bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution and Diversity of Symbiotic Thermophiles,
Symbiobacterium thermophilum
and Related Bacteria, in Natural Environments
Authors: Ueda Kenji , Ohno Michiyo , Yamamoto Kaori , Nara Hanae , Mori Yujiro , Shimada Masafumi , Hayashi Masahiko , Oida Hanako , Terashima Yuko , Nagata Mitsuyo , Beppu Teruhiko
Published: July 2002

Symbiobacterium thermophilum
is a tryptophanase-positive thermophile which shows normal growth only in coculture with its supporting bacteria. Analysis of the 16S rRNA gene (rDNA) indicated that the bacterium belongs to a novel phylogenetic branch at the outermost position of the gram-positive bacterial group without clustering to any other known genus. Here we describe the distribution and diversity of
S. thermophilum
and related bacteria in the environment. Thermostable tryptophanase activity and amplification of the specific 16S rDNA fragment were effectively employed to detect the presence of
Symbiobacterium
. Enrichment with kanamycin raised detection sensitivity. Mixed cultures of thermophiles containing
Symbiobacterium
species were frequently obtained from compost, soil, animal feces, and contents in the intestinal tracts, as well as feeds. Phylogenetic analysis and denaturing gradient gel electrophoresis of the specific 16S rDNA amplicons revealed a diversity of this group of bacteria in the environment.
Desulfovibrio
sp. Genes Involved in the Respiration of Sulfate during Metabolism of Hydrogen and Lactate
Authors: Steger Jennifer L. , Vincent Carr , Ballard Jimmy D. , Krumholz Lee R.
Published: July 2002

To develop a better understanding of respiration by sulfate-reducing bacteria, we examined transcriptional control of respiratory genes during growth with lactate or hydrogen as an electron donor. RNA extracts of
Desulfovibrio desulfuricans
subsp.
aestuarii
were analyzed by using random arbitrarily primed PCR. RNA was reverse transcribed under low-stringency conditions with a set of random primers, and candidate cDNAs were cloned, sequenced, and characterized by BLAST analysis. Putative differentially expressed transcripts were confirmed by Northern blot analysis. Interestingly, dissimilatory bisulfite reductase was upregulated in the presence of hydrogen. To link these transcriptional changes to the physiology of sulfate-reducing bacteria, sulfide was measured during growth of several strains of
Desulfovibrio
on hydrogen or lactate, and this revealed that hydrogen-grown cells produced more sulfide per unit of cell mass than lactate-grown cells. Transcription of other redox proteins was characterized by Northern blotting to determine whether or not they were also transcribed to higher levels in hydrogen-grown cells. Growth on lactate produced greater transcription of [NiFe] hydrogenase. H
2
-grown cells transcribed the adenylylsulfate reductase b subunit and HmcA to higher levels. The results we describe here provide new insight into the continuing debate over how
Desulfovibrio
species utilize redox components to generate membrane potential and to channel electrons to sulfate, the final electron acceptor.
Purification and Characterization of an Arginine Aminopeptidase from
Lactobacillus sakei
Authors: Sanz Yolanda , Toldrá Fidel
Published: July 2002

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from
Lactobacillus sakei.
The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu
2+
, Hg
2+
, and Zn
2+
) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The

K
m

values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for
L. sakei
in the meat environment.
Improved Repetitive-Element PCR Fingerprinting for Resolving Pathogenic and Nonpathogenic Phylogenetic Groups within
Escherichia coli
Authors: Johnson James R. , O'Bryan Timothy T.
Published: July 2002

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for
Escherichia coli
, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15
E. coli
strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic
E. coli
.
Identification of Potentially DiagnosticLeishmania braziliensisAntigens in Human Cutaneous Leishmaniasis by Immunoblot Analysis
Authors: Brito Maria Edileuza F. , Mendonça Mitzi G. , Gomes Yara M. , Jardim Márcio L. , Abath Frederico G. C.
Published: July 2002

The antibody response in patients with American cutaneous leishmaniasis was analyzed by immunoblotting with soluble and insoluble antigens ofLeishmania braziliensis. The recognition of the 27- and/or 30-kDa soluble antigens was considered relevant for the diagnosis of cutaneous leishmaniasis. Immunoblotting was found to be significantly more sensitive and specific than indirect immunofluorescence and enzyme-linked immunosorbent assay.
Influence of Modified Natural or Synthetic Surfactant Preparations on Growth of Bacteria Causing Infections in the Neonatal Period
Authors: Rauprich Petra , Möller Oliver , Walter Gabriele , Herting Egbert , Robertson Bengt
Published: July 2002

