bluuebunny/crossref_metadata_2025 huggingface dataset:
Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic
Actinomyces
spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities
Authors: Li Tong , Khah Massoud Kheir , Slavnic Snjezana , Johansson Ingegerd , Strömberg Nicklas
Published: July 2002

Actinomyces
spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands.
Actinomyces
spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of
Actinomyces naeslundii
genospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (
orf1
- to -
6
and
fimP
) in genospecies 1 and 2 strains, except that
orf5
and
orf3
were divergent in genospecies 1. (ii) In preferential statherin binding, oral
Actinomyces viscosus
strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (
orfA
to -
C
and
fimP
). Bioinformatics suggested sortase (
orfB
,
orf4
, and part of
orf5
), prepilin peptidase (
orfC
and
orf6
), fimbria subunit (
fimP
), and usher- and autotransporter-like (
orfA
and
orf1
to -
3
) functions. Those gene regions corresponding to
orf3
and
orf5
were divergent, those corresponding to
orf2
,
orf1
, and
fimP
were moderately conserved, and those corresponding to
orf4
and
orf6
were highly conserved. Restriction fragment length polymorphism analyses using a
fimP
probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of
A. naeslundii
genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only the
orf4
and
orf6
gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated
Actinomyces
strains.
Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2
Authors: Asai Yasuyuki , Ohyama Yoshinori , Gen Keika , Ogawa Tomohiko
Published: July 2002

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated withPorphyromonas gingivalisfimbriae andStaphylococcus aureuspeptidoglycan, but notEscherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived fromP. gingivalisand similar to a common component of cell wall peptidoglycans in parasitic bacteria,N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), significantly induced IL-8 production and NF-κB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest thatP. gingivalisfimbrial protein and its active peptide are capable of activating HGEC through TLR2.
NovelSalmonella entericaSerovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication
Authors: van der Straaten Tahar , van Diepen Angela , Kwappenberg Kitty , van Voorden Sjaak , Franken Kees , Janssen Riny , Kusters Johannes G. , Granger Donald L. , van Dissel Jaap T.
Published: July 2002

Upon contact with host cells, the intracellular pathogenSalmonella entericaserovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of anS. entericaserovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonellagene designatedsspJthat is located between 54.4 and 64 min of theSalmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104to 107bacteria in C3H/HeN and 101to 104bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carryingsspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, butsspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type bysspJcomplementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.
Enteropathogenic
Escherichia coli
(EPEC) Tir Receptor Molecule Does Not Undergo Full Modification When Introduced into Host Cells by EPEC-Independent Mechanisms
Authors: Kenny Brendan , Warawa Jonathan
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC), like many other gram-negative pathogens, encodes a type III secretion apparatus dedicated to the release of virulence-associated proteins. One such protein, Tir, is translocated into host cells, where it is modified by the addition of phosphate groups, resulting in a number of species with distinct molecular mass. One phosphorylation event, on tyrosine residue 474 of Tir, does not contribute to shifts in molecular mass but is essential for its actin-nucleating function. The role of the nonphosphotyrosine related modifications is unknown. In this paper, we demonstrate, using three different approaches, that Tir does not encode sufficient information to facilitate its complete modification when introduced into host cells in EPEC-independent mechanisms. Each system revealed that Tir is a substrate for a host kinase whose action results in its partial modification to a form similar to one evident in EPEC-infected host cells. Further Tir modification could not be induced by infecting cells with EPEC, suggesting that Tir must be coexpressed with other EPEC factors to enable its full modification within host cells. One approach used
Yersinia
spp. to deliver Tir into host cells, and this system revealed that Tir secretion and translocation can occur in the absence of the Tir chaperone molecule, CesT (formerly known as OrfU). CesT was found to be an efficiency factor which was not required, unlike in EPEC, for Tir stability, indicating that it may function to guide Tir to the translocation apparatus or maintain it in a secretion-competent form.
Flagellar Phase Variation ofSalmonella entericaSerovar Typhimurium Contributes to Virulence in the Murine Typhoid Infection Model but Does Not InfluenceSalmonella-Induced Enteropathogenesis
Authors: Ikeda Jack S. , Schmitt Clare K. , Darnell Stephen C. , Watson Patricia R. , Bispham Jennifer , Wallis Timothy S. , Weinstein Debra L. , Metcalf Eleanor S. , Adams Phillip , O'Connor C. David , O'Brien Alison D.
Published: July 2002

