bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Lsp, a Member of the LraI-Lipoprotein Family inStreptococcus pyogenes, in Eukaryotic Cell Adhesion and Internalization
Authors: Elsner Andrea , Kreikemeyer Bernd , Braun-Kiewnick Andrea , Spellerberg Barbara , Buttaro Bettina A. , Podbielski Andreas
Published: October 2002

Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between themgacore regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein ofStreptococcus pyogenes) and ORF2 exhibited a high degree of homology to thelmb/ORF2 genes ofS. agalactiae(B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistroniclsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase.lspand ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn2+and Cu2+ion concentrations decreasedlspoperon expression, Lsp did not transport divalent cations as described for other LraI-type operons. Thelspmutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, thelspmutant showed decreased transcription ofprtF2encoding the fibronectin-binding protein F2. Both thelspand ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in thelspmutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription ofspeB(cysteine protease) and/or the global regulatorsmga,csrRS, andnra.
Nucleotidase Cascades Are Catalyzed by Secreted Proteins of the Parasitic NematodeTrichinella spiralis
Authors: Gounaris Kleoniki
Published: October 2002

Extracellular nucleotides are signaling molecules whose receptor-mediated effects are involved in a variety of physiological responses in mammalian tissues. An overwhelming body of data indicate that inflammatory and other immune responses can be modulated by the availability and local concentrations of nucleotides via nucleotide receptor signaling, but this is only just beginning to be investigated in the context of infectious disease. Evidence is provided here that the parasitic nematodeTrichinella spiraliscan catalyze the conversion and thus modulate both the availability and concentration of extracellular nucleotides by means of the following secreted exoenzymes: apyrase, 5′-nucleotidase, and adenosine deaminase. These enzymes were characterized in terms of substrate specificity, kinetic behavior, pH, divalent cation preferences, and response to a series of compounds. The secreted 5′-nucleotidase was identified as a protein with an apparent molecular mass of 67 kDa after N-terminal amino acid sequencing of the purified protein. The presence of adenosine deaminase was confirmed in the secreted products by Western blotting with an antibody against a mammalian enzyme, as a protein with an apparent molecular mass of 38 kDa. These secreted proteins constitute an enzymatic cascade which catalyzes the degradation of extracellular nucleotides, with a potential physiological role in the regulation of purinergic signaling.
Identification and Functional Mapping of theMycoplasma fermentansP29 Adhesin
Authors: Leigh Spencer A. , Wise Kim S.
Published: October 2002

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein ofMycoplasma fermentans, binds human HeLa cells and inhibitsM. fermentansbinding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction ofM. fermentanswith a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells byMycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that byMycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).
Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins
Authors: Haake David A. , Matsunaga James
Published: October 2002

The outer membrane (OM) of the mammalian pathogen
Leptospira kirschneri
was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31
LipL45
. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components.
Activation of p38 Mitogen-Activated Protein Kinase AttenuatesLeishmania donovaniInfection in Macrophages
Authors: Junghae Muthoni , Raynes John G.
Published: October 2002

Leishmania-induced macrophage dysfunctions have been correlated with altered signaling events. In this work, we report that SB203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), increasesLeishmania donovanisurvival in human peripheral blood mononuclear macrophages. Consistent with this finding, activation of p38 and c-junN-terminal kinase (JNK) MAPK signaling pathways by anisomycin significantly reduced parasite survival within these cells. However, the majority of the effect was seen in a 50% reduction in the percentage of macrophages infected, with little effect on the highly infected macrophages. The observed effect was likely to be due to the p38 MAPK pathway since SB203580 was able to completely reverse the effect of anisomycin. These findings suggest that the previously reported p38 MAPK inhibition byLeishmaniainfection may be partially overcome by anisomycin. Similar effects were observed in pretreated macrophages or in treatment of infected macrophages. These results suggests that p38 MAPK activation may have a potential therapeutic value in the treatment of visceral leishmaniasis.
The Human Polymeric Immunoglobulin Receptor Facilitates Invasion of Epithelial Cells byStreptococcus pneumoniaein a Strain-Specific and Cell Type-Specific Manner
Authors: Brock Sean C. , McGraw Patricia A. , Wright Peter F. , Crowe James E.
Published: October 2002

