bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of microvascular oxygen pressures during recovery in rat fast‐twitch muscle of differing oxidative capacity
Authors: McDonough Paul , Behnke Brad J. , Padilla Danielle J. , Musch Timothy I. , Poole David C.
Published: April 2007

Whether the speed of recovery of microvascular O2 pressures (
) differs within muscles composed primarily of type II fibres with contrasting oxidative capacity has not been determined. We tested the hypothesis that, following contractions, the recovery of
would be slower in the white (WG; low oxidative capacity) versus the mixed gastrocnemius (MG; comparatively high oxidative capacity). Radiolabelled microsphere and phosphorescence quenching techniques were used to measure muscle blood flow (
, hence O2 delivery,
) and
during contractions (1 Hz twitch) at low (LO, 2.5 V) and high intensities (HI, 4.5 V) in rat (n= 15) MG and WG muscle and during subsequent recovery. Following the LO protocol, end‐contraction
was lower in WG (11.6 ± 0.5 mmHg) than in MG (16.2 ± 0.6 mmHg; P < 0.05) while, contrary to our hypothesis, the initial rate of change in
during recovery (
/dt; MG 0.11 ± 0.01 mmHg s−1 and WG 0.06 ± 0.03 mmHg s−1) and mean response time (MRT; MG 110.3 ± 5.1 s and WG 113.5 ± 8.4 s, P > 0.05) were not different. In contrast, end‐contraction baseline
was not different following the HI protocol (MG 10.3 ± 0.6 mmHg and WG 9.2 ± 0.6 mmHg; P > 0.05) but, in agreement with our hypothesis,
/dt was slower (MG 0.07 ± 0.01 mmHg s−1 and WG 0.03 ± 0.003 mmHg s−1; P < 0.05) and MRT longer (WG 180.8 ± 4.5 s and MG 115.4 ± 6.7 s; P < 0.05) in WG versus MG following the HI protocol. These data suggest that following high‐intensity, though submaximal, muscle contractions,
recovers much faster in the more oxidative mixed gastrocnemius than in the less oxidative white gastrocnemius.
Role of the ABC Transporter PRP1 (ABCC7) in Pentamidine Resistance in
Leishmania
Amastigotes
Authors: Coelho Adriano C. , Messier Nadine , Ouellette Marc , Cotrim Paulo C.
Published: April 2007

Pentamidine is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance mechanism are not well understood. In this work, we show that the intracellular ABC protein PRP1 (pentamidine resistance protein 1) (ABCC7) can confer resistance to pentamidine in
Leishmania
sp. parasites in the intracellular stage.
Characterization of SafC, a Catechol 4-O-Methyltransferase Involved in Saframycin Biosynthesis
Authors: Nelson James T. , Lee Jaeheon , Sims James W. , Schmidt Eric W.
Published: April 2007

Members of the saframycin/safracin/ecteinascidin family of peptide natural products are potent antitumor agents currently under clinical development. Saframycin MX1, fromMyxococcus xanthus, is synthesized by a nonribosomal peptide synthetase, SafAB, and anO-methyltransferase, SafC, although other proteins are likely involved in the pathway. SafC was overexpressed inEscherichia coli, purified to homogeneity, and assayed for its ability to methylate a variety of substrates. SafC was able to catalyze the O-methylation of catechol derivatives but not phenols. Among the substrates tested, the best substrate for SafC wasl-dihydroxyphenylalanine (l-dopa), which was methylated specifically in the 4′-O position (kcat/Km= 5.5 × 103M−1s−1). SafC displayed less activity on other catechol derivatives, including catechol, dopamine, and caffeic acid. The more labilel-5′-methyldopa was an extremely poor substrate for SafC (kcat/Km= ∼2.8 × 10−5M−1s−1).l-Dopa thioester derivatives were also much less reactive thanl-dopa. These results indicate that SafC-catalyzed 4′-O-methylation ofl-dopa occurs prior to 5′-C-methylation, suggesting that 4′-O-methylation is likely the first committed step in the biosynthesis of saframycin MX1. SafC has biotechnological potential as a methyltransferase with unique regioselectivity.
Characterization of
Staphylococcus aureus
Isolates from Buffalo, Bovine, Ovine, and Caprine Milk Samples Collected in Rio de Janeiro State, Brazil
Authors: Aires-de-Sousa Marta , Parente Carlos E. S. R. , Vieira-da-Motta Olney , Bonna Isabel C. F. , Silva Denise A. , de Lencastre Hermínia
Published: April 2007

