bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution ofCandida albicansCell Wall Components to Recognition by and Escape from Murine Macrophages
Authors: McKenzie C. G. J. , Koser U. , Lewis L. E. , Bain J. M. , Mora-Montes H. M. , Barker R. N. , Gow N. A. R. , Erwig L. P.
Published: February 2010

The pathogenicity of the opportunistic human fungal pathogenCandida albicansdepends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects ofC. albicansinnate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion ofC. albicansby macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Δ,pmr1Δ, andmnt3 mnt5Δ), whereas O- and N-linked mannan defects (mnt1Δmnt2Δ andmns1Δ) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Δ,hwp1Δ, andals3Δ) and yeast-locked mutants (clb2Δ,hgc1Δ,cph1Δ,efg1Δ, andefg1Δcph1Δ), was similar to that observed for wild-typeC. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and processC. albicans.
CD1d-Dependent B-Cell Help by NK-Like T Cells Leads to Enhanced and Sustained Production of
Bacillus anthracis
Lethal Toxin-Neutralizing Antibodies
Authors: Devera T. Scott , Aye Lindsay M. , Lang Gillian A. , Joshi Sunil K. , Ballard Jimmy D. , Lang Mark L.
Published: February 2010

The current
Bacillus anthracis
vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against
Bacillus anthracis
but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d
−/−
mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281
−/−
mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response.
In vitro
neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced
in vivo
protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for
Bacillus anthracis
that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.
GroEL and Lipopolysaccharide fromFrancisella tularensisLive Vaccine Strain Synergistically Activate Human Macrophages
Authors: Noah Courtney E. , Malik Meenakshi , Bublitz DeAnna C. , Camenares Devin , Sellati Timothy J. , Benach Jorge L. , Furie Martha B.
Published: February 2010

Francisella tularensis, the causative agent of tularemia, interacts with host cells of innate immunity in an atypical manner. For most Gram-negative bacteria, the release of lipopolysaccharide (LPS) from their outer membranes stimulates an inflammatory response. When LPS from the attenuated live vaccine strain (LVS) or the highly virulent Schu S4 strain ofF. tularensiswas incubated with human umbilical vein endothelial cells, neither species of LPS induced expression of the adhesion molecule E-selectin or secretion of the chemokine CCL2. Moreover, a high concentration (10 μg/ml) of LVS or Schu S4 LPS was required to stimulate production of CCL2 by human monocyte-derived macrophages (huMDM). A screen for alternative proinflammatory factors ofF. tularensisLVS identified the heat shock protein GroEL as a potential candidate. Recombinant LVS GroEL at a concentration of 10 μg/ml elicited secretion of CXCL8 and CCL2 by huMDM through a TLR4-dependent mechanism. When 1 μg of LVS GroEL/ml was added to an equivalent amount of LVS LPS, the two components synergistically activated the huMDM to produce CXCL8. Schu S4 GroEL was less stimulatory than LVS GroEL and showed a lesser degree of synergy when combined with Schu S4 LPS. These findings suggest that the intrinsically low proinflammatory activity ofF. tularensisLPS may be increased in the infected human host through interactions with other components of the bacterium.
Comparative Genomic Analyses of Attenuated Strains of
Mycoplasma gallisepticum
Authors: Szczepanek S. M. , Tulman E. R. , Gorton T. S. , Liao X. , Lu Z. , Zinski J. , Aziz F. , Frasca S. , Kutish G. F. , Geary S. J.
Published: February 2010

Mycoplasma gallisepticum
is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage
M. gallisepticum
strain R (R
low
) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of
M. gallisepticum
. Analysis of the high-passage, attenuated derivative of R
low
, R
high
, indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the
M. gallisepticum
vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of
vlhA
surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R
low
isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent
M. gallisepticum
R
low
. Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter- and intrastrain
M. gallisepticum
genomic variability and the genetic basis of
M. gallisepticum
virulence.
Critical Role of Dispensable Genes inMycoplasma agalactiaeInteraction with Mammalian Cells
Authors: Baranowski Eric , Guiral Sébastien , Sagné Eveline , Skapski Agnès , Citti Christine
Published: February 2010

Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogenMycoplasma agalactiaeand used this library to identify mutants displaying a growth-deficient pheonotype in cell culture.M. agalactiaemutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction ofM. agalactiaewith HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens.
Transmission of Diverse Oral Bacteria to Murine Placenta: Evidence for the Oral Microbiome as a Potential Source of Intrauterine Infection
Authors: Fardini Yann , Chung Peter , Dumm Rochelle , Joshi Nishiant , Han Yiping W.
Published: February 2010