Streptococcus pneumoniaeis a gram-positive bacterial pathogen that causes invasive life-threatening disease worldwide. This organism also commonly colonizes the upper respiratory epithelium in an asymptomatic fashion. To invade, this pathogen must traverse the respiratory epithelial barrier, allowing it to cause disease locally or disseminate hematogenously throughout the body. Previous work has demonstrated thatS. pneumoniaecholine-binding protein A, a pneumococcal surface protein, interacts specifically with the human polymeric immunoglobulin receptor, which is expressed by cells in the respiratory epithelium. Choline-binding protein A is required for efficient colonization of the nasopharynx in vivo. Additionally, a recent study showed that the R6x laboratory strain ofS. pneumoniaeinvades a human pharyngeal cell line in a human polymeric immunoglobulin receptor-dependent manner. These findings raised the possibility that the interaction between choline-binding protein A and human polymeric immunoglobulin receptor may be a key determinant ofS. pneumoniaepathogenesis. However, the strain used in prior invasion studies, R6x, is an unencapsulated, nonpathogenic strain. In the present study we determined the relative ability of strain R6x or pathogenic strains to invade a variety of human polymeric immunoglobulin receptor-expressing epithelial cell lines. The results of this work suggest that human polymeric immunoglobulin receptor-dependent enhanced invasion of epithelial cells byS. pneumoniaeis a limited phenomenon that occurs in a strain-specific and cell type-specific manner.
Attenuated
Salmonella enterica
Serovar Typhi Expressing Urease Effectively Immunizes Mice against
Helicobacter pylori
Challenge as Part of a Heterologous Mucosal Priming-Parenteral Boosting Vaccination Regimen
Authors: Londoño-Arcila Patricia , Freeman Donna , Kleanthous Harry , O'Dowd Aisling M. , Lewis Susan , Turner Arthur K. , Rees Emma L. , Tibbitts Timothy J. , Greenwood Judith , Monath Thomas P. , Darsley Michael J.
Published: October 2002

Recombinant vaccine strains of
Salmonella enterica
serovar Typhi capable of expressing
Helicobacter pylori
urease were generated by transforming strains CVD908 and CVD908-
htrA
with a plasmid harboring the
ureAB
genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNγ) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNγ as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing
S. enterica
serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of
H. pylori
; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against
H. pylori
with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against
H. pylori
and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.
Increased
Trypanosoma cruzi
Invasion and Heart Fibrosis Associated with High Transforming Growth Factor β Levels in Mice Deficient in α
2
-Macroglobulin
Authors: Waghabi M. C. , Coutinho C. M. L. M. , Soeiro M. N. C. , Pereira M. C. S. , Feige J.-J. , Keramidas M. , Cosson A. , Minoprio P. , Van Leuven F. , Araújo-Jorge T. C.
Published: October 2002

Trypanosoma cruzi
proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine α
2
-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to
T. cruzi
infection of mice that are deficient only in α2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine
T. cruzi
infection. We further demonstrated in vitro that human α2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired
T. cruzi
invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor β in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental
T. cruzi
infection.
Histoplasma capsulatumSynthesizes Melanin-Like Pigments In Vitro and during Mammalian Infection
Authors: Nosanchuk Joshua D. , Gómez Beatriz L. , Youngchim Sirida , Díez Soraya , Aisen Philip , Zancopé-Oliveira Rosely M. , Restrepo Angela , Casadevall Arturo , Hamilton Andrew J.
Published: October 2002

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogenHistoplasma capsulatumvar. capsulatumproduced melanin or melanin-like compounds in vitro and during infection. Growth ofH. capsulatummycelia in chemically defined minimal medium produced pigmented conidia. Growth ofH. capsulatumyeast in chemically defined minimal medium withl-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected withH. capsulatumand from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance toH. capsulatumyeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate thatH. capsulatumconidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.
The YeastCandida albicansBinds Complement Regulators Factor H and FHL-1
Authors: Meri T. , Hartmann A. , Lenk D. , Eck R. , Würzner R. , Hellwage J. , Meri S. , Zipfel P. F.
Published: October 2002