Connatal bacterial pneumonia is common in neonates. Animal studies and initial clinical reports indicate that surfactant dysfunction is involved in the pathophysiology of severe neonatal pneumonia. Since respiratory distress syndrome and connatal pneumonia may be difficult to differentiate in the first hours of life, neonates with respiratory failure due to bacterial infections might receive surfactant. Under such conditions surfactant components might be catabolized by bacteria and promote bacterial growth. We therefore investigated the influence of three modified natural (Curosurf, Alveofact, and Survanta) and two synthetic (Exosurf and Pumactant) surfactant preparations on the growth of bacteria frequently cultured from blood or tracheal aspirate fluid in the first days of life. Group B streptococci (GBS),Staphyloccocus aureus, andEscherichia coliwere incubated in a nutrient-free medium (normal saline) for 5 h at 37°C, together with different surfactants at concentrations of 0, 1, 10, and 20 mg/ml. With the exception ofE. coli, incubation in saline alone led to a variable decrease in CFU. In the presence of Alveofact, Exosurf, and Pumactant the decline in bacterial numbers was less marked than in saline alone. Curosurf was bactericidal in a dose-dependent fashion for GBS and had a strong negative impact on the growth of a GBS subtype that lacked the polysaccharide capsule. In contrast, Survanta (10 and 20 mg/ml) significantly promoted the growth ofE. coli, indicating that surfactant components may actually serve as nutrients. We conclude that bacterial growth in different surfactant preparations is influenced by microbial species and the composition and dose of the surfactant. Further studies are necessary to elucidate the mechanisms behind our findings and to evaluate the effects of surfactant on bacterial growth in vivo.
Detection of Phenolic Glycolipid I of
Mycobacterium leprae
in Sera from Leprosy Patients before and after Start of Multidrug Therapy
Authors: Cho Sang-Nae , Cellona Roland V. , Villahermosa Laarni G. , Fajardo Tranquilino T. , Balagon M. Victoria F. , Abalos Rodolfo M. , Tan Esterlina V. , Walsh Gerald P. , Kim Joo-Deuk , Brennan Patrick J.
Published: July 2002

A total of 100 untreated new leprosy patients were recruited prospectively and examined for the presence of phenolic glycolipid I (PGL-I) antigen in their serum specimens by dot enzyme-linked immunosorbent assay (ELISA) using rabbit anti-PGL-I antiserum. The presence of circulating PGL-I antigen was closely related to the bacterial indices (BI) of the patients. The PGL-I antigen was detectable in 27 (93.1%) of 29 patients with a BI of 4.0 or above and in 15 (68.2%) of 22 patients with a BI of 3.0 to 3.9. However, none of the 37 patients with a BI of less than 1.9 had detectable PGL-I antigen by the methods used in this study. The level of PGL-I in serum declined rapidly by about 90% 1 month after the start of multidrug therapy. This study showed clearly that anti-PGL-I IgM antibodies and circulating PGL-I antigen levels reflect the bacterial loads in untreated leprosy patients. The serological parameters based on the PGL-I antigen may therefore be useful in the assessment of leprosy patients at the time of diagnosis and possibly in monitoring patients following chemotherapy.
Distribution of Lymphocyte Subsets in Healthy Human Immunodeficiency Virus-Negative Adult Ethiopians from Two Geographic Locales
Authors: Kassu Afework , Tsegaye Aster , Petros Beyene , Wolday Dawit , Hailu Ermias , Tilahun Tesfaye , Hailu Binyam , Roos Marijke T. L. , Fontanet Arnaud L. , Hamann Dörte , De Wit Tobias F. Rinke
Published: July 2002

Immunological values for 562 factory workers from Wonji, Ethiopia, a sugar estate 114 km southeast of the capital city, Addis Ababa, Ethiopia, were compared to values for 218 subjects from Akaki, Ethiopia, a suburb of Addis Ababa, for whom partial data were previously published. The following markers were measured: lymphocytes, T cells, B cells, NK cells, CD4+T cells, and CD8+T cells. A more in depth comparison was also made between Akaki and Wonji subjects. For this purpose, various differentiation and activation marker (CD45RA, CD27, HLA-DR, and CD38) expressions on CD4+and CD8+T cells were studied in 60 male, human immunodeficiency virus-negative subjects (30 from each site). Data were also compared with Dutch blood donor control values. The results confirmed that Ethiopians have significantly decreased CD4+T-cell counts and highly activated immune status, independent of the geographic locale studied. They also showed that male subjects from Akaki have significantly higher CD8+T-cell counts, resulting in a proportional increase in each of the CD8+T-cell compartments studied: naı̈ve (CD45RA+CD27+), memory (CD45RA−CD27+), cytotoxic effector (CD45RA+CD27−), memory/effector (CD45RA−CD27−), activated (HLA-DR+CD38+), and resting (HLA-DR−CD38−). No expansion of a specific functional subset was observed. Endemic infection or higher immune activation is thus not a likely cause of the higher CD8 counts in the Akaki subjects. The data confirm and extend earlier observations and suggest that, although most lymphocyte subsets are comparable between the two geographical locales, there are also differences. Thus, care should be taken in extrapolating immunological reference values from one population group to another.
Role of Th1 and Th2 Cytokines in Immune Response to UncomplicatedPlasmodium falciparumMalaria
Authors: Torre Donato , Speranza Filippo , Giola Massimo , Matteelli Alberto , Tambini Roberto , Biondi Gilberto
Published: July 2002