AlthoughSalmonella entericaserovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives ofS. entericaserovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that onceS. entericaserovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-typeS. entericaserovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC+phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.
Effects of Superantigen and Lipopolysaccharide on Induction of CD80 through Apoptosis of Human Monocytes
Authors: Takahashi Masahiro , Takahashi Maiko , Shinohara Fumiaki , Takada Haruhiko , Rikiishi Hidemi
Published: July 2002

To investigate the mechanisms underlying superantigen (SAg) stimulation, we analyzed the effect of SAg on monocyte responses with or without lipopolysaccharide (LPS). Addition of gamma interferon (IFN-γ) to unstimulated cultures induced a marked increase in the number of CD80+monocytes, which was inhibited by LPS through the action of interleukin-10. However, CD80+monocytes began to increase before IFN-γ production, observed after 9 h of stimulation with staphylococcal enterotoxin B (SEB). SEB selectively increased the number of apoptotic CD80−monocytes, whereas LPS-treated monocytes were resistant to the apoptotic action of SEB. This SEB-induced killing was abrogated by anti-CD95 monoclonal antibody (MAb) ZB4 and anti-CD95 ligand (CD95L) MAb NOK2, suggesting a CD95-based pathway of apoptosis. Furthermore, the numbers of SEB-induced CD80+monocytes were partially decreased by anti-CD119 (IFN-γ receptor) MAb and by anti-CD95L (NOK2) MAb. The CD30 expression of CD27highT cells induced by SEB was increased by agonistic anti-CD95 (CH11) MAb. Together, our findings showed that SEB-induced monocyte apoptosis is closely associated with the enrichment of CD80+monocytes generated before IFN-γ production, followed by up-regulation of CD80 by IFN-γ, and that LPS has negative effects in both cases. These results also suggested that induction of monocyte apoptosis is an important mechanism by which SAg exerts its anti-inflammatory effects.
Comparison of Pathogenesis and Host Immune Responses toCandida glabrataandCandida albicansin Systemically Infected Immunocompetent Mice
Authors: Brieland Joan , Essig David , Jackson Craig , Frank Doyle , Loebenberg David , Menzel Fred , Arnold Brian , DiDomenico Beth , Hare Roberta
Published: July 2002

Cytokine-mediated host defense againstCandida glabratainfection was compared to that againstC. albicans, using immunocompetent murine models of systemic candidiasis. The pathogenesis of infection was evaluated morphologically and by culture of target organs, while the kinetics of induction of cytokine mRNAs and corresponding proteins were determined in kidneys by real-time reverse transcription-PCR and cytokine-specific murine enzyme-linked immunosorbent assays, respectively. Systemic infection withC. glabrataresulted in a chronic, nonfatal infection with recovery of organisms from kidneys, while intravenous inoculation withC. albicansresulted in rapid mortality with logarithmic growth of organisms in kidneys and recovery ofC. albicansfrom the spleen, liver, and lungs. Survival ofC. glabrata-infected mice was associated with rapid induction of mRNAs and corresponding immunoreactive proteins for the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and gamma interferon (IFN-γ) and the lack of induction of protein for the anti-inflammatory cytokine IL-10. In contrast, mortality inC. albicans-infected mice was associated with induction of mRNA and corresponding protein for IL-10 but delayed (i.e., TNF-α) or absent (i.e., IL-12 and IFN-γ) induction of immunoreactive proinflammatory cytokines. Mice were subsequently treated with cytokine-specific neutralizing monoclonal antibodies (MAbs) to TNF-α, IL-12, or IFN-γ, and the effect on growth ofC. glabratain kidneys was assessed. Neutralization of endogenous TNF-α resulted in a significant increase inC. glabrataorganisms compared to similarly infected mice administered an isotype-matched control MAb, while neutralization of endogenous IL-12 or IFN-γ had no significant effect onC. glabratareplication. These results demonstrate that in response to intravenous inoculation ofC. glabrata, immunocompetent mice develop chronic nonfatal renal infections which are associated with rapid induction of the proinflammatory cytokines TNF-α, IL-12, and IFN-γ. Furthermore, TNF-α plays a key role in host defense against systemic candidiasis caused by eitherC. glabrataorC. albicans, as the absence of endogenous TNF-α activity was associated with enhanced tissue burden in both infection models.
Cellular Mechanisms That Cause Suppressed Gamma Interferon Secretion in Endotoxin-Tolerant Mice
Authors: Varma Tushar K. , Toliver-Kinsky Tracy E. , Lin Cheng Y. , Koutrouvelis Aristides P. , Nichols Joan E. , Sherwood Edward R.
Published: July 2002