The human facultative pathogenic yeastCandida albicanscauses mucocutaneous infections and is the major cause of opportunistic fungal infections in immunocompromised patients.C. albicansactivates both the alternative and classical pathway of the complement system. The aim of this study was to assay whetherC. albicansbinds human complement regulators in order to control complement activation at its surface. We observed binding of two central complement regulators, factor H and FHL-1, from normal human serum toC. albicansby adsorption assays, immunostaining, and fluorescence-activated cell sorter (FACS) analyses. Specificity of acquisition was further confirmed in direct binding assays with purified proteins. The surface-attached regulators maintained their complement regulatory activities and mediated factor I-dependent cleavage of C3b. Adsorption assays with recombinant deletion mutant proteins were used to identify binding domains. Two binding sites were localized. One binding domain common to both factor H and FHL-1 is located in the N-terminal short consensus repeat domains (SCRs) 6 and 7, and the other one located in C-terminal SCRs 19 and 20 is unique to factor H. These data indicate that by surface acquisition of host complement regulators, the human pathogenic yeastC. albicansis able to regulate alternative complement activation at its surface and to inactivate toxic complement activation products.
Borrelia burgdorferi erp
(
ospE
-Related) Gene Sequences Remain Stable during Mammalian Infection
Authors: Stevenson Brian
Published: October 2002

A number of studies have indicated that
Borrelia burgdorferi erp
genes need not vary during vertebrate infection. However, it was recently reported that a
B. burgdorferi
bacterium reisolated from an infected mouse evidenced mutation and recombination events in several
erp
genes. Reexamination of that reisolate indicates that the previously reported changes were no doubt artifacts of the PCR processes originally used to clone those DNAs. Thus, no evidence has been found of
erp
gene variation during mammalian infection.
Shiga Toxin 2 Induces Macrophage-Granulocyte Colonies from Human Bone Marrow and Cord Blood Stem Cells
Authors: Chiyoda Shin , Takeda Tae , Aoki Yosuke
Published: October 2002

Addition of Shiga toxin 2 to human bone marrow or cord blood cell culture induced macrophage-granulocyte colonies. Although Shiga toxin 2 alone induced colonies mainly composed of macrophages, it induced colonies mainly consisting of granulocytes when combined with physiological doses of interleukin-1β, granulocyte colony-stimulating factor, or stem cell factor with interleukin-3.
Neutrophil-Mediated Mycobacteriocidal Immunity in the Lung duringMycobacterium bovisBCG Infection in C57BL/6 Mice
Authors: Fulton S. A. , Reba S. M. , Martin T. D. , Boom W. H.
Published: October 2002

Although neutrophils have been identified as sources of inflammatory cytokines and chemokines, little is known about their immunologic function during mycobacterial infection in the lungs. In this study, we examined the growth ofMycobacterium bovisBCG in the lungs under experimental conditions that altered neutrophil recruitment to the lungs. Depletion and recruitment of neutrophils was associated with respective increases and decreases inM. bovisBCG growth. Thus, neutrophils may enhance mycobacteriocidal immunity in the lungs.
Comparison of Genome Structures of Vibrios, Bacteria Possessing Two Chromosomes
Authors: Tagomori Kenichi , Iida Tetsuya , Honda Takeshi
Published: October 2002

Vibrios are gram-negative γ-proteobacteria which are ubiquitous in marine and estuarine environments. Recently, we demonstrated that some, if not all,
Vibrio
species have two circular chromosomes. The whole genome sequence of
Vibrio cholerae
N16961 has been reported. In this study, we constructed a physical and genetic map of the genome of Kanagawa phenomenon-positive
Vibrio parahaemolyticus
strain KX-V237 and compared it with those of
V. parahaemolyticus
AQ4673 and
V. cholerae
N16961. The genome of KX-V237 comprised two circular chromosomes (3.3 and 1.9 Mb), similar to the structure of the AQ4673 genome. The relative positions of the genes on the genomes were well conserved in the two strains, but a large inversion on the large chromosomes, probably symmetric around the replication origin, was suggested. Although the sizes of the large chromosomes of KX-V237 and
V. cholerae
N16961 were similar, the sizes of the small chromosomes were very different. Unlike N16961, the superintegron of KX-V237 was located on the large chromosome. Comparison of the genetic maps of the chromosomes of KX-V237 and
V. cholerae
N16961 revealed that most of the open reading frames (ORFs) present on the large chromosome of the
V. cholerae
strain had homologues on the large chromosome of the
V. parahaemolyticus
strain and that most of the ORFs on the small chromosome of N16961 were present on the small chromosome of KX-V237. The difference in the orders of the ORFs on the chromosomes of N16961 and KX-V237 implies that numerous and frequent genetic exchanges have occurred intrachromosomally rather than interchromosomally.
icaR
Encodes a Transcriptional Repressor Involved in Environmental Regulation of
ica
Operon Expression and Biofilm Formation in
Staphylococcus epidermidis
Authors: Conlon Kevin M. , Humphreys Hilary , O'Gara James P.
Published: October 2002