Microbial infection of the intrauterine environment is a major cause of preterm birth. The current paradigm indicates that intrauterine infections predominantly originate from the vaginal tract, with the organisms ascending into the sterile uterus. With the improvements in technology, an increasing number of bacterial species have been identified in intrauterine infections that do not belong to the vaginal microflora. We have demonstrated previously that intrauterine infections can originate from the oral cavity following hematogenous transmission. In this study, we begin to systemically examine what proportion of the oral microbiome can translocate to the placenta. Pooled saliva and pooled subgingival plaque samples were injected into pregnant mice through tail veins to mimic bacteremia, which occurs frequently during periodontal infections. The microbial species colonizing the murine placenta were detected using 16S rRNA gene-based PCR and clone analysis. A diverse group of bacterial species were identified, many of which have been associated with adverse pregnancy outcomes in humans although their sources of infection were not determined. Interestingly, the majority of these species were oral commensal organisms. This may be due to a dose effect but may also indicate a unique role of commensal species in intrauterine infection. In addition, a number of species were selectively “enriched” during the translocation, with a higher prevalence in the placenta than in the pooled saliva or subgingival plaque samples. These observations indicate that the placental translocation was species specific. This study provides the first insight into the diversity of oral bacteria associated with intrauterine infection.
Metabolism of nitrogen oxides and hydroxylamine in cells of true denitrifiers and Rhizobium "hedysari" HCNT1
Authors: Casella Sergio , Toffanin Anita , Ciompi Stefania , Rossi Nora , Payne W. J.
Published: February 2010

Cells of several copper-protein denitrifiers that produce nitrite reductase reduced nitrate to gaseous products but were completely or strongly inhibited by the copper chelator diethyldithiocarbamate (DDC), which did not comparably inhibit cells of cytochrome ed1 nitrite reductase producers. Both types of true denitrifiers released NO while reducing nitrite anaerobically in the presence of the uncoupler 3-chlorophenylhydrazonepropanedinitrile (CCCP), as seen previously only in Paracoccus denitrificons. In contrast, the pseudodenitrifier Rhizobium "hedysari" HCNT1, which grows as neither adenitrifier nor a fermenter, failed to reduce nitrate or nitrite or to nitrify ammonia when grown aerobically but reduced nitrite to N2O after growth at low oxygen tension even without any nitrogen oxide in the culture medium. Such oxygen-limited cells also formed N2O when incubated anaerobically with NO and hydroxylamine, and with nitrite and hydroxylamine as well, but not when anaerobic nitrite reduction (i.e., apparent production of NO) was inhibited by DDC. Rhizobium "hedysari" HCNT1 cells released no NO while reducing nitrite even in the presence of CCCP. The use of CCCP may permit differentiation of respiratory from nitrite-detoxifying denitrifying bacteria. Under aeration, but not anoxia, NO and hydroxylamine reacted to form N2O even in the absence of cells, providing a possible indicator of NO production assayable by gas chromatography.Key words: nitrite, nitric oxide, diethyldithiocarbamate (DDC), hydroxylamine, 3-chlorophenylhydrazonepropanedinitrile (CCCP).
Morphogenesis and adhesion of Aureobasidium pullulans
Authors: Andrews John H. , Harris Robin F. , Spear Russell N. , Lau Gee W. , Nordheim Erik V.
Published: February 2010

Two strains of the dimorphic fungus Aureobasidium pullulans were grown in liquid and on solid media varying in carbon and nitrogen content, and on leaf surfaces. Hyphae were observed in all systems but comprised a very low proportion (often below quantitative detection) of the total biomass. In liquid media, hyphae were found sparsely and only in the wash-zone on walls of the culture flasks. Yeast phase growth (blastospores) occurred in pH-buffered media that were nutrient balanced, or continuously carbon-limited (fed-batch culture), or carbon-exhausted (batch culture). Blastospores exposed to conditions with limited nitrogen but sufficient organic carbon, or to acidified media, converted to swollen cells and chlamydospores. The latter morphotypes accumulated carbon internally as lipid granules, and then externally as capsular and soluble extracellular polysaccharide. They were cohesive and also adhered more strongly to cellulose membranes overlying agar media or to leaves than did blastospores. Pullulanase treatment diminished the capsules, cohesion, and adhesion. Addition of soluble extracellular polysaccharide to blastospores enhanced their adhesion to leaves. We conclude that extracellular polysaccharide can play a role in adhesion of A. pullulans.Key words: phylloplane, leaf surface, epiphytic growth, extracellular polysaccharide, cell attachment.
Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation
Authors: Prokop Andreas , Rapp Peter , Wagner Fritz
Published: February 2010

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.
Brown pigmentation of Xanthomonas campestris pv. phaseoli associated with homogentisic acid
Authors: Goodwin P. H. , Sopher C. R.
Published: February 2010

