bluuebunny/crossref_metadata_2025 huggingface dataset:
Dimethylsulfoniopropionate Metabolism by
Pfiesteria
-Associated
Roseobacter
spp
Authors: Miller Todd R. , Belas Robert
Published: June 2004

The
Roseobacter
clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that
Roseobacter
species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in
Pfiesteria
and
Pfiesteria
-like (
Cryptoperidiniopsis
) dinoflagellates, and the identities and metabolic potentials of the associated
Roseobacter
species to degrade DMSP were determined. Both
Pfiesteria piscicida
and
Pfiesteria shumwayae
produce DMSP with an average intracellular concentration of 3.8 μM. Cultures of
P. piscicida
or
Cryptoperidiniopsis
sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 μM DMSP (within 30 h), and the rate of catabolism was much higher for
P. piscicida
cultures than for
P. shumwayae
cultures. The community of bacteria from
P. piscicida
and
Cryptoperidiniopsis
cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the
P. piscicida
cultures and found to be taxonomically related to
Roseobacter
species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated
Roseobacter
spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.
Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts
Authors: Varela Cristian , Pizarro Francisco , Agosin Eduardo
Published: June 2004

Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.
Evolutionary Relationships of Primary Prokaryotic Endosymbionts of Whiteflies and Their Hosts
Authors: Thao MyLo Ly , Baumann Paul
Published: June 2004

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are plant sap-sucking insects that harbor prokaryotic primary endosymbionts (P-endosymbionts) within specialized cells located in their body cavity. Four-kilobase DNA fragments containing 16S-23S ribosomal DNA (rDNA) were amplified from the P-endosymbiont of 24 whiteflies from 22 different species of 2 whitefly subfamilies. In addition, 3-kb DNA fragments containing mitochondrialcytB,nd1, and large-subunit rDNA (LrDNA) were amplified from 17 whitefly species. Comparisons of the P-endosymbiont (16S-23S rDNA) and host (cytB-nd1-LrDNA) phylogenetic trees indicated overall congruence consistent with a single infection of a whitefly ancestor with a bacterium and subsequent cospeciation (cocladogenesis) of the host and the P-endosymbiont. On the basis of both the P-endosymbiont and host trees, the whiteflies could be subdivided into at least five clusters. The major subdivision was between the subfamiliesAleyrodinaeandAleurodicinae. Unlike the P-endosymbionts of may other insects, the P-endosymbionts of whiteflies were related toPseudomonasand possibly to the P-endosymbionts of psyllids. The lineage consisting of the P-endosymbionts of whiteflies is given the designation “CandidatusPortiera” gen. nov., with a single species, “CandidatusPortiera aleyrodidarum” sp. nov.
Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from
Anaerobranca gottschalkii
Authors: Bertoldo Costanzo , Armbrecht Martin , Becker Fiona , Schäfer Thomas , Antranikian Garabed , Liebl Wolfgang
Published: June 2004

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium
Anaerobranca gottschalkii
. In addition, the homologous gene was isolated from a gene library of
Anaerobranca horikoshii
and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria
Fervidobacterium pennivorans
and
Thermotoga maritima
. The pullulanase gene from
A. gottschalkii
(encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in
Escherichia coli
strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in
E. coli
Tuner(DE3)(pLysS), the pullulanase gene from
A. gottschalkii
, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by
E. coli
. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from
A. gottschalkii
is the first thermoalkalistable type I pullulanase that has been described.
Identification of Sigma Factor σB-Controlled Genes and Their Impact on Acid Stress, High Hydrostatic Pressure, and Freeze Survival inListeria monocytogenesEGD-e
Authors: Wemekamp-Kamphuis Henrike H. , Wouters Jeroen A. , de Leeuw Patrick P. L. A. , Hain Torsten , Chakraborty Trinad , Abee Tjakko
Published: June 2004

