bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Characterization of a Putative Transcriptional Regulator Controlling the Expression of Fouling Inhibitors in
Pseudoalteromonas tunicata
Authors: Egan Suhelen , James Sally , Kjelleberg Staffan
Published: July 2002

The dark green pigmented marine bacterium
Pseudoalteromonas tunicata
colonizes living surfaces and produces a range of extracellular compounds that inhibit common fouling organisms, including marine invertebrate larvae, algae, bacteria, and fungi. We have observed a positive correlation between the antifouling activity of
P. tunicata
strain D2 and the expression of pigmentation. To address the hypothesis that pigmentation and antifouling may be jointly regulated in this organism and to begin to identify potential regulatory elements, we used transposon mutagenesis to generate a strain of
P. tunicata
deficient in antifouling activity. The data presented here describe the phenotypic and molecular characterization of a nonpigmented transposon mutant strain of
P. tunicata
(D2W2). Analyses of the antifouling capabilities of D2W2 demonstrate that this strain is deficient in the ability to inhibit each of the target fouling organisms. Genetic analysis of D2W2 identified a gene, designated
wmpR
(white mutant phenotype), with high sequence similarity to transcriptional regulators ToxR from
Vibrio cholerae
and CadC from
Escherichia coli
. Two-dimensional polyacrylamide gel electrophoresis analysis revealed that WmpR is essential for the expression of a significant subset of stationary-phase-induced proteins likely to be important for the synthesis of fouling inhibitors. The identification of a gene involved in the regulation of expression of antifouling phenotypes will contribute to the understanding of the interactions between bacteria and other surface-colonizing organisms in the marine environment.
Genomic Subtraction To Identify and Characterize Sequences of Shiga Toxin-Producing
Escherichia coli
O91:H21
Authors: Pradel Nathalie , Leroy-Setrin Sabine , Joly Bernard , Livrelli Valérie
Published: July 2002

To identify Shiga toxin-producing
Escherichia coli
genes associated with severe human disease, a genomic subtraction technique was used with hemolytic-uremic syndrome-associated O91:H21 strain CH014 and O6:H10 bovine strains. The method was adapted to the Shiga toxin-producing
E. coli
genome: three rounds of subtraction were used to isolate DNA fragments specific to strain CH014. The fragments were characterized by genetic support analysis, sequencing, and hybridization to the genome of a collection of Shiga toxin-producing
E. coli
strains. A total of 42 fragments were found, 19 of which correspond to previously identified unique DNA sequences in the enterohemorrhagic
E. coli
EDL933 reference strain, including 7 fragments corresponding to prophage sequences and others encoding candidate virulence factors, such a SepA homolog protein and a fimbrial usher protein. In addition, the subtraction procedure yielded plasmid-related sequences from
Shigella flexneri
and enteropathogenic and Shiga toxin-producing
E. coli
virulence plasmids. We found that lateral gene transfer is extensive in strain CH014, and we discuss the role of genomic mobile elements, especially bacteriophages, in the evolution and possible transfer of virulence determinants.
Development of Monoclonal Antibodies against
Ureaplasma urealyticum
Serotypes and Their Use for Serotyping Clinical Isolates
Authors: Echahidi Fedoua , Muyldermans Gaëtan , Lauwers Sabine , Naessens Anne
Published: July 2002

Monoclonal antibodies (MAbs) against
Ureaplasma urealyticum
serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of
U. urealyticum
strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.
Molecular Investigation of Transmission of Human Immunodeficiency Virus Type 1 in a Criminal Case
Authors: Machuca Roberto , Jørgensen Louise B. , Theilade Peter , Nielsen Claus
Published: July 2002

Very few criminal cases involving human immunodeficiency virus type 1 (HIV-1) transmission have been described. We report on an HIV-1 transmission case with a child being infected by an HIV-1-positive man. The objective was to determine through molecular epidemiology and phylogenetic analyses whether HIV-1 from the HIV-1-positive man could be the source of infection in the HIV-1-positive child, as claimed by the authorities. We conducted genetic analysis of three different parts of the HIV-1 genome (
gag, pol
, and
env
) by PCR, direct-sequencing, and phylogenetic analyses. We used maximum likelihood, maximum parsimony, and neighbor-joining methods for the phylogenetic analyses to investigate whether the sequences from the man and the child were related. We found that the viral sequences from the man and the child formed separate clusters in all of the phylogenetic analyses compared to the local controls. A unique amino acid deletion was identified in the C2-V3-C3 region of the
env
gene in the virus from the man and the child. These results were used in the criminal court to elucidate whether the virus from the man was related to the virus from the child. In summary, the results from the phylogenetic analyses, the sequence distances between the virus from the man and the virus from the child, and the identification of the unique molecular fingerprint in the
env
gene together indicated that the virus from the man and the virus from the child were epidemiologically linked.
Impaired Bactericidal Activity and Host Resistance toListeria monocytogenesandBorrelia burgdorferiin Rats Administered an Acute Oral Regimen of Ethanol
Authors: Pavia Charles S. , Harris Cynthia M. , Kavanagh Marie
Published: July 2002