The diffusible brown pigment produced by some strains of Xanthomonas campestris pv. phaseoli was found to be due to the secretion and subsequent oxidation of homogentisic acid (2, 5-dihydroxyphenylacetic acid) rather than tyrosinase activity as previously reported. Homogentisic acid is an intermediate in tyrosine catabolism for a number of bacteria. Brown-pigmented strains appeared to be disrupted in tyrosine catabolism and could not use tyrosine as a nutrient. Nonpigmented strains, however, could utilize tyrosine as a nutrient, and although they secreted homogentisic acid, the levels were approximately 1/100th that of brown-pigmented strains. Production of brown pigment was stimulated by tyrosine and repressed by glucose. Growth in glucose resulted in a drop in the pH of the media, and the greatest pigment formation was associated with a shift to alkaline conditions in the culture media. By buffering the media near pH 7.0, brown pigment formation was repressed, even though unoxidized homogentisic acid accumulated in the culture media. The disruption of tyrosine catabolism corresponds to other reports describing differences between brown-pigmented and nonpigmented strains, and provides further support for the retention of a special taxonomic status for the brown-pigmented strains.Key words: homogentisic acid, pigments, Xanthomonas, fuscans.
PID, a new member of the P1 bacteriophage group
Authors: Ackermann Hans-W. , Reynaud Alain , Gayerie Marie C.
Published: February 2010

Phage PID produces particles of essentially uniform head size and differs from P1 in its range and tail length. The dimensions of phage P1 are reassessed. The P1 phage group shows signs of morphological evolution.Key words: bacteriophage P1, evolution, morphology.
Characterization and primary specificity of an extracellular metalloproteinase from Serratia marcescens
Authors: Kim Namsoo , Kim Su Il
Published: February 2010

An extracellular endopeptidase (proteinase) from Serratia marcescens (Serratia marcescens extracellular proteinase, EC 3.4.24.4), purified to homogeneity, was analyzed for enzyme properties. The enzyme has a polypeptide chain molecular mass of 52 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme has an optimal temperature of 40 °C and an optimal pH of 7.0. Enzyme activity was enhanced over two times by the addition of Ca2+ and Mg2+ ions and eliminated almost completely by the presence of 0.2% SDS. The enzyme has broad substrate specificity and contains neither cysteine nor methionine. Low homology was found between the NH2-terminal amino acid sequence of the enzyme of this study and the NH2-terminal sequence of a proteinase from another strain of S. marcescens. Chemical modification with N-bromosuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and 8-anilino-1-naphthalene sulfonic acid and by photooxidation with methylene blue reduced enzyme activity considerably. The enzyme was shown to have broad peptide bond specificity judging from the contribution of 11 amino acids to the carboxyl side of the peptide bonds hydrolyzed.Key words: characterization, primary specificity, extracellular metalloproteinase, Serratia marcescens.
Evaluation and use of a low nutrient medium and reduced incubation temperature to study bacterial contamination in the water supply of dental units
Authors: Williams H. N. , Quinby H. , Romberg E.
Published: February 2010

A low nutrient medium, dilute peptone, and reduced incubation temperatures (25 or 30 °C) were used to recover bacteria from dental unit water supply. Significantly greater numbers of bacterial colony-forming units were recovered on the dilute peptone medium than on the enriched media, blood agar or trypticase soy agar. Lower incubation temperatures yielded greater numbers of colony-forming units on all media. The bacterial population in dental unit water supply following stagnation in the supply lines and flushing of the lines was studied using dilute peptone incubated at 25 °C. No significant differences in the numbers of colony-forming units were found in stagnant water versus fresh water. Flushing the water lines for 10 min did not significantly reduce the numbers of colony-forming units.Key words: dental unit water, water contamination, bacterial contamination.
Sulfonolipid content of Cytophaga and Flexibacter species isolated from soil and cultured under different nutrient and temperature regimes
Authors: Drijber R. A. , McGill W. B.
Published: February 2010

