bluuebunny/crossref_metadata_2025 huggingface dataset:
Insect-Microbe Mutualism without Vertical Transmission: a Stinkbug Acquires a Beneficial Gut Symbiont from the Environment Every Generation
Authors: Kikuchi Yoshitomo , Hosokawa Takahiro , Fukatsu Takema
Published: May 2007

The broad-headed bug
Riptortus clavatus
(Heteroptera: Alydidae) possesses a number of crypts at a posterior midgut region, which house a dense population of a bacterial symbiont belonging to the genus
Burkholderia
. Although the symbiont is highly prevalent (95 to 100%) in the host populations, the symbiont phylogeny did not reflect the host systematics at all. In order to understand the mechanisms underlying the promiscuous host-symbiont relationship despite the specific and prevalent association, we investigated the transmission mode and the fitness effects of the
Burkholderia
symbiont in
R. clavatus
. Inspection of eggs and a series of rearing experiments revealed that the symbiont is not vertically transmitted but is environmentally acquired by nymphal insects. The
Burkholderia
symbiont was present in the soil of the insect habitat, and a culture strain of the symbiont was successfully isolated from the insect midgut. Rearing experiments by using sterilized soybean bottles demonstrated that the cultured symbiont is able to establish a normal and efficient infection in the host insect, and the symbiont infection significantly improves the host fitness. These results indicated that
R. clavatus
postnatally acquires symbiont of a beneficial nature from the environment every generation, uncovering a previously unknown pathway through which a highly specific insect-microbe association is maintained. We suggest that the stinkbug-
Burkholderia
relationship may be regarded as an insect analogue of the well-known symbioses between plants and soil-associated microbes, such as legume-
Rhizobium
and alder-
Frankia
relationships, and we discuss the evolutionary relevance of the mutualistic but promiscuous insect-microbe association.
Identification of Polypeptides Expressed in Response to Vinyl Chloride, Ethene, and Epoxyethane in
Nocardioides
sp. Strain JS614 by Using Peptide Mass Fingerprinting
Authors: Chuang Adina S. , Mattes Timothy E.
Published: May 2007

Enzymes expressed in response to vinyl chloride, ethene, and epoxyethane by
Nocardioides
sp. strain JS614 were identified by using a peptide mass fingerprinting (PMF) approach. PMF provided insight concerning vinyl chloride biodegradation in strain JS614 and extends the use of matrix-assisted laser desorption-ionization time of flight mass spectrometry as a tool to enhance characterization of biodegradation pathways.
Casein Fermentate of
Lactobacillus animalis
DPC6134 Contains a Range of Novel Propeptide Angiotensin-Converting Enzyme Inhibitors
Authors: Hayes M. , Stanton C. , Slattery H. , O'Sullivan O. , Hill C. , Fitzgerald G. F. , Ross R. P.
Published: May 2007

