bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysine and Polyamines Are Substrates for Transglutamination of Rho by the
Bordetella
Dermonecrotic Toxin
Authors: Schmidt Gudula , Goehring Udo-Michael , Schirmer Joerg , Uttenweiler-Joseph Sandrine , Wilm Matthias , Lohmann Mark , Giese Arnd , Schmalzing Guenther , Aktories Klaus
Published: July 2002

Bordetella
dermonecrotic toxin (DNT) catalyzes the transglutamination of glutamine-63/61 of Rho GTPases, thereby constitutively activating Rho proteins. Here we identified second substrates for transglutamination of RhoA by DNT. The enzymatically active fragment of DNT (residues 1136 to 1451, ΔDNT) induced the incorporation of
l
-[
14
C]lysine in RhoA in a concentration-dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by ΔDNT. Transglutamination of the GTPase with
l
-lysine inhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contrast to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT increased the incorporation of
l
-[
14
C]lysine into embryonic bovine lung cells. Microinjection of GST-RhoA together with the enzymatically active DNT fragment into
Xenopus
oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or spermidine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed that lysine is a preferred second substrate at least in vitro.
Role of FliC and FliD Flagellar Proteins of
Clostridium difficile
in Adherence and Gut Colonization
Authors: Tasteyre Albert , Barc Marie-Claude , Collignon Anne , Boureau Helene , Karjalainen Tuomo
Published: July 2002

In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins of
Clostridium difficile
were analyzed. FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus. Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane. The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence.
Mechanisms MediatingPorphyromonas gingivalisGingipain RgpA-Induced Oral Mucosa Inflammation In Vivo
Authors: Rubinstein Israel , Potempa Jan , Travis James , Gao Xiao-Pei
Published: July 2002

Suffusion of gingipain RgpA (GRgpA) elicited a significant concentration-dependent increase in the clearance of macromolecules from in situ hamster cheek pouch which was attenuated by NPC 17647, a selective bradykinin B2receptor antagonist. Leupeptin and a mixture of proteinase inhibitors also attenuated GRgpA-induced responses. These data indicate that GRgpA elicits plasma exudation from in situ oral mucosa in a catalytic site-dependent fashion by elaborating bradykinin.
Infectivity ofChlamydia trachomatisSerovar LGV but Not E Is Dependent on Host Cell Heparan Sulfate
Authors: Taraktchoglou Maria , Pacey Allan A. , Turnbull Jeremy E. , Eley Adrian
Published: July 2002

The ability of heparan sulfate, heparin, and other glycosaminoglycans to inhibit the infectivity ofChlamydia trachomatisserovars E and LGV was examined using a simple competitive inhibition assay with three cell types from the human female reproductive tract, including primary human endosalpingeal cells. With the majority of the glycosaminoglycans tested, LGV was more significantly inhibited than serovar E. We have compared chlamydial infectivity between a wild-type Chinese hamster ovary cell line and two glycosaminoglycan-deficient cell lines. LGV was shown to be unable to infect heparan sulfate-deficient and GAG-deficient Chinese hamster ovary cell lines, whereas the E serovar infected these cells as efficiently as the control (nondeficient) cells. These two sets of experiments confirmed that serovar LGV is more dependent on a heparan sulfate-related mechanism of infectivity than is serovar E. This is further supported by the fact that attempts to purify a heparan sulfate-like molecule from either serovar cultured in glycosaminoglycan-deficient cell lines were nonproductive. Previous reports have suggested that chlamydia are able to produce a heparan sulfate-like molecule that is important for attachment and infectivity. We have attempted to detect possible binding of a specific heparan sulfate antibody toC. trachomatisby flow cytometry. Results showed no binding of the heparan sulfate antibody toC. trachomatisserovar LGV or E. Our results strongly indicate that chlamydiae do not produce a heparan sulfate-like molecule but rather use host cell heparan sulfate in order to infect cells.
Critical Role of CD14 for Production of Proinflammatory Cytokines and Cytokine Inhibitors during Sepsis with Failure To Alter Morbidity or Mortality
Authors: Ebong Samuel J. , Goyert Sanna M. , Nemzek Jean A. , Kim Jiyoun , Bolgos Gerald L. , Remick Daniel G.
Published: July 2002

