encoding the Acidic Endo-β-(1,4)-Xylanase X
24
Authors: Orejas Margarita , MacCabe Andrew P. , Pérez-González JoséAntonio , Kumar Sudeep , Ramón Daniel
Published: July 2002

The
Aspergillus nidulans xlnB
gene, which encodes the acidic endo-β-(1,4)-xylanase X
24
, is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation

creA
d
30

results in considerably elevated levels of
xlnB
mRNA indicates a role for the wide-domain repressor CreA in the repression of
xlnB
promoter (
xlnBp
) activity. Functional analyses of
xlnBp
::
goxC
reporter constructs show that none of the four CreA consensus target sites identified in
xlnBp
are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence
xlnB
expression by acting directly on
xlnB
.
The E1β and E2 Subunits of theBacillus subtilisPyruvate Dehydrogenase Complex Are Involved in Regulation of Sporulation
Authors: Gao Haichun , Jiang Xin , Pogliano Kit , Aronson Arthur I.
Published: July 2002

ThepdhABCDoperon ofBacillus subtilisencodes the pyruvate decarboxylase (E1α and E1β), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of thepdhCgene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1α and E1β. Disruptions of thepdhB,pdhC, andpdhDgenes were isolated, but attempts to construct apdhAmutant were unsuccessful, suggesting that E1α is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. ThepdhBandpdhCmutants sporulated to only 5% of the wild-type level, whereas thepdhDmutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of thepdhBandpdhCmutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of variousspo-lacZfusions revealed that thepdhBmutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas thepdhCmutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1β and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.
ThePseudomonas aeruginosaQuorum-Sensing MoleculeN-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo
Authors: Smith Roger S. , Harris Sarah G. , Phipps Richard , Iglewski Barbara
Published: July 2002

Pseudomonas aeruginosahas two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis ofN-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis ofN-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes inP. aeruginosahad on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of thelasIgene or both thelasIandrhlIgenes greatly diminished the ability ofP. aeruginosato colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C12-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1α (IL-1α) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1β, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems ofP. aeruginosacontribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.
The Global Regulatory
hns
Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown
Escherichia coli
by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production
Authors: Landini Paolo , Zehnder Alexander J. B.
Published: July 2002

The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of
Escherichia coli
W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the
fliC
gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory
hns
gene counteracts increased production of LPS and flagella in response to anoxia and allows
E. coli
W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of
E. coli
W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the
fliC
gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in
E. coli
.
Heat Shock Proteome of
Agrobacterium tumefaciens
: Evidence for New Control Systems
Authors: Rosen Ran , Büttner Knut , Becher Dörte , Nakahigashi Kenji , Yura Takashi , Hecker Michael , Ron Eliora Z.
Published: July 2002

The regulation of
Agrobacterium tumefaciens
heat shock genes involves a transcriptional activator (RpoH) and repressor elements (HrcA-CIRCE). Using proteome analysis and mutants in these control elements, we show that the heat shock induction of 32 (out of 56) heat shock proteins is independent of RpoH and HrcA. These results indicate the existence of additional regulatory factors in the
A
.
tumefaciens
heat shock response.