Gliding bacteria of the genera Cytophaga and Flexibacter contain an unusual sulfonolipid in their outer membrane that may be a potential biochemical marker for these organisms in soil. The precision with which a marker provides information about biomass varies with the range in marker content within the cells of the taxon under study. To evaluate this, Cytophaga and Flexibacter spp. were isolated from earthworm fecal pellets and soil, batch cultured with [35S]sulfate, and extracted for lipids. Sulfonolipid S was measured using isotope dilution techniques and lipid phosphate was determined by colorimetry. Ratios of sulfonolipid S to protein and to phospholipid P were compared among isolates grown under various conditions. Ratios of sulfonolipid S to phospholipid P from 0.22 to 0.98 confirmed sulfonolipids as major cell components of Cytophaga and Flexibacter spp. Two criteria essential to the success of sulfonolipids as a biomarker for these organisms in soil were met: (i) sulfonolipid concentration among the noncellulolytic isolates, with one exception, spanned a narrow range (40–60 μmol S/g cell protein), and (ii) this range in sulfonolipid concentration was maintained under the range of growth conditions studied here. Cellulolytic cytophagas, however, could be clearly differentiated from other Cytophaga and Flexibacter spp. on the basis of sulfonolipid content, 85–164 μmol S/g cell protein. This dichotomy in sulfonolipid content between cellulolytic and noncellulolytic isolates must be considered when interpreting data where sulfonolipids are used as a quantitative biomarker for Cytophaga and Flexibacter spp. in habitats where cellulolytic cytophagas are dominant.Key words: lipid biomarkers, cellulolytic cytophagas, Cytophaga, Flexibacter, sulfonolipids.
Physiological and genetic characterization of the osmotic stress response in Bacillus subtilis
Authors: Ruzal Sandra M. , Sanchez-Rivas Carmen
Published: February 2010

Bacillus subtilis cultures submitted to an osmotic upshock (1.5 M NaCl) lysed unless stationary phase had been reached. Several physiological variations were observed, such as delayed growth (adaptation), a filamentous bacterial appearance, RecA-dependent osmoresistance (SOS), and cross-induction by a previous stress (heat shock). Osmoresistance and sporulation seem to share pathways of regulation such as inhibition in the presence of glucose and glutamine and derepression in a catabolite-resistant mutant such as degUh. However, spores were not obtained on hypertonic media. Mutants of later sporulation stages (spoII, spoIII) presented a response similar to that of the wild-type parent, indicating that both processes probably shared early controls. Null mutations in any of the known key modulators of sporulation (spoOA or degU) resulted in similar levels of osmosensitivity. Sensor mutations in kinA and degS also led to strains with altered responses, the kinA mutant being even more osmosensitive than the degS mutant. Several spoOA mutant phenotypes are due to this gene's control of abrB, a regulator of stationary-phase events, and an abrB mutation relieved the osmosensitivity of the spoOA-containing mutant but had no effect on a wild-type strain.Key words: Bacillus subtilis, osmotic stress, sporulation.
Clarification of the structure of the ampicillin-resistance plasmid RSF0885 from Haemophilus influenzae HR-885 serotype b
Authors: Albritton W. L. , Noble P. A. , Webster K. D.
Published: February 2010

The nonconjugative ampicillin-resistance plasmid RSF0885 has been reported to be as small as 2.9 MDa and as large as 4.1 MDa with at least two restriction enzyme maps reported. In addition, the source of the original plasmid has been reported to be Haemophilus influenzae and Haemophilus parainfluenzae. Characterization of the source strains and sequencing data of the plasmids revealed that H. influenzae serotype b was the original source strain and that ISI-K in the larger plasmid was presumably acquired when the smaller plasmid was maintained in Escherichia coli in S. Falkow's laboratory during the late 1970s.Key words: Haemophilus influenzae, Haemophilus parainfluenzae, RSF0885, ampicillin resistance, ISI-K
The energy dependence of detergent resistance in Enterobacter cloacae: a likely requirement for ATP rather than a proton gradient or a membrane potential
Authors: Aspedon Arden , Nickerson Kenneth W.
Published: February 2010

The enteric bacterium Enterobacter cloacae was grown both aerobically and anaerobically in the presence of up to 1% of the anionic detergent sodium dodecyl sulfate (SDS). A continuous energy supply was necessary to maintain cell integrity and cells grown in SDS (0.1–1%) lysed during carbon-limited stationary phase. The respiratory inhibitor KCN (3 mM) caused rapid lysis when added to aerobic, log phase, SDS-containing cultures growing on glucose as the carbon source. However, when the SDS (0.5%) was added 30 min after KCN, lysis did not occur. The likely reason for this discrepancy concerns the cellular ATP levels. In aerobic cells the ATP levels dropped 10- to 15-fold within 1 min of adding KCN and then increased gradually over the next 30 min. Similarly, the addition of 2 mM iodoacetic acid, an inhibitor of glycolysis, to anaerobic, log phase, SDS-containing cultures caused rapid lysis. However, unlike the situation for KCN-treated aerobic cells, lysis still occurred when SDS (0.5%) was added 30 min after addition of iodoacetic acid. The reason for this difference is that in anaerobic cells, ATP levels dropped 10-to 12-fold within 5 min of the addition of iodoacetic acid and then did not increase over the next 30 min. Evidence that the energy requirement was for ATP was provided by uptake experiments with [14C]benzoic acid and α-[14C] isoaminobutyric acid that showed that the proton gradient (ΔpH) and the membrane potential (Δψ were the same in cells grown in the presence or absence of SDS. Likewise, for both aerobic and anaerobic cultures the absence of a proton gradient (ΔpH = 0) did not cause cell lysis. In carbon-starved stationary phase cells, Δψ was the same in the presence and absence of SDS. Taken together, this evidence suggests that ATP, and not ΔpH or Δψ, is required for SDS resistance.Key words: sodium dodecyl sulfate, bacterial detergent resistance, energy-dependent cell lysis, membrane potential, proton gradient, ATP.
Acid pH tolerance in strains of Rhizobium and Bradyrhizobium, and initial studies on the basis for acid tolerance of Rhizobium tropici UMR1899
Authors: Graham Peter H. , Draeger Kathryn J. , Ferrey Mark L. , Conroy Mark J. , Hammer Bruce E. , Martinez Esperanza , Aarons Sharon R. , Quinto Carmen
Published: February 2010