The relative balance between Th1 and Th2 cytokines appears crucial, since the role of cytokines has been evaluated in several studies by comparison of clinically heterogeneous groups of patients. The aim of this study is to determine the role of proinflammatory Th1 cytokines, interleukin-12 (IL-12) and gamma interferon (IFN-γ), and anti-inflammatory Th2 cytokines, IL-4 and IL-10, in a homogeneous group of patients with uncomplicatedPlasmodium falciparummalaria. Levels of IL-12, IFN-γ, Il-4, and IL-10 in serum for 20 adult patients and 15 healthy control subjects were determined by an immunoenzymatic assay. Serum levels of Th1 cytokines, IL-12 (8.6 ± 2.8 pg/ml; controls, 3.2 ± 0.7 pg/ml) and IFN-γ (39.2 ± 67.6 pg/ml; controls, 8.4 ± 6.3 pg/ml), were significantly increased at admission; 3 days later, levels of IL-12 in serum remained significantly high (8.8 ± 2.6 pg/ml), whereas IFN-γ levels returned to control values. The anti-inflammatory response of Th2 cytokines (IL-10 and IL-4) was distinct. Levels of IL-10 in serum were not significantly increased at day 0 and day 3 (306.6 ± 200.4 pg/ml and 56.6 ± 38.4 pg/ml, respectively; controls, 17.4 ± 9.0 pg/ml). In contrast, levels of IL-4 in serum were not increased on admission (3.4 ± 1.2 pg/ml; controls, 2.4 ± 0.8 pg/ml), but at day 3 a moderate and significant increase of IL-4 levels was observed (4.5 ± 1.7 pg/ml). In conclusion, the increase of Th1 cytokine IL-12 and IFN-γ levels during the acute phase of uncomplicatedP. falciparummalaria may reflect an early and effective immune response regulated by proinflammatory Th1 cytokines, and in particular IFN-γ may play a role in limiting progression from uncomplicated malaria to severe and life-threatening complications.
Low-Dose UVB Contributes to Host Resistance againstLeishmania amazonensisInfection in Mice through Induction of Gamma Interferon and Tumor Necrosis Factor Alpha Cytokines
Authors: Khaskhely Noor Mohammad , Maruno Motoyoshi , Uezato Hiroshi , Takamiyagi Atsushi , Ramzi Saeef Taher , Kasem Khan Mohammad Al , Kariya Ken-ichi , Toda Takayoshi , Hashiguchi Yoshihisa , Landires Eduardo A. Gomez , Nonaka Shigeo
Published: July 2002

UV radiation suppresses the immune response, a fact which raises the question of whether the phenomenon may find practical applications in the outcome of infectious diseases. In this study, BALB/c mice were exposed to low-dose UVB (250 J/m2) from Dermaray M-DMR-100 for 4 consecutive days. Twelve hours after the last UV exposure, groups of mice were injected with 2 × 106Leishmania amazonensispromastigotes. The development of skin lesions, as assessed by measurement of visible cutaneous lesions, was significantly suppressed in low-dose UVB-irradiated mice compared to nonirradiated controls. In order to characterize the cytokines involved in this phenomenon, BALB/c mice were irradiated with identical doses of UVB, and gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 4 cytokine levels in blood serum and skin were examined at different times by a sandwich enzyme-linked immunosorbent assay, immunohistochemical analysis, and reverse transcription (RT)-PCR. Upregulated expression of serum IFN-γ and TNF-α was observed from 6 to 24 h. Positive results for IFN-γ and TNF-α in UVB-irradiated mice were obtained by immunohistochemical analysis. By RT-PCR, the mRNA expression of both IFN-γ and TNF-α cytokines was detected in a time-dependent manner only in UVB-irradiated mice. Histopathological analysis and electron microscopy revealed that cellular infiltration, tissue parasitism, and parasitophorus vacuoles in irradiated mice were markedly less noticeable than those in nonirradiated controls. These results suggested that low-dose UVB irradiation played a pathogen-suppressing role inLeishmania-susceptible BALB/c mice via systemic and local upregulation of Th1 (IFN-γ and TNF-α) cytokines.
New Fimbrial Gene Cluster of S-Fimbrial Adhesin Family
Authors: Babai Reuven , Stern Baruch E. , Hacker Joerg , Ron Eliora Z.
Published: July 2002