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as
Staphylococcus aureus
by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE),
spa
typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A,
spa
type t359, sequence type 747 [ST747]; PFGE B,
spa
type t1180, ST750; PFGE C,
spa
type t605, ST126; PFGE D,
spa
type t127, ST751; and PFGE F,
spa
type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with
S. aureus
from a human origin suggested that all but one clone found in the present study might be animal specific.
Nitric Oxide Reductase-Targeted Real-Time PCR Quantification of Denitrifier Populations in Soil
Authors: Dandie C. E. , Miller M. N. , Burton D. L. , Zebarth B. J. , Trevors J. T. , Goyer C.
Published: April 2007

The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (
cnorB
P
[
Pseudomonas mandelii
and closely related strains] and
cnorB
B
[
Bosea
,
Bradyrhizobium
, and
Ensifer
spp.]) in agricultural soils based on functional
cnorB
(nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (
r
= 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the
cnorB
P
population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The
cnorB
B
guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions.
cnorB
P
populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and
cnorB
P
population densities increased significantly with increasing rates of glucose addition.
cnorB
B
guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.
Plant Cell Wall Degradation by Saprophytic
Bacillus subtilis
Strains: Gene Clusters Responsible for Rhamnogalacturonan Depolymerization
Authors: Ochiai Akihito , Itoh Takafumi , Kawamata Akiko , Hashimoto Wataru , Murata Kousaku
Published: April 2007

Plant cell wall degradation is a premier event when
Bacillus subtilis
, a typical saprophytic bacterium, invades plants. Here we show the degradation system of rhamnogalacturonan type I (RG-I), a component of pectin from the plant cell wall, in
B. subtilis
strain 168. Strain 168 cells showed a significant growth on plant cell wall polysaccharides such as pectin, polygalacturonan, and RG-I as a carbon source. DNA microarray analysis indicated that three gene clusters (
yesOPQRSTUVWXYZ
,
ytePQRST
, and
ybcMOPST-ybdABDE
) are inducibly expressed in strain 168 cells grown on RG-I. Cells of an industrially important bacterium,
B. subtilis
strain natto, fermenting soybeans also express the gene cluster including the
yes
series during the assimilation of soybean used as a carbon source. Among proteins encoded in the
yes
cluster, YesW and YesX were found to be novel types of RG lyases releasing disaccharide from RG-I. Genetic and enzymatic properties of YesW and YesX suggest that strain 168 cells secrete YesW, which catalyzes the initial cleavage of the RG-I main chain, and the resultant oligosaccharides are converted to disaccharides through the extracellular exotype YesX reaction. The disaccharide is finally degraded into its constituent monosaccharides through the reaction of intracellular unsaturated galacturonyl hydrolases YesR and YteR. This enzymatic route for RG-I degradation in strain 168 differs significantly from that in plant-pathogenic fungus
Aspergillus aculeatus
. This is, to our knowledge, the first report on the bacterial system for complete RG-I main chain degradation.
Faster, Simpler, More-Specific Methods for Improved Molecular Detection of
Phytophthora ramorum
in the Field
Authors: Tomlinson J. A. , Barker I. , Boonham N.
Published: April 2007

Phytophthora ramorum
is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of
P. ramorum
in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of
P. ramorum
in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.
Risk of Handling as a Route of Exposure to Infectious Waterborne
Cryptosporidium parvum
Oocysts via Atlantic Blue Crabs (
Callinectes sapidus
)
Authors: Graczyk Thaddeus K. , McOliver Cynthia , Silbergeld Ellen K. , Tamang Leena , Roberts Jennifer D.
Published: April 2007

