bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoimmune Dilated Cardiomyopathy in PD-1 Receptor-Deficient Mice
Authors: Nishimura Hiroyuki , Okazaki Taku , Tanaka Yoshimasa , Nakatani Kazuki , Hara Masatake , Matsumori Akira , Sasayama Shigetake , Mizoguchi Akira , Hiai Hiroshi , Minato Nagahiro , Honjo Tasuku
Published: July 2002

Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2
−/−
mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1
−/−
mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.
Variation Among Biomes in Temporal Dynamics of Aboveground Primary Production
Authors: Knapp Alan K. , Smith Melinda D.
Published: July 2002

Interannual variability in aboveground net primary production (ANPP) was assessed with long-term (mean = 12 years) data from 11 Long Term Ecological Research sites across North America. The greatest interannual variability in ANPP occurred in grasslands and old fields, with forests the least variable. At a continental scale, ANPP was strongly correlated with annual precipitation. However, interannual variability in ANPP was not related to variability in precipitation. Instead, maximum variability in ANPP occurred in biomes where high potential growth rates of herbaceous vegetation were combined with moderate variability in precipitation. In the most dynamic biomes, ANPP responded more strongly to wet than to dry years. Recognition of the fourfold range in ANPP dynamics across biomes and of the factors that constrain this variability is critical for detecting the biotic impacts of global change phenomena.
Interactions of Bacterial Cationic Peptide Antibiotics with Outer and Cytoplasmic Membranes of
Pseudomonas aeruginosa
Authors: Zhang Lijuan , Dhillon Pawandeep , Yan Hong , Farmer Susan , Hancock Robert E. W.
Published: July 2002

Polymyxins B and E1 and gramicidin S are bacterium-derived cationic antimicrobial peptides. The polymyxins were more potent than gramicidin S against
Pseudomonas aeruginosa
, with MICs of 0.125 to 0.25 and 8 μg/ml, respectively. These peptides differed in their affinities for binding to lipopolysaccharide, but all were able to permeabilize the outer membrane of wild-type
P. aeruginosa
PAO1 strain H103, suggesting differences in their mechanisms of self-promoted uptake. Gramicidin S caused rapid depolarization of the bacterial cytoplasmic membrane at concentrations at which no killing was observed within 30 min, whereas, conversely, the concentrations of the polymyxins that resulted in rapid killing resulted in minimal depolarization. These data indicate that the depolarization of the cytoplasmic membrane by these peptides did not correlate with bacterial cell lethality.
In Vitro Activities of DU-1102, a New Trioxaquine Derivative, againstPlasmodium falciparumIsolates
Authors: Basco Leonardo K. , Dechy-Cabaret Odile , Ndounga Mathieu , Meche Fleurette Solange , Robert Anne , Meunier Bernard
Published: July 2002

The antimalarial trioxaquine derivative DU-1102, synthesized by covalent linkage between aminoquinoline and trioxane moieties, was highly active against Cameroonian isolates (mean 50% inhibitory concentration of 43 nmol/liter) ofPlasmodium falciparum. There was no correlation between the responses to DU-1102 and chloroquine and only a low correlation between the responses to DU-1102 and pyrimethamine, suggesting an independent mode of action of the trioxaquine against the parasites.
Antibacterial Properties of Dermaseptin S4 Derivatives with In Vivo Activity
Authors: Navon-Venezia Shiri , Feder Rina , Gaidukov Leonid , Carmeli Yehuda , Mor Amram
Published: July 2002

Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents. Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K
4
K
20
-S4 and two shorter versions, K
4
-S4(1-16) and K
4
-S4(1-13). The range of MICs of K
4
K
20
-S4 against clinical isolates of
Staphylococcus aureus
,
Pseudomonas aeruginosa,
and
Escherichia coli
were, respectively, 1 to 4, 1 to 4, and 1 to 16 μg/ml. MICs of the short derivatives were rather similar or two to fourfold higher. Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either
E. coli
or
S. aureus
by 6 log units in 30 min or less. Compared with MSI-78 or PG-1, K
4
-S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes. Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the
l
- or
d
isomer of either of the dermaseptin derivatives. The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with
P. aeruginosa
. Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K
4
-S4(1-16) or K
4
-S4(1-13), respectively. In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K
4
-S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h. Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.
Antimicrobial Peptides Protect Coho Salmon from
Vibrio anguillarum
Infections
Authors: Jia X. , Patrzykat A. , Devlin R. H. , Ackerman P. A. , Iwama G. K. , Hancock R. E. W.
Published: July 2002