Acid pH limits the persistence of Rhizobium strains in soil, and the nodulation and nitrogen fixation of legumes. To identify acid-tolerant strains, we tested the ability of 45 Rhizobium, Azorhizobium, and Bradyrhizobium strains to produce isolated colonies on agar medium of pH 4.00 to pH 7.00. Only Rhizobium tropici UMR1899 (=CIAT899) grew at pH 4.00 in unbuffered medium, though 6 strains of R. tropici and 3 Bradyrhizobium strains grew at pH 4.25, and 15 strains grew at pH 4.50. Tolerance to acid pH in R. tropici UMR1899 was not an adaptive response, nor was it plasmid mediated, correlated with the production of extracellular polysaccharide, or related to synthesis of polyamines in the cell. When UMR1899 was grown in buffered medium at acid pH, it maintained a ΔpH (measured using 31P-NMR) of up to 1.7 pH units. However, when this strain was subjected to acid shock, it showed only limited ability to regulate cytoplasmic pH in the short term. Cells of UMR1899 accumulated glutamate under pH stress, and were markedly hydrophobic and resistant to the effects of crystal violet, the latter traits raising the possibility that outer membrane composition and structure could also be a factor in pH tolerance.Key words: Rhizobium, pH tolerance, glutamate, hydrophobicity, 31P-NMR.
Genetic characterization of Rhizobium sp. strain TAL1145 that nodulates tree legumes
Authors: George M. L. C. , Young J. P. W. , Borthakur D.
Published: February 2010

Rhizobium sp. strain TALI 145 nodulates Leucaena ieucocephaia and Phaseolus vulgaris, in addition to a wide range of tropical tree legumes. Six overlapping clones that complemented nodulation defects in leucaena and bean rhizobia were isolated and a 40-kb map of the symbiosis region was constructed. The common nod and nifA genes were situated approximately 17 kb apart, with the nodlJ genes in between. These clones enabled a derivative of TAL1145 carrying a partially deleted pSym to form ineffective nodules on both leucaena and bean, and a similar derivative of Rhizobium etli TAL182 to form ineffective nodules on bean. When two representative clones, pUHR9 and pUHR114, were each transferred to wild-type rhizobial strains, they allowed ineffective nodulation by Rhizobium meliloti on both leucaena and bean and by Rhizobium leguminosarum bv. viciae on bean. Transconjugants of R. leguminosarum bv. trifolii formed effective nodules on leucaena and ineffective nodules on bean. Tn5 mutagenesis of the symbiosis region resulted in a variety of nodulation and fixation phenotypes on leucaena and bean. On the basis of 16S rRNA sequences, TAL1145 was found to be distinct from both R. tropici and NGR234, the two groups of leucaena symbionts that were previously described.Key words: Rhizobium, Leucaena leucocephala, nodulation, nitrogen fixation.
Detection of enterotoxigenic Escherichia coli in water by polymerase chain reaction amplification and hybridization
Authors: Tamanai-Shacoori Z. , Jolivet-Gougeon A. , Pommepuy M. , Cormier M. , Colwell R. R.
Published: February 2010

Enterotoxigenic Escherichia coli was studied in waste water, river water, and seawater from six locations along the west coast of Normandy by using the polymerase chain reaction (PCR) to amplify the heat labile (LT) gene. Cellular DNA was extracted from centrifugation pellets and amplified using PCR. The PCR products were detected by gel electrophoresis and confirmed by hybridization assay, using an 850 base pair HindIII DNA fragment probe from pEWD299 conjugated to digoxigenin and specific for the LT gene. Results of the PCR amplification were compared with those of GM1 enzyme-linked immunosorbent assay, latex agglutination, and colony hybridization. The PCR method was found to be more precise and less time consuming, especially when compared with methods requiring culture of isolates for enumeration of enterotoxigenic E. coli in water.Key words: enterotoxigenic Escherichia coli, PCR, environmental water, digoxigenin.
Biotransformation of 2,4,6-trinitrotoluene (TNT) by a Methanococcus sp. (strain B) isolated from a lake sediment
Authors: Boopathy R. , Kulpa C. f.
Published: February 2010