A rat model was used to examine how ethanol ingestion may interfere with antimicrobial immunity both in vitro and in vivo. Nonimmune Long-Evans rats were given a short-course treatment orally with excessive amounts of ethanol. Their spleens were removed at the time of sacrifice, and separate spleen cell suspensions were prepared and tested in vitro for their ability to kill two bacterial pathogens,Listeria monocytogenesandBorrelia burgdorferi.After the bacteria were mixed separately with various concentrations of spleen cells, it was found that spleen cells from the ethanol-treated rats killed fewer bacteria than matching pair-fed controls, based on counts of the number of cultured CFU (forListeria) or based on microscopic examination (forBorrelia). For the in vivo studies, ethanol-treated and control rats were infected intraperitoneally withListeria, and then, 1 to 3 days later, they were assessed for systemic infection based on the numbers of organisms present in their livers and spleens. Numbers of bacterial CFU for both organs were significantly higher in the group fed ethanol for the first 2 days after listerial challenge. These results support the concept that acute exposure to high levels of ethanol can impair host defense mechanisms, especially those expressed at the cellular level, which could lead to increased susceptibility to certain types of infections.
Attachment ofToxoplasma gondiito a Specific Membrane Fraction of CHO Cells
Authors: Dutta Chaitali , Grimwood Jane , Kasper Lloyd H.
Published: July 2002

We have observed previously that attachment ofToxoplasma gondiito synchronized host cells is considerably increased at the mid-S phase (4 h postrelease). Synchronized CHO host cells at the mid-S phase were fractionated by molecular weight, and the antigens were used to produce a panel of polyclonal mouse antisera. The polyclonal antisera raised against fraction 4 with molecular mass ranging approximately from 18 to 40 kDa significantly reduced attachment to mid-S-phase host cells. Immunofluorescence assays demonstrated strong reactivity to mid-S-phase host cells and identified a number of potential receptors on Western blots. These data indicate that there is a specific host membrane receptor for parasite attachment that is upregulated during the mid-S phase of the host cell cycle.
Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen ofMycobacterium bovisBCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis
Authors: Lefèvre Philippe , Denis Olivier , De Wit Lucas , Tanghe Audrey , Vandenbussche Paul , Content Jean , Huygen Kris
Published: July 2002

Using spleen cells from mice vaccinated with liveMycobacterium bovisBCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen anM. bovisBCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with theM. tuberculosisLppx protein and that it contained a sequence 94% identical with theM. leprae38-mer polypeptide 13B3 recognized by T cells from killedM. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenousM. tuberculosischallenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenousM. tuberculosischallenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test.
Construction and Characterization of aSalmonella entericaSerovar Typhimurium Clone Expressing a Salivary Adhesin ofStreptococcus mutansunder Control of the Anaerobically InduciblenirBPromoter
Authors: Huang Yan , Hajishengallis George , Michalek Suzanne M.
Published: July 2002

AttenuatedSalmonella entericaserovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) ofStreptococcus mutansantigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the induciblenirBpromoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing ofSalmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains thenirBpromoter. The resulting plasmid was introduced into serovar Typhimurium by electroporation. Production of the SBR-CTA2/B chimeric protein under anaerobic conditions was verified by enzyme-linked immunosorbent assay of whole-cell lysates on plates coated with GM1ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium undernirBcontrol. Anaerobic expression of SBR was confirmed by Western blotting of whole-cell lysates probed with anti-SBR antibodies. The resulting serovar Typhimurium strains were administered by either the oral or the intranasal route to mice, and colonization was assessed by microbiologic analysis of dissociated spleens, Peyer's patches (PP), and nasal tissues. High numbers of the recombinant strains persisted in PP and spleen for at least 21 days following oral challenge. A single intranasal administration of theSalmonellaclones to mice also resulted in the colonization of the nasal tissues by the recombinant bacteria. Salmonellae were recovered from nasal lymphoid tissues, superficial lymph nodes, internal jugular lymph nodes, PP, and spleens of mice for at least 21 days after challenge. This study provides quantitative evidence for colonization bySalmonellastrains expressing a recombinant protein under the control of the induciblenirBpromoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.
Identification and Genetic Characterization of
Haemophilus influenzae
Genetic Island 1
Authors: Chang Chih-Ching , Gilsdorf Janet R. , DiRita Victor J. , Marrs Carl F.
Published: July 2002