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen
Vibrio anguillarum
. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with
V. anguillarum
, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of
V. anguillarum
at a dose that would kill 50 to 90% of the population. Fish receiving 200 μg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 μg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.
Detection and Identification of
Leishmania
DNA within Naturally Infected Sand Flies by Seminested PCR on Minicircle Kinetoplastic DNA
Authors: Aransay Ana M. , Scoulica Efstathia , Tselentis Yannis
Published: July 2002

A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of
Leishmania
species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most
Leishmania
species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight
Leishmania
species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of
Leishmania
spp. in areas of endemicity.
Anaerobic Conversion of Lactic Acid to Acetic Acid and 1,2-Propanediol by
Lactobacillus buchneri
Authors: Oude Elferink Stefanie J. W. H. , Krooneman Janneke , Gottschal Jan C. , Spoelstra Sierk F. , Faber Folkert , Driehuis Frank
Published: July 2002

The degradation of lactic acid under anoxic conditions was studied in several strains of
Lactobacillus buchneri
and in close relatives such as
Lactobacillus parabuchneri
,
Lactobacillus kefir
, and
Lactobacillus hilgardii
. Of these lactobacilli,
L. buchneri
and
L. parabuchneri
were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1,2-propanediol-dependent oxidoreductase (530 to 790 nmol min
−1
mg of protein
−1
), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by
L. buchneri
does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by
L. buchneri
is not certain, but some results indicate that it plays a role in maintaining cell viability.
Pigment and Virulence Deficiencies Associated with Mutations in the
aroE
Gene of
Xanthomonas oryzae
pv. oryzae
Authors: Goel Ajay Kumar , Rajagopal Lakshmi , Sonti R. V.
Published: July 2002

Xanthomonadins are yellow, membrane-bound pigments produced by members of the genus
Xanthomonas
. We identified an ethyl methanesulfonate-induced
Xanthomonas oryzae
pv. oryzae mutant (BXO65) that is deficient for xanthomonadin production and virulence on rice, as well as auxotrophic for aromatic amino acids (Pig
−
Vir
−
Aro
−
). Reversion analysis indicated that these multiple phenotypes are due to a single mutation. A genomic library of the wild-type strain was used to isolate a 7.0-kb clone that complements BXO65. By transposon mutagenesis, marker exchange, sequence analysis, and subcloning, the complementing activity was localized to a 849-bp open reading frame (ORF). This ORF is homologous to the
aroE
gene, which encodes shikimate dehydrogenase in various bacterial species. Shikimate dehydrogenase activity was present in the wild-type strain and the mutant with the complementing clone, whereas no activity was found in BXO65. This clone also complemented an
Escherichia coli aroE
mutant for prototrophy, indicating that
aroE
is functionally conserved in
X. oryzae
pv. oryzae and
E. coli
. The nucleotide sequence of the 2.9-kb region containing
aroE
revealed that a putative DNA helicase gene is located adjacent to
aroE
. Our results indicate that
aroE
is required for normal levels of virulence and xanthomonadin production in
X. oryzae
pv. oryzae.
Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed
Authors: Smalla K. , Wieland G. , Buchner A. , Zock A. , Parzy J. , Kaiser S. , Roskot N. , Heuer H. , Berg G.
Published: July 2002

The bacterial rhizosphere communities of three host plants of the pathogenic fungus
Verticillium dahliae
, field-grown strawberry (
Fragaria ananassa
Duch.), oilseed rape (
Brassica napus
L.), and potato (
Solanum tuberosum
L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times.
Bacillus megaterium
and
Arthrobacter
sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria.
Nocardia
populations were identified as strawberry-specific bands.
Identification of Mur, an Atypical Peptidoglycan Hydrolase Derived fromLeuconostoc citreum
Authors: Cibik Recep , Tailliez Patrick , Langella Philippe , Chapot-Chartier Marie-Pierre
Published: July 2002