This work evaluated the angiotensin-converting-enzyme (ACE)-inhibitory activities of a bovine sodium caseinate fermentate generated using the proteolytic capabilities of the porcine small intestinal isolate
Lactobacillus animalis
DPC6134 (NCIMB deposit 41355). The crude 10-kDa
L. animalis
DPC6134 fermentate exhibited ACE-inhibitory activity of 85.51% (±15%) and had a 50% inhibitory concentration (IC
50
) of 0.8 mg protein/ml compared to captopril, which had an IC
50
value of 0.005 mg/ml. Fractionation of the crude
L. animalis
DPC6134 fermentate by membrane filtration and reversed-phase high-performance liquid chromatography (HPLC) generated three bioactive fractions from a total of 72 fractions. Fractions 10, 19, and 43 displayed ACE-inhibitory activity percentages of 67.53 (±15), 83.71 (±19), and 42.36 (±11), respectively, where ACE inhibition was determined with 80 μl of the fractions with protein concentrations of 0.5 mg/ml. HPLC and mass spectrometry analysis identified 25 distinct peptide sequences derived from α-, β-, and κ-caseins. In silico predictions, based on the C-terminal tetrapeptide sequences, suggested that peptide NIPPLTQTPVVVPPFIQ, corresponding to β-casein f(73-89); peptide IGSENSEKTTMP, corresponding to α
s1
-casein f(201212); peptide SQSKVLPVPQ, corresponding to β-casein f(166-175); peptide MPFPKYPVEP, corresponding to β-casein f(124133); and peptide EPVLGPVRGPFP, corresponding to β-casein f(210-221), contained ACE-inhibitory activities. These peptides were chosen for chemical synthesis to confirm the ACE-inhibitory activity of the fractions. Chemically synthesized peptides displayed IC
50
values in the range of 92 μM to 790 μM. Additionally, a simulated gastrointestinal digestion confirmed that the ACE-inhibitory 10-kDa
L. animalis
DPC6134 fermentation was resistant to a cocktail of digestive enzymes found in the gastrointestinal tract.
Molecular Diversity of Methanogens in Feedlot Cattle from Ontario and Prince Edward Island, Canada
Authors: Wright André-Denis G. , Auckland Clare H. , Lynn Denis H.
Published: May 2007

The molecular diversity of rumen methanogens in feedlot cattle and the composition of the methanogen populations in these animals from two geographic locations were investigated using 16S rRNA gene libraries prepared from pooled PCR products from 10 animals in Ontario (127 clones) and 10 animals from Prince Edward Island (114 clones). A total of 241 clones were examined, with
Methanobrevibacter ruminantium
accounting for more than one-third (85 clones) of the clones identified. From these 241 clones, 23 different 16S rRNA phylotypes were identified. Feedlot cattle from Ontario, which were fed a corn-based diet, revealed 11 phylotypes (38 clones) not found in feedlot cattle from Prince Edward Island, whereas the Prince Edward Island cattle, which were fed potato by-products as a finishing diet, had 7 phylotypes (42 clones) not found in cattle from Ontario. Five sequences, representing the remaining 161 clones (67% of the clones), were common in both herds. Of the 23 different sequences, 10 sequences (136 clones) were 89.8 to 100% similar to those from cultivated methanogens belonging to the orders
Methanobacteriales
,
Methanomicrobiales
, and
Methanosarcinales
, and the remaining 13 sequences (105 clones) were 74.1 to 75.8% similar to those from
Thermoplasma volcanium
and
Thermoplasma acidophilum
. Overall, nine possible new species were identified from the two clone libraries, including two new species belonging to the order
Methanobacteriales
and a new genus/species within the order
Methanosarcinales
. From the present survey, it is difficult to conclude whether the geographical isolation between these two herds or differences between the two finishing diets directly influenced community structure in the rumen. Further studies are warranted to properly assess the differences between these two finishing diets.
Application of Flow Cytometry to Segregated Kinetic Modeling Based on the Physiological States of Microorganisms
Authors: Quirós Covadonga , Herrero Mónica , García Luis A. , Díaz Mario
Published: May 2007

Flow cytometry (FC) has been introduced to characterize and to assess the physiological states of microorganisms in conjunction with the classical plate-counting method. To show the applicability of the technique, in particular for the development of kinetic models, pure culture fermentation experiments were followed over time, using both prokaryotic (
Lactobacillus hilgardii
) and eukaryotic (
Saccharomyces cerevisiae
) microorganisms growing in standard culture media (MRS and YPD). The differences observed between the active and viable cells determined by FC and CFU, respectively, allowed us to determine that a large number of cells were in a viable but nonculturable (VBNC) state, which resulted in a subpopulation much larger than the damaged-cell (double-stained) subpopulation. Finally, the determination of the evolution of viable, the VBNC, and the dead cells allowed us to develop a segregated kinetic model to describe the yeast and the bacteria population dynamics and glucose consumption in batch cultures. This model, more complete than that which is traditionally used, based only on viable cell measurements, describes better the behavior and the functionality of the cultures, giving a deeper knowledge in real time about the status and the course of the bioprocesses.
Genomic Methylation: a Tool for Typing
Helicobacter pylori
Isolates
Authors: Vale Filipa F. , Vítor Jorge M. B.
Published: May 2007

The genome sequences of three
Helicobacter pylori
strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the
H. pylori
genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing
H. pylori
isolates. We studied the expression of
H. pylori
methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction.
Responses of a Thermophilic
Synechococcus
Isolate from the Microbial Mat of Octopus Spring to Light
Authors: Kilian Oliver , Steunou Anne-Soisig , Fazeli Fariba , Bailey Shaun , Bhaya Devaki , Grossman Arthur R.
Published: May 2007

Thermophilic cyanobacteria of the genus
Synechococcus
are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park.
Synechococcus
OS-B′ was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 μmol photons m
−2
s
−1
but dies when the irradiance is increased to 400 μmol photons m
−2
s
−1
. At 200 μmol photons m
−2
s
−1
(high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid.
Synechococcus
OS-B′ synthesizes the carotenoids zeaxanthin and β,β-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that
Synechococcus
OS-B′ accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (
psbAI
and
psbAII
/
psbAIII
, respectively) were also regulated according to the light regimen. The results are discussed in the context of how
Synechococcus
OS-B′ may cope with high light irradiances in the high-temperature environment of the microbial mat.
Urban Feral Pigeons (
Columba livia
) as a Source for Air- and Waterborne Contamination with
Enterocytozoon bieneusi
Spores
Authors: Graczyk Thaddeus K. , Sunderland Deirdre , Rule Ana M. , da Silva Alexandre J. , Moura Iaci N. S. , Tamang Leena , Girouard Autumn S. , Schwab Kellogg J. , Breysse Patrick N.
Published: May 2007

This study demonstrated that a person with 30 min of occupational or nonoccupational exposure to urban feral pigeons, such as exposure through the cleaning of surfaces contaminated with pigeon excrement, could inhale approximately 3.5 × 10
3
Enterocytozoon bieneusi
spores and that 1.3 × 10
3
spores could be inhaled by a nearby person.
Molecular Identification and Genetic Analysis of Norovirus Genogroups I and II in Water Environments: Comparative Analysis of Different Reverse Transcription-PCR Assays
Authors: La Rosa G. , Fontana S. , Di Grazia A. , Iaconelli M. , Pourshaban M. , Muscillo M.
Published: May 2007

Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.
Use of Selective Capture of Transcribed Sequences To Identify Genes Preferentially Expressed byStreptococcus suisupon Interaction with Porcine Brain Microvascular Endothelial Cells
Authors: Fittipaldi Nahuel , Gottschalk Marcelo , Vanier Ghyslaine , Daigle France , Harel Josée
Published: May 2007

By using the selective capture of transcribed sequences (SCOTS) approach, we identified 28 genes preferentially expressed by the major swine pathogen and zoonotic agentStreptococcus suisupon interaction with porcine brain microvascular endothelial cells. Several of these genes may be considered newS. suiscandidate virulence factors. Results from this study demonstrate the suitability of SCOTS for the elucidation of gene expression in streptococcal species and may contribute to a better understanding of the pathogenesis ofS. suisinfections.
Pichia acaciae
Killer System: Genetic Analysis of Toxin Immunity
Authors: Paluszynski John P. , Klassen Roland , Meinhardt Friedhelm
Published: May 2007

The gene responsible for self-protection in the
Pichia acaciae
killer plasmid system was identified by heterologous expression in
Saccharomyces cerevisiae
. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2
ORF4
and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.
Mass Spectrometry-Based Strategy for Direct Detection and Quantification of Some Mycotoxins Produced by
Stachybotrys
and
Aspergillus
spp. in Indoor Environments
Authors: Bloom Erica , Bal Karol , Nyman Eva , Must Aime , Larsson Lennart
Published: May 2007