Endotoxin (lipopolysaccharide [LPS]) tolerance is a state of altered immunity characterized, in part, by suppression of LPS-induced gamma interferon (IFN-γ) expression. However, the cellular mediators regulating LPS-induced production of IFN-γ in normal mice and the effect of LPS tolerance on these mediators has not been well characterized. Our studies show that macrophage dysfunction is the primary factor causing suppressed IFN-γ expression in LPS-tolerant mice. Specifically, LPS-tolerant macrophages have a markedly impaired ability to induce IFN-γ secretion by T cells and NK cells obtained from either control or LPS-tolerant mice. However, T cells and NK cells isolated from LPS-tolerant mice produce normal levels of IFN-γ when cocultured with control macrophages or exogenous IFN-γ-inducing factors. Assessment of important IFN-γ-regulating factors showed that interleukin-12 (IL-12) and costimulatory signals provided by IL-15, IL-18, and CD86 are largely responsible for LPS-induced IFN-γ expression in control mice. IL-10 is an inhibitor of IFN-γ production in both the control and LPS-tolerant groups. Expression of IL-12 and the IL-12 receptor β1 (IL-12Rβ1) and IL-12Rβ2 subunits are suppressed in the spleens of LPS-tolerant mice. LPS-tolerant splenocytes also exhibit decreased production of IL-15 and IL-15Rα. However, expression of IL-18 and the B7 proteins CD80 and CD86 are unchanged or increased compared to controls after induction of LPS tolerance. CD28, a major receptor for B7 proteins, is also increased in the spleens of LPS-tolerant mice. Expression of the inhibitory cytokine IL-10 and the IL-10R are sustained after induction of LPS tolerance. These data show that suppression of IFN-γ production in LPS-tolerant mice is largely due to macrophage dysfunction and provide insight into the cellular alterations that occur in LPS tolerance. This study also better defines the factors that mediate LPS-induced IFN-γ production in normal mice.
Protective Efficacy of a DNA Vaccine Encoding Antigen 85A from
Mycobacterium bovis
BCG against Buruli Ulcer
Authors: Tanghe Audrey , Content Jean , Van Vooren Jean-Paul , Portaels Françoise , Huygen Kris
Published: July 2002

Buruli ulcer, caused by
Mycobacterium ulcerans
, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine
M. bovis
BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the
M. ulcerans
homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in
M. ulcerans
, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of
M. ulcerans
- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of
M. ulcerans
- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.
Genetic Immunization with the Region Encoding the α-Helical Domain of PspA Elicits Protective Immunity againstStreptococcus pneumoniae
Authors: Bosarge Joseph R. , Watt James M. , McDaniel D. Olga , Swiatlo Edwin , McDaniel Larry S.
Published: July 2002