A mesophilic, irregular coccoid methanogen, which shows close resemblance to Methanococcus sp., was isolated from a sediment sample of St. Joseph Lake located in the University of Notre Dame campus. Formate or hydrogen plus carbon dioxide served as substrate for methanogenesis in a mineral salt medium. This organism was studied for its ability to metabolize 2,4,6-trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm of TNT within 40–60 days of incubation at 30 °C. The main intermediate produced was 2,4-diamino-6-nitrotoluene. The TNT transformation rates were higher in cells grown in hydrogen plus carbon dioxide than in cells grown in formate. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.2. The optimal growth conditions for this isolate are described.Key words: biotransformation, methanogens, bioremediation, nitroaromatics, TNT, anaerobic process.
Relative fitness in vitro and in planta of Pseudomonas syringae strains containing copper and streptomycin resistance plasmids
Authors: Sundin George W. , Bender Carol L.
Published: February 2010

The effect of resistance plasmids encoding copper resistance (Cur), streptomycin resistance (Smr), and both Cur and Smr on competitive fitness of Pseudomonas syringae pv. syringae was studied in vitro and in planta. The Cur Smr plasmid pPSR1 provided a selective advantage to its bacterial host (Pseudomonas syringae pv. syringae FF5.1) on Pyrus calleryana leaves that were treated weekly with copper and (or) streptomycin bactericides. However, populations of the plasmid-free Cus Sms FF5.1 were reduced 10- to 1000-fold over a 12-week period on trees treated with bactericides. The resistance plasmids pPSR4 (Cur), pPSR5 (Smr), and pPSR4::Tn 5393 (Cur Smr) were highly stable for over 200 generations of growth in glucose-limited batch culture. Results of competition experiments in vitro indicated that Pseudomonas syringae pv. syringae FF5 containing pPSR4, pPSR5, or pPSR4::Tn5393 was reduced to less than 5% of the total culture in competition with wild-type FF5. In growth chamber studies, the resistance plasmids studied did not have an impact on epiphytic fitness of Pseudomonas syringae pv. syringae. Our data suggest that resistance plasmids will persist in populations of Pseudomonas syringae pv. syringae following their initial selection regardless of the bactericidal spray regime.Key words: competitive fitness, epiphytic fitness, copper and streptomycin resistance.
Expression and detection of pap-, sfa-, and afa-encoded fimbrial adhesin systems among uropathogenic Escherichia coli
Authors: Daigle France , Harel Josée , Fairbrother John M. , Lebel Pierre
Published: February 2010

Thirty-five Escherichia coli isolates from young children and women with pyelonephritis, cystitis, or asymptomatic bacteriuria were characterized genotypically and phenotypically. The isolates were examined genotypically by using DNA probes specific for the hemolysin gene and for the pap, sfa, and afa adhesin systems. Genes for the adhesin systems were also detected by polymerase chain reaction, using multigene amplification. The isolates were serotyped, tested for hemolysin production, and classified for their adhesion specificity by hemagglutination and by binding specificity assays. Twenty-seven of the 35 isolates were pap positive. Results showed that pap-positive isolates expressing class I or class II G adhesins were more frequent in cases of pyelonephritis than in cases of cystitis and asymptomatic bacteriuria. Expression of the class III G adhesins was more frequent in isolates from cystitis and asymptomatic bacteriuria than in isolates from pyelonephritis. Multiple adhesin systems and hemolysin were more frequently found in isolates from cases of pyelonephritis than in isolates from cases of cystitis and asymptomatic bacteriuria. There was perfect correlation between the results obtained by polymerase chain reaction and and those obtained by hybridization.Key words: Escherichia coli, UTI, fimbriae, PCR, virulence factors.
Urokinase enhances the growth of Pseudomonas spp. in vitro under nonshaking (oxygen limited) conditions
Authors: Hart David A. , Woods Donald E.
Published: February 2010