Biofilm formation in
Staphylococcus epidermidis
is dependent upon the
ica
operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The
icaR
gene, located adjacent to the
ica
operon, appears to be a member of the
tetR
family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the
ica
operon by IS
256
accounts for 25 to 33% of phase variants. In this study,
icaA
and
icaR
regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS
256
is absent. Predictably,
ica
operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS
256
-generated phase variants. In contrast, the
icaR
gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An
icaR
::Em
r
insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in
ica
operon expression but did not significantly alter regulation of the
icaR
gene itself. Activation of
ica
operon transcription by ethanol in CSF41498 was
icaR
dependent. In contrast, a small but significant induction of
ica
by NaCl and glucose (NaCl-glucose) was observed in the
icaR
::Em
r
mutant. In addition, transcription of the
icaR
gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the
ica
operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (
icaR
+
) but not in RP62A variants (
icaR
deficient). These data indicate that
icaR
encodes a repressor of
ica
operon transcription required for ethanol but not NaCl-glucose activation of
ica
operon expression and biofilm formation.
Developmental Regulation of theStreptomyces lividans ramGenes: Involvement of RamR in Regulation of theramCSABOperon
Authors: Keijser Bart J. F. , van Wezel Gilles P. , Canters Gerard W. , Vijgenboom Erik
Published: October 2002

Streptomycetes are filamentous soil bacteria that produce spores through a complex process of morphological differentiation. Theramcluster plays an important part during the development. Theramgenes encode a membrane-bound kinase (RamC), a small protein (RamS), components of an ABC transporter (RamAB), and a response regulator (RamR). While the introduction of an extra copy of theramcluster accelerates development inStreptomyces lividans,ramABRdisruption mutants are unable to produce aerial hyphae and spores. The developmental regulation oframgene transcription was analyzed. Transcription of theramgenes occurred only on solid rich media and not on minimal media. TheramRgene is transcribed from a single promoter during all growth stages, with the highest levels during aerial growth. TheramCSABgenes comprise one operon and are transcribed from one principal promoter, P1, directly upstream oframC. Transcription oframCSABwas already observed during vegetative growth, but was strongly upregulated upon initiation of formation of aerial hyphae and was decreased during late stages of development. A large inverted repeat located downstream oframSterminated the majority of transcripts. The introduction oframRon a multicopy vector inS. lividansstrongly induced P1 activity, while disruption of this regulator eliminated all P1 promoter activity. This shows thatramRis a crucial activator oframCSABtranscription. Importantly, inbldA,bldB,bldD, orbldHmutants,ramRandramCSABare not transcribed, whileramgene transcription was observed in the earliestwhimutant,whiG. This indicates that the transcription of theramgenes marks the transition from vegetative to aerial growth.
Purification, Overproduction, and Partial Characterization of β-RFAP Synthase, a Key Enzyme in the Methanopterin Biosynthesis Pathway†
Authors: Scott Joseph W. , Rasche Madeline E.
Published: October 2002

Methanopterin is a folate analog involved in the C
1
metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme β-ribofuranosylaminobenzene 5′-phosphate synthase (β-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of
p
-aminobenzoic acid with phosphoribosylpyrophosphate to form β-RFAP, CO
2
, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding β-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of β-RFAP synthase. The enzyme was purified from the methane-producing archaeon
Methanosarcina thermophila
, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen
Methanococcus jannaschii
and the sulfate-reducing archaeon
Archaeoglobus fulgidus
. The putative β-RFAP synthase gene from
A. fulgidus
was expressed in
Escherichia coli
, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the β-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C
1
metabolism by the bacterium
Methylobacterium extorquens
. The identification of a gene encoding a potential β-RFAP synthase in
M. extorquens
is the first report of a putative methanopterin biosynthetic gene found in the
Bacteria
and provides evidence that the pathways of methanopterin biosynthesis in
Bacteria
and
Archaea
are similar.
DksA Affects ppGpp Induction of RpoS at a Translational Level
Authors: Brown Larissa , Gentry Daniel , Elliott Thomas , Cashel Michael
Published: October 2002