Fimbrial adhesins that mediate attachment to host cells are produced by most virulent
Escherichia coli
isolates. These virulence factors play an important role in the initial stages of bacterial colonization and also in determination of the host and tissue specificity. Isolates belonging to serotype O78 are known to cause a large variety of clinical syndromes in farm animals and humans and have been shown to produce several types of adherence fimbriae. We studied the fimbrial adhesin from an avian septicemic
E. coli
isolate of serotype O78. Analysis of the genetic organization of the
fac
(fimbria of avian
E. coli
) gene cluster indicates that it belongs to the S-fimbrial adhesin family. Seven open reading frames coding for major and minor structural subunits were identified, and most of them showed a high degree of homology to the corresponding Sfa and Foc determinants. The least-conserved open reading frame was
facS
, encoding a protein known to play an important role in determining adherence specificity in other S-fimbrial gene clusters.
Enterotoxin-Specific Immunoglobulin E Responses in Humans after Infection or Vaccination with Diarrhea-Causing Enteropathogens
Authors: Qadri Firdausi , Asaduzzaman Muhammad , Wennerås Christine , Mohi Golam , Albert M. John , Abdus Salam Mohammad , Sack R. Bradley , Jertborn Marianne , R. McGhee Jerry , Sack David A. , Holmgren Jan
Published: July 2002

Cholera toxin (CT)-specific antibody responses of the immunoglobulin E (IgE) isotype in the sera of adult patients suffering from infection with eitherVibrio choleraeO1,V. choleraeO139, or enterotoxigenicEscherichia coli(ETEC) were analyzed and compared with those in the sera of volunteers immunized with a bivalent B subunit O1/O139 whole-cell cholera vaccine. A significant IgE response to CT was observed in 90% of the patients withV. choleraeO1 infection (18 of 20;P= <0.001) and 95% of the patients withV. choleraeO139 infection (19 of 20;P= <0.001). Similarly, the majority of the patients with ETEC diarrhea (83%; 13 of 15) showed a positive IgE response to CT. Eight of 10 North American volunteers (80%) orally challenged withV. choleraeO1 showed CT-specific IgE responses (P= 0.004). In contrast, Swedish volunteers immunized with the oral cholera vaccine showed no IgE responses to CT (Pvalue not significant). During the study period, total IgE levels in the sera of the diarrheal patients, the North American volunteers, and the Swedish cholera vaccinees alike remained unchanged. However, the total IgE levels in the sera of patients and healthy Bangladeshi controls were on average 89-fold higher than those in the sera of the healthy Swedish volunteers and 34-fold higher than those in the sera of the North American volunteers.
Localization of Dysfunctional Tight Junctions in
Salmonella enterica
Serovar Typhimurium-Infected Epithelial Layers
Authors: Jepson Mark A. , Schlecht Hélène B. , Collares-Buzato Carla B.
Published: July 2002

Infection of polarized MDCK epithelial layers by
Salmonella enterica
serovar Typhimurium is accompanied by increased tight junction permeability and by contraction of perijunctional actinomyosin. We localized dysfunctional tight junctions in serovar Typhimurium-infected MDCK layers by imaging apical-basolateral intramembrane diffusion of fluorescent lipid and found that loss of the apical-basolateral diffusion barrier (tight junction fence function) was most marked in areas of prominent perijunctional contraction. The protein kinase inhibitor staurosporine prevented perijunctional contraction but did not reverse the effects of serovar Typhimurium on tight junction barrier function. Hence, perijunctional contraction is not required for
Salmonella
-induced tight junction dysfunction and this epithelial response to infection may be multifactorial.
Jejunal Brush Border Microvillous Alterations inGiardia muris-Infected Mice: Role of T Lymphocytes and Interleukin-6
Authors: Scott K. G.-E. , Logan M. R. , Klammer G. M. , Teoh D. A. , Buret A. G.
Published: July 2002

Intestinal colonization with the protozoanGiardiacauses diffuse brush border microvillous alterations and disaccharidase deficiencies, which in turn are responsible for intestinal malabsorption and maldigestion. The role of T cells and/or cytokines in the pathogenesis ofGiardia-induced microvillous injury remains unclear. The aim of this study was to assess the role of T cells and interleukin-6 (IL-6) in the brush border pathophysiology of acute murine giardiasis in vivo. Athymic nude (nu−/nu−) CD-1 mice and isogenic immunocompetent (nu+/nu+) CD-1 mice (4 weeks old) received an axenicGiardia muristrophozoite inoculum or vehicle (control) via orogastric gavage. Weight gain and food intake were assessed daily. On day 6, segments of jejunum were assessed for parasite load, brush border ultrastructure, IL-6 content, maltase and sucrase activities, villus-crypt architecture, and intraepithelial lymphocyte (IEL) infiltration. Despite similar parasitic loads on day 6, infected immunocompetent animals, but not infected nude mice, showed a diffuse loss of brush border microvillous surface area, which was correlated with a significant reduction in maltase and sucrase activities and a decrease in jejunal IL-6 concentration. In both athymic control and infected mice, jejunal brush border surface area and disaccharidases were high, but levels of tissue IL-6 were low and comparable to the concentration measured in immunocompetent infected animals. In both immunocompetent and nude mice, infection caused a small but significant increase in the numbers of IELs. These findings suggest that the enterocyte brush border injury and malfunction seen in giardiasis is, at least in part, mediated by thymus-derived T lymphocytes and that suppressed jejunal IL-6 does not necessarily accompany microvillous shortening.
Immune Response Genes Modulate Serologic Responses toVibrio choleraeTcpA Pilin Peptides
Authors: Meeks Michael D. , Wade Terri K. , Taylor Ronald K. , Wade William F.
Published: July 2002