Urokinase is a proteinase that normally functions as a plasminogen activator. It is detected in a number of tissues and can be expressed by inflammatory cells such as macrophages and polymorphonuclear leucocytes. Addition of human urokinase to cultures of mucoid or nonmucoid variants of Pseudomonas aeruginosa (strain PAO and clinical isolates from patients with cystic fibrosis) or Pseudomonas cepacia incubated in a minimal medium under nonshaking (oxygen limited) conditions led to dose-dependent enhancement of bacterial growth. The enzyme exhibited a minimal effect on the growth of bacteria when cultured under more intense aeration conditions. This enhancement of bacterial growth by urokinase required the presence of active enzyme and was not detected with inactivated enzyme or noncatalytic domains of the enzyme. Enhancement of bacterial growth was not observed following incubation of P. aeruginosa with other proteinases including thrombin, neutrophil elastase, trypsin, chymotrypsin, or pseudomonas elastase and pseudomonas alkaline proteinase. Therefore, the observed effect of urokinase was relatively specific for this enzyme. As urokinase is a natural constituent of the lung, this enzyme could contribute to bacterial growth during pulmonary infections, particularly in an inflammatory environment in which the oxygen tension may be reduced.Key words: plasminogen activators, Pseudomonas aeruginosa, bacterial growth, urokinase, host proteinases.
Double-stranded RNA mycoviruses in section Nigri of the Aspergillus genus
Authors: Varga János , Kevei Ferenc , Vágvölgyi Csaba , Vriesema Aldwin , Croft James H.
Published: February 2010

Double-stranded RNA bands were detected electrophoretically in about 7% of natural isolates and in 13 of 51 collection strains belonging in section Nigri of the genus Aspergillus. The identity of these bands was proved by S1 nuclease and RNase treatment. Most of the virus-containing natural isolates came from Indonesia. Electron microscopic examination of the strains revealed the presence of virus-like particles in the mycelia of the strains examined. All of the virus-like particles were isometric and their size was around 30–35 nm, while some Indonesian isolates also contained virus-like particles in the size range 23–25 nm. It was possible to cure some of these strains of virus-like particles by mutagenic treatment. The four strains tested lost their virus-like particles and also their 'arginine–proline leaky' phenotype and became prototrophic. Virus transfer was possible among these four strains by protoplast fusion. It also proved possible to transfer mycoviruses into a more distantly related Aspergillus tubingensis strain by prolonged incubation of the polyethylene glycol treated protoplasts in osmotically stabilized medium. In spite of the finding that all Aspergillus foetidus and both Aspergillus heteromorphus strains examined contained double-stranded RNA segments and virus-like particles, no phenotypes related to the presence of these VLPs have been observed so far.Key words: Aspergillus niger, dsRNA, mycovirus, virus-like particles (VLPs).
[methyl-3H]Thymidine and [3H]leucine incorporation in Vibrio spp. grown in nutrient-limited continuous cultures
Authors: Snyder Richard A. , Robarts Richard D. , Caldwell Douglas E.
Published: February 2010

Vibrio alginolyticus, Vibrio logei, Vibrio natriegens, and Vibrio neries were grown in nutrient-limited continuous cultures at generation times (TD) of 5–135 h on complex media with cell yields of 0.8–12 × 106 bacteria/mL. Average cell volume, as determined by image analysis of video fluorescence microscopy, decreased for V. logei and V. neries, did not change for V. alginolyticus, and increased for V. natriegens with increasing TD. The increase in cell volume observed for V. natriegens was due to the development of filamentous cells. Batch cultures were grown on media with 10 times the nutrient concentration of continuous cultures. Tritiated thymidine incorporation was measured using phenol–chloroform extractions; leucine incorporation was measured in trichloroacetic acid precipitates. At concentrations of exogenous thymidine high enough to inhibit de novo synthesis of thymidine, the number of bacteria produced per mole of thymidine incorporated did not vary with changing generation time, or between batch and continuous cultures examined in this study. However, the number of bacteria produced per mole of leucine incorporated decreased per unit production with increasing TD for all four vibrios. A significant difference in the bacterial production conversion factor (bacteria produced per mole of label incorporated) for thymidine was found for V. neries relative to the three other Vibrio species, but no significant differences were found between growth conditions within species. Corrections for biovolume differences between species and growth rates reduced variability in conversion factors, and also yielded a significantly different conversion factor for V. neries. Conversion factors for leucine incorporation spanned three orders of magnitude, from 1015 to 1018 bacteria/mol of leucine incorporated.Key words: leucine, thymidine, bacterial production, chemostats.
Production of 2-aminobutyrate by Megasphaera elsdenii
Authors: Furtado A. F. , McAllister T. A. , Cheng K. -J. , Milligan L. P.
Published: February 2010

Production of the amino acid 2-aminobutyrate was studied in four strains of Megasphaera elsdenii grown on a lactate-based growth medium containing Bacto-casamino acids and yeast extract. Supplementation with threonine increased the production of 2-aminobutyrate in three of the four strains, but no substantial increase in production was noted with serine, methionine, or aspartate, all of which are potential sources for the precursor of 2-aminobutyrate, 2-oxobutyrate. L-Cycloserine, an inhibitor of alanine transaminases, decreased both alanine and 2-aminobutyrate production, suggesting that 2-aminobutyrate synthesis may share the same metabolic pathway as alanine synthesis or that 2-oxobutyrate can act as a substrate for alanine transaminases. Decreases in the production of 2-aminobutyrate were associated with a reduction in the catabolism of branched-chain amino acids in two of the four strains.Key words: Megasphaera, amino acid, degradation, 2-aminobutyrate, rumen.
Comparative physiology of phenanthrene degradation by two dissimilar pseudomonads isolated from a creosote-contaminated soil
Authors: Stringfellow William T. , Aitken Michael D.
Published: February 2010