The gene encoding the alternative sigma factor σBinListeria monocytogenesis induced upon exposure of cells to several stresses. In this study, we investigated the impact of asigBnull mutation on the survival ofL. monocytogenesEGD-e at low pH, during high-hydrostatic-pressure treatment, and during freezing. The survival of ΔsigBmutant exponential-phase cells at pH 2.5 was 10,000-fold lower than the survival of EGD-e wild-type cells. Moreover, the ΔsigBmutant failed to show an acid tolerance response. Upon preexposure for 1 h to pH 4.5, the survival at pH 2.5 was 100,000-fold lower for the ΔsigBmutant than for the wild type. The glutamate decarboxylase (GAD) acid resistance system is important in survival and adaptation ofL. monocytogenesin acidic conditions. The σBdependence of thegadgenes (gadA,gadB,gadC,gadD, andgadE) was analyzed in silico. Putative σB-dependent promoter sites were found upstream of thegadCBoperon (encoding a glutamate/γ-aminobutyrate antiporter and a glutamate decarboxylase, respectively) and thelmo2434gene (gadD, encoding a putative glutamate decarboxylase). Reverse transcriptase PCR revealed that expression of thegadCBoperon and expression ofgadDare indeed σBdependent. In addition, a proteomics approach was used to analyze the protein expression profiles upon acid exposure. Although the GAD proteins were not recovered, nine proteins accumulated in the wild type but not in the ΔsigBstrain. These proteins included Pfk, GalE, ClpP, and Lmo1580. Exposure to pH 4.5, in order to preload cells with active σBand consequently with σB-dependent general stress proteins, also provided considerable protection against high-hydrostatic-pressure treatment and freezing. The combined data argue that the expression of σB-dependent genes providesL. monocytogeneswith nonspecific multiple-stress resistance that may be relevant for survival in the natural environment as well as during food processing.
Syntrophic-Methanogenic Associations along a Nutrient Gradient in the Florida Everglades
Authors: Chauhan Ashvini , Ogram Andrew , Reddy K. R.
Published: June 2004

Nutrient runoff from the Everglades Agricultural Area resulted in a well-documented gradient of phosphorus concentrations in soil and water, with concomitant ecosystem-level changes, in the northern Florida Everglades. It was recently reported that sulfate-reducing prokaryote assemblage composition, numbers, and activities are dependent on position along the gradient (H. Castro, K. R. Reddy, and A. Ogram, Appl. Environ. Microbiol.
68:
6129-6137, 2002). The present study utilized a combination of culture- and non-culture-based approaches to study differences in composition of assemblages of syntrophic and methanogenic microbial communities in eutrophic, transition, and oligotrophic areas along the phosphorus gradient. Methanogenesis rates were much higher in eutrophic and transition regions, and sequence analysis of 16S rRNA gene clone libraries constructed from samples taken from these regions revealed differences in composition and activities of syntroph-methanogen consortia. Methanogens from eutrophic and transition regions were almost exclusively composed of hydrogenotrophic methanogens, with approximately 10,000-fold-greater most probable numbers of hydrogenotrophs than of acetotrophs. Most cultivable strains from eutrophic and transition regions clustered within novel lineages. In non-culture-based studies to enrich syntrophs, most bacterial and archaeal clones were either members of novel lineages or closely related to uncultivated environmental clones. Novel cultivable
Methanosaeta
sp. and fatty acid-oxidizing bacteria related to the genera
Syntrophomonas
and
Syntrophobacter
were observed in microcosms containing soil from eutrophic regions, and different lines of evidence indicated the existence of novel syntrophic association in eutrophic regions.
Detection and Quantification of Airborne Conidia of
Fusarium circinatum
, the Causal Agent of Pine Pitch Canker, from Two California Sites by Using a Real-Time PCR Approach Combined with a Simple Spore Trapping Method
Authors: Schweigkofler Wolfgang , O'Donnell Kerry , Garbelotto Matteo
Published: June 2004

Pinus radiata
(Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production. A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by
Fusarium circinatum
(
Hypocreales
). We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air. The assay is also useful for diagnosing the presence of the pathogen on plants. The test is based on the
F. circinatum
specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon. Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of
F. circinatum
spores. The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium. A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002. Spore counts were in the range of ca. 1 × 10
3
to ca. 7 × 10
5
/m
2
, with the highest spore counts in the fall, suggesting a seasonal fluctuation.
Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi
Authors: Ye Min , Qu Guiqin , Guo Hongzhu , Guo Dean
Published: June 2004

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12β-position by the fungusAlternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12β-hydroxyl cinobufagin, 12β-hydroxyl desacetylcinobufagin, 3-oxo-12β-hydroxyl cinobufagin, 3-oxo-12β-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12α-hydroxyl cinobufagin. The last five products are new compounds. 12β-Hydroxylation of cinobufagin byA. alternatais a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12β also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12β-hydroxyl cinobufagin and 3-oxo-12α-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12β-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.
Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (
Picea mariana
) Seedlings Grown in a Nursery
Authors: Filion M. , Hamelin R. C. , Bernier L. , St-Arnaud M.
Published: June 2004

Bacterial and fungal populations associated with the rhizosphere of healthy black spruce (
Picea mariana
) seedlings and seedlings with symptoms of root rot were characterized by cloned rRNA gene sequence analysis. Triplicate bacterial and fungal rRNA gene libraries were constructed, and 600 clones were analyzed by amplified ribosomal DNA restriction analysis and grouped into operational taxonomical units (OTUs). A total of 84 different bacterial and 31 different fungal OTUs were obtained and sequenced. Phylogenetic analyses indicated that the different OTUs belonged to a wide range of bacterial and fungal taxa. For both groups, pairwise comparisons revealed that there was greater similarity between replicate libraries from each treatment than between libraries from different treatments. Significant differences between pooled triplicate samples from libraries of genes from healthy seedlings and pooled triplicate samples from libraries of genes from diseased seedlings were also obtained for both bacteria and fungi, clearly indicating that the rhizosphere-associated bacterial and fungal communities of healthy and diseased
P. mariana
seedlings were different. The communities associated with healthy and diseased seedlings also showed distinct ecological parameters as indicated by the calculated diversity, dominance, and evenness indices. Among the main differences observed at the community level, there was a higher proportion of
Acidobacteria
,
Gammaproteobacteria
, and Homobasidiomycetes clones associated with healthy seedlings, while the diseased-seedling rhizosphere harbored a higher proportion of
Actinobacteria
, Sordariomycetes, and environmental clones. The methodological approach described in this study appears promising for targeting potential rhizosphere-competent biological control agents against root rot diseases occurring in conifer nurseries.
Modeling the Rate of Attachment of
Listeria monocytogenes
,
Pantoea agglomerans
, and
Pseudomonas fluorescens
to, and the Probability of Their Detachment from, Potato Tissue at 10°C
Authors: Garrood M. J. , Wilson P. D. G. , Brocklehurst T. F.
Published: June 2004

The rate of attachment of bacteria to, and their subsequent detachment from, the cut surface of raw potato tissue was measured and modeled by using mathematical approaches that allowed detailed objective comparisons of adhesion processes under different conditions. Attachment was rapid and reached equilibrium after contact for 60 min. A new method to measure the probability of detachment was developed and modeled, revealing that the probability of detachment for
Pseudomonas fluorescens
remained unchanged for contact times between less than 5 s and 60 min.
Listeria monocytogenes
, however, was more easily removed initially, with the probability of detachment decreasing over the first 2 min of contact but remaining constant and equivalent to that for
Pseudomonas fluorescens
thereafter. For all of the bacteria tested, the number of bacteria attached after 2 min of contact was proportional to the inoculum concentration raised to the power of 0.79.
Characterization of Bacterial Communities in Feces from Healthy Elderly Volunteers and Hospitalized Elderly Patients by Using Real-Time PCR and Effects of Antibiotic Treatment on the Fecal Microbiota
Authors: Bartosch Sabine , Fite Alemu , Macfarlane George T. , McMurdo Marion E. T.
Published: June 2004

Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years;n= 35) living in the local community, elderly hospitalized patients (age, 66 to 103;n= 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100;n= 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in theBacteroides-Prevotellagroup following hospitalization. Reductions in bifidobacteria,Desulfovibriospp.,Clostridium clostridiiforme, andFaecalibacterium prausnitziiwere also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria,Desulfovibriospp.,C. clostridiiforme, andF. prausnitziidid not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic speciesEnterococcus faecalisproliferated.
Vertical Transmission of Endobacteria in the Arbuscular Mycorrhizal FungusGigaspora margaritathrough Generation of Vegetative Spores
Authors: Bianciotto V. , Genre A. , Jargeat P. , Lumini E. , Bécard G. , Bonfante P.
Published: June 2004

Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences, these endobacteria have recently been identified as a new taxon,CandidatusGlomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungal reproduction (asexual), a system based on transformed carrot roots and single-spore inocula ofGigaspora margaritawas used. Under these in vitro sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored, and it was shown, by using primers designed for both 16S and 23S ribosomal DNAs, to occur through several vegetative spore generations (SG0 to SG4). A method of confocal microscopy for quantifying the density of endobacteria in spore cytoplasm was designed and applied; endobacteria were consistently found in all of the spore generations, although their number rapidly decreased from SG0 to SG4. The study demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus, and suggests that these bacteria are obligate endocellular components of their AM fungal host.
Unusual Microbial Xylanases from Insect Guts
Authors: Brennan YaLi , Callen Walter N. , Christoffersen Leif , Dupree Paul , Goubet Florence , Healey Shaun , Hernández Myrian , Keller Martin , Li Ke , Palackal Nisha , Sittenfeld Ana , Tamayo Giselle , Wells Steve , Hazlewood Geoffrey P. , Mathur Eric J. , Short Jay M. , Robertson Dan E. , Steer Brian A.
Published: June 2004

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.
Discovery of the Novel Candidate Phylum “
Poribacteria
” in Marine Sponges
Authors: Fieseler Lars , Horn Matthias , Wagner Michael , Hentschel Ute
Published: June 2004

Marine sponges (
Porifera
) harbor large amounts of commensal microbial communities within the sponge mesohyl. We employed 16S rRNA gene library construction using specific PCR primers to provide insights into the phylogenetic identity of an abundant sponge-associated bacterium that is morphologically characterized by the presence of a membrane-bound nucleoid. In this study, we report the presence of a previously unrecognized evolutionary lineage branching deeply in the domain
Bacteria
that is moderately related to the
Planctomycetes
,
Verrucomicrobia
, and
Chlamydia
lines of decent. Because members of this lineage showed <75% 16S rRNA gene sequence similarity to known bacterial phyla, we suggest the status of a new candidate phylum, named “
Poribacteria
”, to acknowledge the affiliation of the new bacterium with sponges. The affiliation of the morphologically conspicuous sponge bacterium with the novel phylogenetic lineage was confirmed by fluorescence in situ hybridization with newly designed probes targeting different sites of the poribacterial 16S rRNA. Consistent with electron microscopic observations of cell compartmentalization, the fluorescence signals appeared in a ring-shaped manner. PCR screening with “
Poribacteria
”-specific primers gave positive results for several other sponge species, while samples taken from the environment (seawater, sediments, and a filter-feeding tunicate) were PCR negative. In addition to a report for
Planctomycetes
, this is the second report of cell compartmentalization, a feature that was considered exclusive to the eukaryotic domain, in prokaryotes.
Methods for Enhanced Culture Recovery of
Francisella tularensis
Authors: Petersen Jeannine M. , Schriefer Martin E. , Gage Kenneth L. , Montenieri John A. , Carter Leon G. , Stanley Miles , Chu May C.
Published: June 2004

Francisella tularensis
is found in a wide variety of hosts and extrahost environments, making culture recovery a diagnostic challenge. Here we demonstrate improved recovery times and good sensitivity (90%) when cultures were inoculated on the site of an investigation using fresh tissues. For contaminated specimens, antibiotic supplementation of enriched cysteine heart agar blood culture medium improved recovery of
F. tularensis
by 81.1%. For transport of tissues, immediate freezing yielded culture recovery rates as high as 94%.
Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry
Authors: Roldán M. , Thomas F. , Castel S. , Quesada A. , Hernández-Mariné M.
Published: June 2004

A new imaging technique for the analysis of fluorescent pigments from a single cell is reported. It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods. The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages.
Bacterial Strains Isolated from Different Niches Can Exhibit Different Patterns of Adhesion to Substrata
Authors: Bakker Dewi P. , Postmus Bart R. , Busscher Henk J. , van der Mei Henny C.
Published: June 2004

Various mechanisms have been demonstrated to be operative in bacterial adhesion to surfaces, but whether bacterial adhesion to surfaces can ever be captured in one generally valid mechanism is open to question. Although many papers in the literature make an attempt to generalize their conclusions, the majority of studies of bacterial adhesion comprise only two or fewer strains. Here we demonstrate that three strains isolated from a medical environment have a decreasing affinity for substrata with increasing surface free energy, whereas three strains from a marine environment have an increasing affinity for substrata with increasing surface free energy. Furthermore, adhesion of the marine strains related positively with substratum elasticity, but such a relation was absent in the strains from the medical environment. This study makes it clear that strains isolated from a given niche, whether medical or marine, utilize different mechanisms in adherence, which hampers the development of a generalized theory for bacterial adhesion to surfaces.
Enhancement of Cry19Aa Mosquitocidal Activity against
Aedes aegypti
by Mutations in the Putative Loop Regions of Domain II
Authors: Abdullah Mohd Amir F. , Dean Donald H.
Published: June 2004

Improvements in the mosquitocidal activity of
Bacillus thuringiensis
Cry19Aa were achieved by protein engineering of putative surface loop residues in domain II through rational design. The improvement of
Aedes
toxicity in Cry19Aa was 42,000-fold and did not affect its toxicity against
Anopheles
or
Culex
.
Short-Duration Low-Direct-Current Electrical Field Treatment Is a Practical Tool for Considerably Reducing Counts of Gram-Negative Bacteria Entrapped in Gel Beads
Authors: Zvitov R. , Zohar-Perez C. , Nussinovitch A.
Published: June 2004

Application of a direct-current electrical field for very short times can serve as a practical nonthermal procedure to reduce or modify the microbial distribution in gel beads. The viability of
Escherichia coli
and
Serratia marcescens
entrapped in alginate and agarose beads decreases as the field intensity and duration of electrical field increase.
Differences between Betaproteobacterial Ammonia-Oxidizing Communities in Marine Sediments and Those in Overlying Water
Authors: Freitag Thomas E. , Prosser James I.
Published: June 2004

To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments.
Nitrosospira
cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples. Detailed phylogenetic analysis of marine
Nitrosospira
cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes.
Nocardia arthritidis
sp. nov., a New Pathogen Isolated from a Patient with Rheumatoid Arthritis in Japan
Authors: Kageyama Akiko , Torikoe Kimiaki , Iwamoto Masahiro , Masuyama Jun-Ichi , Shibuya Yasuhiro , Okazaki Hitoaki , Yazawa Katsukiyo , Minota Seiji , Kroppenstedt Reiner M. , Mikami Yuzuru
Published: June 2004

Two different bacterial strains with different drug susceptibilities were isolated from the sputum and an inflammatory discharge from a swelling in the left thigh of a patient with rheumatoid arthritis. Both bacterial strains were provisionally assigned to the genus
Nocardia
on the basis of their morphological and chemotaxonomic characteristics and were further studied in order to establish their taxonomic status. One strain (IFM 10034) was identified as
Nocardia farcinica
on the basis of its physiological characteristics. The other strain, which was designated
Nocardia
sp. strain IFM 10035
T
, revealed a unique pattern of phenotypic properties that distinguished it from other representatives of established
Nocardia
species. Comparative 16S rRNA gene sequence studies of
Nocardia
sp. strain IFM 10035
T
also showed that the bacterium was closely related to the species
Nocardia beijingensis
. Determination of DNA-DNA relatedness, however, indicated that
Nocardia
sp. strain IFM 10035
T
could be delineated from
N. beijingensis
. The genotypic and phenotypic data combined indicated that the bacterium merits description as a new
Nocardia
species. The name proposed for the new species is
Nocardia arthritidis
sp. nov., the type strain being IFM 10035
T
(NBRC 100137
T
, JCM 12120
T
, DSM44731
T
). The present study suggests that
Nocardia
infections can be caused by multiple species of the bacterium.
Implications of Molecular Genotyping of
Helicobacter pylori
Isolates from Different Human Populations by Genomic Fingerprinting of Enterobacterial Repetitive Intergenic Consensus Regions for Strain Identification and Geographic Evolution
Authors: Hussain M. Abid , Kauser Farhana , Khan Aleem A. , Tiwari Santosh , Habibullah Chittoor M. , Ahmed Niyaz
Published: June 2004

Biogeographic partitioning of the genome is typical of the gastric pathogen
Helicobacter pylori
. Such population-specific evolution could serve as a model for understanding host-pathogen interaction and the impact of genetic drift and recombination on insular populations. With a total of 320 isolates from six geographic regions (Japan, India, England, Spain, Ireland, Africa, and Peru) analyzed by enterobacterial repetitive intergenic consensus (ERIC)-based genotyping, we examined genetic affinities among various
H. pylori
populations in the world. Several strain-specific and region-specific differences were observed by ERIC-based typing. Polymorphic ERIC patterns indicated that the ERIC sequences are in fact dispersed in the
H. pylori
chromosome at different locations separated by various distances. Phylogenetic analysis of 61 representative isolates revealed three distinct genetic clusters populated by isolates with shared ERIC types independent of the
cag
right-junction motif type and
vacA
allele status. Among the notable genetic relationships were the genotypic similarities between Irish and Japanese and between Peruvian and Japanese isolates. Insular genotypic characteristics of Irish isolates amid genetic similarity to East Asian, as well as North European, strains have been once again proved in this study. Peruvian genotypes were more similar to those of Japanese isolates than to those of Iberian or European isolates. Given the current debate on the origin and age of present-day
H. pylori
, this is a significant finding that supports the possibility of ancient colonization of Amerindians with East Asian strains. Genotypic data presented here will be additionally helpful in realizing the importance of
H. pylori
geographical genomics in the development of gastroduodenal pathology.
Specific T-Cell Epitopes for Immunoassay-Based Diagnosis ofMycobacterium tuberculosisInfection
Authors: Brock Inger , Weldingh Karin , Leyten Eliane M. S. , Arend Sandra M. , Ravn Pernille , Andersen Peter
Published: June 2004

The currently used method for immunological detection of tuberculosis infection, the tuberculin skin test, has low specificity. Antigens specific forMycobacterium tuberculosisto replace purified protein derivative are therefore urgently needed. We have performed a rigorous assessment of the diagnostic potential of four recently identified antigens (Rv2653, Rv2654, Rv3873, and Rv3878) from genomic regions that are lacking from theMycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine strains as well as from the most common nontuberculous mycobacteria. The fine specificity of potential epitopes in these molecules was evaluated by sensitive testing of the T-cell responses of peripheral blood mononuclear cells derived fromM. bovisBCG-vaccinated healthy individuals to synthesized overlapping peptides. Three of the four molecules contained regions with significant specificity problems (Rv2653, Rv3873, and Rv3878). We selected and combined the specific peptide stretches from the four proteins not recognized byM. bovisBCG-vaccinated individuals. These peptide stretches were tested with peripheral blood mononuclear cells obtained from patients with microscopy- or culture-confirmed tuberculosis and from healthyM. bovisBCG-vaccinated controls. The combination of the most promising stretches from this analysis showed a sensitivity level (57%) comparable to the level found with the two well-knownM. tuberculosis-specific proteins ESAT-6 and CFP-10 (75 and 66%, respectively). The combination of ESAT-6, CFP-10, and the novel specific peptide stretches gave an overall sensitivity of 84% at a specificity of 97%. In a validation experiment with new experimental groups, the sensitivities obtained were 57% for the combination of peptides and 90% for the combination of the peptides, ESAT-6, and CFP-10. This combination gave a specificity of 95%.
Development of a Multiplex PCR-Ligase Detection Reaction Assay for Diagnosis of Infection by the Four Parasite Species Causing Malaria in Humans
Authors: McNamara David T. , Thomson Jodi M. , Kasehagen Laurin J. , Zimmerman Peter A.
Published: June 2004

The diagnosis of infections caused by
Plasmodium
species is critical for understanding the nature of malarial disease, treatment efficacy, malaria control, and public health. The demands of field-based epidemiological studies of malaria will require faster and more sensitive diagnostic methods as new antimalarial drugs and vaccines are explored. We have developed a multiplex PCR-ligase detection reaction (LDR) assay that allows the simultaneous diagnosis of infection by all four parasite species causing malaria in humans. This assay exhibits sensitivity and specificity equal to those of other PCR-based assays, identifying all four human malaria parasite species at levels of parasitemias equal to 1 parasitized erythrocyte/μl of blood. The multiplex PCR-LDR assay goes beyond other PCR-based assays by reducing technical procedures and by detecting intraindividual differences in species-specific levels of parasitemia. Application of the multiplex PCR-LDR assay will provide the sensitivity and specificity expected of PCR-based diagnostic assays and will contribute new insight regarding relationships between the human malaria parasite species and the human host in future epidemiological studies.
Development of a Species-Specific Probe forPythium insidiosumand the Diagnosis of Pythiosis
Authors: Schurko Andrew M. , Mendoza Leonel , de Cock Arthur W. A. M. , Bedard James E. J. , Klassen Glen R.
Published: June 2004

Pythium insidiosum, the only species in the genus that infects mammals, is the etiological agent of pythiosis, a granulomatous disease characterized by cutaneous and subcutaneous lesions and vascular diseases. Accurate diagnosis of pythiosis and identification of its causal agent are often inconsistent with current immunological diagnostic methods. A species-specific DNA probe was constructed by using a 530-bp HinfI fragment from the ribosomal DNA intergenic spacer ofP. insidiosum.When the probe was incubated with dot blots of genomic DNA from 104Pythiumspecies, it hybridized only to the DNA ofP. insidiosumandP. destruens—two species that have been considered conspecific. The probe also hybridized to DNA from 22P. insidiosumisolates in this study, regardless of their geographic origin or animal host. When tested against genomic DNA from other pathogenic organisms (Aspergillus fumigatus,Basidiobolus ranarum,Conidiobolus coronatus,Lagenidium giganteum,Paracoccidioides brasiliensis, andPrototheca wickerhamii), no cross-hybridization of the probe was detected. The specificity of the probe to hybridize to genomic DNA from all isolates ofP. insidiosumand not cross-react with DNA from otherPythiumspecies or pathogens that cause symptoms similar to pythiosis in their hosts makes it a powerful tool for the accurate diagnosis of pythiosis. In addition, the probe has the potential for pathological and environmental diagnostic applications.
Analysis of the Allelic Diversity of the Mycobacterial Interspersed Repetitive Units inMycobacterium tuberculosisStrains of the Beijing Family: Practical Implications and Evolutionary Considerations
Authors: Mokrousov Igor , Narvskaya Olga , Limeschenko Elena , Vyazovaya Anna , Otten Tatiana , Vyshnevskiy Boris
Published: June 2004

A study set comprised 44Mycobacterium tuberculosisstrains of the Beijing family selected for their representativeness among those previously characterized by IS6110-RFLP and spoligotyping (Northwest Russia, 1997 to 2003). In the present study, these strains were subjected to mycobacterial interspersed repetitive units (MIRU) typing to assess a discriminatory power of the 12-MIRU-loci scheme (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). The 44 Russian Beijing strains were subdivided into 12 MIRU types with identical profiles: 10 unique strains and two major types shared by 10 and 24 strains. Thus, basically, two distinct sublineages appear to shape the evolution of the Beijing strains in Russia. Most of the MIRU loci were found to be (almost) monomorphic in the Russian Beijing strains; the Hunter-Gaston discriminatory index (HGDI) for all 12 loci taken together was 0.65, whereas MIRU26 (the most variable in our study) showed a moderate level of discrimination (0.49). The results were compared against all available published MIRU profiles of Beijing strains from Russia (3 strains) and other geographic areas (51 strains in total), including South Africa (38 strains), East Asia (7 strains), and the United States (4 strains). A UPGMA (unweighted pair-group method with arithmetic averages)-based tree was constructed. Interestingly, no MIRU types were shared by Russian and South African strains (the two largest samples in this analysis), whereas both major Russian types included also isolates from other locations (United States and/or East Asia). This implies the evolution of the Beijing genotype to be generally strictly clonal, although a possibility of a convergent evolution of the MIRU loci cannot be excluded. We propose a dissemination of the prevailing local Beijing clones to have started earlier in South Africa rather than in Russia since more monomorphic loci were identified in Russian samples than in South African samples (mean HGDI scores, 0.08 versus 0.17). To conclude, we suggest to use a limited number of MIRUs for preliminary subdivision of Beijing strains in Russian (loci 26 + 31), South African (10 + 26 + 39), and global settings (10 + 26 + 39).
Hepatitis B Virus Genotypes and Precore and Core Mutants in Brazilian Patients
Authors: Sitnik Roberta , Pinho João Renato Rebello , Bertolini Dennis Armando , Bernardini Antonio Plinio , da Silva Luiz Caetano , Carrilho Flair José
Published: June 2004