We investigated the immunopathophysiologic responses during sepsis induced by cecal ligation and puncture (CLP) in CD4-deficient (CD14 knockout [CD14KO]) mice. Our studies were designed to specifically test the role of CD14 in the inflammatory response to sepsis and to ascertain if alterations would improve morbidity or mortality. Sepsis was induced using the CLP model with appropriate antibiotic treatment. The severity of sepsis increased in the CD14KO mice with increasing puncture size (18 gauge [18G], 21G, and 25G). Following CLP, body temperature (at 12 h) and gross motor activity levels of the sham and 25G CLP groups recovered to normal, while the 21G and 18G CLP groups exhibited severe hypothermia coupled with decreased gross motor activity and body weight. There were no significant differences in survival, temperature, body weight, or activity levels between CD14KO and control mice after 21G CLP. However, CD14KO mice expressed two- to fourfold less pro-inflammatory (interleukin-1β [IL-1β], tumor necrosis factor [TNF], and IL-6) and anti-inflammatory (IL-10, IL-1 receptor antagonist, and TNF receptors I and II) cytokines in the blood after 21G CLP. Plasma levels of the chemokines macrophage inflammatory protein 2α and KC were similarly reduced in CD14KO mice. A similar trend of decreased cytokine and cytokine inhibitor levels was observed in the peritoneal cavity of CD14KO mice. Our results indicate that the CD14 pathway of activation plays a critical role in the production of both pro-inflammatory cytokines and cytokine inhibitors but has minimal impact on the morbidity or mortality induced by the CLP model of sepsis.
Apoptotic Signaling Pathway Activated by
Helicobacter pylori
Infection and Increase of Apoptosis-Inducing Activity under Serum-Starved Conditions
Authors: Shibayama Keigo , Doi Yohei , Shibata Naohiro , Yagi Tetsuya , Nada Toshi , Iinuma Yoshitsugu , Arakawa Yoshichika
Published: July 2002

The enhanced gastric epithelial cell apoptosis observed during infection with
Helicobacter pylori
has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by
H. pylori
infection, human gastric epithelial cells were incubated with
H. pylori
for up to 72 h.
H. pylori
infection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressed
H. pylori
-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that
H. pylori
induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of
H. pylori
was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce
H. pylori
-mediated pathogenesis, leading to a variety of clinical outcomes.
Complement Contributes to Protective Immunity against Reinfection byPlasmodium chabaudi chabaudiParasites
Authors: Taylor Philip R. , Seixas Elsa , Walport Mark J. , Langhorne Jean , Botto Marina
Published: July 2002

We have studied the impact of deficiency of the complement system on the progression and control of the erythrocyte stages of the malarial parasitePlasmodium chabaudi chabaudi. C1q-deficient mice and factor B- and C2-deficient mice, deficient in the classical complement pathway and in both the alternative and classical complement activation pathways, respectively, exhibited only a slight delay in the resolution of the acute phase of parasitemia. Complement-deficient mice showed a transiently elevated level of gamma interferon (IFN-γ) in the plasma at the time of the acute parasitemia compared with that of wild-type mice. Although there was a trend for increased precursor frequencies in CD4+T cells from C1q-deficient mice producing IFN-γ in response to malarial antigens in vitro, intracellular cytokine staining of spleen cells ex vivo showed no difference in the numbers of IFN-γ+splenic CD4+and CD8+cells. In contrast, C1q-deficient animals were significantly more susceptible to a second challenge with the same parasite. C1q-deficient animals showed a reduced level of anti-malarial immunoglobulin G2a (IgG2a) antibody 100 days after primary infection. However, following a significantly higher parasitemia, C1q-deficient mice had increased levels of IgM and IgG2a anti-malarial antibodies. In summary, this study indicates that while complement plays only a minor role in the control of the acute phase of parasitemia of a primary infection, it does contribute to parasite control in reinfection.
Deficiency of
d
-Erythroascorbic Acid Attenuates Hyphal Growth and Virulence of
Candida albicans
Authors: Huh Won-Ki , Kim Seong-Tae , Kim Hyungsoo , Jeong Gajin , Kang Sa-Ouk
Published: July 2002

In some lower eukaryotes,
d
-erythroascorbic acid, a five-carbon analog of
l
-ascorbic acid, is present instead of
l
-ascorbic acid. We have cloned
ALO1
, the gene encoding
d
-arabinono-1,4-lactone oxidase, which catalyzes the final step of
d
-erythroascorbic acid biosynthesis in
Candida albicans
. The
ALO1
gene contained a continuous open reading frame of 1,671 bp that encodes a polypeptide consisting of 557 amino acids with a calculated molecular mass of 63,428 Da. To investigate the functional roles of
d
-erythroascorbic acid in
C. albicans
, we disrupted or overexpressed the
ALO1
gene. In the
alo1/alo1
null mutants, the activity of
d
-arabinono-1,4-lactone oxidase was completely lost and
d
-erythroascorbic acid could not be detected. When
ALO1
on a multicopy plasmid was transformed in
C. albicans
, the enzyme activity and the intracellular
d
-erythroascorbic acid level were increased up to 3.4-fold and 4.0-fold, respectively. The
alo1/alo1
null mutants of
C. albicans
showed increased sensitivity towards oxidative stress. Overexpression of
ALO1
made the cells more resistant to the same stress. The
alo1/alo1
mutants showed defective hyphal growth and attenuated virulence. Taken together, our results suggest that
d
-erythroascorbic acid functions as an important antioxidant and can be considered one of the virulence factors enhancing the pathogenicity of
C. albicans
.
Stress-Induced ClpP Serine Protease ofListeria monocytogenesIs Essential for Induction of Listeriolysin O-Dependent Protective Immunity
Authors: Gaillot Olivier , Bregenholt Søren , Jaubert Francis , Di Santo James P. , Berche Patrick
Published: July 2002

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogenListeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, aclpPdeletion mutant ofL. monocytogeneswas used to study the generation of anti-Listeriaprotective immunity. We found that ClpP is required for the intracellular growth ofL. monocytogenesin resident macrophages in vivo. Mice infected with doses as high as 106clpPmutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disruptedhlygene, this lack of protection was overcome by inoculation of very high infecting doses ofclpPmutant bacteria (5 × 108), thus producing sufficient amounts of LLO to stimulate anti-Listeriaimmunity. The role of ClpP was confirmed by showing that anti-Listeriaimmunity was restored in mice infected with aclpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response againstL. monocytogenes.
Independent and Coordinate Effects of ADP-Ribosyltransferase and GTPase-Activating Activities of Exoenzyme S on HT-29 Epithelial Cell Function
Authors: Fraylick Jennifer E. , La Rocque Jeannine R. , Vincent Timothy S. , Olson Joan C.
Published: July 2002

Type III-mediated translocation of exoenzyme S (ExoS) into HT-29 epithelial cells by
Pseudomonas aeruginosa
causes complex alterations in cell function, including inhibition of DNA synthesis, altered cytoskeletal structure, loss of readherence, microvillus effacement, and interruption of signal transduction. ExoS is a bifunctional protein having both GTPase-activating (GAP) and ADP-ribosyltransferase (ADPRT) functional domains. Comparisons of alterations in HT-29 cell function caused by
P. aeruginosa
strains that translocate ExoS having GAP or ADPRT mutations allowed the independent and coordinate functions of the two activities to be assessed. An E381A ADPRT mutation revealed that ExoS ADPRT activity was required for effects of ExoS on DNA synthesis and long-term cell rounding. Conversely, the R146A GAP mutation appeared to have little impact on the cellular effects of ExoS. While transient cell rounding was detected following exposure to the E381A mutant, this rounding was eliminated by an E379A-E381A ADPRT double mutation, implying that residual ADPRT activity, rather than GAP activity, was effecting transient cell rounding by the E381A mutant. To explore this possibility, E381A and R146A-E381A mutants were examined for their ability to ADP-ribosylate Ras in vitro or in vivo. While no ADP-ribosylation of Ras was detected by either mutant in vitro, both mutants were able to modify Ras when translocated by the bacteria, with the R146A-E381A mutant causing more efficient modification than the E381A mutant, in association with increased inhibition of DNA synthesis. Comparisons of Ras ADP-ribosylation by wild-type and E381A mutant ExoS by two-dimensional electrophoresis found the former to ADP-ribosylate Ras at two sites, while the latter modified Ras only once. These studies draw attention to the key role of ExoS ADPRT activity in causing the effects of bacterially translocated ExoS on DNA synthesis and cell rounding. In addition, the studies provide insight into the enhancement of ExoS ADPRT activity within the eukaryotic cell microenvironment and into possible modulatory roles that the GAP and ADPRT domains might have on the function of each other.
Adherent Invasive
Escherichia coli
Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death
Authors: Glasser Anne-Lise , Boudeau Jerome , Barnich Nicolas , Perruchot Marie-Helene , Colombel Jean-Frederic , Darfeuille-Michaud Arlette
Published: July 2002

Escherichia coli
strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive
E. coli
(AIEC) strains isolated from patients with CD. All the 15 AIEC strains tested were able to replicate extensively within J774-A1 cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at 1 h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A1 cells, even after 24 h postinfection. LF82-infected J774-A1 cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-α) than cells stimulated with 1 μg of lipopolysaccharide (LPS)/ml. No release of interleukin-1β was observed with LPS-prestimulated J774-A1 cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A1 cells release high levels of TNF-α. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions.
M+Group A Streptococci Are Phagocytized and Killed in Whole Blood by C5a-Activated Polymorphonuclear Leukocytes
Authors: DeMaster Eric , Schnitzler Norbert , Cheng Qi , Cleary Patrick
Published: July 2002

Historically, resistance to phagocytosis has been determined by incubating group A streptococci in human blood and comparing the numbers of CFU before and after incubation. Utilizing a flow cytometry-based technique, we have investigated the phagocytosis of M+group A streptococci by polymorphonuclear leukocytes (PMNs) in heparinized human peripheral whole blood. Intracellular labeling of streptococci with a nontoxic fluorescent dye allowed us to quantify the association and phagocytosis of M+streptococci by PMNs in whole blood in the presence or absence of C5a, a physiologically important chemotactic activator of PMNs. We found that wild-type strains of group A streptococci that are resistant to phagocytosis (determined by the classical Lancefield method) readily associate with C5a-activated whole-blood PMNs. In the absence of opsonizing M-type-specific antibodies, the M+streptococci associated with PMNs are phagocytized and killed. In addition, blockade of the β2integrin, CD11b/CD18, with anti-human CD11b monoclonal antibody inhibited association between M+streptococci and C5a-activated PMNs. These findings establish a new relationship between M+streptococci and PMNs, in which C5a-activated PMNs have the capacity to kill M+streptococci in whole blood through a receptor-mediated phagocytic mechanism.
p53-Independent Expression of p21CIP1/WAF1in Plasmacytic Cells during G2Cell Cycle Arrest Induced byActinobacillus actinomycetemcomitansCytolethal Distending Toxin
Authors: Sato Tsuyoshi , Koseki Takeyoshi , Yamato Kenji , Saiki Keitarou , Konishi Kiyoshi , Yoshikawa Masanosuke , Ishikawa Isao , Nishihara Tatsuji
Published: July 2002

The cytolethal distending toxin (CDT) fromActinobacillus actinomycetemcomitanshas been shown to induce cell cycle arrest in the G2/M phase in HeLa cells. In the present study, the mechanism of CDT-induced cell cycle arrest was investigated by using HS-72 cells, a murine B-cell hybridoma cell line. Using flow cytometric analysis, we found that the recombinant CDT (rCDT) fromA. actinomycetemcomitansinduced G2cell cycle arrest in HS-72 cells and that rCDT upregulated expression of the cyclin-dependent kinase inhibitor p21CIP1/WAF1and the tumor suppressor protein p53. HS-72 cells transfected with the E6/E7 gene of human papillomavirus type 16, which lacked rCDT-induced accumulation of p53, exhibited expression of p21CIP1/WAF1or G2cell cycle arrest upon exposure to rCDT. Furthermore, ectopic expression of a dominant negative p53 mutant did not inhibit rCDT-mediated p21CIP1/WAF1expression or G2cell cycle arrest in HS-72 cells. These results suggest that the CDT fromA. actinomycetemcomitansinduces p21CIP1/WAF1expression and G2cell cycle arrest in B-lineage cells by p53-independent pathways. Together with additional observations made with HeLa cells and COS-1 cells cultured with the rCDT fromA. actinomycetemcomitans,the results of this study indicate that CDT-induced p53 accumulation may not be required for G2cell cycle arrest and that an increased level of p21CIP1/WAF1may be important for sustaining G2cell cycle arrest in several mammalian cells.
Membrane Localization of the S1 Subunit of Pertussis Toxin in
Bordetella pertussis
and Implications for Pertussis Toxin Secretion
Authors: Farizo Karen M. , Fiddner Stefanie , Cheung Anissa M. , Burns Drusilla L.
Published: July 2002