The type b capsule of pathogenic
Haemophilus influenzae
is a critical factor for
H. influenzae
survival in the blood and the establishment of invasive infections. Other pathogenic factors associated with type b strains may also play a role in invasion and sustained bacteremia, leading to the seeding of deep tissues. The gene encoding haemocin is the only noncapsular gene found to be specific to type b strains until now. Here we report the discovery of an approximately 16-kb genetic locus, HiGI1, that is present primarily in type b strains. Pulsed-field gel electrophoresis and Southern hybridization were used to map this new locus between
secG
(HI0445) and
fruA
(HI0446), which are contiguous in Rd, a nonpathogenic derivative of a serotype d strain. It is inserted at the 3′ end of tRNA
4
Leu
and has regions whose G+C content differs from the average genomic G+C content of
H. influenzae
. An integrase gene, which encodes a CP4-57 like integrase, is located downstream of tRNA
4
Leu
. Hybridization probes based on the sequences within the HiGI1 locus have been used to screen 61
H. influenzae
strains (2 type a, 22 type b, 2 type c, 1 type d, 3 type e, 7 type f, and 21 nontypeable
H. influenzae
[NTHi]) from our collection. This HiGI1 locus exists in all 22 type b strains and two NTHi strains and is likely to have been acquired by an ancestral type b strain.
The Metalloproteinase Inhibitor GI5402 Inhibits Endotoxin-Induced Soluble CD27 and CD16 Release in Healthy Humans
Authors: Dekkers Pascale E. P. , ten Hove Tessa , Lauw Fanny N. , Koene Harry R. , Lumley Philip , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

Metalloproteinases have been implicated in the cleavage of a number of cell surface immune receptors. Oral administration of the metalloproteinase inhibitor GI5402 attenuated the release of soluble CD27 and CD16 into the circulation after intravenous endotoxin injection in healthy humans.
Construction and Characterization of
Haemophilus ducreyi
Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats
Authors: Filiatrault Melanie J. , Gibson Bradford W. , Schilling Birgit , Sun Shuhua , Munson Robert S. , Campagnari Anthony A.
Published: July 2002

To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of
Haemophilus ducreyi
35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of
H. ducreyi
35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of
Escherichia coli
. A second ORF had homology to the LgtF (glucosyltransferase) of
Neisseria meningitidis
. Individual isogenic mutants lacking expression of the putative
H. ducreyi
heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in
trans
to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-
N
-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel
H. ducreyi
mutants provide important tools for studying the regulation of LOS assembly and biosynthesis.
Antigen Specificity of T-Cell Response toMycobacterium aviumInfection in Mice
Authors: Pais Teresa F. , Cunha Joana Feijó , Appelberg Rui
Published: July 2002

T cells fromMycobacterium avium-infected C57BL/6 mice reacted to culture filtrate, envelope, and cytosol proteins and to fractions obtained from these proteins. Multiple targets were recognized, such as 29- to 45-kDa and <21-kDa antigens of the culture filtrate, antigens of around 30 kDa in the envelope and cytosol, and 45- to 116-kDa proteins in the envelope.
Role ofNramp1Deletion inChlamydiaInfection in Mice
Authors: Pal Sukumar , Peterson Ellena M. , de la Maza Luis M.
Published: July 2002

Elicited macrophages from 129sv mice with a functional deletion of the natural-resistance-associated macrophage protein 1 gene (Nramp1) were shown to be as susceptible as wild-type mice to infection with theChlamydia trachomatismouse pneumonitis and L3 serovars and toChlamydia pneumoniae. Furthermore, the two groups of mice were shown to be similarly susceptible to an intranasal infection with these microorganisms. In conclusion, theNramp1gene does not appear to play a major role in the regulation of the susceptibility of mice to a chlamydial infection.
The Major Acute-Phase Protein, Serum Amyloid P Component, in Mice Is Not Involved in Endogenous Resistance against Tumor Necrosis Factor Alpha-Induced Lethal Hepatitis, Shock, and Skin Necrosis
Authors: Van Molle Wim , Hochepied Tino , Brouckaert Peter , Libert Claude
Published: July 2002