A method for genotyping hepatitis B virus by partial HBsAg gene sequencing with primers common to all known genotypes was developed. Mutations related to anti-HBs resistance are also detected with this method. Samples from 103 Brazilian patients were analyzed. Precore and core region of these viruses were also sequenced in 101 patients. Genotypes A, B, C, D, and F were found with frequencies of 49.5, 2.9, 13.6, 24.3, and 9.7%, respectively. Genotypes B and C were found only in Asian patients, whereas genotypes A, D, and F were more common in patients without an Asian background. Precore mutants were found in 32 (31.7%) of 101 patients, with a higher frequency in those infected with genotype D (22 of 25 [88.0%]). Analysis of nucleotide 1858 showed presence of thymine in all patients with genotypes B, C, and D and in a few patients with genotypes A (10.0%) and F (30.0%), who showed more frequently the presence of cytosine. This nucleotide was closely related to the presence of precore mutants. Mutations in the basal core promoter were found in 64 of 101 (63.4%) samples. These mutations were more frequent in patients infected with genotype F (90.0%) and less frequent in patients infected with genotype B (33.3%). Deletions in this region were found in two genotype C-infected patients.
Human Papillomavirus Testing with the Hybrid Capture 2 Assay and PCR as Screening Tools
Authors: Kulmala S.-M. , Syrjänen S. , Shabalova I. , Petrovichev N. , Kozachenko V. , Podistov J. , Ivanchenko O. , Zakharenko S. , Nerovjna R. , Kljukina L. , Branovskaja M. , Grunberga V. , Juschenko A. , Tosi P. , Santopietro R. , Syrjänen K.
Published: June 2004

The recognition of high-risk human papillomaviruses (HPVs) as etiological agents of cervical cancer has increased the demands to use testing for HPV for the detection of abnormal cervical smears and for cervical cancer screening. The present study compared the performance of the Hybrid Capture 2 (HC2) assay with that of PCR for the detection of significant cervical lesions in 1,511 women with different risks for HPV infections in three New Independent States of the former Soviet Union. The results showed that the level of agreement between the HC2 assay and PCR was substantial, with a kappa (Cohen) value of 0.669 (95% confidence interval [CI], 0.629 to 0.709). Of the 228 samples with discrepant results, 92 were positive by the HC2 assay but negative by PCR, whereas 136 samples were PCR positive but HC2 assay negative. The positive predictive values (PPVs) of the HC2 assay and PCR in detecting high-grade intraepithelial lesions (HSILs) were 4.5% (95% CI, 3.5 to 5.5%) and 3.6% (95% CI, 2.7 to 4.5%), respectively, and the negative predictive values (NPVs) were 99.6% (95% CI, 99.3 to 99.9%) and 99.3% (95% CI, 98.9 to 99.7%), respectively. The sensitivities of the HC2 assay and PCR for the detection of HSILs were 85.2 and 74.0%, respectively, and the specificities were 67.2 and 64.1%, respectively. In receiver operating characteristic (ROC) analysis, the performance of the HC2 assay for the detection of HSILs was excellent (
P
= 0.0001); the area under the ROC analysis curve was 0.858 (95% CI, 0.811 to 0.905), and the optimal balance between sensitivity (86.5%) and specificity (80%) was obtained with an HC2 assay cutoff level of 15.6 relative light units/positive control. Use of this cutoff would increase the specificity of the HC2 assay to 80.0% without compromising sensitivity. In conclusion, the results of PCR and the HC2 assay were concordant for 85% of samples, resulting in substantial reproducibility. Both tests had low PPVs, equal specificities, and equal (almost 100%) NPVs for the detection of HSILs; but the sensitivity of the HC2 assay was slightly better.
Multilocus Sequence Typing Is a Reliable Alternative Method to DNA Fingerprinting for Discriminating among Strains of
Candida albicans
Authors: Robles Juan C. , Koreen Larry , Park Steven , Perlin David S.
Published: June 2004

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for
Candida albicans
have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of
C. albicans
, we applied it to a previously well-characterized set of 29
C. albicans
isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.
Quantification of Intestinal Bacterial Populations by Real-Time PCR with a Universal Primer Set and Minor Groove Binder Probes: a Global Approach to the Enteric Flora
Authors: Ott Stephan J. , Musfeldt Meike , Ullmann Uwe , Hampe Jochen , Schreiber Stefan
Published: June 2004

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10
1
to 10
3
bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.