Two species of bacteria, identified as Pseudomonas stutzeri (P-16) and Pseudomonas saccharophila (P-15) by fatty acid methyl ester analysis, were found in a phenanthrene enrichment culture of a creosote-contaminated soil. The organisms are shown to be physiologically dissimilar, and their genetic relatedness is discussed. Phenanthrene degradation by both organisms followed Michaelis–Menten kinetics, allowing for the determination of half-saturation (Ks) and maximum activity coefficients, using nonlinear regression. Both organisms utilized kinetically similar enzymes for phenanthrene uptake and oxidation, as evidenced by similar Ks coefficients of approximately 0.2 mg/L and temperature optima of 40 °C, but levels of expression differed with different media. Each organism degraded phenanthrene via salicylic acid, but patterns of intermediate metabolism were shown to differ. P-15 excreted 1-hydroxy-2-naphthoic acid during growth on phenanthrene and demonstrated Michaelis–Menten kinetics for the oxidation of 1-hydroxy-2-naphthoic acid by resting cells. P-16 excreted only trace amounts of 1-hydroxy-2-naphthoic acid and demonstrated linear kinetics in response to 1-hydroxy-2-naphthoic acid concentration. P-15 was found to form thick biofilms on phenanthrene crystals and was characterized by a hydrophobic cell surface, whereas P-16 grew mostly in suspension and was hydrophilic. Neither organism produced significant amounts of biosurfactants when grown on phenanthrene. The implications of these findings for the design of systems to remediate contaminated soil are discussed.Key words: phenanthrene, 1-hydroxy-2-naphthoic acid, biodegradation, kinetics, polycyclic aromatic hydrocarbons.
Biodegradation of naphthenic acids by microbial populations indigenous to oil sands tailings
Authors: Herman David C. , Fedorak Phillip M. , MacKinnon Mike D. , Costerton J. W.
Published: February 2010

Organic acids, similar in structure to naphthenic acids, have been associated with the acute toxicity of tailings produced by the oil sands industry in northeastern Alberta, Canada. Bacterial cultures enriched from oil sands tailings were found to utilize as their sole carbon source both a commercial mixture of naphthenic acids and a mixture of organic acids extracted from oil sands tailings. Gas chromatographic analysis of both the commercial naphthenic acids and the extracted organic acids revealed an unresolved "hump" formed by the presence of many overlapping peaks. Microbial activity directed against the commercial mixture of naphthenic acids converted approximately 50% of organic carbon into CO2 and resulted in a reduction in many of the gas chromatographic peaks associated with this mixture. Acute toxicity testing utilizing the Microtox test revealed a complete absence of detectable toxicity following the biodegradation of the naphthenic acids. Microbial activity mineralized approximately 20% of the organic carbon present in the extracted organic acids mixture, although there was no indication of a reduction in any gas chromatographic peaks with biodegradation. Microbial attack on the organic acids mixture reduced acute toxicity to approximately one half of the original level. Respirometric measurements of microbial activity within microcosms containing oil sands tailings were used to provide further evidence that the indigenous microbial community could biodegrade naphthenic acids and components within the extracted organic acids mixture.Key words: naphthenic acids, biodegradation, oil sands tailings, toxicity testing.
Possible roles of competition for thiamine, production of inhibitory compounds, and hyphal interactions in suppression of the take-all fungus by a sterile red fungus
Authors: Shankar M. , Kurtböke D. I. , Gillespie-Sasse L. M. J. , Rowland C. Y. , Sivasithamparam K.
Published: February 2010

Possible mechanisms involved in the suppression of the take-all fungus (Gaeumannomyces graminis var. tritici) by a sterile red fungus were examined. Studies on the effects of foliar application of thiamine to plants showed a reduction in take-all suppression by the sterile red fungus thereby indicating that competition for this particular vitamin, required by both fungi, could be one of the mechanisms of antagonism. Treatment with the exudate produced by the sterile red fungus on solid substrates resulted in lysis of the hyphae of the take-all fungus. Exudate produced on sterilised wheat seeds was more inhibitory to the take-all fungus than that produced on ryegrass seeds. Fungal inhibition increased as the duration of exposure to the exudate increased. The primary effects of hyphal interactions between the sterile red fungus and the take-all fungus were evident as coiling and contact lysis. It is possible that a combination of mechanisms including the above may be involved in suppression of the take-all fungus by the sterile red fungus.Key words: take-all suppression, sterile red fungus.