The RpoS sigma factor (also called σSor σ38) is known to regulate at least 50 genes in response to environmental sources of stress or during entry into stationary phase. Regulation of RpoS abundance and activity is complex, with many factors participating at multiple levels. One factor is the nutritional stress signal ppGpp. The absence of ppGpp blocks or delays the induction ofrpoSduring entry into stationary phase. Artificially inducing ppGpp, without starvation, is known to inducerpoSduring the log phase 25- to 50-fold. Induction of ppGpp is found to have only minor effects onrpoStranscript abundance or on RpoS protein stability; instead, the efficiency ofrpoSmRNA translation is increased by ppGpp as judged by both RpoS pulse-labeling and promoter-independent effects onlacZfusions. DksA is found to affect RpoS abundance in a manner related to ppGpp. DeletingdksAblocksrpoSinduction by ppGpp. Overproduction of DksA inducesrpoSbut not ppGpp. DeletingdksA neither alters regulation of ppGpp in response to amino acid starvation nor nullifies the inhibitory effects of ppGpp on stable RNA synthesis. Although this suggests thatdksAis epistatic to ppGpp, inducing ppGpp does not induce DksA. AdksAdeletion does display a subset of the same multiple-amino-acid requirements found for ppGpp0mutants, but overproducing DksA does not satisfy ppGpp0requirements. Sequenced spontaneous extragenic suppressors ofdksApolyauxotrophy are frequently the same T563PrpoBallele that suppresses a ppGpp0phenotype. We propose that DksA functions downstream of ppGpp but indirectly regulatesrpoSinduction.
Suppressive Subtractive Hybridization Detects Extensive Genomic Diversity in
Thermotoga maritima
Authors: Nesbø Camilla L. , Nelson Karen E. , Doolittle W. Ford
Published: October 2002

Comparisons between genomes of closely related bacteria often show large variations in gene content, even between strains of the same species. Such studies have focused mainly on pathogens; here, we examined
Thermotoga maritima,
a free-living hyperthermophilic bacterium, by using suppressive subtractive hybridization. The genome sequence of
T. maritima
MSB8 is available, and DNA from this strain served as a reference to obtain strain-specific sequences from
Thermotoga
sp. strain RQ2, a very close relative (∼96% identity for orthologous protein-coding genes, 99.7% identity in the small-subunit rRNA sequence). Four hundred twenty-six RQ2 subtractive clones were sequenced. One hundred sixty-six had no DNA match in the MSB8 genome. These differential clones comprise, in sum, 48 kb of RQ2-specific DNA and match 72 genes in the GenBank database. From the number of identical clones, we estimated that RQ2 contains 350 to 400 genes not found in MSB8. Assuming a similar genome size, this corresponds to 20% of the RQ2 genome. A large proportion of the RQ2-specific genes were predicted to be involved in sugar transport and polysaccharide degradation, suggesting that polysaccharides are more important as nutrients for this strain than for MSB8. Several clones encode proteins involved in the production of surface polysaccharides. RQ2 encodes multiple subunits of a V-type ATPase, while MSB8 possesses only an F-type ATPase. Moreover, an RQ2-specific MutS homolog was found among the subtractive clones and appears to belong to a third novel archaeal type MutS lineage. Southern blot analyses showed that some of the RQ2 differential sequences are found in some other members of the order
Thermotogales
, but the distribution of these variable genes is patchy, suggesting frequent lateral gene transfer within the group.
Phosphorylation of thePseudomonas aeruginosaResponse Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility
Authors: Whitchurch Cynthia B. , Erova Tatiana E. , Emery Jacqui A. , Sargent Jennifer L. , Harris Jonathan M. , Semmler Annalese B. T. , Young Michael D. , Mattick John S. , Wozniak Daniel J.
Published: October 2002

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility inPseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutantalgRalleles were introduced into the chromosome ofP. aeruginosastrains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function.algDmutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation inP. aeruginosa.
Microarray Transcription Profiling of a
Shewanella oneidensis etrA
Mutant
Authors: Beliaev Alex S. , Thompson Dorothea K. , Fields Matthew W. , Wu Liyou , Lies Douglas P. , Nealson Kenneth H. , Zhou Jizhong
Published: October 2002