A gene encoding a protein homologous to known bacterialN-acetyl-muramidases has been cloned fromLeuconostoc citreumby a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases,L. citreumMur protein does not contain amino acid repeats involved in cell wall binding. The purifiedL. citreumMur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion ofL. citreumMur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin ofLactococcus lactis. Expression of the Mur-cA fusion protein was able to complement anacmAmutation inL. lactis;normal cell separation after cell division was restored by Mur-cA expression.
Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced byVibrio tubiashii
Authors: Kothary Mahendra H. , Delston Rachel B. , Curtis Sherill K. , McCardell Barbara A. , Tall Ben D.
Published: July 2002

An extracellular cytolysin fromVibrio tubiashiiwas purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of theVibrio vulnificuscytolysin.
Microbial Population Structures in Soil Particle Size Fractions of a Long-Term Fertilizer Field Experiment
Authors: Sessitsch Angela , Weilharter Alexandra , Gerzabek Martin H. , Kirchmann Holger , Kandeler Ellen
Published: July 2002

Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil particle size fractions have been well studied, little information is available on the structure of microbial populations in microhabitats. In this study, topsoil samples of different fertilizer treatments of a long-term field experiment were analyzed. Size fractions of 200 to 63 μm (fine sand fraction), 63 to 2 μm (silt fraction), and 2 to 0.1 μm (clay fraction) were obtained by a combination of low-energy sonication, wet sieving, and repeated centrifugation. Terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes were used to compare bacterial community structures in different particle size fractions. The microbial community structure was significantly affected by particle size, yielding higher diversity of microbes in small size fractions than in coarse size fractions. The higher biomass previously found in silt and clay fractions could be attributed to higher diversity rather than to better colonization of particular species. Low nutrient availability, protozoan grazing, and competition with fungal organisms may have been responsible for reduced diversities in larger size fractions. Furthermore, larger particle sizes were dominated by α-
Proteobacteria
, whereas high abundance and diversity of bacteria belonging to the
Holophaga/Acidobacterium
division were found in smaller size fractions. Although very contrasting organic amendments (green manure, animal manure, sewage sludge, and peat) were examined, our results demonstrated that the bacterial community structure was affected to a greater extent by the particle size fraction than by the kind of fertilizer applied. Therefore, our results demonstrate specific microbe-particle associations that are affected to only a small extent by external factors.
Distribution of Antibody againstErysipelothrix rhusiopathiaein Cattle
Authors: Sawada Takuo , Hassanein Raafat , Yamamoto Tohru , Yoshida Takaharu
Published: July 2002

Serum samples collected from 854 cattle in nine prefectures of Japan, from Hokkaido to Okinawa, between 1988 and 1992 were examined for presence of antibodies againstErysipelothrix rhusiopathiaeby growth agglutination test. Most of the sera showed positive reactions, and the antibody titers ranged from below 4 to above 128. Seventy-six percent of the sera showed titers of 32 or above, and 34% showed titers of 128 or above. The titers had a tendency to be higher in the south and lower in the north and were clearly low in sera from areas with no swine industry. These results indicated that Japanese cattle had been infected withE. rhusiopathiaeand that clinical cases of the disease were possible.
Reduced Levels of Nitric Oxide Metabolites in Cerebrospinal Fluid Are Associated with Equine Protozoal Myeloencephalitis
Authors: Njoku Chinedu J. , Saville William J. A. , Reed Stephen M. , Oglesbee Michael J. , Rajala-Schultz Päivi J. , Stich Roger W.
Published: July 2002