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) induces lethal hepatitis when injected intod-(+)-galactosamine-sensitized mice on the one hand or systemic inflammatory response syndrome (SIRS) in normal mice on the other hand. We studied whether serum amyloid P component (SAP), the major acute-phase protein in mice, plays a protective role in both lethal models. For this purpose, we used SAP0/0mice generated by gene targeting. We studied the lethal response of SAP0/0or SAP+/+mice to both lethal triggers but found no differences in the sensitivity of both types of mice. We also investigated whether SAP is involved in establishing two types of endogenous protection: one using a single injection of interleukin-1β (IL-1β) for desensitization and clearly involving a liver protein, the other by tolerizing mice for 5 days using small doses of human TNF-α. Although after IL-1β or after tolerization the SAP levels in the serum had risen fourfold in the control mice and not in the SAP0/0mice, the same extents of desensitization and tolerization were achieved. Finally, we observed that the induction of hemorrhagic necrosis in the skin of mice by two consecutive local injections with TNF-α was not altered in SAP0/0mice. We conclude that the presence or absence of SAP has no influence on the sensitivity of mice to TNF-α-induced hepatitis, SIRS, and hemorrhagic necrosis or on the endogenous protective mechanisms of desensitization or tolerization.
Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits, and Unrelated Adjuvants
Authors: Scharton-Kersten Tanya , Yu Jian-mei , Vassell Russell , O'Hagan Derek , Alving Carl R. , Glenn Gregory M.
Published: July 2002

We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 μg. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1β [IL-1β] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application.
Random Transposon Mutagenesis of
Campylobacter jejuni
Authors: Golden Neal J. , Camilli Andrew , Acheson David W. K.
Published: July 2002

Genetic studies of
Campylobacter jejuni
have been limited due to the lack of a transposon mutagenesis method. Here, we describe a novel technique for random transposon mutagenesis using a
mariner
-based transposon into
C. jejuni
strain 480. Insertions were random, as demonstrated by Southern blot analysis and insertional junction sequencing. We have demonstrated, for the first time, random in vivo transposon mutagenesis of
C. jejuni
.
Identity of Activation Molecule 3 on Superantigen-Stimulated Bovine Cells Is CD26
Authors: Lee Sang-Un , Ferens Witold , Davis William C. , Hamilton Mary Jo , Park Yong-Ho , Fox Lawrence K. , Naessens Jan , Bohach Gregory A.
Published: July 2002

Most CD8+T cells in cultures of bovine mononuclear cells stimulated with staphylococcal enterotoxin C1 develop an unusual phenotype characterized by expression of activation molecule 3 (ACT3). This superantigen-dependent phenotype may be relevant to immunopathogenesis mediated by certain microbial toxins. The size and N-terminal sequence of immunoprecipitated ACT3 indicate that ACT3 is the bovine orthologue of CD26.
Identification, Characterization, and Variable Expression of a Naturally Occurring Inhibitor Protein of IS
1106
Transposase in Clinical Isolates of
Neisseria
meningitidis
Authors: Salvatore Paola , Pagliarulo Caterina , Colicchio Roberta , Zecca Patrizia , Cantalupo Giuseppina , Tredici Maurizio , Lavitola Alfredo , Bucci Cecilia , Bruni Carmelo B. , Alifano Pietro
Published: July 2002

Transposition plays a role in the epidemiology and pathogenesis of
Neisseria
meningitidis
. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS
1106
controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS
1106
transposase lacking the NH
2
-terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS
1106
, as shown by gel shift and in vitro transposition assays. IS
1106
Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.
Escherichia coli
Strain RDEC-1 AF/R1 Endogenous Fimbrial Glycoconjugate Receptor Molecules in Rabbit Small Intestine
Authors: Ryu Hyoik , Kim Young S. , Grange Philippe A. , Cassels Frederick J.
Published: July 2002

Escherichia coli
strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease. The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for
E. coli
diarrheal diseases. In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and β-galactosyl residues apparently involved. The AF/R1-mediated adherence activity of [
14
C]-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli. Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence. These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS). This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex. Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli. Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Galβ1-1Cer) in the lipid extract of rabbit small intestinal brush borders. RDEC-1 adherence to Galβ1-1Cer was partially inhibited in the presence of galactose. These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.
HLA-A*01-Restricted Cytotoxic T-Lymphocyte Epitope from thePlasmodium falciparumCircumsporozoite Protein
Authors: Kumar Anita , Kumar Sanjai , Le Thong P. , Southwood Scott , Sidney John , Cohen Joe , Sette Alessandro , Hoffman Stephen L.
Published: July 2002