Pneumococcal surface protein A (PspA) is a pneumococcal virulence factor capable of eliciting protection against pneumococcal infection in mice. Previous studies have demonstrated that the protection is antibody mediated. Here we examined the ability ofpspAto elicit a protective immune response following genetic immunization of mice. Mice were immunized by intramuscular injections with a eukaryotic expression vector encoding the α-helical domain of PspA/Rx1. Immunization induced a PspA-specific serum antibody response, and immunized mice survived pneumococcal challenge. Survival and antibody responses occurred in a dose-dependent manner, the highest survival rates being seen with doses of 10 μg or greater. The ability of genetic immunization to elicit cross-protection was demonstrated by the survival of immunized mice challenged with pneumococcal strains differing in capsule and PspA types. Also, immunized mice were protected from intravenous and intratracheal challenges with pneumococci. Similar to the results seen with immunization with PspA, the survival of mice genetically immunized withpspAwas antibody mediated. There was no decline in the level of protection 7 months after immunization. These results support the use of genetic immunization to elicit protective immune responses against extracellular pathogens.
Central Importance of Immunoglobulin A in Host Defense againstGiardiaspp
Authors: Langford T. Dianne , Housley Michael P. , Boes Marianne , Chen Jianzhu , Kagnoff Martin F. , Gillin Frances D. , Eckmann Lars
Published: July 2002

The protozoan pathogenGiardiais an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controllingGiardiainfection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls withGiardia murisorGiardia lambliaGS/M-83-H7. We found that IgA-deficient mice could not eradicate eitherG. murisorG. lambliainfection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clearG. murisinfections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies clearedG. murisinfection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to controlG. murisinfection, show that IgA-dependent host defenses are central for eradicatingGiardiaspp. Moreover, B-cell-dependent but IgA-independent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.
Reduced Infectivity of aLeishmania donovaniBiopterin Transporter Genetic Mutant and Its Use as an Attenuated Strain for Vaccination
Authors: Papadopoulou Barbara , Roy Gaétan , Breton Marie , Kündig Christoph , Dumas Carole , Fillion Isabelle , Singh Ajay K. , Olivier Martin , Ouellette Marc
Published: July 2002

Pterins are essential for the growth ofLeishmaniaspecies, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated theLeishmania donovanibiopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. TheL. donovani BT1null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against aL. donovanichallenge. Splenocytes isolated from mice immunized with theBT1null mutant parasites produced significant amounts of interferon gamma following stimulation withL. donovanipromastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport inL. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.
Transient Requirement of the PrrA-PrrB Two-Component System for Early Intracellular Multiplication of
Mycobacterium tuberculosis
Authors: Ewann Fanny , Jackson Mary , Pethe Kevin , Cooper Andrea , Mielcarek Nathalie , Ensergueix Danielle , Gicquel Brigitte , Locht Camille , Supply Philip
Published: July 2002

Adaptive regulation of gene expression in response to environmental changes is a general property of bacterial pathogens. By screening an ordered transposon mutagenesis library of
Mycobacterium tuberculosis
, we have identified three mutants containing a transposon in the coding sequence or in the 5′ regions of genes coding for two-component signal transduction systems (
trcS
,
regX3
,
prrA
). The intracellular multiplication capacity of the three mutants was investigated in mouse bone marrow-derived macrophages. Only the
prrA
mutant showed a defect in intracellular growth during the early phase of infection, and this defect was fully reverted when the mutant was complemented with
prrA-prrB
wild-type copies. The mutant phenotype was transient, as after 1 week this strain recovered full growth capacity to reach levels similar to that of the wild type at day 9. Moreover, a transient induction of
prrA
promoter activity was observed during the initial phase of macrophage infection, as shown by a
prrA
promoter
-gfp
fusion in
M. bovis
BCG infecting the mouse macrophages. The concordant transience of the
prrA
mutant phenotype and
prrA
promoter activity indicates that the PrrA-PrrB two-component system is involved in the environmental adaptation of
M. tuberculosis
, specifically in an early phase of the intracellular growth, and that, similar to other facultative intracellular parasites,
M. tuberculosis
can use genes temporarily required at different stages in the course of macrophage infection.
Biofilm Formation and Interaction with the Surfaces of Gallstones by
Salmonella
spp
Authors: Prouty A. M. , Schwesinger W. H. , Gunn J. S.
Published: July 2002