Equine protozoal myeloencephalitis (EPM) is a disease of horses that is primarily associated with infection with the apicomplexanSarcocystis neurona. Infection with this parasite alone is not sufficient to induce the disease, and the mechanism of neuropathogenesis associated with EPM has not been reported. Nitric oxide (NO) functions as a neurotransmitter, a vasodilator, and an immune effector and is produced in response to several parasitic protozoa. The purpose of this work was to determine if the concentration of NO metabolites (NOx−) in the cerebrospinal fluid (CSF) is correlated with the development of EPM. CSF NOx−levels were measured before and after transport-stressed, acclimated, or dexamethasone-treated horses (n= 3 per group) were experimentally infected withS. neuronasporocysts. CSF NOx−levels were also compared between horses that were diagnosed with EPM after natural infection withS. neuronaand horses that did not have clinical signs of disease or that showed no evidence of infection with the parasite (n= 105). Among the experimentally infected animals, the mean CSF NOx−levels of the transport-stressed group, which had the most severe clinical signs, was reduced after infection, while these values were found to increase after infection in the remaining groups that had less severe signs of EPM. Under natural conditions, horses with EPM (n= 65) had a lower mean CSF NOx−concentration than clinically normal horses with antibodies (Abs) againstS. neurona(n= 15) in CSF, and horses that developed ataxia (n= 81) had a significantly lower mean CSF NOx−concentration than horses that did not have neurologic signs (n= 24). In conclusion, lower CSF NOx−levels were associated with clinical EPM, suggesting that measurement of CSF NOx−levels could improve the accuracy of diagnostic tests that are based upon detection ofS. neurona-specific Abs in CSF alone and that reduced NO levels could be causatively related to the development of EPM.
Identification and Characterization of the
scl
Gene Encoding a Group A
Streptococcus
Extracellular Protein Virulence Factor with Similarity to Human Collagen
Authors: Lukomski Slawomir , Nakashima Kazumitsu , Abdi Iman , Cipriano Vincent J. , Ireland Robin M. , Reid Sean D. , Adams Gerald G. , Musser James M.
Published: July 2002

Group A
Streptococcus
(GAS) expresses cell surface proteins that mediate important biological functions such as resistance to phagocytosis, adherence to plasma and extracellular matrix proteins, and degradation of host proteins. An open reading frame encoding a protein of 348 amino acid residues was identified by analysis of the genome sequence available for a serotype M1 strain. The protein has an LPATGE sequence located near the carboxy terminus that matches the consensus sequence (LPXTGX) present in many gram-positive cell wall-anchored molecules. Importantly, the central region of this protein contains 50 contiguous Gly-X-X triplet amino acid motifs characteristic of the structure of human collagen. The structural gene (designated
scl
for streptococcal collagen-like) was present in all 50 GAS isolates tested, which together express 21 different M protein types and represent the breadth of genomic diversity in the species. DNA sequence analysis of the gene in these 50 isolates found that the number of contiguous Gly-X-X motifs ranged from 14 in serotype M6 isolates to 62 in a serotype M41 organism. M1 and M18 organisms had the identical allele, which indicates very recent horizontal gene transfer. The gene was transcribed abundantly in the logarithmic but not stationary phase of growth, a result consistent with the occurrence of a DNA sequence with substantial homology with a consensus Mga binding site immediately upstream of the
scl
open reading frame. Two isogenic mutant M1 strains created by nonpolar mutagenesis of the
scl
structural gene were not attenuated for mouse virulence as assessed by intraperitoneal inoculation. In contrast, the isogenic mutant derivative made from the M1 strain representative of the subclone most frequently causing human infections was significantly less virulent when inoculated subcutaneously into mice. In addition, both isogenic mutant strains had significantly reduced adherence to human A549 epithelial cells grown in culture. These studies identify a new extracellular GAS virulence factor that is widely distributed in the species and participates in adherence to host cells and soft tissue pathology.
In Vitro Responsiveness of γδ T Cells fromMycobacterium bovis-Infected Cattle to Mycobacterial Antigens: Predominant Involvement of WC1+Cells
Authors: Smyth Allister J. , Welsh Michael D. , Girvin R. Martyn , Pollock John M.
Published: July 2002