DNA microarrays were used to examine the effect of an insertional mutation in the
Shewanella oneidensis etrA
(electron transport regulator) locus on gene expression under anaerobic conditions. The mRNA levels of 69 genes with documented functions in energy and carbon metabolism, regulation, transport, and other cellular processes displayed significant alterations in transcript abundance in an
etrA
-mutant genetic background. This is the first microarray study indicating a possible involvement of EtrA in the regulation of gene expression in
S. oneidensis
MR-1.
One Repeat of the Cell Wall Binding Domain Is Sufficient for Anchoring the
Lactobacillus acidophilus
Surface Layer Protein
Authors: Smit Egbert , Pouwels Peter H.
Published: October 2002

The N-terminal repeat (SAC1) of the S-protein of
Lactobacillus acidophilus
bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not. Treatment of CWFs with hydrofluoric acid, but not phenol, prevented binding. Apparently, SAC1 is necessary and sufficient for cell wall binding. Our data suggest that SAC anchors the S-protein to a cell wall teichoic acid.
Hierarchy of Carbon Source Selection inParacoccus pantotrophus: Strict Correlation between Reduction State of the Carbon Substrate and Aerobic Expression of thenapOperon
Authors: Ellington M. J. K. , Bhakoo K. K. , Sawers G. , Richardson D. J. , Ferguson S. J.
Published: October 2002

Paracoccus pantotrophuscan express a periplasmic nitrate reductase (Nap) during aerobic growth. A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates. To investigate the regulation ofnapexpression, a transcriptional fusion between thenappromoter region ofP. pantotrophusand thelacZgene was constructed. When this fusion was used, analyses showed that transcription from thenappromoter increases as the average reduction state of the carbon atoms increases. Thus, β-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate. This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum. If two carbon sources were presented together, the β-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone. This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium. Analysis of culture medium by1H nuclear magnetic resonance showed that for aerobic growthP. pantotrophusstrictly selected its carbon source in the order succinate-acetate-butyrate. This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates. The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of thenapoperon.
Identification by Heterologous Expression and Gene Disruption of VisA as
l
-Lysine 2-Aminotransferase Essential for Virginiamycin S Biosynthesis in
Streptomyces virginiae
Authors: Namwat Wises , Kinoshita Hiroshi , Nihira Takuya
Published: October 2002

The
visA
gene of
Streptomyces virginiae
has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the
visA
gene was achieved in
Escherichia coli
by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear
l
-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35°C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts
l
-lysine into Δ
1
-piperidine 2-carboxylic acid. A
visA
deletion mutant of
S. virginiae
was created by homologous recombination, and the in vivo function of the
visA
gene was studied by phenotypic comparison between the wild type and the
visA
deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that
visA
is not involved in primary metabolism or morphological differentiation. However, the
visA
mutant failed to produce VS while maintaining the production of virginiamycin M
1
at a level comparable to that of the parental wild-type strain, demonstrating that
visA
is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from
l
-lysine.
Bacillus subtilis
Mutant LicT Antiterminators Exhibiting Enzyme I- and HPr-Independent Antitermination Affect Catabolite Repression of the
bglPH
Operon
Authors: Lindner Cordula , Hecker Michael , Le Coq Dominique , Deutscher Josef
Published: October 2002

The
Bacillus subtilis
antiterminator LicT regulates the expression of
bglPH
and
bglS
, which encode the enzymes for the metabolism of aryl-β-glucosides and the β-glucanase BglS. The N-terminal domain of LicT (first 55 amino acids) prevents the formation of ρ-independent terminators on the respective transcripts by binding to target sites overlapping these terminators. Proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulate the antitermination activity of LicT by phosphorylating histidines in its two PTS regulation domains (PRDs). Phosphorylation at His-100 in PRD-1 requires the PTS proteins enzyme I and HPr and the phosphorylated permease BglP and inactivates LicT. During transport and phosphorylation of aryl-β-glucosides, BglP is dephosphorylated, which renders LicT active and thus leads to
bglPH
and
bglS
induction. In contrast, phosphorylation at His-207 and/or His-269 in PRD-2, which requires only enzyme I and HPr, is absolutely necessary for LicT activity and
bglPH
and
bglS
expression. We isolated spontaneous
licT
mutants expressing
bglPH
even when enzyme I and HPr were absent (as indicated by the designation “Pia” [PTS-independent antitermination]). Introduced in a
ptsHI
+
strain, two classes of
licT
(Pia) mutations could be distinguished. Mutants synthesizing LicT(Pia) antiterminators altered in PRD-2 still required induction by aryl-β-glucosides, whereas mutations affecting PRD-1 caused constitutive
bglPH
expression. One of the two carbon catabolite repression (CCR) mechanisms operative for
bglPH
requires the ρ-independent terminator and is probably prevented when LicT is activated by P∼His-HPr-dependent phosphorylation in PRD-2 (where the prefix “P∼” stands for “phospho”). During CCR, the small amount of P∼His-HPr present in cells growing on repressing PTS sugars probably leads to insufficient phosphorylation at PRD-2 of LicT and therefore to reduced
bglPH
expression. In agreement with this concept, mutants synthesizing a P∼His-HPr-independent LicT(Pia) had lost LicT-modulated CCR.
Effects of the P1 Plasmid Centromere on Expression of P1 Partition Genes
Authors: Hao Jian-Jiang , Yarmolinsky Michael
Published: October 2002