Dampness in buildings has been linked to adverse health effects, but the specific causative agents are unknown. Mycotoxins are secondary metabolites produced by molds and toxic to higher vertebrates. In this study, mass spectrometry was used to demonstrate the presence of mycotoxins predominantly produced by
Aspergillus
spp. and
Stachybotrys
spp. in buildings with either ongoing dampness or a history of water damage. Verrucarol and trichodermol, hydrolysis products of macrocyclic trichothecenes (including satratoxins), and trichodermin, predominately produced by
Stachybotrys chartarum
, were analyzed by gas chromatography-tandem mass spectrometry, whereas sterigmatocystin (mainly produced by
Aspergillus versicolor
), satratoxin G, and satratoxin H were analyzed by high-performance liquid chromatography-tandem mass spectrometry. These mycotoxin analytes were demonstrated in 45 of 62 building material samples studied, in three of eight settled dust samples, and in five of eight cultures of airborne dust samples. This is the first report on the use of tandem mass spectrometry for demonstrating mycotoxins in dust settled on surfaces above floor level in damp buildings. The direct detection of the highly toxic sterigmatocystin and macrocyclic trichothecene mycotoxins in indoor environments is important due to their potential health impacts.
EnterohemorrhagicEscherichia coliBiofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin
Authors: Lee Jintae , Bansal Tarun , Jayaraman Arul , Bentley William E. , Wood Thomas K.
Published: May 2007

Since indole is present at up to 500 μM in the stationary phase and is an interspecies biofilm signal (J. Lee, A. Jayaraman, and T. K. Wood, BMC Microbiol. 7:42, 2007), we investigated hydroxyindoles as biofilm signals and found them also to be nontoxic interspecies biofilm signals for enterohemorrhagicEscherichia coliO157:H7 (EHEC),E. coliK-12, andPseudomonas aeruginosa. The genetic basis of EHEC biofilm formation was also explored, and notably, virulence genes in biofilm cells were repressed compared to those in planktonic cells. In Luria-Bertani medium (LB) on polystyrene with quiescent conditions, 7-hydroxyindole decreased EHEC biofilm formation 27-fold and decreased K-12 biofilm formation 8-fold without affecting the growth of planktonic cells. 5-Hydroxyindole also decreased biofilm formation 11-fold for EHEC and 6-fold for K-12. In contrast, isatin (indole-2,3-dione) increased biofilm formation fourfold for EHEC, while it had no effect for K-12. When continuous-flow chambers were used, confocal microscopy revealed that EHEC biofilm formation was reduced 6-fold by indole and 10-fold by 7-hydroxyindole in LB. Whole-transcriptome analysis revealed that isatin represses indole synthesis by repressingtnaABC7- to 37-fold in EHEC, and extracellular indole levels were found to be 20-fold lower. Furthermore, isatin repressed the AI-2 transporterslsrABCDFGKR, while significantly inducing the flagellar genesflgABCDEFGHIJKandfliAEFGILMNOPQ(which led to a 50% increase in motility). 7-Hydroxyindole induces the biofilm inhibitor/stress regulatorycfRand repressescysADIJPU/fliC(which led to a 50% reduction in motility) andpurBCDEFHKLMNRT. Isogenic mutants showed that 7-hydroxyindole inhibitsE. colibiofilm through cysteine metabolism. 7-Hydroxyindole (500 μM) also stimulatesP. aeruginosaPAO1 biofilm formation twofold; therefore, hydroxyindoles are interspecies bacterial signals, and 7-hydroxyindole is a potent EHEC biofilm inhibitor.
Diversity of Anaerobic Microorganisms Involved in Long-Chain Fatty Acid Degradation in Methanogenic Sludges as Revealed by RNA-Based Stable Isotope Probing
Authors: Hatamoto Masashi , Imachi Hiroyuki , Yashiro Yuto , Ohashi Akiyoshi , Harada Hideki
Published: May 2007

Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with
13
C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect
13
C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in
13
C-labeled and unlabeled rRNA fractions. The bacterial populations in the
13
C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family
Syntrophaceae
, clone cluster MST belonging to the class
Deltaproteobacteria
,
Clostridium
clusters III and IV, phylum
Bacteroidetes
, phylum
Spirochaetes
, and family
Syntrophomonadaceae
. Although
Syntrophomonadaceae
species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.
Quantitative Evaluation of the Impact of Bather Density on Levels of Human-Virulent Microsporidian Spores in Recreational Water
Authors: Graczyk Thaddeus K. , Sunderland Deirdre , Tamang Leena , Shields Timothy M. , Lucy Frances E. , Breysse Patrick N.
Published: May 2007

Microsporidial gastroenteritis, a serious disease of immunocompromised people, can have a waterborne etiology. During summer months, samples of recreational bathing waters were tested weekly for human-virulent microsporidian spores and water quality parameters in association with high and low bather numbers during weekends and weekdays, respectively.
Enterocytozoon bieneusi
spores were detected in 59% of weekend (
n
= 27) and 30% of weekday (
n
= 33) samples, and
Encephalitozoon intestinalis
spores were concomitant in a single weekend sample; the overall prevalence was 43%. The numbers of bathers, water turbidity levels, prevalences of spore-positive samples, and concentrations of spores were significantly higher for weekend than for weekday samples;
P
values were <0.001, <0.04, <0.03, and <0.04, respectively. Water turbidity and the concentration of waterborne spores were significantly correlated with bather density, with
P
values of <0.001 and <0.01, respectively. As all water samples were collected on days deemed acceptable for bathing by fecal bacterial standards, this study reinforces the scientific doubt about the reliability of bacterial indicators in predicting human waterborne pathogens. The study provides evidence that bathing in public waters can result in exposure to potentially viable microsporidian spores and that body contact recreation in potable water can play a role in the epidemiology of microsporidiosis. The study indicates that resuspension of bottom sediments by bathers resulted in elevated turbidity values and implies that the microbial load from both sediments and bathers can act as nonpoint sources for the contamination of recreational waters with
Enterocytozoon bieneusi
spores. Both these mechanisms can be considered for implementation in predictive models for contamination with microsporidian spores.
Specific Detection and Real-Time PCR Quantification of Potentially Mycophagous Bacteria Belonging to the Genus
Collimonas
in Different Soil Ecosystems
Authors: Höppener-Ogawa Sachie , Leveau Johan H. J. , Smant Wiecher , van Veen Johannes A. , de Boer Wietse
Published: May 2007

The bacterial genus
Collimonas
has the remarkable characteristic that it grows at the expense of living fungal hyphae under laboratory conditions. Here, we report the first field inventory of the occurrence and abundance of
Collimonas
in soils (
n
= 45) with naturally different fungal densities, which was performed in order to test the null hypothesis that there is a relationship between the presence of
Collimonas
and fungal biomass. Estimates of fungal densities were based on ergosterol measurements. Each soil was also characterized in terms of its physical and chemical properties and vegetation and management types. Culturable
Collimonas
was identified in plate-spread soil samples by its ability to clear colloidal chitin, in combination with a
Collimonas
-specific restriction fragment length polymorphism analysis of 16S rRNA PCR amplified from individual colonies. Using this approach, we found culturable collimonads only in (semi)natural grasslands. A real-time PCR assay for the specific quantification of
Collimonas
16S rRNA in total soil DNA was developed.
Collimonas
was detectable in 80% of the soil samples, with densities up to 10
5
cells g
−1
(dry weight) soil. The numbers of
Collimonas
cells per gram of soil were consistently lowest in fungus-poor arable soils and, surprisingly, also in fungus-rich organic layers of forest soils. When all soils were included, no significant correlation was observed between the number of
Collimonas
cells and ergosterol-based soil fungal biomass. Based on this result, we rejected our null hypothesis, and possible explanations for this were addressed.
Dekkera bruxellensisandLactobacillus viniForm a Stable Ethanol-Producing Consortium in a Commercial Alcohol Production Process
Authors: Passoth Volkmar , Blomqvist Johanna , Schnürer Johan
Published: May 2007

The ethanol production process of a Swedish alcohol production plant was dominated byDekkera bruxellensisandLactobacillus vini, with a high number of lactic acid bacteria. The product quality, process productivity, and stability were high; thus,D. bruxellensisandL. vinican be regarded as commercial ethanol production organisms.
Possible Origins of CTnBST, a Conjugative Transposon Found Recently in a Human Colonic
Bacteroides
Strain
Authors: Schlesinger David J. , Shoemaker Nadja B. , Salyers Abigail A.
Published: May 2007

A previous survey of
Bacteroides
isolates suggested that the
ermB
gene entered
Bacteroides
spp. recently. Previously,
ermB
had been found almost exclusively in gram-positive bacteria. In one
Bacteroides
strain,
ermB
was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of
Bacteroides fragilis
YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the
ermB
gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the
ermB
gene was 100% identical to an
ermB
region found in the genome of the gram-positive bacterium
Arcanobacterium pyogenes
. A screen of
Bacteroides
isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by
Bacteroides
spp. We have also identified other
Bacteroides
isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a
Bacteroides
CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from
Bacteroides
spp. or some related species.
Mycorrhizal and Rhizobial Colonization of Genetically Modified and Conventional Soybeans
Authors: Powell Jeff R. , Gulden Robert H. , Hart Miranda M. , Campbell Rachel G. , Levy-Booth David J. , Dunfield Kari E. , Pauls K. Peter , Swanton Clarence J. , Trevors Jack T. , Klironomos John N.
Published: May 2007

We grew plants of nine soybean varieties, six of which were genetically modified to express transgenic
cp4-epsps
, in the presence of
Bradyrhizobium japonicum
and arbuscular mycorrhizal fungi. Mycorrhizal colonization and nodule abundance and mass differed among soybean varieties; however, in no case was variation significantly associated with the genetic modification.
Screening of Exopolysaccharide-Producing
Lactobacillus
and
Bifidobacterium
Strains Isolated from the Human Intestinal Microbiota
Authors: Ruas-Madiedo Patricia , Moreno José Antonio , Salazar Nuria , Delgado Susana , Mayo Baltasar , Margolles Abelardo , de los Reyes-Gavilán Clara G.
Published: May 2007

Using phenotypic approaches, we have detected that 17% of human intestinal
Lactobacillus
and
Bifidobacterium
strains could be exopolysaccharide (EPS) producers. However, PCR techniques showed that only 7% harbored genes related to the synthesis of heteropolysaccharides. This is the first work to screen the human intestinal ecosystem for the detection of EPS-producing strains.
Simple and Rapid F+ Coliphage Culture, Latex Agglutination, and Typing Assay To Detect and Source Track Fecal Contamination
Authors: Love David C. , Sobsey Mark D.
Published: May 2007

Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters.
Comparison of Aerobic Methanotrophic Communities in Littoral and Profundal Sediments of Lake Constance by a Molecular Approach
Authors: Rahalkar Monali , Schink Bernhard
Published: May 2007

Analysis of
pmoA
and 16S rRNA gene clone libraries of methanotrophic bacteria in Lake Constance revealed an overall dominance of type I methanotrophs in both littoral and profundal sediments. The sediments exhibited minor differences in their methanotrophic community structures. Type X methanotrophs made up a significant part of the clone libraries only in the profundal sediment and were also found only there as a prominent peak by T-RFLP analyses.
Inhibition of
Escherichia coli
Biofilm Formation by Self-Assembled Monolayers of Functional Alkanethiols on Gold
Authors: Hou Shuyu , Burton Erik A. , Simon Karen A. , Blodgett Dustin , Luk Yan-Yeung , Ren Dacheng
Published: May 2007

Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation.
Escherichia coli
was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl,
l
-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to
E. coli
biofilm formation; e.g., it repressed biofilm formation by
E. coli
DH5α by 99.5% ± 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs,
E. coli
formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.
Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution
Authors: Trefzer Axel , Jungmann Volker , Molnár István , Botejue Ajit , Buckel Dagmar , Frey Gerhard , Hill D. Steven , Jörg Mario , Ligon James M. , Mason Dylan , Moore David , Pachlatko J. Paul , Richardson Toby H. , Spangenberg Petra , Wall Mark A. , Zirkle Ross , Stege Justin T.
Published: May 2007

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.
Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples
Authors: Hill Vincent R. , Kahler Amy M. , Jothikumar Narayanan , Johnson Trisha B. , Hahn Donghyun , Cromeans Theresa L.
Published: May 2007

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for φX174 bacteriophage, MS2 bacteriophage,
Enterococcus faecalis
,
Clostridium perfringens
spores, and
Cryptosporidium parvum
oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance.
Characterization of the Zinc-Containing Metalloprotease Encoded by
zpx
and Development of a Species-Specific Detection Method for
Enterobacter sakazakii
Authors: Kothary M. H. , McCardell B. A. , Frazar C. D. , Deer D. , Tall B. D.
Published: May 2007

Enterobacter sakazakii
causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused “rounding” of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37°C and a pH range of 5 to 7. Proteolytic activity is inhibited by
ortho
-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride,
N
-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by
Serratia proteamaculans
,
Pectobacterium carotovorum
, and
Anabaena
sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135
E. sakazakii
strains possessed the metalloprotease gene,
zpx
, and 25 non-
E. sakazakii
strains did not. The cloned
zpx
gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.
Tracking Host Sources of
Cryptosporidium
spp. in Raw Water for Improved Health Risk Assessment
Authors: Ruecker Norma J. , Braithwaite Shannon L. , Topp Edward , Edge Thomas , Lapen David R. , Wilkes Graham , Robertson Will , Medeiros Diane , Sensen Christoph W. , Neumann Norman F.
Published: May 2007

Recent molecular evidence suggests that different species and/or genotypes of
Cryptosporidium
display strong host specificity, altering our perceptions regarding the zoonotic potential of this parasite. Molecular forensic profiling of the small-subunit rRNA gene from oocysts enumerated on microscope slides by U.S. Environmental Protection Agency method 1623 was used to identify the range and prevalence of
Cryptosporidium
species and genotypes in the South Nation watershed in Ontario, Canada. Fourteen sites within the watershed were monitored weekly for 10 weeks to assess the occurrence, molecular composition, and host sources of
Cryptosporidium
parasites impacting water within the region.
Cryptosporidium andersoni
,
Cryptosporidium
muskrat genotype II,
Cryptosporidium
cervine genotype,
C. baileyi
,
C. parvum
,
Cryptosporidium
muskrat genotype I, the
Cryptosporidium
fox genotype, genotype W1, and genotype W12 were detected in the watershed. The molecular composition of the
Cryptosporidium
parasites, supported by general land use analysis, indicated that mature cattle were likely the main source of contamination of the watershed. Deer, muskrats, voles, birds, and other wildlife species, in addition to sewage (human or agricultural) may also potentially impact water quality within the study area. Source water protection studies that use land use analysis with molecular genotyping of
Cryptosporidium
parasites may provide a more robust source-tracking tool to characterize fecal impacts in a watershed. Moreover, the information is vital for assessing environmental and human health risks posed by water contaminated with zoonotic and/or anthroponotic forms of
Cryptosporidium
.
Evaluation of Liquid- and Fog-Based Application of Sterilox Hypochlorous Acid Solution for Surface Inactivation of Human Norovirus
Authors: Park Geun Woo , Boston Deyanna M. , Kase Julie A. , Sampson Mark N. , Sobsey Mark D.
Published: May 2007

Noroviruses (NVs) are the most frequent cause of outbreaks of gastroenteritis in common settings, with surface-mediated transfer via contact with fecally contaminated surfaces implicated in exposure. NVs are environmentally stable and persistent and have a low infectious dose. Several disinfectants have been evaluated for efficacy to control viruses on surfaces, but the toxicity and potential damage to treated materials limits their applicability. Sterilox hypochlorous acid (HOCl) solution (HAS) has shown broad-spectrum antimicrobial activity while being suitable for general use. The objectives of this study were to evaluate the efficacy of HAS to reduce NV both in aqueous suspensions and on inanimate carriers. HOCl was further tested as a fog to decontaminate large spaces. HOCl effectiveness was evaluated using nonculturable human NV measured by reverse transcriptase PCR (RT-PCR) and two surrogate viruses, coliphage MS2 and murine NV, that were detected by both infectivity and RT-PCR. Exposing virus-contaminated carriers of ceramic tile (porous) and stainless steel (nonporous) to 20 to 200 ppm of HOCl solution resulted in ≥99.9% (≥3 log
10
) reductions of both infectivity and RNA titers of tested viruses within 10 min of exposure time. HOCl fogged in a confined space reduced the infectivity and RNA titers of NV, murine NV, and MS2 on these carriers by at least 99.9% (3 log
10
), regardless of carrier location and orientation. We conclude that HOCl solution as a liquid or fog is likely to be effective in disinfecting common settings to reduce NV exposures and thereby control virus spread via fomites.
Self-Formed Adaptor PCR: a Simple and Efficient Method for Chromosome Walking
Authors: Wang Shiming , He Jian , Cui Zhongli , Li Shunpeng
Published: May 2007

We developed a self-formed adaptor PCR (termed SEFA PCR) which can be used for chromosome walking. Most of the amplified flanking sequences were longer than 2.0 kb, and some were as long as 6.0 kb. SEFA PCR is simple and efficient and should have broad applications in the isolation of unknown sequences in complex genomes.
Isolation of Key Methanogens for Global Methane Emission from Rice Paddy Fields: a Novel Isolate Affiliated with the Clone Cluster Rice Cluster I
Authors: Sakai Sanae , Imachi Hiroyuki , Sekiguchi Yuji , Ohashi Akiyoshi , Harada Hideki , Kamagata Yoichi
Published: May 2007

Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H2) continuously provided by heterotrophic H2-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H2-producing syntroph,Syntrophobacter fumaroxidans, as the H2supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.
Protein Arginine Methylation in
Candida albicans
: Role in Nuclear Transport
Authors: McBride Anne E. , Zurita-Lopez Cecilia , Regis Anthony , Blum Emily , Conboy Ana , Elf Shannon , Clarke Steven
Published: May 2007

Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In
Candida albicans
, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison,
C. albicans
candidate PRMTs display similarity to
Saccharomyces cerevisiae
Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between
C. albicans
and
S. cerevisiae
: CaHmt1 supports growth of
S. cerevisiae
strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in
S. cerevisiae
. In
C. albicans
strains lacking CaHmt1, asymmetric dimethylarginine and ω-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major
C. albicans
type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in
C. albicans
. CaNpl3 allows partial growth of
S. cerevisiae npl3
Δ strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in
C. albicans
strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the
C. albicans
Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an
rmt2
Δ/
rmt2
Δ strain, as well as biochemical evidence for additional cryptic PRMTs.
Phosphatidylinositol 4-Kinase III-β Is Required for Golgi Maintenance and Cytokinesis in
Trypanosoma brucei
Authors: Rodgers Melissa J. , Albanesi Joseph P. , Phillips Margaret A.
Published: May 2007

The parasitic protozoan
Trypanosoma brucei
contains two type III phosphatidylinositol 4-kinases (α and β). We have cloned the gene encoding the
T. brucei
type III phosphatidylinositol 4-kinase β (TbPI4KIII-β), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-β in procyclic
T. brucei
by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-β have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-β is an essential protein in procyclic
T. brucei
, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.