It is generally accepted that protective immunity against tuberculosis is generated through the cell-mediated immune (CMI) system, and a greater understanding of such responses is required if better vaccines and diagnostic tests are to be developed. γδ T cells form a major proportion of the peripheral blood mononuclear cells (PBMC) in the ruminant system and, considering data from other species, may have a significant role in CMI responses in bovine tuberculosis. This study compared the in vitro responses of αβ and γδ T cells fromMycobacterium bovis-infected and uninfected cattle. The results showed that, following 24 h of culture of PBMC withM. bovis-derived antigens, the majority of γδ T cells from infected animals became highly activated (upregulation of interleukin-2R), while a lower proportion of the αβ T-cell population showed activation. Similar responses were evident to a lesser degree in uninfected animals. Study of the kinetics of this response showed that γδ T cells remained significantly activated for at least 7 days in culture, while activation of αβ T cells declined during that period. Subsequent analysis revealed that the majority of activated γδ T cells expressed WC1, a 215-kDa surface molecule which is not expressed on human or murine γδ T cells. Furthermore, in comparison with what was found for CD4+T cells,M. bovisantigen was found to induce strong cellular proliferation but relatively little gamma interferon release by purified WC1+γδ T cells. Overall, while the role of these cells in protective immunity remains unclear, their highly activated status in response toM. bovissuggests an important role in antimycobacterial immunity, and the ability of γδ T cells to influence other immune cell functions remains to be elucidated, particularly in relation to CMI-based diagnostic tests.
Limited Mycobacterial Infection of the Liver as a Consequence of Its Microanatomical Structure Causing Restriction of Mycobacterial Growth to Professional Phagocytes
Authors: Seiler Peter , Schwendener Reto A. , Bandermann Silke , Brinkmann Volker , Grode Leander , Kaufmann Stefan H. E. , Aichele Peter
Published: July 2002

Among sites of extrapulmonary growth ofMycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded byM. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.
PerR Controls Oxidative Stress Resistance and Iron Storage Proteins and Is Required for Virulence in
Staphylococcus aureus
Authors: Horsburgh Malcolm J. , Clements Mark O. , Crossley Howard , Ingham Eileen , Foster Simon J.
Published: July 2002

The
Staphylococcus aureus
genome encodes three ferric uptake regulator (Fur) homologues: Fur, PerR, and Zur. To determine the exact role of PerR, we inactivated the gene by allelic replacement using a kanamycin cassette, creating strain MJH001 (
perR
). PerR was found to control transcription of the genes encoding the oxidative stress resistance proteins catalase (KatA), alkyl hydroperoxide reductase (AhpCF), bacterioferritin comigratory protein (Bcp), and thioredoxin reductase (TrxB). Furthermore, PerR regulates transcription of the genes encoding the iron storage proteins ferritin (Ftn) and the ferritin-like Dps homologue, MrgA. Transcription of
perR
was autoregulated, and PerR repressed transcription of the iron homeostasis regulator Fur, which is a positive regulator of catalase expression. PerR functions as a manganese-dependent, transcriptional repressor of the identified regulon. Elevated iron concentrations produced induction of the PerR regulon. PerR may act as a peroxide sensor, since addition of external hydrogen peroxide to 8325-4 (wild type) resulted in increased transcription of most of the PerR regulon, except for
fur
and
perR
itself. The PerR-regulated
katA
gene encodes the sole catalase of
S. aureus,
which is an important starvation survival determinant but is surprisingly not required for pathogenicity in a murine skin abscess model of infection. In contrast, PerR is not necessary for starvation survival but is required for full virulence (
P
< 0.005) in this model of infection. PerR of
S. aureus
may act as a redox sentinel protein during infection, analogous to the in vitro activities of OxyR and PerR of
Escherichia coli
and
Bacillus subtilis
, respectively. However, it differs in its response to the metal balance within the cell and has the added capability of regulating iron uptake and storage.
Relationships between
Staphylococcus aureus
Genetic Background, Virulence Factors,
agr
Groups (Alleles), and Human Disease
Authors: Jarraud Sophie , Mougel Christophe , Thioulouse Jean , Lina Gerard , Meugnier Hélène , Forey Françoise , Nesme Xavier , Etienne Jerome , Vandenesch François
Published: July 2002