The partition operon of P1 plasmid encodes two proteins, ParA and ParB, required for the faithful segregation of plasmid copies to daughter cells. The operon is followed by a centromere analog,parS, at which ParB binds. ParA, a weak ATPase, represses theparpromoter most effectively in its ADP-bound form. ParB can recruit ParA toparS, stimulate its ATPase, and significantly stimulate the repression. We report here thatparSalso participates in the regulation of expression of thepargenes. A single chromosomalparSwas shown to augment repression of several copies of theparpromoter by severalfold. The repression increase was sensitive to the levels of ParA and ParB and to their ratio. The increase may be attributable to a conformational change in ParA mediated by theparS-ParB complex, possibly acting catalytically. We also observed an inciseffect ofparSwhich enhanced expression ofparB, presumably due to a selective modulation of the mRNA level. Although ParB had been earlier found to spread into and silence genes flankingparS, silencing of theparoperon by ParB spreading was not significant. Based upon analogies between partitioning and septum placement, we speculate that the regulatory switch controlled by theparS-ParB complex might be essential for partitioning itself.
Carbonic Anhydrase Is Essential for Growth of
Ralstonia eutropha
at Ambient CO
2
Concentrations
Authors: Kusian Bernhard , Sültemeyer Dieter , Bowien Botho
Published: October 2002

Mutant strain 25-1 of the facultative chemoautotroph
Ralstonia eutropha
H16 had previously been shown to exhibit an obligately high-CO
2
-requiring (HCR) phenotype. Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO
2
. In the present study, a gene designated
can
and encoding a β-carbonic anhydrase (CA) was identified as the site altered in strain 25-1. The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can. A
can
deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1. Overexpression of
can
in
Escherichia coli
and mass spectrometric determination of CA activity demonstrated that
can
encodes a functional CA. The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO
4
for maximal activity. Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO
2
concentrations. The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO
2
concentrations requires sufficient CA activity rather than the specific function of Can. The metabolic function(s) depending on CA activity remains to be identified.
Isoprenoid Biosynthesis in
Synechocystis
sp. Strain PCC6803 Is Stimulated by Compounds of the Pentose Phosphate Cycle but Not by Pyruvate or Deoxyxylulose-5-Phosphate
Authors: Ershov Yuri V. , Gantt R. Raymond , Cunningham, Francis X. , Gantt Elisabeth
Published: October 2002

The photosynthetic cyanobacterium
Synechocystis
sp. strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-
C
-methyl-
d
-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in
Escherichia coli
, or by deoxyxylulose-5-phosphate, the first pathway intermediate in
E. coli
. However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds. Fosmidomycin (at 1 μM and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect [
14
C]IPP incorporation stimulated by DHAP plus GA3P. To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium. The combined results suggest that the MEP pathway, as described for
E. coli
, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in
Synechocystis
sp. strain PCC6803. Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.
Characterization ofbcsAMutations That Bypass Two Distinct Signaling Requirements forMyxococcus xanthusDevelopment
Authors: Cusick John K. , Hager Elizabeth , Gill Ronald E.
Published: October 2002

The BsgA protease is required for starvation-induced development inMyxococcus xanthus.Bypass suppressors of absgAmutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5mutagenesis of absgAmutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Ω951 insertion was localized to a gene,bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism. Mutations inbcsAbypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling. Bypass of the B-signaling requirement by thebcsAmutation was accompanied by restored expression of a subset of developmentally inducedlacZfusions to the BsgA protease-deficient strain.bcsAmutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (Ω4427), suggesting that thebcsAgene product may normally act to inhibit development in a cell-density-regulated fashion. Bypass of the requirements for both B and C signaling bybcsAmutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements.