bluuebunny/crossref_metadata_2025 huggingface dataset:
Composition and Gene Expression of the
cag
Pathogenicity Island in
Helicobacter pylori
Strains Isolated from Gastric Carcinoma and Gastritis Patients in Costa Rica
Authors: Occhialini Alessandra , Marais Armelle , Urdaci Maria , Sierra Rafaela , Muñoz Nubia , Covacci Antonello , Mégraud Francis
Published: July 2002

The composition and in vitro expression of the
cag
pathogenicity island genes in a group of
Helicobacter pylori
strains obtained from patients suffering from chronic gastritis-associated dyspepsia (
n
= 26) or gastric carcinoma (
n
= 17) were analyzed. No significant difference in the distribution of the 10 studied regions was found between the cases and the controls. Nine strains did not harbor any of the selected regions: eight (30.8%) isolated from patients with gastritis only and one (5.9%) from a patient with gastric carcinoma. No association was found between the number of repeated sequences at the 3′ end of the
cagA
gene or the presence of tyrosine phosphorylation motifs and the clinical origin of the strains. The
virB10
homolog gene was the sole gene studied to be significantly expressed more often in cancer strains than in gastritis strains (
P
= 0.03).
Identification of Stress-Responsive Genes in
Streptococcus mutans
by Differential Display Reverse Transcription-PCR
Authors: Chia Jean-San , Lee Ya-Yu , Huang Peng-Tu , Chen Jen-Yang
Published: July 2002

Streptococcus mutans
, which causes dental caries in the human oral cavity and occasionally causes infective endocarditis in the heart, withstands adverse environmental stress through diverse alterations in protein synthesis. Differential gene expression in response to environmental stress was analyzed by RNA fingerprinting using arbitrarily primed PCR with a panel of 11mer primers designed for differential display in
Enterobacteriaceae
. Dot and Northern blot hybridization confirmed that the transcription of several genes was up- or down-regulated following exposure to acid shock from pH 7.5 to 5.5. RNA of a gene designated AP-185 (acid-stress protein) was induced specifically by acid treatment, while RNA of GSP-781 (general-stress protein) was up-regulated significantly when bacteria were exposed to high osmolarity and temperature, as well as low pH. The deduced amino acid sequence of AP-185 shares homology (78% identity) with branched-chain amino acid aminotransferase. Cloning and sequence analysis of GSP-781 revealed a potential secreted protein of a molecular mass of about 43 kDa and with a pI predicted to be 5.5. Transcriptional levels of another gene, designated AR-186 (acid-repressed protein), which encodes putative aconitase, were repressed by acid treatment but were enhanced by plasma or serum components. Analogous results were identified in
icd
and
citZ
genes, and repression of these genes, along with AR-186, was also observed when they were exposed to high osmolarity and temperature. These results indicate that differential regulation of specific genes at the transcriptional level is triggered by different stress and that genes responsible for glutamate biosynthesis in the citrate pathway are coordinately regulated during the stress response of
S. mutans
.
Elevated Cytokine and Chemokine Levels and Prolonged Pulmonary Airflow Resistance in a Murine
Mycoplasma pneumoniae
Pneumonia Model: a Microbiologic, Histologic, Immunologic, and Respiratory Plethysmographic Profile
Authors: Hardy Robert D. , Jafri Hasan S. , Olsen Kurt , Wordemann Meike , Hatfield Jeanine , Rogers Beverly B. , Patel Padma , Duffy Lynn , Cassell Gail , McCracken George H. , Ramilo Octavio
Published: July 2002

Because
Mycoplasma pneumoniae
is hypothesized to play an important role in reactive airway disease/asthma, a comprehensive murine model of
M. pneumoniae
lower respiratory infection was established. BALB/c mice were intranasally inoculated once with
M. pneumoniae
and sacrificed at 0 to 42 days postinoculation. All mice became infected and developed histologic evidence of acute pulmonary inflammation, which cleared by 28 days postinoculation. By contrast,
M. pneumoniae
persisted in the respiratory tract for the entire 42 days studied. Tumor necrosis factor alpha, gamma interferon, interleukin-6 (IL-6), KC (functional IL-8), MIP-1α, and MCP-1/JE concentrations were significantly elevated in bronchoalveolar lavage samples, whereas IL-4 and IL-10 concentrations were not significantly elevated. Pulmonary airflow resistance, as measured by plethysmography, was detected 1 day postinoculation and persisted even after pulmonary inflammation had resolved at day 28. Serum anti-
M. pneumoniae
immunoglobulin G titers were positive in all mice by 35 days. This mouse model provides a means to investigate the immunopathogenesis of
M. pneumoniae
infection and its possible role in reactive airway disease/asthma.
Analysis of the Capsule Biosynthetic Locus of
Mannheimia
(
Pasteurella
)
haemolytica
A1 and Proposal of a Nomenclature System
Authors: Lo Reggie Y. C. , McKerral Linda J. , Hills Tanya L. , Kostrzynska Magdalena
Published: July 2002

A 16-kbp DNA region that contains genes involved in the biosynthesis of the capsule of
Mannheimia
(
Pasteurella
)
haemolytica
A1 has been characterized. The gene cluster can be divided into three regions like those of the typical group II capsule biosynthetic clusters in gram-negative bacteria. Region 1 contains four genes (
wzt
,
wzm
,
wzf
, and
wza
) which code for an ATP-binding cassette transport apparatus for the secretion of the capsule materials across the membranes. The
M. haemolytica
A1
wzt
and
wzm
genes were able to complement
Escherichia coli kpsT
and
kpsM
mutants, respectively. Further, the ATP binding activity of Wzt was demonstrated by its affinity for ATP-agarose, and the lipoprotein nature of Wza was supported by [
3
H]palmitate labeling. Region 2 contains six genes; four genes (
orf1
/
2
/
3
/
4
) code for unique functions for which no homologues have been identified to date. The remaining two genes (
nmaA
and
nmaB
) code for homologues of UDP–
N
-acetylglucosamine-2-epimerase and UDP–
N
-acetylmannosamine dehydrogenase, respectively. These two proteins are highly homologous to the
E. coli
WecB and WecC proteins (formerly known as RffE and RffD), which are involved in the biosynthesis of enterobacterial common antigen (ECA). Complementation of an
E. coli rffE/D
mutant with the
M. haemolytica
A1
nmaA/B
genes resulted in the restoration of ECA biosynthesis. Region 3 contains two genes (
wbrA
and
wbrB
) which are suggested to be involved in the phospholipid modification of capsular materials.
Normal IncA Expression and Fusogenicity of Inclusions in
Chlamydia trachomatis
Isolates with the
incA
I47T Mutation
Authors: Pannekoek Yvonne , van der Ende Arie , Eijk Paul P. , van Marle Jan , de Witte Moniek A. , Ossewaarde Jacobus M. , van den Brule Adriaan J. C. , Morré Servaas A. , Dankert Jacob
Published: July 2002

To investigate the correlation between the
incA
I47T mutation in
Chlamydia trachomatis
and the nonfusogenic phenotype, the
incA
genes of 25 isolates were sequenced. Four major sequence types were identified. Seven isolates (28%) had the I47T mutation. Isolates representing the four sequence types expressed IncA in the membrane of one large single inclusion. In conclusion, the
incA
I47T mutation is not associated with the nonfusogenic phenotype.
Production of Matrix Metalloproteinases in Response to Mycobacterial Infection
Authors: Quiding-Järbrink Marianne , Smith Debbie A. , Bancroft Gregory J.
Published: July 2002

Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viableMycobacterium bovisBCG orMycobacterium tuberculosisH37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG orM. tuberculosisin vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-α), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-α or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-γ), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-γ treatment increased macrophage secretion of TNF-α but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-α available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG orM. tuberculosisinduced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-α, IL-18, and IFN-γ. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.
Lipopolysaccharides from Periodontopathic BacteriaPorphyromonas gingivalisandCapnocytophaga ochraceaAre Antagonists for Human Toll-Like Receptor 4
Authors: Yoshimura Atsutoshi , Kaneko Takashi , Kato Yoshifumi , Golenbock Douglas T. , Hara Yoshitaka
Published: July 2002

Toll-like receptors (TLRs) 2 and 4 have recently been identified as possible signal transducers for various bacterial ligands. To investigate the roles of TLRs in the recognition of periodontopathic bacteria by the innate immune system, a Chinese hamster ovary (CHO) nuclear factor-κB (NF-κB)-dependent reporter cell line, 7.7, which is defective in both TLR2- and TLR4-dependent signaling pathways was transfected with human CD14 and TLRs. When the transfectants were exposed to freeze-dried periodontopathic bacteria,Actinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Capnocytophaga ochracea, andFusobacterium nucleatum, and a non-oral bacterium,Escherichia coli, all species of the bacteria induced NF-κB-dependent CD25 expression in 7.7/huTLR2 cells. Although freeze-driedA.actinomycetemcomitans,F.nucleatum, andE.colialso induced CD25 expression in 7.7/huTLR4 cells, freeze-driedP.gingivalisdid not. Similarly, lipopolysaccharides (LPS) extracted fromA.actinomycetemcomitans,F.nucleatum, andE.coliinduced CD25 expression in 7.7/huTLR4 cells, but LPS fromP.gingivalisandC.ochraceadid not. Furthermore, LPS fromP.gingivalisandC.ochraceaattenuated CD25 expression in 7.7/huTLR4 cells induced by repurified LPS fromE.coli. LPS fromP.gingivalisandC.ochraceaalso inhibited the secretion of interleukin-6 (IL-6) from U373 cells, the secretion of IL-1β from human peripheral blood mononuclear cells, and ICAM-1 expression in human gingival fibroblasts induced by repurified LPS fromE.coli. These findings indicated that LPS fromP.gingivalisandC.ochraceaworked as antagonists for human TLR4. The antagonistic activity of LPS from these periodontopathic bacteria may be associated with the etiology of periodontal diseases.
Production of Prostaglandins and Leukotrienes by Pathogenic Fungi
Authors: Noverr Mairi C. , Toews Galen B. , Huffnagle Gary B.
Published: July 2002

These studies demonstrate that pathogenic fungi (dermatophytic, subcutaneous, and systemic) have the ability to produce eicosanoids both from simple metabolites and from arachidonic acid. Host-derived eicosanoids have been previously demonstrated to enhance fungal colonization and atopic disease development. Thus, fungus-derived eicosanoids represent a potential class of novel virulence factors.
Cell Differentiation Is a Key Determinant of Cathelicidin LL-37/Human Cationic Antimicrobial Protein 18 Expression by Human Colon Epithelium
Authors: Hase Koji , Eckmann Lars , Leopard John D. , Varki Nissi , Kagnoff Martin F.
Published: July 2002

Antimicrobial peptides are highly conserved evolutionarily and are thought to play an important role in innate immunity at intestinal mucosal surfaces. To better understand the role of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) in intestinal mucosal defense, we characterized the regulated expression and production of this peptide by human intestinal epithelium. LL-37/hCAP18 is shown to be expressed within epithelial cells located at the surface and upper crypts of normal human colon. Little or no expression was seen within the deeper colon crypts or within epithelial cells of the small intestine. Paralleling its expression in more differentiated epithelial cells in vivo, LL-37/hCAP18 mRNA and protein expression was upregulated in spontaneously differentiating Caco-2 human colon epithelial cells and in HCA-7 human colon epithelial cells treated with the cell differentiation-inducing agent sodium butyrate. LL-37/hCAP18 expression by colon epithelium does not require commensal bacteria, since LL-37/hCAP18 is produced with a similar expression pattern by epithelial cells in human colon xenografts that lack a luminal microflora. LL-37/hCAP18 mRNA was not upregulated in response to tumor necrosis factor alpha, interleukin 1α (IL-1α), gamma interferon, lipopolysaccharide, or IL-6, nor did the expression patterns and levels of LL-37/hCAP18 in the epithelium of the normal and inflamed colon differ. On the other hand, infection of HCA-7 cells withSalmonella entericaserovar Dublin or enteroinvasiveEscherichia colimodestly upregulated LL-37/hCAP18 mRNA expression. We conclude that differentiated human colon epithelium expresses LL-37/hCAP18 as part of its repertoire of innate defense molecules and that the distribution and regulated expression of LL-37/hCAP18 in the colon differs markedly from that of other enteric antimicrobial peptides, such as defensins.
Genomic Localization of a T Serotype Locus to a Recombinatorial Zone Encoding Extracellular Matrix-Binding Proteins in
Streptococcus pyogenes
Authors: Bessen Debra E. , Kalia Awdhesh
Published: July 2002

Streptococcus pyogenes
is an important bacterial pathogen afflicting humans. A striking feature is its extraordinary biological diversity, evident in the wide range of diseases it can cause and the antigenic heterogeneity present on its surface. The T antigens form the basis of a major serological typing scheme that is often used as an alternative or supplement to M typing. Unlike M typing, the genetic basis for T typing is poorly understood. In this report, the
tee6
gene is localized to a position ≈3.3 kb downstream from
prtF1
(or
sfbI
), which encodes the Fn-binding protein, protein F, a key virulence factor. Comparison of this portion of the genome with those of four additional strains reveals the presence of genes encoding a collagen-binding protein (Cpa) and a second Fn-binding protein (PrtF2 or PfbpI). This chromosomal region—here designated the FCT region—is ≈11 to 16 kb in length and is flanked at both ends by long stretches of highly conserved sequence. For each of the five strains, the FCT region contains a unique combination of semiconserved loci, indicative of extensive intergenomic recombination. The data provide evidence that the highly recombinatorial FCT region of the
S. pyogenes
genome is under strong selection for change in response to the host environment.
Monoclonal Antibody against thePlasmodium falciparumChitinase, PfCHT1, Recognizes a Malaria Transmission-Blocking Epitope inPlasmodium gallinaceumOokinetes Unrelated to the Chitinase PgCHT1
Authors: Langer Rebecca C. , Li Fengwu , Popov Vsevolod , Kurosky Alexander , Vinetz Joseph M.
Published: July 2002

To initiate invasion of the mosquito midgut,Plasmodiumookinetes secrete chitinases that are necessary to cross the chitin-containing peritrophic matrix en route to invading the epithelial cell surface. To investigate chitinases as potential immunological targets of blocking malaria parasite transmission to mosquitoes, a monoclonal antibody (MAb) was identified that neutralized the enzymatic activity of the sole chitinase ofPlasmodium falciparum, PfCHT1, identified to date. This MAb, designated 1C3, previously shown to react with an apical structure ofP. falciparumookinetes, also reacts with a discrete apical structure ofP. gallinaceumookinetes. In membrane feeding assays, MAb 1C3 markedly inhibitedP. gallinaceumoocyst development in mosquito midguts. MAb 1C3 affinity isolated an ∼210-kDa antigen which, under reducing conditions, became a 35-kDa antigen. This isolated 35-kDa protein cross-reacted with an antiserum raised against a synthetic peptide derived from theP. gallinaceumchitinase active site, PgCHT1, even though MAb 1C3 did not recognize native or recombinant PgCHT1 on Western blot. Therefore, this affinity-purified 35-kDa antigen appears similar to a previously identified protein, PgCHT2, a putative second chitinase ofP. gallinaceum. Epitope mapping indicated MAb 1C3 recognized a region of PfCHT1 that diverges from a homologous amino acid sequence conserved within sequenced chitinases ofP. berghei, P. yoelii, andP. gallinaceum(PgCHT1). A synthetic peptide derived from the mapped 1C3 epitope may be useful as a component of a subunit transmission-blocking vaccine.
Porphyromonas gingivalisLipopolysaccharide Is Both Agonist and Antagonist for p38 Mitogen-Activated Protein Kinase Activation
Authors: Darveau Richard P. , Arbabi Saman , Garcia Iris , Bainbridge Brian , Maier Ronald V.
Published: July 2002

Lipopolysaccharide (LPS) is a key inflammatory mediator. It has been proposed to function as an important molecule that alerts the host of potential bacterial infection. Although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained fromPorphyromonas gingivalis, an etiologic agent for periodontitis, evokes a highly unusual host cell response. Human monocytes respond to this LPS by the secretion of a variety of different inflammatory mediators, while endothelial cells do not. In addition,P. gingivalisLPS inhibits endothelial cell expression of E-selectin and interleukin 8 (IL-8) induced by other bacteria. In this report the ability ofP. gingivalisLPS to activate p38 mitogen-activated protein (MAP) kinase was investigated. It was found that p38 MAP kinase activation occurred in response toP. gingivalisLPS in human monocytes. In contrast, no p38 MAP kinase activation was observed in response toP. gingivalisLPS in human endothelial cells or CHO cells transfected with human Toll-like receptor 4 (TLR-4). In addition,P. gingivalisLPS was an effective inhibitor ofEscherichia coli-induced p38 MAP kinase phosphorylation in both endothelial cells and CHO cells transfected with human TLR-4. These data demonstrate thatP. gingivalisLPS activates the LPS-associated p38 MAP kinase in monocytes and that it can be an antagonist forE. coliLPS activation of p38 MAP kinase in endothelial and CHO cells. These data also suggest that although LPS is generally considered a bacterial component that alerts the host to infection, LPS fromP. gingivalismay selectively modify the host response as a means to facilitate colonization.
Role of Gamma Interferon inHelicobacter pyloriInduction of Inflammatory Mediators during Murine Infection
Authors: Obonyo Marygorret , Guiney Donald G. , Harwood Julia , Fierer Joshua , Cole Sheri P.
Published: July 2002

Gamma interferon (IFN-γ) has been proposed to play an important role inHelicobacter-related gastritis. Using the IFN-γ gene knockout (IFN-γ−/−) mouse model and a murine gastric epithelial cell line, GSM06, we demonstrated thatHelicobacter pylorimaximally induced macrophage inflammatory protein-2 (MIP-2) and inducible nitric oxide synthase (iNOS) mRNA only in wild-type mice. MIP-2 and iNOS mRNA were also induced byH. pyloriin GSM06 cells. Induction of cyclooxygenase 2 mRNA through IFN-γ was demonstrated in GSM06 cells. These data indicate that IFN-γ mediates the induction of MIP-2 and iNOS mRNA expression byH. pyloriin mice.
Role of Signal Sequence in Vaccine-Induced Protection against Experimental Coccidioidomycosis
Authors: Jiang Chengyong , Magee D. Mitchell , Ivey F. Douglas , Cox Rebecca A.
Published: July 2002

The vaccine efficacy of the gene sequence encoding the signal peptide of the antigen known as antigen 2 or proline-rich antigen (Ag2/PRA), an immunodominant antigen present in the cell wall of the fungal pathogenCoccidioides immitis, was investigated in a murine model of coccidioidomycosis. Expression plasmids for Ag2/PRA(1-18) DNA (signal sequence), Ag2/PRA(19-194) DNA (lacking the signal sequence), and Ag2/PRA(1-194) DNA (full length) were inserted in the pVR1012 vector, and the constructs were used to vaccinate the highly susceptible BALB/c mouse strain. Immunization with the signal gene sequence significantly reduced the fungal burden in the lungs and spleens of mice 12 days after intraperitoneal challenge with a lethal dose of 2,500C. immitisarthroconidia, to a level comparable to the protection induced in mice immunized with the full-length Ag2/PRA(1-194) DNA. The Ag2/PRA(19-194) gene protected mice but to a significantly lower level than the signal sequence or the full-length Ag2 gene. The immunizing capacity of Ag2/PRA(1-18) was not attributable to a nonspecific immunostimulatory effect of DNA, as evidenced by the fact that mice immunized with a frameshift mutation of Ag2/PRA(1-18) were not protected against challenge. Furthermore, a synthetic peptide corresponding to the translated sequence of Ag2/PRA(1-18) DNA protected mice, albeit at a lower level than the Ag2/PRA(1-18) DNA vaccine. The protection induced with the signal gene vaccine correlated with the production of gamma interferon when splenocytes from Ag2/PRA(1-18)-immunized mice were stimulated with recombinant full-length Ag2 and was not associated with the production of anti-Coccidioidesimmunoglobulin G antibody. This is the first study to establish that a signal peptide sequence alone, administered as a gene vaccine or synthetic peptide, can induce protective immunity against a microbial pathogen.
Cellular and Molecular Regulation of Vaccination with Heat Shock Protein 60 fromHistoplasma capsulatum
Authors: Deepe, Jr., George S. , Gibbons Reta S.
Published: July 2002

Vaccination with heat shock protein 60 (Hsp60) fromHistoplasma capsulatuminduces a protective immune response in mice. We explored the cellular and molecular requirements for the efficacy of recombinant Hsp60 in mice. Depletion of CD4+, but not CD8+, cells during the inductive phase of vaccination abolished protection, as assessed by survival and by the fungal burden in lungs and spleens. In the expressive phase, the elimination of CD4+or CD8+cells after immunization did not significantly alter fungal recovery or survival from a lethal challenge. Depletion of both subpopulations after Hsp60 vaccination resulted in a failure to control a lethal infection and a higher fungal burden in lungs and spleens. Cytokine release by spleen cells from mice vaccinated with Hsp60 produced substantially more gamma interferon and interleukin-10 and -12 than that of cells from mice immunized with eitherH. capsulatumrecombinant Hsp70 or bovine serum albumin. The generation of gamma interferon, but not of interleukin-10, was dependent on T cells, in particular CD4+cells. Treatment of Hsp60-immunized mice with monoclonal antibody to gamma interferon or interleukin-10 or -12 in the inductive phase of vaccination was accompanied by increased recovery of yeast cells from lungs and spleens and 100% mortality. Likewise, the neutralization of gamma interferon or interleukin-12 abolished the protective effect of Hsp60 in the expressive phase. These results delineate the complexity of the regulatory elements necessary for vaccination against this fungus.
Hierarchy of Susceptibility of Dendritic Cell Subsets to Infection byLeishmania major: Inverse Relationship to Interleukin-12 Production
Authors: Henri Sandrine , Curtis Joan , Hochrein Hubertus , Vremec David , Shortman Ken , Handman Emanuela
Published: July 2002

Dendritic cells (DCs) are professional antigen-presenting cells which initiate and regulate T-cell immune responses. Here we show that murine splenic DCs can be ranked on the basis of their ability to phagocytose and harbor the obligately intracellular parasiteLeishmania major.CD4+CD8−DCs are the most permissive host cells forL. majoramastigotes, followed by CD4−CD8−DCs; CD4−CD8+cells are the least permissive. However, the least susceptible CD4−CD8+DC subset was the best interleukin-12 producer in response to infection. Infection did not induce in any DC subset production of the proinflammatory cytokine gamma interferon and nitric oxide associated with the induction of Th1 responses. The number of parasites phagocytosed by DCs was low, no more than 3 organisms per cell, compared to more than 10 organisms per macrophage. In infected DCs, the parasites are located in a parasitophorous vacuole containing both major histocompatibility complex (MHC) class II and lysosome-associated membrane protein 1 molecules, similar to their location in the infected macrophage. The parasite-driven redistribution of MHC class II to this compartment indicates that infected DCs should be able to present parasite antigen.
Efficient Spore Synthesis in
Bacillus subtilis
Depends on the CcdA Protein
Authors: Schiött Torbjörn , Hederstedt Lars
Published: July 2002

CcdA is known to be required for the synthesis of
c
-type cytochromes in
Bacillus subtilis
, but the exact function of this membrane protein is not known. We show that CcdA also plays a role in spore synthesis. The expression of
ccdA
and the two downstream genes
yneI
and
yneJ
was analyzed. There is a promoter for each gene, but there is only one transcription terminator, located after the
yneJ
gene. The promoter for
ccdA
was found to be weak and was active mainly during the transition from exponential growth to stationary phase. The promoters for
yneI
and
yneJ
were both active in the exponential growth phase. The levels of the CcdA and YneJ proteins in the membrane were consistent with the observed promoter activities. The
ccdA
promoter activity was independent of whether the
ccdA-yneI-yneJ
gene products were absent or overproduced in the cell. It is shown that the four known cytochromes
c
in
B. subtilis
and the YneI and YneJ proteins are not required for sporulation. The combined data from analysis of sporulation-specific sigma factor activity, resistance properties of spores, and spore morphology indicate that CcdA deficiency affects stage V in sporulation. We conclude that CcdA, YneI, and YneJ are functionally unrelated proteins and that the role of CcdA in cytochrome
c
and spore synthesis probably relates to sulfhydryl redox chemistry on the outer surface of the cytoplasmic membrane.
Cross-Pathway Regulation in
Saccharomyces cerevisiae
: Activation of the Proline Utilization Pathway by Gal4p In Vivo
Authors: D'Alessio Michael , Brandriss Marjorie C.
Published: July 2002

The Put3p and Gal4p transcriptional activators are members of a distinct class of fungal regulators called the Cys
6
Zn(II)
2
binuclear cluster family. This family includes over 50 different
Saccharomyces cerevisiae
proteins that share a similar domain organization. Gal4p activates the genes of the galactose utilization pathway permitting the use of galactose as the sole source of carbon and energy. Put3p controls the expression of the proline utilization pathway that allows yeast cells to grow on proline as the sole nitrogen source. We report that Gal4p can activate the
PUT
structural genes in a strain lacking Put3p. We also show that the activation of
PUT2
by Gal4p depends on the presence of the inducer galactose and the Put3p binding site and that activation increases with increased dosage of Gal4p. Put3p cannot activate the
GAL
genes in the absence of Gal4p. Our in vivo results confirm previously published in vitro data showing that Gal4p is more promiscuous than Put3p in its DNA binding ability. The results also suggest that under appropriate circumstances, Gal4p may be able to function in place of a related family member to activate expression.
Defining a
rob
Regulon in
Escherichia coli
by Using Transposon Mutagenesis
Authors: Bennik Marjon H. J. , Pomposiello Pablo J. , Thorne Derek F. , Demple Bruce
Published: July 2002

The Rob protein of
Escherichia coli
is a member of the AraC-XylS family of prokaryotic transcriptional regulators and is expressed constitutively. Deletion of the
rob
gene increases susceptibility to organic solvents, while overexpression of Rob increases tolerance to organic solvents and resistance to a variety of antibiotics and to the superoxide-generating compound phenazine methosulfate. To determine whether constitutive levels of Rob regulate basal gene expression, we performed a MudJ transposon screen in a
rob
deletion mutant containing a plasmid that allows for controlled
rob
gene expression. We identified eight genes and confirmed that seven are transcriptionally activated by normal expression of Rob from the chromosomal
rob
gene (
inaA
,
marR
,
aslB
,
ybaO
,
mdlA
,
yfhD
, and
ybiS
). One gene,
galT
, was repressed by Rob. We also demonstrated by Northern analysis that basal expression of
micF
is significantly higher in wild-type
E. coli
than in a
rob
deletion mutant. Rob binding to the promoter regions of most of these genes was substantiated in electrophoretic mobility shift assays. However, Mu insertions in individual Rob-regulated genes did not affect solvent sensitivity. This phenotype may depend on changes in the expression of several of these Rob-regulated genes or on other genes that were not identified. Rob clearly affects the basal expression of genes with a broad range of functions, including antibiotic resistance, acid adaptation, carbon metabolism, cell wall synthesis, central intermediary metabolism, and transport. The magnitudes of Rob's effects are modest, however, and the protein may thus play a role as a general transcription cofactor.
Transcription of the
nfrA-ywcH
Operon from
Bacillus subtilis
Is Specifically Induced in Response to Heat
Authors: Moch Christine , Schrögel Oliver , Allmansberger Rudolf
Published: July 2002

The NfrA protein, an oxidoreductase from the soil bacterium
Bacillus subtilis
, is synthesized during the stationary phase and in response to heat. Analysis of promoter mutants revealed that the
nfrA
gene belongs to the class III heat shock genes in
B. subtilis
. An approximate 10-fold induction at both the transcriptional and the translational levels was found after thermal upshock. This induction resulted from enhanced synthesis of mRNA. Genetic and Northern blot analyses revealed that
nfrA
and the gene downstream of
nfrA
are transcribed as a bicistronic transcriptional unit. The unstable full-length transcript is processed into two short transcripts encoding
nfrA
and
ywcH
. The
nfrA-ywcH
operon is not induced by salt stress or by ethanol. According to previously published data, the transcription of class III genes in general is activated in response to the addition of these stressors. However, this conclusion is based on experiments which lacked a valid control. Therefore, it seems possible that the transcription of all class III genes is specifically induced by heat shock.
Metabolic Pathways for Cytotoxic End Product Formation from Glutamate- and Aspartate-Containing Peptides by
Porphyromonas gingivalis
Authors: Takahashi Nobuhiro , Sato Takuichi , Yamada Tadashi
Published: July 2002

Metabolic pathways involved in the formation of cytotoxic end products by
Porphyromonas gingivalis
were studied. The washed cells of
P. gingivalis
ATCC 33277 utilized peptides but not single amino acids. Since glutamate and aspartate moieties in the peptides were consumed most intensively, a dipeptide of glutamate or aspartate was then tested as a metabolic substrate of
P. gingivalis. P. gingivalis
cells metabolized glutamylglutamate to butyrate, propionate, acetate, and ammonia, and they metabolized aspartylaspartate to butyrate, succinate, acetate, and ammonia. Based on the detection of metabolic enzymes in the cell extracts and stoichiometric calculations (carbon recovery and oxidation/reduction ratio) during dipeptide degradation, the following metabolic pathways were proposed. Incorporated glutamylglutamate and aspartylaspartate are hydrolyzed to glutamate and aspartate, respectively, by dipeptidase. Glutamate is deaminated and oxidized to succinyl-coenzyme A (CoA) by glutamate dehydrogenase and 2-oxoglutarate oxidoreductase. Aspartate is deaminated into fumarate by aspartate ammonia-lyase and then reduced to succinyl-CoA by fumarate reductase and acyl-CoA:acetate CoA-transferase or oxidized to acetyl-CoA by a sequential reaction of fumarase, malate dehydrogenase, oxaloacetate decarboxylase, and pyruvate oxidoreductase. The succinyl-CoA is reduced to butyryl-CoA by a series of enzymes, including succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and butyryl-CoA oxidoreductase. A part of succinyl-CoA could be converted to propionyl-CoA through the reactions initiated by methylmalonyl-CoA mutase. The butyryl- and propionyl-CoAs thus formed could then be converted into acetyl-CoA by acyl-CoA:acetate CoA-transferase with the formation of corresponding cytotoxic end products, butyrate and propionate. The formed acetyl-CoA could then be metabolized further to acetate.
Genetic and Biochemical Characterization of 4-Carboxy-2-Hydroxymuconate-6-Semialdehyde Dehydrogenase and Its Role in the Protocatechuate 4,5-Cleavage Pathway in
Sphingomonas paucimobilis
SYK-6
Authors: Masai Eiji , Momose Kiyotaka , Hara Hirofumi , Nishikawa Seiji , Katayama Yoshihiro , Fukuda Masao
Published: July 2002

Protocatechuate (PCA) is the key intermediate metabolite in the lignin degradation pathway of
Sphingomonas paucimobilis
SYK-6 and is metabolized to pyruvate and oxaloacetate via the PCA 4,5-cleavage pathway. We characterized the 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase gene (
ligC
). CHMS is the 4,5-cleavage product of PCA and is converted into 2-pyrone-4,6-dicarboxylate (PDC) by LigC. We found that
ligC
was located 295 bp downstream of
ligB
, which encodes the large subunit of the PCA 4,5-dioxygenase. The
ligC
gene consists of a 945-bp open reading frame encoding a polypeptide with a molecular mass of 34,590 Da. The deduced amino acid sequence of
ligC
showed 19 to 20% identity with 3-chlorobenzoate
cis
-dihydrodiol dehydrogenase of
Alcaligenes
sp. strain BR60 and phthalate
cis
-dihydrodiol dehydrogenases of
Pseudomonas putida
NMH102-2 and
Burkholderia cepacia
DBO1, which are unrelated to group I, II, and III microbial alcohol dehydrogenases (M. F. Reid and C. A. Fewson, Crit. Rev. Microbiol. 20:13–56, 1994). The
ligC
gene was expressed in
Escherichia coli
and LigC was purified to near homogeneity. Production of PDC from CHMS catalyzed by LigC was confirmed in the presence of NADP
+
by electrospray ionization-mass spectrometry and gas chromatography-mass spectrometry. LigC is a homodimer. The isoelectric point, optimum pH, and optimum temperature were estimated to be 5.3, 8.0, and 25°C, respectively. The

K
m

for NADP
+
was estimated to be 24.6 ± 1.5 μM, which was approximately 10 times lower than that for NAD
+
(252 ± 3.9 μM). The

K
m

s for CHMS in the presence of NADP
+
and NAD
+
are 26.0 ± 0.5 and 20.6 ± 1.0 μM, respectively. Disruption of
ligC
in
S. paucimobilis
SYK-6 prevented growth with vanillate. Only PCA was accumulated during the incubation of vanillate with the whole cells of the
ligC
insertion mutant (DLC), indicating a lack of PCA 4,5-dioxygenase activity in DLC. However, the introduction of
ligC
into DLC restored its ability to grow on vanillate. PDC was suggested to be an inducer for
ligAB
gene expression.
Interdependence of Activation at
rhaSR
by Cyclic AMP Receptor Protein, the RNA Polymerase Alpha Subunit C-Terminal Domain, and RhaR
Authors: Holcroft Carolyn C. , Egan Susan M.
Published: July 2002

The
Escherichia coli rhaSR
operon encodes two AraC family transcription activators, RhaS and RhaR, and is activated by RhaR in the presence of
l
-rhamnose. β-Galactosidase assays of various
rhaS-lacZ
promoter fusions combined with mobility shift assays indicated that a cyclic AMP receptor protein (CRP) site located at −111.5 is also required for full activation of
rhaSR
expression. To address the mechanisms of activation by CRP and the RNA polymerase α-subunit C-terminal domain (α-CTD) at
rhaSR
, we tested the effects of alanine substitutions in CRP activating regions 1 and 2, overexpression of a truncated version of α (α-Δ235), and alanine substitutions throughout α-CTD. We found that DNA-contacting residues in α-CTD are required for full activation, and for simplicity, we discuss α-CTD as a third activator of
rhaSR
. CRP and RhaR could each partially activate transcription in the absence of the other two activators, and α-CTD was not capable of activation alone. In the case of CRP, this suggests that this activation involves neither an α-CTD interaction nor cooperative binding with RhaR, while in the case of RhaR, this suggests the likelihood of direct interactions with core RNA polymerase. We also found that CRP, RhaR, and α-CTD each have synergistic effects on activation by the others, suggesting direct or indirect interactions among all three. We have some evidence that the α-CTD–CRP and α-CTD–RhaR interactions might be direct. The magnitude of the synergistic effects was usually greater with just two activators than with all three, suggesting possible redundancies in the mechanisms of activation by CRP, α-CTD, and RhaR.
CTX Prophages in Classical Biotype
Vibrio cholerae
: Functional Phage Genes but Dysfunctional Phage Genomes
Authors: Davis Brigid M. , Moyer Kathryn E. , Boyd E. Fidelma , Waldor Matthew K.
Published: July 2002

CTXφ is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by
Vibrio cholerae
. CTX prophages in O1 El Tor and O139 strains of
V. cholerae
are found within arrays of genetically related elements integrated at a single locus within the
V. cholerae
large chromosome. The prophages of O1 El Tor and O139 strains generally yield infectious CTXφ. In contrast, O1 classical strains of
V. cholerae
do not produce CTXφ, although they produce cholera toxin and they contain CTX prophages integrated at two sites. We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally. The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXφ. Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages. RS1, a genetic element that is closely related to CTXφ and is often interspersed with CTX prophages in El Tor strains, was not detected in classical
V. cholerae
. Our model for CTXφ production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction. Thus, failure of O1 classical strains of
V. cholerae
to produce CTXφ is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.
Characterization of a Major Cluster ofnif,fix, and Associated Genes in a Sugarcane Endophyte,Acetobacter diazotrophicus
Authors: Lee Sunhee , Reth Alexander , Meletzus Dietmar , Sevilla Myrna , Kennedy Christina
Published: July 2002

A major 30.5-kb cluster ofnifand associated genes ofAcetobacter diazotrophicus(syn.Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguousnif-fixand associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of thenif-fixcluster inAzospirillum brasilense, while the individual gene products are more similar to those in species ofRhizobiaceaeor inRhodobacter capsulatus.
Cytochrome
c
′ from
Rhodobacter capsulatus
Confers Increased Resistance to Nitric Oxide
Authors: Cross Richard , Aish Joanne , Paston Samantha J. , Poole Robert K. , Moir James W. B.
Published: July 2002

We report the cloning and sequencing of the gene containing cytochrome
c
′ (
cycP
) from the photosynthetic purple bacterium
Rhodobacter capsulatus
and the regions flanking that gene. Mutant strains unable to synthesize cytochrome
c
′ had increased sensitivity to nitrosothiols and to nitric oxide (which binds to the heme moiety of cytochrome
c
′).
Cloning and Characterization of the Lipooligosaccharide Galactosyltransferase II Gene of
Haemophilus ducreyi
Authors: Sun Shuhua , Schilling Birgit , Tarantino Laurie , Tullius Michael V. , Gibson Bradford W. , Munson Robert S.
Published: July 2002

Haemophilus ducreyi
is the etiologic agent of chancroid, a genital ulcer disease. The lipooligosaccharide (LOS) is considered to be a major virulence determinant and has been implicated in the adherence of
H. ducreyi
to keratinocytes. Strain A77, an isolate from the Paris collection, is serum sensitive, poorly adherent to fibroblasts, and deficient in microcolony formation. Structural analysis indicates that the LOS of strain A77 lacks the galactose residue found in the
N
-acetyllactosamine portion of the strain 35000HP LOS as well as the sialic acid substitution. From an
H. ducreyi
35000HP genomic DNA library, a clone complementing the defect in A77 was identified by immunologic screening with monoclonal antibody (MAb) 3F11, a MAb which recognizes the
N
-acetyllactosamine portion of strain 35000HP LOS. The clone contained a 4-kb insert that was sequenced. One open reading frame which encodes a protein with a molecular weight of 33,400 was identified. This protein has homology to glycosyltransferases of
Haemophilus influenzae
,
Haemophilus somnus
,
Neisseria
species, and
Pasteurella haemolytica
. The putative
H. ducreyi
glycosyltransferase gene was insertionally inactivated, and an isogenic mutant of strain 35000HP was constructed. The most complex LOS glycoform produced by the mutant has a mobility on sodium dodecyl sulfate-polyacrylamide gel identical to that of the LOS of strain A77 and lacks the 3F11-binding epitope. Structural studies confirm that the most complex glycoform of the LOS isolated from the mutant lacks the galactose residue found in the
N
-acetyllactosamine portion of the strain 35000HP LOS. Although previously published data suggested that the serum-sensitive phenotype of A77 was due to the LOS mutation, we observed that the complemented A77 strain retained its serum-sensitive phenotype and that the galactosyltransferase mutant retained its serum-resistant phenotype. Thus, the serum sensitivity of strain A77 cannot be attributed to the galactosyltransferase mutation in strain A77.
Identification of
ccdA
in
Paracoccus pantotrophus
GB17: Disruption of
ccdA
Causes Complete Deficiency in
c
-Type Cytochromes
Authors: Bardischewsky Frank , Friedrich Cornelius G.
Published: July 2002

A transposon Tn
5-mob
insertional mutant of
Paracoccus pantotrophus
GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional
c-
type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a
c
-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn
5-mob
-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA,
ccdA
, and ORFB, with the latter being oriented divergently. ORFA and
ccdA
were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn
5-mob
was inserted into
ccdA
. The deduced ORFA product showed no similarity to any protein in databases. However, the
ccdA
gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome
c
biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of
P. pantotrophus
is 42% identical to a protein predicted by ORF2, and its location within the
sox
gene cluster coding for lithotrophic sulfur oxidation suggested a different function.
In Vitro Processing of the 16S rRNA of the Thermophilic Archaeon
Sulfolobus solfataricus
Authors: Ciammaruconi Andrea , Londei Paola
Published: July 2002

In this paper we have analyzed the processing in vitro of the 16S rRNA of the thermophilic archaeon
Sulfolobus solfataricus
, using pre-rRNA substrates transcribed in vitro and different protein preparations as the source of processing enzymes. We show that the 5′ external transcribed spacer of the
S. solfataricus
pre-rRNA transcript contains a target site for a specific endonuclease, which recognizes a conserved sequence also existing in the early A0 and 0 processing sites of
Saccharomyces cerevisiae
and vertebrates. This site is present in other members of the kingdom
Crenarchaeota
but apparently not in the
Euryarchaeota
. Furthermore,
S. solfataricus
pre-16S RNA is processed within the double-helical stem formed by the inverted repeats flanking the 16S RNA sequence, in correspondence with a bulge-helix-bulge motif. The endonuclease responsible for this cleavage is present in both the
Crenarchaeota
and the
Euryarchaeota
. The processing pattern remained the same when the substrate was a 30S ribonucleoprotein particle instead of the naked RNA. Maturation of either the 5′ or the 3′ end of the 16S RNA molecule was not observed, suggesting either that maturation requires conditions not easily reproducible in vitro or that the responsible endonucleases are scarcely represented in cell extracts.
Transcription-Induced Cytosine-to-Thymine Mutations Are Not Dependent on Sequence Context of the Target Cytosine
Authors: Beletskii Anton , Bhagwat Ashok S.
Published: July 2002

We showed previously that transcription of a plasmid-borne
kan
allele increases C-to-T mutations in the nontranscribed strand. Using two new plasmid-borne
kan
alleles, one
cmp
allele, and a chromosomal
kan
allele, we found in this study that transcription-induced mutations are not limited to specific genes, alleles, or locations and are likely to be a general property of transcript elongation in
Escherichia coli
.