The expression of most
Staphylococcus aureus
virulence factors is controlled by the
agr
locus, which encodes a two-component signaling pathway whose activating ligand is an
agr
-encoded autoinducing peptide (AIP). A polymorphism in the amino acid sequence of the AIP and of its corresponding receptor divides
S. aureus
strains into four major groups. Within a given group, each strain produces a peptide that can activate the
agr
response in the other member strains, whereas the AIPs belonging to different groups are usually mutually inhibitory. We investigated a possible relationship between
agr
groups and human
S. aureus
disease by studying 198
S. aureus
strains isolated from 14 asymptomatic carriers, 66 patients with suppurative infection, and 114 patients with acute toxemia. The
agr
group and the distribution of 24 toxin genes were analyzed by PCR, and the genetic background was determined by means of amplified fragment length polymorphism (AFLP) analysis. The isolates were relatively evenly distributed among the four
agr
groups, with 61 strains belonging to
agr
group I, 49 belonging to group II, 43 belonging to group III, and 45 belonging to group IV. Principal coordinate analysis performed on the AFLP distance matrix divided the 198 strains into three main phylogenetic groups, AF1 corresponding to strains of
agr
group IV, AF2 corresponding to strains of
agr
groups I and II, and AF3 corresponding to strains of
agr
group III. This indicated that the
agr
type was linked to the genetic background. A relationship between genetic background,
agr
group, and disease type was observed for several toxin-mediated diseases: for instance,
agr
group IV strains were associated with generalized exfoliative syndromes, and phylogenetic group AF1 strains with bullous impetigo. Among the suppurative infections, endocarditis strains mainly belonged to phylogenetic group AF2 and
agr
groups I and II. While these results do not show a direct role of the
agr
type in the type of human disease caused by
S. aureus
, the
agr
group may reflect an ancient evolutionary division of
S. aureus
in terms of this species’ fundamental biology.
Molecular Cloning of thefurGene fromActinobacillus actinomycetemcomitans
Authors: Haraszthy V. I. , Lally E. T. , Haraszthy G. G. , Zambon J. J.
Published: July 2002

In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through thefurgene product. Initial experiments showed that a clonedEscherichia coli furgene probe hybridized to Southern blots ofActinobacillus actinomycetemcomitansstrain JP2 (serotype b) chromosomal DNA. TheA. actinomycetemcomitans furgene was then cloned utilizing partial functional complementation of thefurmutant inE. colistrain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity toHaemophilus influenzaeFur and 62% identity toE. coliFur. The pUCAafurgene probe (generated from JP2 serotype b) hybridized to representatives from all fiveA. actinomycetemcomitansserotypes as well as to two strains derived from monkeys, suggesting thatfuris widely distributed inA. actinomycetemcomitans.Open reading frames having >70% identity with theE. coliandH. influenzae flavodoxinandgyrase Agenes, respectively, were found. Expression of theA. actinomycetemcomitans furgene product repressedfiuexpression and siderophore production inE. coli. A gel shift assay demonstrated that the expressedA. actinomycetemcomitansFur protein bound the bacterialfurconsensus sequence. Further characterization of thefurgene product inA. actinomycetemcomitansmay improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.
Identification of the
Vibrio cholerae
Enterobactin Receptors VctA and IrgA: IrgA Is Not Required for Virulence
Authors: Mey Alexandra R. , Wyckoff Elizabeth E. , Oglesby Amanda G. , Rab Eva , Taylor Ronald K. , Payne Shelley M.
Published: July 2002

The gram-negative enteric pathogen
Vibrio cholerae
requires iron for growth.
V. cholerae
has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make. One siderophore that
V. cholerae
transports, but does not make, is enterobactin. Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA. In this study, two candidate enterobactin receptor genes,
irgA
(VC0475) and
vctA
(VCA0232), were identified by analysis of the
V. cholerae
genomic sequence. A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin. When either
irgA
or
vctA
was supplied on a plasmid, the ability of the
irgA vctA
double mutant to use enterobactin was restored. This indicates that both VctA and IrgA transport enterobactin. We also identify the genes
vctPDGC
, which are linked to
vctA
and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230). An
irgA
::Tn
phoA
mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of
V. cholerae
infection (M. B. Goldberg, V. J. DiRita, and S. B. Calderwood, Infect. Immun. 58:55-60, 1990). In this work, a new
irgA
mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose. These data indicate that the virulence defect in MBG40 is not due to the loss of
irgA
function and that
irgA
is unlikely to be an important virulence factor.
Identification of the Gene Encoding Sulfopyruvate Decarboxylase, an Enzyme Involved in Biosynthesis of Coenzyme M
Authors: Graupner Marion , Xu Huimin , White Robert H.
Published: July 2002