Cholera is an enteric disease caused byVibrio cholerae.Toxin-coregulated pilus (TCP), a type 4 pilus expressed byV. cholerae, is a cholera virulence factor that is required for host colonization. The TCP polymer is composed of subunits of TcpA pilin. Antibodies directed against TcpA are protective in animal models of cholera. While natural or recombinant forms of TcpA are difficult to purify to homogeneity, it is anticipated that synthesized TcpA peptides might serve as immunogens in a subunit vaccine. We wanted to assess the potential for effects of the immune response (Ir) gene that could complicate a peptide-based vaccine. Using a panel of mice congenic at theH-2locus we tested the immunogenicity of TcpA peptide sequences (peptides 4 to 6) found in the carboxyl termini of both the classical (Cl) and El Tor (ET) biotypes of TCP. Cl peptides have been shown to be immunogenic in CD-1 mice. Our data clearly establish that there are effects of the Ir gene associated with both biotypes of TcpA. These effects are dynamic and dependent on the biotype of TcpA and the haplotypes of the host. In addition to the effects of the classic class II Ir gene, class I (D, L) or nonclassical class I (Qa-2) may also affect immune responses to TcpA peptides. To overcome the effects of the class II Ir gene, multiple TcpA peptides similar to peptides 4, 5, and 6 could be used in a subunit vaccine formulation. Identification of the most protective B-cell epitopes of TcpA within a particular peptide and conjugation to a universal carrier may be the most effective method to eliminate the effects of the class II and class I Ir genes.
Bacterial Lipopolysaccharide and Tumor Necrosis Factor Alpha Synergistically Increase Expression of Human Endothelial Adhesion Molecules through Activation of NF-κB and p38 Mitogen-Activated Protein Kinase Signaling Pathways
Authors: Jersmann Hubertus P. A. , Hii Charles S. T. , Ferrante Judith V. , Ferrante Antonio
Published: July 2002

One of the recognized associations of bacterial infection with cardiovascular events is the activation of endothelium and upregulation of adhesion molecules. The two major proinflammatory mediators implicated in the causation of cardiovascular events, bacterial lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF), were found to cooperate to enhance the adhesive properties of endothelial cells. These caused synergistic upregulation of intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells as determined by flow cytometry analysis and enzyme-linked immunosorbent assay. This synergism was not due to TNF causing an upregulation of CD14 expression. Treatment with both LPS and TNF resulted in a marked increase in the translocation of NF-κB into the nucleus. The activity of p38 mitogen-activated protein kinase was also synergistically enhanced, while the activity of c-junN-terminal kinase was increased in an additive manner. The results demonstrate that LPS and TNF act synergistically to upregulate the expression of endothelial cell adhesion molecules, possibly by amplification of signaling pathways upstream of transcription. These findings have implications for the understanding of the acceleration of atherosclerotic events seen in low-grade infections with gram-negative organisms.
Circumvention of Herd Immunity during an Outbreak of Meningococcal Disease Could Be Correlated to Escape Mutation in the
porA
Gene of
Neisseria meningitidis
Authors: Taha Muhamed-Kheir , Bichier Edouard , Perrocheau Anne , Alonso Jean-Michel
Published: July 2002

Meningococcal strains isolated during an outbreak were shown to belong to the ET-5 complex and to harbor a mutation in the VR2 region of the
porA
gene. They were less susceptible to the bactericidal effect of normal human serum than was the ET-5 wild-type strain. These results are of concern, as PorA is a potential target in vaccine design.
Human Monoclonal Antibodies againstPseudomonas aeruginosaLipopolysaccharide Derived from Transgenic Mice Containing Megabase Human Immunoglobulin Loci Are Opsonic and Protective against Fatal Pseudomonas Sepsis
Authors: Hemachandra Sonali , Kamboj Kulwant , Copfer Janna , Pier Gerald , Green Larry L. , Schreiber John R.
Published: July 2002

Pseudomonas aeruginosais a significant human pathogen, and no vaccine is commercially available. Passive antibody prophylaxis using monoclonal antibodies (MAb) against protectiveP. aeruginosaepitopes is an alternative strategy for preventingP. aeruginosainfection, but mouse MAb are not suitable for use in humans. Polyclonal human antibodies from multiple donors have variable antibody titers, and human MAb are difficult to make. We used immunoglobulin-inactivated transgenic mice reconstituted with megabase-size human immunoglobulin loci to generate a human MAb against the polysaccharide (PS) portion of the lipopolysaccharide O side chain of a common pathogenic serogroup ofP. aeruginosa, 06ad. The anti-PS human immunoglobulin G2 MAb made from mice immunized with heat-killedP. aeruginosawas specific for serogroup 06ad pseudomonas. The MAb was highly opsonic for the uptake and killing ofP. aeruginosaby human polymorphonuclear leukocytes in the presence of human complement. In addition, 25 μg of the MAb protected 100% of neutropenic mice from fatalP. aeruginosasepsis. DNA sequence analysis of the genes encoding the MAb revealed VH3 and Vκ2/A2 variable-region genes, similar to variable-region genes in humans immunized with bacterial PS and associated with high-avidity anti-PS antibodies. We conclude that human MAb toP. aeruginosamade in these transgenic mice are highly protective and that these mice mimic the antibody response seen in humans immunized with T-cell-independent antigens such as bacterial PS.
Pasteurella multocida
Gene Expression in Response to Iron Limitation
Authors: Paustian Michael L. , May Barbara J. , Kapur Vivek
Published: July 2002

Pasteurella multocida
is the causative agent of a wide range of diseases in avian and mammalian hosts. Gene expression in response to low iron conditions was analyzed in
P. multocida
using whole-genome microarrays. The analysis shows that the expression of genes involved in energy metabolism and electron transport generally decreased 2.1- to 6-fold while that of genes used for iron binding and transport increased 2.1- to 7.7-fold in
P. multocida
during the first 2 h of growth under iron-limiting conditions compared with controls. Notably, 27% of the genes with significantly altered expression had no known function, illustrating the limitations of using publicly available databases to identify genes involved in microbial metabolism and pathogenesis. Taken together, the results of our investigations demonstrate the utility of whole-genome microarray analyses for the identification of genes with altered expression profiles during varying growth conditions and provide a framework for the detailed analysis of the molecular mechanisms of iron acquisition and metabolism in
P. multocida
and other gram-negative bacteria.
Fresh Isolates from Children with SeverePlasmodium falciparumMalaria Bind to Multiple Receptors
Authors: Heddini Andreas , Pettersson Fredrik , Kai Oscar , Shafi Juma , Obiero Jack , Chen Qijun , Barragan Antonio , Wahlgren Mats , Marsh Kevin
Published: July 2002

The sequestration ofPlasmodium falciparum-infected erythrocytes (pRBC) away from the peripheral circulation is a property of all field isolates. Here we have examined the pRBC of 111 fresh clinical isolates from children with malaria for a number of adhesive features in order to study their possible coexpression and association with severity of disease. A large number of adhesion assays were performed studying rosetting, giant rosetting, and binding to CD36, intercellular adhesion molecule 1, platelet endothelial cell adhesion molecule 1, thrombospondin, heparin, blood group A, and immunoglobulins. Suspension assays were performed at the actual parasitemia of the isolate, while all the static adhesion assays were carried out at an equal adjusted parasitemia. The ability to bind to multiple receptors, as well as the ability to form rosettes and giant rosettes, was found to be more frequent among isolates from children with severe versus mild malaria (P= 0.0015). Rosettes and giant rosettes were more frequent for children with severe malaria, and the cell aggregates were larger and tighter, than for those with mild disease (P= 0.0023). Binding of immunoglobulins (97% of isolates) and of heparin (81% of isolates) to infected erythrocytes was common, and binding to heparin and blood group A was associated with severity of disease (P= 0.011 andP= 0.031, respectively). These results support the idea that isolates that bind to multiple receptors are involved in the causation of severe malaria and that several receptor-ligand interactions work synergistically in bringing about severe disease.
Identification of a Transcytosis Epitope on Staphylococcal Enterotoxins
Authors: Shupp Jeffrey W. , Jett Marti , Pontzer Carol H.
Published: July 2002

Staphylococcal enterotoxins (SE) are exoproteins produced byStaphylococcus aureusthat act as superantigens and have been implicated as a leading cause of food-borne disease and toxic shock. Little is known about how these molecules penetrate the gut lining and gain access to both local and systemic immune tissues. To model movement in vitro of staphylococcal enterotoxins, we have employed a monolayer system composed of crypt-like human colonic T-84 cells. SEB and SEA showed comparable dose-dependent transcytosis in vitro, while toxic shock syndrome toxin (TSST-1) exhibited increased movement at lower doses. Synthetic peptides corresponding to specific regions of the SEB molecule were tested in vitro to identify the domain of the protein involved in the transcytosis of SE. A toxin peptide of particular interest contains the amino acid sequence KKKVTAQELD, which is highly conserved across all SE. At a toxin-to-peptide ratio of 1:10, movement of SEB across the monolayers was reduced by 85%. Antisera made against the SEB peptide recognized native SEB and also inhibited SEB transcytosis. Finally, the conserved 10-amino-acid peptide inhibited transcytosis of multiple staphylococcal enterotoxins, SEA, SEE, and TSST-1. These data demonstrate that this region of the staphylococcal enterotoxins plays a distinct role in toxin movement across epithelial cells. It has implications for the prevention of staphylococcal enterotoxin-mediated disease by design of a peptide vaccine that could reduce systemic exposure to oral or inhaled superantigens. Since the sequence identified is highly conserved, it allows for a single epitope blocking the transcytosis of multiple SE.
Human Secretory Immune Response to Fatty Acid-Binding Protein Fraction from
Giardia lamblia
Authors: Hasan S. M. T. , Maachee M. , Córdova O. M. , Diaz de la Guardia R. , Martins M. , Osuna A.
Published: July 2002

The secretory immune response in humans infected with
Giardia lamblia
was studied by using saliva samples and an 8-kDa antigen capable of binding fatty acids. This antigen was not recognized by saliva samples from healthy individuals. The antigen may be useful in diagnostic studies of
G. lamblia
infection.
Inactivation of
gltB
Abolishes Expression of the Assimilatory Nitrate Reductase Gene (
nasB
) in
Pseudomonas putida
KT2442
Authors: Eberl Leo , Ammendola Aldo , Rothballer Michael H. , Givskov Michael , Sternberg Claus , Kilstrup Mogens , Schleifer Karl-Heinz , Molin Søren
Published: July 2002

By using mini-Tn
5
transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase,
luxAB
, to genes of
Pseudomonas putida
KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (
nasB
) of
P. putida
KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking
nasB
suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of
nasB
expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the
gltB
gene of
P. putida
KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the
gltB
mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.
Role of the
yiaR
and
yiaS
Genes of
Escherichia coli
in Metabolism of Endogenously Formed
l
-Xylulose
Authors: Ibañez Ester , Gimenez Rosa , Pedraza Tomas , Baldoma Laura , Aguilar Juan , Badia Josefa
Published: July 2002

Genes
yiaP
and
yiaR
of the
yiaKLMNOPQRS
cluster of
Escherichia coli
are required for the metabolism of the endogenously formed
l
-xylulose, whereas
yiaS
is required for this metabolism only in
araD
mutants. Like AraD, YiaS was shown to have
l
-ribulose-5-phosphate 4-epimerase activity. Similarity of YiaR to several 3-epimerases suggested that this protein could catalyze the conversion of
l
-xylulose-5-phosphate into
l
-ribulose-5-phosphate, thus completing the pathway between
l
-xylulose and the general metabolism.
RNase III Processing of Intervening Sequences Found in Helix 9 of 23S rRNA in the Alpha Subclass of
Proteobacteria
Authors: Evguenieva-Hackenberg Elena , Klug Gabriele
Published: July 2002

We provide experimental evidence for RNase III-dependent processing in helix 9 of the 23S rRNA as a general feature of many species in the alpha subclass of
Proteobacteria
(alpha-
Proteobacteria
). We investigated 12
Rhodobacter
,
Rhizobium
,
Sinorhizobium
,
Rhodopseudomonas
, and
Bartonella
strains. The processed region is characterized by the presence of intervening sequences (IVSs). The 23S rDNA sequences between positions 109 and 205 (
Escherichia coli
numbering) were determined, and potential secondary structures are proposed. Comparison of the IVSs indicates very different evolutionary rates in some phylogenetic branches, lateral genetic transfer, and evolution by insertion and/or deletion. We show that the IVS processing in
Rhodobacter capsulatus
in vivo is RNase III-dependent and that RNase III cleaves additional sites in vitro. While all IVS-containing transcripts tested are processed in vitro by RNase III from
R. capsulatus
,
E. coli
RNase III recognizes only some of them as substrates and in these substrates frequently cleaves at different scissile bonds. These results demonstrate the different substrate specificities of the two enzymes. Although RNase III plays an important role in the rRNA, mRNA, and bacteriophage RNA maturation, its substrate specificity is still not well understood. Comparison of the IVSs of helix 9 does not hint at sequence motives involved in recognition but reveals that the “antideterminant” model, which represents the most recent attempt to explain the
E. coli
RNase III specificity in vitro, cannot be applied to substrates derived from alpha-
Proteobacteria
.
Calcium and Malate Are Sporulation-Promoting Factors ofPhysarum polycephalum
Authors: Renzel Stefan , Esselborn Sigrid , Sauer Helmut W. , Hildebrandt Armin
Published: July 2002

Fruiting body formation (sporulation) is a distinctive, irreversible differentiation process in the life cycle of the slime moldPhysarum polycephalum. The most important requirement for sporulation ofPhysarumis a period of starvation, and normally sporulation proceeds in the light. It is shown here that by omitting the liquid sporulation medium and elevating the temperature from 21 to 25°C, sporulation can occur routinely in the dark. It is further shown that this autocrine signaling in the dark requires calcium ions and malate. A putative sporulation control factor was detected in conditioned media derived from plasmodia starved in the dark, which was then identified as polymalate. As an additional role for this previously detected polyanion, specific for the plasmodial state ofPhysarum, it is suggested that the secreted compound serves as a source for both malate and calcium ions and thus promotes sporulation without light signaling.
Effects of Calcium and Calcium Chelators on Growth and Morphology of
Escherichia coli
L-Form NC-7
Authors: Onoda T. , Enokizono J. , Kaya H. , Oshima A. , Freestone P. , Norris V.
Published: July 2002

Growth of a wall-less, L-form of
Escherichia coli
specifically requires calcium, and in its absence, cells ceased dividing, became spherical, swelled, developed large vacuoles, and eventually lysed. The key cell division protein, FtsZ, was present in the L-form at a concentration five times less than that in the parental strain. One interpretation of these results is that the L-form possesses an enzoskeleton partly regulated by calcium.
Genetic and Biochemical Characterization of the Pathway in
Pantoea citrea
Leading to Pink Disease of Pineapple
Authors: Pujol Catherine J. , Kado Clarence I.
Published: July 2002

Pink disease of pineapple, caused by
Pantoea citrea
, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of
P. citrea
that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of
c
-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2-ketogluconate, resulting in an inability to produce the compound 2,5-diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by
P. citrea
appears to be responsible for the dark color characteristic of the pink disease of pineapple.
A Common Regulator for the Operons Encoding the Enzymes Involved in
d
-Galactarate,
d
-Glucarate, and
d
-Glycerate Utilization in
Escherichia coli
Authors: Monterrubio Rafael , Baldoma Laura , Obradors Nuria , Aguilar Juan , Badia Josefa
Published: July 2002

Genes for
d
-galactarate (
gar
) and
d
-glucarate (
gud
) metabolism in
Escherichia coli
are organized in three transcriptional units:
garD
,
garPLRK
, and
gudPD
. Two observations suggested a common regulator for the three operons. (i) Their expression was triggered by
d
-galactarate,
d
-glucarate, and
d
-glycerate. (ii) Metabolism of the three compounds was impaired by a single Tn
5
insertion mapped in the
yaeG
gene (proposed name,
sdaR
), outside the
d
-galactarate and
d
-glucarate systems. Expression of the
sdaR
gene is autogenously regulated.
p
-Hydroxyphenylacetic Acid Metabolism in
Pseudomonas putida
F6
Authors: O'Connor Kevin E. , Witholt Bernard , Duetz Wouter
Published: July 2002

Pseudomonas putida
F6 was found to metabolize
p
-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of
P. putida
F6 catalyze the NAD(P)H-independent hydroxylation of
p
-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.
Properties and Mutation Analysis of the CelK Cellulose-Binding Domain from the
Clostridium thermocellum
Cellulosome
Authors: Kataeva Irina A. , Seidel Ronald D. , Li Xin-Liang , Ljungdahl Lars G.
Published: July 2002

The family IV cellulose-binding domain of
Clostridium thermocellum
CelK (CBD
CelK
) was expressed in
Escherichia coli
and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 μmol/g of cellulose and relative affinities (
K
r
) of 2.33 and 9.87 liters/g, respectively. The CBD
CelK
is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD
CelK
also binds to the soluble polysaccharides lichenin, glucomannan, and barley β-glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD
CelK
contains 1 mol of calcium per mol. The CBD
CelK
has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD
CelK
four highly conserved aromatic residues (Trp
56
, Trp
94
, Tyr
111
, and Tyr
136
) and Asp
192
were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N
CelK
. The CBD-N
CelK
and D192A retained binding parameters close to that of the intact CBD
CelK
, W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and
K
r
and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD
CelK
led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD
CelK
and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD
CelK
are not important for binding. Tyr136, corresponding to
Cellulomonas fimi
CenC CBD
N1
Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD
CelK
correctly folded.
Roles of
rpoN
,
fliA,
and
flgR
in Expression of Flagella in
Campylobacter jejuni
Authors: Jagannathan Aparna , Constantinidou Chrystala , Penn Charles W.
Published: July 2002

Three potential regulators of flagellar expression present in the genome sequence of
Campylobacter jejuni
NCTC 11168, the genes
rpoN
,
flgR,
and
fliA,
which encode the alternative sigma factor ς
54
, the ς
54
-associated transcriptional activator FlgR, and the flagellar sigma factor ς
28
, respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated in
C. jejuni
strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that the
rpoN
and
flgR
mutants were nonflagellate and that the
fliA
mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of
rpoN
,
flgR,
and
fliA
in the expression of flagellin.
Site-Specific Integrative Elements of Rhizobiophage
16-3
Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera
Authors: Semsey Szabolcs , Blaha Béla , Köles Krisztián , Orosz László , Papp Péter P.
Published: July 2002

The integrase protein of the
Rhizobium meliloti
41 phage
16-3
has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (
attB
) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the
16-3
phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the
attP
element and the integrase gene (
int
) of the phage can integrate into the chromosomes of
R. meliloti
1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the
att
core was different from that expected previously. The minimal
attB
was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.