Commercial Atlantic blue crabs (
Callinectes sapidus
) were exposed to 2.0 × 10
4
infectious waterborne oocysts of
Cryptosporidium parvum
. The study demonstrated that blue crabs can transfer
C. parvum
oocysts to persons involved in handling or preparing crabs and that they may contaminate other surfaces or products during storage.
Identification of a Gene Cluster EnablingLactobacillus caseiBL23 To Utilizemyo-Inositol
Authors: Yebra María Jesús , Zúñiga Manuel , Beaufils Sophie , Pérez-Martínez Gaspar , Deutscher Josef , Monedero Vicente
Published: April 2007

Genome analysis ofLactobacillus caseiBL23 revealed that, compared toL. caseiATCC 334, it carries a 12.8-kb DNA insertion containing genes involved in the catabolism of the cyclic polyolmyo-inositol (MI). Indeed,L. caseiATCC 334 does not ferment MI, whereas strain BL23 is able to utilize this carbon source. The inserted DNA consists of aniolRgene encoding a DeoR family transcriptional repressor and a divergently transcribediolTABCDG1G2EJKoperon, encoding a complete MI catabolic pathway, in which theiolKgene probably codes for a malonate semialdehyde decarboxylase. The presence ofiolKsuggests thatL. caseihas two alternative pathways for the metabolism of malonic semialdehyde: (i) the classical MI catabolic pathway in which IolA (malonate semialdehyde dehydrogenase) catalyzes the formation of acetyl-coenzyme A from malonic semialdehyde and (ii) the conversion of malonic semialdehyde to acetaldehyde catalyzed by the product ofiolK. The function of theiolgenes was verified by the disruption ofiolA,iolT, andiolD, which provided MI-negative strains. By contrast, the disruption ofiolKresulted in a strain with no obvious defect in MI utilization. Transcriptional analyses conducted with different mutant strains showed that theiolTABCDG1G2EJKcluster is regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor IolR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This is the first example of an operon for MI utilization in lactic acid bacteria and illustrates the versatility of carbohydrate utilization inL. caseiBL23.
Protein Engineering of Wzc To Generate New Emulsan Analogs
Authors: Dams-Kozlowska Hanna , Kaplan David L.
Published: April 2007

Acinetobacter venetianusRag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modifiedwzcvariants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant, exhibiting decreased adherence to polystyrene, and inhibited biofilm formation. The results described in this report show the functional effect of Wzc modification on the molecular weight of a high-molecular-weight polysaccharide. Moreover, in the present study we developed a genetic system to control polymerization of apoemulsan. The use of selective exogenous fatty acid feeding strategies, as well as genetic manipulation of sugar backbone chain length, is a promising new approach for bioengineering emulsan analogs.
Trehalose Biosynthesis in
Rhizobium leguminosarum
bv.
trifolii
and Its Role in Desiccation Tolerance
Authors: McIntyre Helen J. , Davies Holiday , Hore Timothy A. , Miller Simon H. , Dufour Jean-Pierre , Ronson Clive W.
Published: April 2007