The products of two adjacent genes in the chromosome of
Methanococcus jannaschii
are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in
Streptomyces wedmorensis
. These two
M. jannaschii
genes were recombinantly expressed in
Escherichia coli
, and their gene products were tested for the ability to catalyze the decarboxylation of a series of α-ketoacids. Both subunits are required to form an α
6
β
6
dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The
M. jannaschii
sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.
Swarming of
Pseudomonas aeruginosa
Is Dependent on Cell-to-Cell Signaling and Requires Flagella and Pili
Authors: Köhler Thilo , Curty Lasta Kocjancic , Barja Francisco , van Delden Christian , Pechère Jean-Claude
Published: July 2002

We describe swarming in
Pseudomonas aeruginosa
as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in
P. aeruginosa
is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a
fliC
mutant of
P. aeruginosa
displayed slightly diminished swarming, a
pilR
and a
pilA
mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the
las
cell-to-cell signaling system showed diminished swarming behavior, while
rhl
mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of
P. aeruginosa
in this type of surface motility.
Identification of an Operon Required for Ferrichrome Iron Utilization in
Vibrio cholerae
Authors: Rogers Marc B. , Sexton Jessica A. , DeCastro G. Joel , Calderwood Stephen B.
Published: July 2002

Mutagenesis of
Vibrio cholerae
with Tn
phoA
, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to
Escherichia coli
FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the
E. coli fhu
operon, the
V. cholerae
ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.
Biofilm Formation by the Fungal Pathogen
Candida albicans
: Development, Architecture, and Drug Resistance
Authors: Chandra Jyotsna , Kuhn Duncan M. , Mukherjee Pranab K. , Hoyer Lois L. , McCormick Thomas , Ghannoum Mahmoud A.
Published: July 2002

Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant
Candida albicans
biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that
C. albicans
biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (
ALS
) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of
C. albicans
to form biofilms contrasts sharply with that of
Saccharomyces cerevisiae
, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of
Candida
biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.
One of Two
hemN
Genes in
Bradyrhizobium japonicum
Is Functional during Anaerobic Growth and in Symbiosis
Authors: Fischer Hans-Martin , Velasco Leonardo , Delgado Maria J. , Bedmar Eulogio J. , Schären Simon , Zingg Daniel , Göttfert Michael , Hennecke Hauke
Published: July 2002

Previously, we screened the symbiotic gene region of the
Bradyrhizobium japonicum
chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472–1480, 2000). Here we report more details on one of the genes identified, a
hemN
-like gene (now called
hemN
1
) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that
B. japonicum
possesses a second
hemN
-like gene (
hemN
2
), which was then cloned by using
hemN
1
as a probe. The
hemN
2
gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of
nirK
, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN
1
and HemN
2
, respectively) and share 53% identical (68% similar) amino acids. Expression of both
hemN
genes was monitored with the help of chromosomally integrated translational
lacZ
fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (≥20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK
2
. In addition, maximal anaerobic
hemN
1
expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual
hemN
genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the
hemN
1
mutant, strains lacking a functional
hemN
2
gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several
c
-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that
B. japonicum hemN
2
, but not
hemN
1
, encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a
Salmonella enterica
serovar Typhimurium
hemF hemN
mutant with
hemN
2
only.
Ribonuclease M5 Has Few, If Any, mRNA Substrates inBacillus subtilis
Authors: Condon Ciarán , Rourera Jordi , Brechemier-Baey Dominique , Putzer Harald
Published: July 2002