Salmonellae can exist in an asymptomatic carrier state in the human gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against
Salmonella enterica
serovar Typhi in carriers with gallstones. Therefore, we hypothesized that
Salmonella
spp. form biofilms on the surfaces of gallstones, where the bacteria are protected from high concentrations of bile and antibiotics. A number of methods were utilized to examine biofilm formation on human gallstones and glass coverslips in vitro, including confocal, light, and scanning electron microscopy. In our assays, salmonellae formed full biofilms on the surfaces of gallstones within 14 days and appeared to excrete an exopolysaccharide layer that bound them to the surfaces and to other bacteria. Efficient biofilm formation on gallstones was dependent upon the presence of bile, as a biofilm did not form on gallstones within 14 days in Luria-Bertani broth alone. The biofilms formed by a
Salmonella enterica
serovar Typhi Vi antigen mutant, as well as strains with mutations in genes that eliminate production of four different fimbriae, were indistinguishable from the biofilms formed by the parents. Mutants with an incomplete O-antigen, mutants that were nonmotile, and mutants deficient in quorum sensing were unable to develop complete biofilms. In addition, there appeared to be selectivity in salmonella binding to the gallstone surface that did not depend on the topology or surface architecture. These studies should aid in the understanding of the
Salmonella
carrier state, an important but underresearched area of typhoid fever pathogenesis. If the basis of carrier development can be understood, it may be possible to identify effective strategies to prevent or treat this chronic infection.
Segmented Filamentous Bacteria Interact with Intraepithelial Mononuclear Cells
Authors: Meyerholz David K. , Stabel Thomas J. , Cheville Norman F.
Published: July 2002

Segmented filamentous bacteria (SFB) are found in multiple species and play an important role in the development of mucosal immunity. The mechanism by which the bacteria interact with the immune system has not been well defined. We provide morphologic evidence of direct interaction between SFB and intraepithelial mononuclear cells.
mRNA Composition and Control of Bacterial Gene Expression
Authors: Liang S.-T. , Xu Y.-C. , Dennis P. , Bremer H.
Published: July 2002

The expression of any given bacterial protein is predicted to depend on (i) the transcriptional regulation of the promoter and the translational regulation of its mRNA and (ii) the synthesis and translation of total (bulk) mRNA. This is because total mRNA acts as a competitor to the specific mRNA for the binding of initiation-ready free ribosomes. To characterize the effects of mRNA competition on gene expression, the specific activity of β-galactosidase expressed from three different promoter-
lacZ
fusions (P
spc
-
lacZ
, P
RNAI
-
lacZ
, and P
RNAII
-
lacZ
) was measured (i) in a
relA
+
background during exponential growth at different rates and (ii) in
relA
+
and Δ
relA
derivatives of
Escherichia coli
B/r after induction of a mild stringent or a relaxed response to raise or lower, respectively, the level of ppGpp. Expression from all three promoters was stimulated during slow exponential growth or at elevated levels of ppGpp and was reduced during fast exponential growth or at lower levels of ppGpp. From these observations and from other considerations, we propose (i) that the concentration of free, initiation-ready ribosomes is approximately constant and independent of the growth rate and (ii) that bulk mRNA made during slow growth and at elevated levels of ppGpp is less efficiently translated than bulk mRNA made during fast growth and at reduced levels of ppGpp. These features lead to an indirect enhancement in the expression of LacZ (or of any other protein) during growth in media of poor nutritional quality and at increased levels of ppGpp.
The Alternative Sigma Factor RpoN Is Required for
hrp
Activity in
Pseudomonas syringae
pv. Maculicola and Acts at the Level of
hrpL
Transcription
Authors: Hendrickson Erik L. , Guevera Pablo , Ausubel Frederick M.
Published: July 2002

β-Glucuronidase (
uidA
) reporter gene fusions were constructed for the
hrpZ
,
hrpL
, and
hrpS
genes from the phytopathogen
Pseudomonas syringae
pv. maculicola strain ES4326. These reporters, as well as an
avrRpt2-uidA
fusion, were used to measure transcriptional activity in ES4326 and a ES4326
rpoN
mutant.
rpoN
was required for the expression of
avrRpt2
,
hrpZ
, and
hrpL
in vitro in minimal media and in vivo when infiltrated into
Arabidopsis thaliana
leaves. In contrast, the expression of
hrpS
was essentially the same in wild-type and
rpoN
mutant strains. Constitutive expression of
hrpL
in an
rpoN
mutant restored
hrpZ
transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (
Nicotiana tobacum
), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into
Arabidopsis
leaves. These data indicate that
rpoN
-mediated control of
hrp
gene expression acts at the level of
hrpL
and that in planta growth of
P. syringae
is not required for the elicitation of disease symptoms.
The FtsH Protein Accumulates at the Septum of
Bacillus subtilis
during Cell Division and Sporulation
Authors: Wehrl Wolfgang , Niederweis Michael , Schumann Wolfgang
Published: July 2002

The
ftsH
gene encodes an ATP- and Zn
2+
-dependent metalloprotease which is anchored to the cytoplasmic membrane via two transmembrane segments in such a way that the very short amino- and the long carboxy termini are exposed to the cytoplasm. Deletion of the
ftsH
gene in
Bacillus subtilis
results in a pleiotropic phenotype such as filamentous growth. This observation prompted us to ask whether
ftsH
is involved in cell division. A translational fusion was constructed between the complete coding region of
ftsH
and
gfp
+
the latter carrying five point mutations to obtain enhanced fluorescence. We detected that the FtsH protein accumulates in the midcell septum of dividing cells, and during sporulation first in the asymmetrically located septa of sporulating cells and later in the membrane which engulfs the forespore. These observations revealed a new function of FtsH.
Identification of
Escherichia coli dnaE
(
polC
) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine
Authors: Hiratsuka Koji , Reha-Krantz Linda J.
Published: July 2002

Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679–680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the
dnaE
(
polC
) gene of
Escherichia coli
that alter sensitivity to 2′,3′-dideoxyadenosine (ddA). Two
dnaE
(
polC
) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the α subunit, the protein product of the
dnaE
(
polC
) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative
dnaE
(
polC
) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the α subunit of the DNA polymerase III holoenzyme.
Expansion of the Clavulanic Acid Gene Cluster: Identification and In Vivo Functional Analysis of Three New Genes Required for Biosynthesis of Clavulanic Acid by
Streptomyces clavuligerus
Authors: Li Rongfeng , Khaleeli Nusrat , Townsend Craig A.
Published: July 2002

Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strain
Streptomyces clavuligerus
were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236,
In
V. P. Gullo et al., ed.,
Development in industrial microbiology series
, 1993). In contrast, we report the identification of three new genes,
orf10
(
cyp
),
orf11
(
fd
), and
orf12
, that are required for clavulanic acid biosynthesis as indicated by gene replacement and
trans
-complementation analysis in
S. clavuligerus
. These genes are contained within a 3.4-kb DNA fragment located directly downstream of
orf9
(
cad
) in the clavulanic acid cluster. While the
orf10
(
cyp
) and
orf11
(
fd
) proteins show homologies to other known
CYP-150
cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by
orf12
shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.
Construction and Use of Derivatives of Transposon Tn
4001
That Function in
Mycoplasma pulmonis
and
Mycoplasma arthritidis
Authors: Dybvig Kevin , French C. Todd , Voelker LeRoy L.
Published: July 2002

Previous attempts to introduce transposon Tn
4001
into
Mycoplasma pulmonis
and
Mycoplasma arthritidis
have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn
4001C
and Tn
4001T
were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the
tetM
tetracycline resistance determinant into Tn
4001
. Both Tn
4001C
and Tn
4001T
transposed in
M. pulmonis
, and Tn
4001T
transposed in
M. arthritidis
. The incorporation of a Tn
4001T
derivative that contained
lacZ
into either
Mycoplasma
species resulted in transformants with readily detectable LacZ activity. Tn
4001T
may be of general utility for use as a mycoplasma cloning vehicle because
tetM
functions in all species of
Mycoplasma
examined thus far.
Regulation of Quorum Sensing by RpoS in
Pseudomonas aeruginosa
Authors: Whiteley Marvin , Parsek Matthew R. , Greenberg E. P.
Published: July 2002

The LasR-LasI and RhlR-RhlI quorum-sensing systems are global regulators of gene expression in the opportunistic pathogen
Pseudomonas aeruginosa
. Previous studies suggest that the RhlR-RhlI system activates expression of
rpoS
. We constructed merodiploid strains of
P. aeruginosa
containing the native
rpoS
gene and an
rpoS-lacZ
fusion. Studies of
lacZ
transcription in these strains indicated that
rpoS
was not regulated by RhlR-RhlI. We also generated an
rpoS
null mutant. This
rpoS
mutant showed elevated levels of
rhlI
(but not
rhlR
) transcription, elevated levels of the RhlI-generated acylhomoserine lactone quorum-sensing signal, and elevated levels of RhlR-RhlI-regulated gene transcription. These findings indicate that there is a relationship between RpoS and quorum sensing, but rather than the RhlR-RhlI system influencing the expression of
rpoS
, it appears that RpoS regulates
rhlI
.
Phosphate Starvation-Inducible Proteins of
Bacillus subtilis
: Proteomics and Transcriptional Analysis
Authors: Antelmann Haike , Scharf Christian , Hecker Michael
Published: July 2002

The phosphate starvation response in
Bacillus subtilis
was analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ς
B
, the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a
sigB
mutant and a
phoR
mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of
glpQ
and
ydhF
confirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that
glpQ
is transcribed monocistronically from a ς
A
promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore,
ydhF
might be cotranscribed with
phoB
initiating from the
phoB
promoter. Only a small group of proteins remained phosphate starvation inducible in both
phoR
and
sigB
mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ς
B
-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ς
B
-dependent manner and in the
sigB
mutant probably via ς
H
. YxiE was induced by phosphate starvation independently of ς
B
and PhoPR.
Stationary-Phase Variation Due to Transposition of Novel Insertion Elements in
Xanthomonas oryzae
pv. oryzae
Authors: Rajeshwari R. , Sonti R. V.
Published: July 2002

Xanthomonas oryzae
pv. oryzae causes bacterial leaf blight, a serious disease of rice. Spontaneous mutants which are deficient for virulence and extracellular polysaccharide (Eps) production accumulate in large numbers in stationary-phase cultures of this bacterium, a phenomenon which we have called stationary-phase variation. A clone (pSD1) carrying the Eps biosynthetic gene (
gum
) cluster of
X. oryzae
pv. oryzae restored Eps production and virulence to several
spv
(for stationary-phase variation) mutants. Data from localized recombination analysis, Southern hybridization, PCR amplification, and sequence analysis showed that the mutations are due to insertion of either one of two novel endogenous insertion sequence (IS) elements, namely, IS
Xo1
and IS
Xo2
, into
gumM
, the last gene of the
gum
gene cluster. The results of Southern analysis indicate the presence of multiple copies of both IS elements in the genome of
X. oryzae
pv. oryzae. These results demonstrate the role of IS elements in stationary-phase variation in
X. oryzae
pv. oryzae.
Changes in
cspL
,
cspP
, and
cspC
mRNA Abundance as a Function of Cold Shock and Growth Phase in
Lactobacillus plantarum
Authors: Derzelle Sylviane , Hallet Bernard , Francis Kevin P. , Ferain Thierry , Delcour Jean , Hols Pascal
Published: July 2002

An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in
Lactobacillus plantarum
. In addition to the two previously reported
cspL
and
cspP
genes, a third gene,
cspC
, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of
cspL
mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of
cspC
and
cspP
remain unchanged or increase only slightly (about two- to threefold). Cold induction of
cspL
mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three
csp
mRNAs contrasts with that observed for four unrelated non-
csp
genes, which all showed a sharp decrease in mRNA level, followed in one case (
bglH
) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three
csp
mRNAs was also found to vary during growth at optimal temperature (28°C).
cspC
and
cspP
mRNA levels are maximal during the lag period, whereas the abundance of the
cspL
transcript is highest during late-exponential-phase growth. The differential expression of the three
L. plantarum csp
genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.
Binding of Ferric Enterobactin by theEscherichia coliPeriplasmic Protein FepB
Authors: Sprencel Cathy , Cao Zhenghua , Qi Zengbiao , Scott Daniel C. , Montague Marjorie A. , Ivanoff Nora , Xu Jide , Raymond Kenneth M. , Newton Salete M. C. , Klebba Phillip E.
Published: July 2002

The periplasmic protein FepB ofEscherichia coliis a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay theKdof the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (Kd= 30 nM) and ferric enantioenterobactin (Kd= 15 nM), the left-handed stereoisomer of the naturalE. colisiderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (Kd= 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.
Spermidine Acetyltransferase Is Required To Prevent Spermidine Toxicity at Low Temperatures in
Escherichia coli
Authors: Limsuwun Kornvika , Jones Pamela G.
Published: July 2002

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In
Escherichia coli
, the
speG
gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of
E. coli
at low temperature was examined using a
speG
mutant. At 37°C, growth of the
speG
mutant was normal in the presence of 0.5 or 1 mM spermidine. However, following a shift to 7°C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7°C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg
2+
restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg
2+
, resulting in ribosome inactivation at low temperatures.
Genes Involved in Anaerobic Metabolism of Phenol in the Bacterium
Thauera aromatica
Authors: Breinig Sabine , Schiltz Emile , Fuchs Georg
Published: July 2002

Genes involved in the anaerobic metabolism of phenol in the denitrifying bacterium
Thauera aromatica
have been studied. The first two committed steps in this metabolism appear to be phosphorylation of phenol to phenylphosphate by an unknown phosphoryl donor (“phenylphosphate synthase”) and subsequent carboxylation of phenylphosphate to 4-hydroxybenzoate under release of phosphate (“phenylphosphate carboxylase”). Both enzyme activities are strictly phenol induced. Two-dimensional gel electrophoresis allowed identification of several phenol-induced proteins. Based on N-terminal and internal amino acid sequences of such proteins, degenerate oligonucleotides were designed to identify the corresponding genes. A chromosomal DNA segment of about 14 kbp was sequenced which contained 10 genes transcribed in the same direction. These are organized in two adjacent gene clusters and include the genes coding for five identified phenol-induced proteins. Comparison with sequences in the databases revealed the following similarities: the gene products of two open reading frames (ORFs) are each similar to either the central part and N-terminal part of phosphoenolpyruvate synthases. We propose that these ORFs are components of the phenylphosphate synthase system. Three ORFs showed similarity to the
ubiD
gene product, 3-octaprenyl-4-hydroxybenzoate carboxy lyase; UbiD catalyzes the decarboxylation of a 4-hydroxybenzoate analogue in ubiquinone biosynthesis. Another ORF was similar to the
ubiX
gene product, an isoenzyme of UbiD. We propose that (some of) these four proteins are involved in the carboxylation of phenylphosphate. A 700-bp PCR product derived from one of these ORFs cross-hybridized with DNA from different
Thauera
and
Azoarcus
strains, even from those which have not been reported to grow with phenol. One ORF showed similarity to the
mutT
gene product, and three ORFs showed no strong similarities to sequences in the databases. Upstream of the first gene cluster, an ORF which is transcribed in the opposite direction codes for a protein highly similar to the DmpR regulatory protein of
Pseudomonas putida
. DmpR controls transcription of the genes of aerobic phenol metabolism, suggesting a similar regulation of anaerobic phenol metabolism by the putative regulator.