Pertussis toxin is secreted from
Bordetella pertussis
with the assistance of the Ptl transport system, a member of the type IV family of macromolecular transporters. The S1 subunit and the B oligomer combine to form the holotoxin prior to export from the bacterial cell, although the site of assembly is not known. To better understand the pathway of pertussis toxin assembly and secretion, we examined the subcellular location of the S1 subunit, expressed with or without the B oligomer and the Ptl proteins. In wild-type
B. pertussis
, the majority of the S1 subunit that remained cell associated localized to the bacterial membranes. In mutants of
B. pertussis
that do not express pertussis toxin and/or the Ptl proteins, full-length S1, expressed from a plasmid, partitioned almost entirely to the bacterial membranes. Several lines of evidence strongly suggest that the S1 subunit localizes to the outer membrane of
B. pertussis
. First, we found that membrane-bound full-length S1 was almost completely insoluble in Triton X-100. Second, recombinant S1 previously has been shown to localize to the outer membrane of
Escherichia coli
(J. T. Barbieri, M. Pizza, G. Cortina, and R. Rappuoli, Infect. Immun. 58:999-1003, 1990). Third, the S1 subunit possesses a distinctive amino acid motif at its carboxy terminus, including a terminal phenylalanine, which is highly conserved among bacterial outer membrane proteins. By using site-directed mutagenesis, we determined that the terminal phenylalanine is critical for stable expression of the S1 subunit. Our findings provide evidence that prior to assembly with the B oligomer and independent of the Ptl proteins, the S1 subunit localizes to the outer membrane of
B. pertussis.
Thus, outer membrane-bound S1 may serve as a nucleation site for assembly with the B oligomer and for interactions with the Ptl transport system.
Virulence of
Streptococcus pneumoniae
: PsaA Mutants Are Hypersensitive to Oxidative Stress
Authors: Tseng Hsing-Ju , McEwan Alastair G. , Paton James C. , Jennings Michael P.
Published: July 2002

psaA
encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex.
Streptococcus pneumoniae
D39
psaA
mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that
psaA
and
psaD
mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in
psaA
and
psaD
mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.
Immunization with the RgpA-Kgp Proteinase-Adhesin Complexes ofPorphyromonas gingivalisProtects against Periodontal Bone Loss in the Rat Periodontitis Model
Authors: Rajapakse P. Sunethra , O'Brien-Simpson Neil M. , Slakeski Nada , Hoffmann Brigitte , Reynolds Eric C.
Published: July 2002

A major virulence factor ofPorphyromonas gingivalisis the extracellular noncovalently associated complexes of Arg-X- and Lys-X-specific cysteine proteinases and adhesins designated the RgpA-Kgp complexes. In this study we investigated the ability of RgpA-Kgp as an immunogen to protect againstP. gingivalis-induced periodontal bone loss in the rat. Specific-pathogen-free Sprague-Dawley rats were immunized with either formalin-killed wholeP. gingivalisATCC 33277 cells with incomplete Freund's adjuvant, RgpA-Kgp with incomplete Freund's adjuvant, or incomplete Freund's adjuvant alone. The animals were then challenged by oral inoculation with liveP. gingivalisATCC 33277 cells. Marked periodontal bone loss was observed in animals immunized with incomplete Freund's adjuvant alone; this bone loss was significantly (P< 0.05) greater than that detected in animals immunized with formalin-killed whole cells or RgpA-Kgp or in unchallenged animals. There was no significant difference in periodontal bone loss between animals immunized with formalin-killed whole cells and those immunized with RgpA-Kgp. The bone loss in these animals was also not significantly different from that in unchallenged animals. DNA probe analysis of subgingival plaque samples showed that 100% of the animals immunized with incomplete Freund's adjuvant alone and challenged withP. gingivalisATCC 33277 were positive for the bacterium. However,P. gingivalisATCC 33277 could not be detected in subgingival plaque samples from animals immunized with formalin-killed whole cells or with RgpA-Kgp. Immunization with formalin-killed whole cells or RgpA-Kgp induced a high-titer serum immunoglobulin G2a response. Western blot analysis of RgpA-Kgp using pooled protective antisera taken from rats immunized with RgpA-Kgp revealed immunodominant bands at 44, 39, and 27 kDa. In conclusion, immunization with RgpA-Kgp restricted colonization byP. gingivalisand periodontal bone loss in the rat.
Point Mutations in a Peptidoglycan Biosynthesis Gene Cause Competence Induction in
Haemophilus influenzae
Authors: Ma Caixia , Redfield Rosemary J.
Published: July 2002

We have identified three new
Haemophilus influenzae
mutations causing cells to exhibit extreme hypercompetence at all stages of growth. The mutations are in
murE
, which encodes the
meso
-diaminopimelate-adding enzyme of peptidoglycan synthesis. All are point mutations causing nonconservative amino acid substitutions, two at a poorly conserved residue (G
435
→R and G
435
→W) and the third at a highly conserved leucine (L
361
→S). The mutant strains have very similar phenotypes and do not exhibit any defects in cell growth, permeability, or sensitivity to peptidoglycan antibiotics. Cells retain the normal specificity of DNA uptake for the
H. influenzae
uptake signal sequence. The mutations do not bypass genes known to be needed for competence induction but do dramatically increase expression of genes required for the normal pathway of DNA uptake. We conclude that the mutations do not act by increasing cell permeability but by causing induction of the normal competence pathway via a previously unsuspected signal.
Genetic Antagonism and Hypermutability in
Mycobacterium smegmatis
Authors: Karunakaran Ponniah , Davies Julian
Published: July 2002

Multidrug-resistant strains of
Mycobacterium tuberculosis
are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and
M. tuberculosis
strains that are resistant to both streptomycin and rifampin contain mutations in the
rpsL
and
rpoB
genes, respectively. Coexisting mutations of this kind in
Escherichia coli
have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084–2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157–1161, 1977). We investigated this possibility in
Mycobacterium smegmatis
by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of
M. smegmatis
under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in
M. smegmatis
showed that the frequencies were enhanced up to 10
4
-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.
ComP, a Pilin-Like Protein Essential for Natural Competence in
Acinetobacter
sp. Strain BD413: Regulation, Modification, and Cellular Localization
Authors: Porstendörfer Dirk , Gohl Olivia , Mayer Frank , Averhoff Beate
Published: July 2002

We recently identified a pilin-like competence factor, ComP, which is essential for natural transformation of the gram-negative soil bacterium
Acinetobacter
sp. strain BD413. Here we demonstrate that transcription and synthesis of the pilin-like competence factor ComP are maximal in the late stationary growth phase, whereas competence is induced immediately after inoculation of a stationary-phase culture into fresh medium. Western blot analyses revealed three forms of ComP, one with an apparent molecular mass of 15 kDa, which correlates with the molecular mass deduced from the DNA sequence, one 20-kDa form, which was found to be glycosylated, and one 23-kDa form. The glycosylation of ComP was not required for its function in DNA binding and uptake. The 20-kDa form was present in the cytoplasmic membrane, the periplasm, and the outer membrane, whereas the 23-kDa form was located in the outer membrane and might be due to a further modification. Immunological data suggest that ComP is not a subunit of the pilus structures. Possible functions of ComP in the DNA transformation machinery of
Acinetobacter
sp. strain BD413 are discussed.
Genetic Analysis of an Incomplete
mutS
Gene from
Pseudomonas putida
Authors: Kurusu Yasurou , Narita Tomoaki , Suzuki Makoto , Watanabe Taeko
Published: July 2002

We genetically characterized the
Pseudomonas putida mutS
gene and found that it encodes a smaller MutS protein than do the genes of other bacteria. This gene is able to function in the
mutS
mutants of
Escherichia coli
and
Bacillus subtilis
. A
P. putida mutS
mutant has a mutation frequency 1,000-fold greater than that of the wild-type strain.
Identification of a Serine Hydrolase Which Cleaves the Alicyclic Ring of Tetralin
Authors: Hernáez M. J. , Andújar E. , Ríos J. L. , Kaschabek S. R. , Reineke W. , Santero E.
Published: July 2002

A gene designated
thnD
, which is required for biodegradation of the organic solvent tetralin by
Sphingomonas macrogoltabidus
strain TFA, has been identified. Sequence comparison analysis indicated that
thnD
codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65°C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl
meta
-cleavage pathway. However, it is not active towards the equivalent intermediates of
meta
-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of
meta
-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.
Identification of a Copper-Responsive Two-Component System on the Chromosome of
Escherichia coli
K-12
Authors: Munson George P. , Lam Deborah L. , Outten F. Wayne , O'Halloran Thomas V.
Published: July 2002

Using a genetic screen we have identified two chromosomal genes,
cusRS
(
ylcA ybcZ
), from
Escherichia coli
K-12 that encode a two-component, signal transduction system that is responsive to copper ions. This regulatory system is required for copper-induced expression of
pcoE
, a plasmid-borne gene from the
E. coli
copper resistance operon
pco
. The closest homologs of CusR and CusS are plasmid-borne two-component systems that are also involved in metal responsive gene regulation: PcoR and PcoS from the
pco
operon of
E. coli
; CopR and CopS from the
cop
operon, which provides copper resistance to
Pseudomonas syringae
; and SilR and SilS from the
sil
locus, which provides silver ion resistance to
Salmonella enterica
serovar Typhimurium. The genes
cusRS
are also required for the copper-dependent expression of at least one chromosomal gene, designated
cusC
(
ylcB
), which is allelic to the recently identified virulence gene
ibeB
in
E. coli
K1. The
cus
locus may comprise a copper ion efflux system, because the expression of
cusC
is induced by high concentrations of copper ions. Furthermore, the translation products of
cusC
and additional downstream genes are homologous to known metal ion antiporters.
Nonspecific Adherence byActinobacillus actinomycetemcomitansRequires Genes Widespread inBacteriaandArchaea
Authors: Kachlany Scott C. , Planet Paul J. , Bhattacharjee Mrinal K. , Kollia Evyenia , DeSalle Rob , Fine Daniel H. , Figurski David H.
Published: July 2002

The gram-negative coccobacillus,Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates ofA. actinomycetemcomitansform tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence ofA. actinomycetemcomitansto surfaces. The recently developed transposon IS903φkanwas used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad−). Unlike wild-type cells, Tad−mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that thetadgenes are involved in secretion of factors required for tight adherence ofA. actinomycetemcomitans. Remarkably, complete and highly conservedtadgene clusters are present in the genomes of the bubonic plague bacillusYersinia pestisand the human and animal pathogenPasteurella multocida. Partialtadloci also occur in strikingly diverseBacteriaandArchaea. Our results show that thetadgenes are required for tight adherence ofA. actinomycetemcomitansto surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose thattad-like genes have a significant role in microbial colonization.
Vancomycin Resistance Is Associated with Serine-Containing Peptidoglycan in
Enterococcus gallinarum
Authors: Grohs Patrick , Gutmann Laurent , Legrand Raymond , Schoot Bernard , Mainardi Jean Luc
Published: July 2002

In
Enterococcus gallinarum
SC1, a low-level vancomycin-resistant strain, only monomeric muropentapeptides with a C-terminal
d
-alanine were detected after growth without vancomycin. In contrast, in SC1 induced by vancomycin, as well as in AIB39, a constitutive vancomycin-resistant strain, monomeric and dimeric muropentapeptides with a C-terminal
d
-serine were detected.
Environmental Regulation of Exopolysaccharide Production in
Sinorhizobium meliloti
Authors: Mendrygal Kiprian E. , González Juan E.
Published: July 2002

Exopolysaccharide production by
Sinorhizobium meliloti
is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners.
S. meliloti
wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by
S. meliloti
. The low-phosphate conditions normally found in the soil (1 to 10 μM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by
S. meliloti
in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.
Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon
Pyrobaculum islandicum
Authors: Kähler Markus , Antranikian Garabed
Published: July 2002

In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon
Pyrobaculum islandicum
. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom
Crenarchaeota
,
Pyrodictium occultum
and
Aeropyrum pernix
, and several members of the kingdom
Euryarchaeota
. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3′-5′ exonuclease motifs were also found. The gene was expressed in
Escherichia coli
, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from
P. islandicum
, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations and
N
-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90°C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3′-5′ exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify λ DNA fragments of up to 1,500 bp.
Insertion Mutagenesis of
wca
Reduces Acid and Heat Tolerance of Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Mao Ying , Doyle Michael P. , Chen Jinru
Published: July 2002

Strains of enterohemorrhagic
Escherichia coli
(EHEC) serotype O157:H7 produce under stress copious amounts of exopolysaccharide (EPS) composed of colanic acid (CA). Studies were performed to evaluate the association of production of CA with survival of EHEC under adverse environmental conditions. A CA-deficient mutant, M4020, was obtained from a CA-proficient parental strain,
E. coli
O157:H7 W6-13, by inserting a kanamycin resistance gene cassette (
kan
) into
wcaD
and
wcaE
, 2 of the 21 genes required for CA biosynthesis. M4020 was defective in CA production as determined from the ratio of uronic acid to protein (UA/P) of cells grown from 1 to 4 days at 25°C on minimal glucose agar (MGA), MacConkey agar, and sorbitol-MacConkey agar, and by colony morphology on MGA. The results of stress treatment revealed that M4020 was substantially less tolerant to acid (pH 4.5 and 5.5) and heat (55 and 60°C) in comparison to W6-13, indicating that CA confers on
E. coli
O157:H7 a protective effect from the environmental stresses of acid and heat.
Signal Transduction Cascade for Regulation of RpoS: Temperature Regulation of DsrA
Authors: Repoila F. , Gottesman S.
Published: July 2002

Many environmental parameters modulate the amount of the RpoS sigma factor in
Escherichia coli
. Temperature control of RpoS depends on the untranslated RNA DsrA. DsrA activates RpoS translation by pairing with the leader of the mRNA. We find that temperature affects both the rate of transcription initiation of the
dsrA
gene and the stability of DsrA RNA. Both are increased at low temperature (25°C) compared to 37 or 42°C. The combination of these results is 25-fold-less DsrA at 37°C and 30-fold less at 42°C than at 25°C. Using an adapted
lacZ
-based reporter system, we show that temperature control of transcription initiation of
dsrA
requires only the minimal promoter of 36 bp. Overall, transcription responses to temperature lead to a sixfold increase in DsrA synthesis at 25°C over that at 42°C. Furthermore, two activating regions and a site for LeuO negative regulation were identified in the
dsrA
promoter. The activating regions also activate transcription in vitro. DsrA decays with a half-life of 23 min at 25°C and 4 min at 37 and 42°C. These results demonstrate that the
dsrA
promoter and the stability of DsrA RNA are the thermometers for RpoS temperature sensing. Multiple inputs to DsrA accumulation allow sensitive modulation of changes in the synthesis of the downstream targets of DsrA such as RpoS.
Energy-Dependent Conformational Change in the TolA Protein ofEscherichia coliInvolves Its N-Terminal Domain, TolQ, and TolR
Authors: Germon Pierre , Ray Marie-Céline , Vianney Anne , Lazzaroni Jean Claude
Published: July 2002

TolQ, TolR, and TolA inner membrane proteins ofEscherichia coliare involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in ΔtolQ, ΔtolR,andtolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such aslpp,pal,and Δrfamutants: the behavior of TolA inlppmutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Δrfamutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.
A Program of
Yersinia enterocolitica
Type III Secretion Reactions Is Activated by Specific Signals
Authors: Lee Vincent T. , Mazmanian Sarkis K. , Schneewind Olaf
Published: July 2002

Successful establishment of
Yersinia
infections requires the type III machinery, a protein transporter that injects virulence factors (Yops) into macrophages. It is reported here that the
Yersinia
type III pathway responds to environmental signals by transporting proteins to distinct locations.
Yersinia enterocolitica
cells sense an increase in extracellular amino acids (glutamate, glutamine, aspartate, and asparagine) that results in the activation of the type III pathway. Another signal, provided by serum proteins such as albumin, triggers the secretion of YopD into the extracellular medium. The third signal, a decrease in calcium concentration, appears to be provided by host cells and causes
Y. enterocolitica
to transport YopE and presumably other virulence factors across the eukaryotic plasma membrane. Mutations in several genes encoding regulatory molecules (
lcrG
,
lcrH
,
tyeA
,
yopD
,
yopN
,
yscM1
, and
yscM2
) bypass the signal requirement of the type III pathway. Together these results suggest that yersiniae may have evolved distinct secretion reactions in response to environmental signals.
Killing Effect and Antitoxic Activity of the
Leptospira interrogans
Toxin-Antitoxin System in
Escherichia coli
Authors: Picardeau Mathieu , Ren Shuangxi , Saint Girons Isabelle
Published: July 2002

We report the first evidence of a chromosome-encoded toxin-antitoxin locus in spirochetes. This locus has been found in the pathogenic spirochete
Leptospira interrogans
and exhibits homologies with the
pem
/
chp
loci. The
L. interrogans chp
locus consists of two genes:
chpK
(for “killer protein”) and its upstream partner
chpI
(for “inhibitory protein”). Expression of ChpK in
Escherichia coli
results in the inhibition of bacterial growth. The coexpression of ChpI neutralizes ChpK toxicity. By Southern blot analysis,
chp
homologs were found in all representative pathogenic strains of
L. interrogans
.
IS
186
Insertion at a Hot Spot in the
lon
Promoter as a Basis for Lon Protease Deficiency of
Escherichia coli
B: Identification of a Consensus Target Sequence for IS
186
Transposition
Authors: SaiSree L. , Reddy Manjula , Gowrishankar J.
Published: July 2002

The radiation sensitivity of
Escherichia coli
B was first described more than 50 years ago, and the genetic locus responsible for the trait was subsequently identified as
lon
(encoding Lon protease). We now show that both
E. coli
B and the first reported
E. coli
K-12
lon
mutant, AB1899, carry IS
186
insertions in opposite orientations at a single site in the
lon
promoter region and that this site represents a natural hot spot for transposition of the insertion sequence (IS) element. Our analysis of deposited sequence data for a number of other IS
186
insertion sites permitted the deductions that (i) the consensus target site sequence for IS
186
transposition is 5′-(G)
≥4
(N)
3–6
(C)
≥4
-3′, (ii) the associated host sequence duplication varies within the range of 6 to 12 bp and encompasses the N
(3–6)
sequence, and (iii) in a majority of instances, at least one end of the duplication is at the G-N (or N-C) junction. IS
186
-related sequences were absent in closely related bacterium
Salmonella enterica
serovar Typhimurium, indicating that this IS element is a recent acquisition in the evolutionary history of
E. coli.