Here, we report the identification of a novel CD8+cytotoxic T-lymphocyte epitope on thePlasmodium falciparumcircumsporozoite protein (3D7; amino acids 310 to 319 [EPSDKHIKEY]) that is restricted by HLA-A*01 and is recognized by human volunteers immunized with irradiatedP. falciparumsporozoites. HLA-A*01 is the second most common HLA allele among Caucasians.
Comparison of the Fibronectin-Binding Protein FNE from
Streptococcus equi
Subspecies
equi
with FNZ from
S. equi
Subspecies
zooepidemicus
Reveals a Major and Conserved Difference
Authors: Lindmark Hans , Nilsson Martin , Guss Bengt
Published: July 2002

The gene
fnz
from
Streptococcus equi
subspecies
zooepidemicus
encodes a cell surface protein that binds fibronectin (Fn). Fifty tested isolates of
S. equi
subspecies
equi
all contain DNA sequences with similarity to
fnz
. This work describes the cloning and sequencing of a gene, designated
fne
, with similarity to
fnz
from two
S. equi
subspecies
equi
isolates. The DNA sequences were found to be identical in the two strains, and sequence comparison of the
fne
and
fnz
genes revealed only minor differences. However, one base deletion was found in the middle of the
fne
gene and eight base pairs downstream of the altered reading frame there is a stop codon. An Fn-binding protein was purified from the growth medium of a subspecies
equi
culture. Determination of the NH
2
-terminal amino acid sequence and molecular mass, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the purified protein is the gene product of the 5′-terminal half of
fne
. Fn-binding activity has earlier only been found in the COOH-terminal half of FNZ. By the use of a purified recombinant protein containing the NH
2
half of FNZ, we provide here evidence that this half of the protein also harbors an Fn-binding domain.
Association of Interleukin-10 Cytokine Expression Status with HLA Non-DRB1*02 andMycobacterium bovisBCG Scar-Negative Status in South Indian Pulmonary Tuberculosis Patients
Authors: Dheenadhayalan V. , Shanmugalakshmi S. , Vani S. , Muthuveeralakshmi P. , Arivarignan G. , Nageswari A. D. , Pitchappan R M.
Published: July 2002

HLA DRB1*02 and its subtypes predispose individuals for a far-advanced sputum-positive pulmonary tuberculosis transcending ethnic boundaries.Mycobacterium bovisBCG does not afford the desired protection against adult pulmonary tuberculosis, and a spectrum of immune reactivity exists in controls and hospital contacts. All of these findings have been identified and demonstrated in areas of endemicity. Skewing of immunity from protective to pathogenic may involve a shift in the Th1-Th2 paradigm. To elaborate these ideas, we studied gamma interferon (IFN-γ), interleukin-4 (IL-4), and IL-10 cytokine expression in 71 adult pulmonary tuberculosis patients and 74 controls from areas of endemicity in south India by 48-h microculture and reverse transcription-PCR. Most of the patients and controls expressed IFN-γ de novo, and in the presence of purified protein derivative (PPD), all of them expressed significantly higher levels of IFN-γ, suggesting a PPD-specific recall memory. HLA DRB1* allele-dependent IFN-γ expression was identified only in controls, suggesting a skewing of the immune response in patients. In contrast to the case for IFN-γ, only some patients and controls expressed IL-4 or IL-10 (Th2 profile); thus, the Th1 profile was identifiable only by a nonexpression of IL-4 or IL-10 in this area of endemicity. The Th2 profile was associated with HLA non-DRB1*02 and BCG scar-negative status in patients, attributing a significant risk (odds ratio = 2.074; 95% confidence interval = 0.612 to 7.07). It is possible thatMycobacterium tuberculosis(PPD)-specific IL-10 is expressed preemptively in unvaccinated (BCG scar-negative) individuals with a non-DR2 genetic background by chronic exposure in this area of endemicity and leads to pulmonary tuberculosis of adults.
Genetic Analysis of the
rgg-gtfG
Junctional Region and Its Role in
Streptococcus gordonii
Glucosyltransferase Activity
Authors: Vickerman M. M. , Minick P. E.
Published: July 2002

Glucans synthesized by glucosyltransferase enzymes of oral streptococci facilitate bacterial accumulation on surfaces. The
Streptococcus gordonii
glucosyltransferase gene,
gtfG,
is positively regulated by
rgg,
which encodes a putative cytoplasmic protein. The
gtfG
promoter and ribosomal binding sequences are located within a DNA inverted repeat immediately downstream of
rgg
. Polycistronic
rgg-gtfG
as well as
rgg
- and
gtfG
-specific transcripts are associated with this chromosomal region. Previous studies have shown that the
rgg
product acts in
trans
near the
gtfG
promoter to increase the level of
gtfG
transcript, but it does not affect the level of
rgg-gtfG
transcript. To further analyze regulation by
rgg
, a series of strain Challis derivatives was constructed and glucosyltransferase activities were determined. Strains in which
rgg
was separated from
gtfG
by integrated vector sequences had decreased levels of glucosyltransferase activity; plasmid-borne
rgg
could not increase activity to parental levels. As expected, strains with chromosomal deletions involving the
rgg
structural gene and either the
rgg
or
gtfG
promoter also showed decreased glucosyltransferase activity. Plasmid-borne
rgg
could increase glucosyltransferase activity only in strains which had a 36-bp chromosomal region beginning 72 nucleotides upstream of the
gtfG
transcriptional start site. Results suggest that these nucleotides, located within the 3′ end of
rgg
, are necessary, either by direct involvement in binding or by indirectly affecting secondary structure, for Rgg to increase glucosyltransferase activity. Surprisingly, the presence of the
rgg
promoter upstream of this 36-bp region significantly increased the effects of plasmid-borne
rgg
. Implications for glucosyltransferase regulation and applicability to other
rgg
-like determinants are considered.
Antigen-Presenting Cell Function duringPlasmodium yoeliiInfection
Authors: Luyendyk James , Olivas O. Renee , Ginger Lisa A. , Avery Anne C.
Published: July 2002

Antigen-presenting cells (APC) play a key role in orchestrating immune responses. T-cell proliferative responses are inhibited during the erythrocyte stages of malaria infection, and a number of studies have suggested that APC are responsible for this phenomenon. In the present studies we examine individual components of the T-cell-activating function of APC: expression of costimulatory and major histocompatibility complex (MHC) class II proteins, the ability to process and present antigen to T cells, and the ability to support cytokine production. We find that during the acute phases ofPlasmodium yoeliierythrocyte stage infection, APC upregulate the expression of class II MHC and CD80, maintain expression of CD86, process and present antigen, and support gamma interferon production. However the CD11b+subpopulation produces a soluble factor or factors that specifically inhibit interleukin-2 (IL-2) production by responding CD4 T cells. This factor is distinct from prostaglandin E2, NO, or transforming growth factor β. The data suggest that IL-2 suppression observed during malaria infection is not due to functional defects of APC but is triggered by production of a factor(s) that actively suppresses production of IL-2 by T cells.
Mutually Exclusive Utilization of P
R
and P
RM
Promoters in Bacteriophage 434 O
R
Authors: Xu Jian , Koudelka Gerald B.
Published: July 2002

Establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the P
RM
promoter and repress transcription from the divergent P
R
promoter. Several lines of evidence indicate that the 434 repressor activates initiation of P
RM
transcription by occupying a binding site adjacent to the P
RM
promoter and directly contacting RNA polymerase. The overlapping architecture of the P
RM
and P
R
promoters suggests that an RNA polymerase bound at P
R
may repress P
RM
transcription initiation. Hence, part of the stimulatory effect of the 434 repressor may be relief of interference between RNA polymerase binding to the P
RM
promoter and to the P
R
promoter. Consistent with this proposal, we show that the repressor cannot activate P
RM
transcription if RNA polymerase binds at P
R
prior to addition of the 434 repressor. However, unlike the findings with the related λ phage, formation of RNA polymerase promoter complexes at P
RM
and at P
R
apparently are mutually exclusive. We find that the RNA polymerase-mediated inhibition of repressor-stimulated P
RM
transcription requires the presence of an open complex at P
R
. Taken together, these results indicate that establishment of an open complex at P
R
directly prevents formation of an RNA polymerase-P
RM
complex.
DNA Polymerase I Is Essential for Growth of
Methylobacterium dichloromethanicum
DM4 with Dichloromethane
Authors: Kayser Martin F. , Stumpp Michael T. , Vuilleumier Stéphane
Published: July 2002

Methylobacterium dichloromethanicum
DM4 grows with dichloromethane as the unique carbon and energy source by virtue of a single enzyme, dichloromethane dehalogenase–glutathione
S
-transferase. A mutant of the dichloromethane-degrading strain
M. dichloromethanicum
DM4, strain DM4-1445, was obtained by mini-Tn
5
transposon mutagenesis that was no longer able to grow with dichloromethane. Dichloromethane dehalogenase activity in this mutant was comparable to that of the wild-type strain. The site of mini-Tn
5
insertion in this mutant was located in the
polA
gene encoding DNA polymerase I, an enzyme with a well-known role in DNA repair. DNA polymerase activity was not detected in cell extracts of the
polA
mutant. Conjugation of a plasmid containing the intact DNA polymerase I gene into the
polA
mutant restored growth with dichloromethane, indicating that the
polA
gene defect was responsible for the observed lack of growth of this mutant with dichloromethane. Viability of the DM4-1445 mutant was strongly reduced upon exposure to both UV light and dichloromethane. The
polA′-lacZ
transcriptional fusion resulting from mini-Tn
5
insertion was constitutively expressed at high levels and induced about twofold after addition of 10 mM dichloromethane. Taken together, these data indicate that DNA polymerase I is essential for growth of
M. dichloromethanicum
DM4 with dichloromethane and further suggest an important role of the DNA repair machinery in the degradation of halogenated, DNA-alkylating compounds by bacteria.
DNA-Binding Proteins Possibly Involved in Regulation of the Post-Logarithmic-Phase Expression of Lipoprotein P35 in
Borrelia burgdorferi
Authors: Indest Karl J. , Philipp Mario T.
Published: July 2002

Previously, we have shown that the transcription of
p35
, a lipoprotein gene of
Borrelia burgdorferi
, is upregulated or initiated during the post-logarithmic bacterial growth phase in vitro. To identify potential regulatory factors, we examined the formation of DNA-protein complexes by electromobility shift assay, using a 157-bp DNA fragment that spans the
p35
promoter region and cell-free extracts of spirochetes harvested from both logarithmic and stationary growth phases. The binding properties of the extracts with the promoter region of the
flaB
gene, a constitutively expressed, growth-phase-independent gene, were also compared. The results from these experiments demonstrate that
B. burgdorferi
stationary-phase cell-free extracts have a growth-phase-dependent DNA binding protein that interacts specifically with the
p35
promoter region. We show, in addition, that a segment from the 157-bp
p35
promoter region which contains both a T-rich stretch and an inverted repeat is able to compete off the stationary-phase-specific complex when the segment is present in molar excess.
Motility and Flagellum Synthesis in
Halobacillus halophilus
Are Chloride Dependent
Authors: Roeßler Markus , Wanner Gerhard , Müller Volker
Published: July 2002

The motility of
Halobacillus halophilus
as observed on swarm agar plates was strictly dependent on the chloride concentration. Cl
−
was apparently not used as the coupling ion for flagellar rotation. Cells grown in the absence of chloride were devoid of flagella, but flagellation was restored upon the addition of chloride. These experiments indicate that chloride is involved in synthesis of flagella in
H. halophilus
.
Identification of an Extradiol Dioxygenase Involved in Tetralin Biodegradation: Gene Sequence Analysis and Purification and Characterization of the Gene Product
Authors: Andújar Eloísa , Hernáez María José , Kaschabek Stefan R. , Reineke Walter , Santero Eduardo
Published: July 2002

A genomic region involved in tetralin biodegradation was recently identified in
Sphingomonas
strain TFA. We have cloned and sequenced from this region a gene designated
thnC
, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene was found to be the metabolite accumulated by a
thnC
insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (
V
max
, 40.5 U mg
−1
;

K
m

, 18.6 μM). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity toward 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1,2-dihydroxynaphthalene dioxygenases.
Spirochaeta aurantia
Has Diacetyl Chloramphenicol Esterase Activity
Authors: Sohaskey Charles D. , Barbour Alan G.
Published: July 2002

The free-living spirochete
Spirochaeta aurantia
was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol.
S. aurantia
extracts also hydrolyzed other esterase substrates, and two of these, α-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in
Streptomyces
species, but in this study their activity was weaker than that of
S. aurantia
. The
S. aurantia
esterase could reduce the effectiveness of
cat
as either a selectable marker or a reporter gene in this species.
Stability of
Mycoplasma pneumoniae
Cytadherence-Accessory Protein HMW1 Correlates with Its Association with the Triton Shell
Authors: Balish Mitchell F. , Hahn Tae-Wook , Popham Phillip L. , Krause Duncan C.
Published: July 2002

Mycoplasma pneumoniae
adsorbs to host respiratory epithelium primarily by its attachment organelle, the proper function of which depends upon mycoplasma adhesin and cytoskeletal proteins. Among the latter are the cytadherence-associated proteins HMW1 and HMW2, whose specific roles in this process are unknown. In the
M. pneumoniae
cytadherence mutant I-2, loss of HMW2 results in accelerated turnover of HMW1 and other cytadherence-accessory proteins, probably by proteolysis. However, both the mechanism of degradation and the means by which these proteins are rendered susceptible to it are not understood. In this study, we addressed whether HMW1 degradation is a function of its presence among specific subcellular fractions and established that HMW1 is a peripheral membrane protein that is antibody accessible on the outer surfaces of both wild-type and mutant I-2
M. pneumoniae
but to a considerably lesser extent in the mutant. Quantitation of HMW1 in Triton X-100-fractionated extracts from cells pulse-labeled with [
35
S]methionine indicated that HMW1 is synthesized in a Triton X-100-soluble form that exists in equilibrium with an insoluble (cytoskeletal) form. Pulse-chase analysis demonstrated that over time, HMW1 becomes stabilized in the cytoskeletal fraction and associated with the cell surface in wild-type
M. pneumoniae
. The less efficient transition to the cytoskeleton and mycoplasma cell surface in mutant I-2 leads to accelerated degradation of HMW1. These data suggest a role for HMW2 in promoting export of HMW1 to the cell surface, where it is stable and fully functional.
Conserved Promoter Motif Is Required for Cell Cycle Timing of
dnaX
Transcription in
Caulobacter
Authors: Keiler Kenneth C. , Shapiro Lucy
Published: July 2002

Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium
Caulobacter crescentus
, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including
dnaX
,
dnaA
,
dnaN
,
gyrB
, and
dnaK
, are induced at the G
1
/S-phase transition. By analyzing mutations in the
dnaX
promoter, we identified a motif between the −10 and −35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis,
Caulobacter
extracts contain an activity that binds specifically to the RRF in vitro.
Involvement of the XpsN Protein in Formation of the XpsL-XpsM Complex in
Xanthomonas campestris
pv. campestris Type II Secretion Apparatus
Authors: Lee Hsien-Ming , Tyan Shiaw-Wei , Leu Wei-Ming , Chen Ling-Yun , Chen David Chanhen , Hu Nien-Tai
Published: July 2002

The
xps
gene cluster is required for the second step of type II protein secretion in
Xanthomonas campestris
pv. campestris. Deletion of the entire gene cluster caused accumulation of secreted proteins in the periplasm. By analyzing protein abundance in the chromosomal mutant strains, we observed mutual dependence for normal steady-state levels between the XpsL and the XpsM proteins. The XpsL protein was undetectable in total lysate prepared from the
xpsM
mutant strain, and vice versa. Introduction of the wild-type
xpsM
gene carried on a plasmid into the
xpsM
mutant strain was sufficient for reappearance of the XpsL protein, and vice versa. Moreover, both XpsL and XpsM proteins were undetectable in the
xpsN
mutant strain. They were recovered either by reintroducing the wild-type
xpsN
gene or by introducing extra copies of wild-type
xpsL
or
xpsM
individually. Overproduction of wild-type XpsL and -M proteins simultaneously, but not separately, in the wild-type strain of
X. campestris
pv. campestris caused inhibition of secretion. Complementation of an
xpsL
or
xpsM
mutant strain with a plasmid-borne wild-type gene was inhibited by coexpression of XpsL and XpsM. The presence of the
xpsN
gene on the plasmid along with the
xpsL
and the
xpsM
genes caused more severe inhibition in both cases. Furthermore, complementation of the
xpsN
mutant strain was also inhibited. In both the wild-type strain and a strain with the
xps
gene cluster deleted (XC17433), carrying pCPP-LMN, which encodes all three proteins, each protein coprecipitated with the other two upon immunoprecipitation. Expression of pairwise combinations of the three proteins in XC17433 revealed that the XpsL-XpsM and XpsM-XpsN pairs still coprecipitated, whereas the XpsL-XpsN pair no longer coprecipitated.