InBacillus subtilis, maturation of 5S rRNA is catalyzed by an enzyme called RNase M5. We searched for potential mRNA substrates for RNase M5 by gene array technology, based on the premise that most endonucleolytic cleavages have an effect on the stability of RNA and hence on steady-state levels of expression. Only a handful of genes had significantly altered expression inrnmVmutants compared to wild-type strains that could subsequently be confirmed by Northern blotting. The effect of RNase M5 on the expression of the best candidates, theodhABandsucCDoperons, is indirect, by a mechanism we do not yet understand. We show that an effect of RNase M5 on the expression of the remaining candidate,ctsR, is due to the failure to process the 5S rRNA contained in therrnWlying directly upstream. We thus conclude that RNase M5 has very few or possibly no mRNA substrates inB. subtilis.
Regulation ofSalmonella entericaSerovar TyphimuriummntHTranscription by H2O2, Fe2+, and Mn2+
Authors: Kehres David G. , Janakiraman Anu , Slauch James M. , Maguire Michael E.
Published: July 2002

MntH, a bacterial homolog of mammalian natural resistance associated macrophage protein 1 (Nramp1), is a primary transporter for Mn2+influx inSalmonellaentericaserovar Typhimurium andEscherichia coli. S. entericaserovar Typhimurium MntH contributes to H2O2resistance and is important for full virulence. Consistent with its phenotype and function,mntHis regulated at the transcriptional level by both H2O2and substrate cation. We have now identified threetrans-acting regulatory factors and the three correspondingcis-actingmntHpromoter motifs that mediate this regulation. In the presence of hydrogen peroxide,mntHis activated by OxyR, acting through an OxyR-binding motif centered just upstream of the likely −35 RNA polymerase-binding site. In the presence of Fe2+,mntHis repressed primarily by Fur, acting through a Fur-binding motif overlapping the −35 region. In the presence of Mn2+,mntHis repressed primarily by theSalmonellaequivalent ofE. colib0817, a distant homolog of theBacillus subtilismanganese transport repressor, MntR, acting through an inverted-repeat motif located between the likely −10 polymerase binding site and the ribosome binding site.E. colib0817 was recently shown to bind the identical inverted-repeat motif in theE. coli mntHpromoter and hence has been renamed MntR (S. I. Patzer and K. Hantke, J. Bacteriol.183:4806-4813, 2001). Using Δfur, ΔmntR, and ΔfurΔmntRmutant strains as well as mutations in the Fur- and MntR-binding motif elements, we found that Fe2+can also mediate repression through the Mn2+repressor MntR.
mig
-
14
Is a
Salmonella
Gene That Plays a Role in Bacterial Resistance to Antimicrobial Peptides
Authors: Brodsky Igor E. , Ernst Robert K. , Miller Samuel I. , Falkow Stanley
Published: July 2002

It was previously demonstrated that the
mig
-
14
gene of
Salmonella enterica
serovar Typhimurium is necessary for bacterial proliferation in the liver and spleen of mice following intragastric inoculation and that
mig
-
14
expression, which is induced within macrophages, is under the control of the global regulator PhoP. Here we demonstrate that the
mig
-
14
promoter is induced by growth in minimal medium containing low magnesium or acidic pH, consistent with regulation by PhoP. In addition,
mig
-
14
is strongly induced by polymyxin B, protamine, and the mammalian antimicrobial peptide protegrin-1. While
phoP
is necessary for the induction of
mig
-
14
in response to protamine and protegrin,
mig
-
14
is still induced by polymyxin B in a
phoP
background. We also demonstrate that
mig
-
14
is necessary for resistance of
S. enterica
serovar Typhimurium to both polymyxin B and protegrin-1. Gram-negative resistance to a variety of antimicrobial peptides has been correlated with modifications of lipopolysaccharide structure. However, we show that
mig
-
14
is not required for one of these modifications, the addition of 4-aminoarabinose to lipid A. Additionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of wild-type and
mig
-
14
lipopolysaccharide also shows no detectable differences between the two strains. Therefore,
mig
-
14
contributes to
Salmonella
resistance to antimicrobial peptides by a mechanism that is not yet fully understood.