Rhizobium leguminosarum
bv.
trifolii
forms nitrogen-fixing root nodules on the pasture legume
Trifolium repens
, and
T. repens
seed is often coated with a compatible
R. leguminosarum
bv.
trifolii
strain prior to sowing. However, significant losses in bacterial viability occur during the seed-coating process and during storage of the coated seeds, most likely due to desiccation stress. The disaccharide trehalose is known to function as an osmoprotectant, and trehalose accumulation due to de novo biosynthesis is a common response to desiccation stress in bacteria. In this study we investigated the role of endogenous trehalose synthesis in desiccation tolerance in
R. leguminosarum
bv.
trifolii
strain NZP561. Strain NZP561 accumulated trehalose as it entered the stationary phase due to the combined actions of the TreYZ and OtsAB pathways. Mutants deficient in either pathway showed near-wild-type levels of trehalose accumulation, but double
otsA treY
mutants failed to accumulate any trehalose. The double mutants were more sensitive to the effects of drying, and their survival was impaired compared to that of the wild type when glass beads were coated with the organisms and stored at relative humidities of 5 and 32%. The
otsA treY
mutants were also less competitive for nodule occupancy. Gene expression studies showed that the
otsA
and
treY
genes were expressed constitutively and that expression was not influenced by the growth phase, suggesting that trehalose accumulation is controlled at the posttranscriptional level or by control of trehalose breakdown rates. Our results indicate that accumulated trehalose plays an important role in protecting
R. leguminosarum
bv.
trifolii
cells against desiccation stress and against stress encountered during nodulation.
Quantitative Assessment of Contamination of Fresh Food Produce of Various Retail Types by Human-Virulent Microsporidian Spores
Authors: Jedrzejewski Szymon , Graczyk Thaddeus K. , Slodkowicz-Kowalska Anna , Tamang Leena , Majewska Anna C.
Published: April 2007

This study demonstrated that fresh food produce, such as berries, sprouts, and green-leafed vegetables, sold at the retail level can contain potentially viable microsporidian spores of human-virulent species, such as
Enterocytozoon bieneusi
,
Encephalitozoon intestinalis
, and
Encephalitozoon cuniculi
, at quantities representing a threat of food-borne infection.
Desensitization of Feedback Inhibition of the
Saccharomyces cerevisiae
γ-Glutamyl Kinase Enhances Proline Accumulation and Freezing Tolerance
Authors: Sekine Tomoko , Kawaguchi Akari , Hamano Yoshimitsu , Takagi Hiroshi
Published: April 2007

In response to osmotic stress, proline is accumulated in many bacterial and plant cells as an osmoprotectant. The yeast
Saccharomyces cerevisiae
induces trehalose or glycerol synthesis but does not increase intracellular proline levels during various stresses. Using a proline-accumulating mutant, we previously found that proline protects yeast cells from damage by freezing, oxidative, or ethanol stress. This mutant was recently shown to carry an allele of
PRO1
which encodes the Asp154Asn mutant γ-glutamyl kinase (GK), the first enzyme of the proline biosynthetic pathway. Here, enzymatic analysis of recombinant proteins revealed that the GK activity of
S. cerevisiae
is subject to feedback inhibition by proline. The Asp154Asn mutant was less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation. To improve the enzymatic properties of GK, PCR random mutagenesis in
PRO1
was employed. The mutagenized plasmid library was introduced into an
S. cerevisiae
non-proline-utilizing strain, and proline-overproducing mutants were selected on minimal medium containing the toxic proline analogue azetidine-2-carboxylic acid. We successfully isolated several mutant GKs that, due to extreme desensitization to inhibition, enhanced the ability to synthesize proline better than the Asp154Asn mutant. The amino acid changes were localized at the region between positions 142 and 154, probably on the molecular surface, suggesting that this region is involved in allosteric regulation. Furthermore, we found that yeast cells expressing Ile150Thr and Asn142Asp/Ile166Val mutant GKs were more tolerant to freezing stress than cells expressing the Asp154Asn mutant.
The Early Response to Acid Shock inLactobacillus reuteriInvolves the ClpL Chaperone and a Putative Cell Wall-Altering Esterase
Authors: Wall Torun , Båth Klara , Britton Robert A. , Jonsson Hans , Versalovic James , Roos Stefan
Published: April 2007

To be able to function as a probiotic, bacteria have to survive the passage through the gastrointestinal tract. We have examined survival and gene expression ofLactobacillus reuteriATCC 55730 after a sudden shift in environmental acidity to a pH close to the conditions in the human stomach. More than 80% of theL. reutericells survived at pH 2.7 for 1 h. A genomewide expression analysis experiment using microarrays displayed 72 differentially expressed genes at this pH. The early response to severe acid shock inL. reuteridiffered from long-term acid adaptation to milder acid stress studied in other lactic acid bacteria. The genes induced included the following:clpL, genes putatively involved in alterations of the cell membrane and the cell wall; genes encoding transcriptional regulators; phage genes; and genes of unknown function. Two genes,clpL, encoding an ATPase with chaperone activity, and lr1516, encoding a putative esterase, were selected for mutation analyses. The mutants were significantly more sensitive to acid than the wild type was. Thus, these genes could contribute to the survival ofL. reuteriin the gastrointestinal tract.
DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of
Escherichia coli
Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease
Authors: Han Weiqing , Liu Bin , Cao Boyang , Beutin Lothar , Krüger Ulrike , Liu Hongbo , Li Yayue , Liu Yanqun , Feng Lu , Wang Lei
Published: April 2007

Escherichia coli
strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8
E. coli
serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186
E. coli
and
Shigella
O-serogroup reference strains, 13
E. coli
reference strains for virulence markers, 43
E. coli
clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10
3
CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated
E. coli
in porcine feces samples.
Swarming Behavior of and Hemolysin BL Secretion by
Bacillus cereus
Authors: Ghelardi Emilia , Celandroni Francesco , Salvetti Sara , Ceragioli Mara , Beecher Douglas J. , Senesi Sonia , Wong Amy C. L.
Published: April 2007

An association between swarming and hemolysin BL secretion was observed in a collection of 42
Bacillus cereus
isolates (
P
= 0.029). The highest levels of toxin were detected in swarmers along with swarm cell differentiation (
P
= 0.021), suggesting that swarming
B. cereus
strains may have a higher virulence potential than nonswarming strains.
Diversity and Function of
Chloroflexus
-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration
Authors: Bachar Ami , Omoregie Enoma , de Wit Rutger , Jonkers Henk M.
Published: April 2007

We studied the diversity of
Chloroflexus
-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum
Chloroflexi
resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family
Chloroflexaceae
. Photopigment analysis revealed the presence of bacteriochlorophyll
c
(BChl
c
), BChl
d
, and γ-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of
Chloroflexus aurantiacus
. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.
Kinetic Constant Variability in Bacterial Oxidation of Elemental Sulfur
Authors: Pokorna Blanka , Mandl Martin , Borilova Sarka , Ceskova Pavla , Markova Romana , Janiczek Oldrich
Published: April 2007

Wide ranges of growth yields on sulfur (from 2.4 × 10
10
to 8.1 × 10
11
cells g
−1
) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter
−1
h
−1
) of an
Acidithiobacillus ferrooxidans
strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.
Structure and Function of Methanogens along a Short-Term Restoration Chronosequence in the Florida Everglades
Authors: Smith Jason M. , Castro Hector , Ogram Andrew
Published: April 2007

The removal of plants and soil to bedrock to eradicate exotic invasive plants within the Hole-in-the-Donut (HID) region, part of the Everglades National Park (Florida), presented a unique opportunity to study the redevelopment of soil and the associated microbial communities in the context of short-term primary succession and ecosystem restoration. The goal of this study was to identify relationships between soil redevelopment and activity and composition of methanogenic assemblages in HID soils. Methane production potentials indicated a general decline in methanogenic activity with restoration age. Microcosm incubations strongly suggested hydrogenotrophic methanogenesis as the most favorable pathway for methane formation in HID soils from all sites. Culture-independent techniques targeting methyl coenzyme M reductase genes (
mcrA
) were used to assess the dynamics of methanogenic assemblages. Clone libraries were dominated by sequences related to hydrogenotrophic methanogens of the orders
Methanobacteriales
and
Methanococcales
and suggested a general decline in the relative abundance of
Methanobacteriales mcrA
with time since restoration. Terminal restriction fragment length polymorphism analysis indicated methanogenic assemblages remain relatively stable between wet and dry seasons. Interestingly, analysis of soils across the restoration chronosequence indicated a shift in
Methanobacteriales
populations with restoration age, suggesting genotypic shifts due to site-specific factors.
Quantitative Detection of
Clostridium tyrobutyricum
in Milk by Real-Time PCR
Authors: López-Enríquez Lorena , Rodríguez-Lázaro David , Hernández Marta
Published: April 2007

We developed a real-time PCR assay for the quantitative detection of
Clostridium tyrobutyricum
, which has been identified as the major causal agent of late blowing in cheese. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent in 40% of the reactions. The quantification was linear (
R
2
> 0.9995) over a 5-log dynamic range, down to 10 genome equivalents, with a PCR efficiency of >0.946. With optimized detergent treatment and enzymatic pretreatment of the sample before centrifugation and nucleic acid extraction, the assay counted down to 300
C. tyrobutyricum
spores, with a relative accuracy of 82.98 to 107.68, and detected as few as 25 spores in 25 ml of artificially contaminated raw or ultrahigh-temperature-treated whole milk.
Shedding of
Escherichia coli
O157:H7 in Calves Is Reduced by Prior Colonization with the Homologous Strain
Authors: Naylor Stuart W. , Flockhart Allen , Nart Pablo , Smith David G. E. , Huntley John , Gally David L. , Low J. Christopher
Published: April 2007

Enterohemorrhagic
Escherichia coli
O157:H7 has a natural reservoir in the intestinal tracts of cattle. Colonization is asymptomatic and transient, but it is not clear if protective immunity is induced. This study demonstrates that prior colonization induces humoral immune responses to bacterial antigens and reduces bacterial shedding after experimental challenge with the homologous strain.
Genomic and Phenotypic Diversity of Coastal
Vibrio cholerae
Strains Is Linked to Environmental Factors
Authors: Keymer Daniel P. , Miller Michael C. , Schoolnik Gary K. , Boehm Alexandria B.
Published: April 2007

Studies of
Vibrio cholerae
diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of
V. cholerae
. Comparative genome hybridization using microarrays constructed from the fully sequenced
V. cholerae
O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence
V. cholerae
population structure in aquatic environments.
Coprinus comatus
Damages Nematode Cuticles Mechanically with Spiny Balls and Produces Potent Toxins To Immobilize Nematodes
Authors: Luo Hong , Liu Yajun , Fang Lin , Li Xuan , Tang Ninghua , Zhang Keqin
Published: April 2007

We reported recently a unique fungal structure, called the spiny ball, on the vegetative hyphae of
Coprinus comatus
(O. F. Müll.:Fr.) Pers. Although some observations regarding the role of this structure were presented, its function remained largely unknown. In this study, we showed that purified (isolated and washed) spiny balls could immobilize and kill the free-living nematode
Panagrellus redivivus
Goodey highly efficiently. Scanning electron microscopy studies illustrated that the spiny structure damaged the nematode cuticle, suggesting the presence of a mechanical force during the process of nematode immobilization. Severe injuries on nematode cuticles caused the leakage of inner materials of the nematodes. When these structures were ground in liquid nitrogen, their killing efficacy against nematodes was lost, indicating that the shape and the complete structure of the spiny balls are indispensable for their function. However, extraction with organic solvents never lowered their activity against
P. redivivus
, and the extracts showed no obvious effect on the nematode. We also investigated whether
C. comatus
was able to produce toxins which would aid in the immobilization of nematodes. In total, we identified seven toxins from
C. comatus
that showed activity to immobilize the nematodes
P. redivivus
and
Meloidogyne incognita
(Kofoid et White) Chitwood. The chemical structures of these toxins were identified with nuclear magnetic resonance, mass spectrometry, infrared, and UV spectrum analysis. Two compounds were found to be novel. The toxins found in
C. comatus
are O-containing heterocyclic compounds.
Diversity and Mechanisms of Alkali Tolerance in Lactobacilli
Authors: Sawatari Yuki , Yokota Atsushi
Published: April 2007

We determined the maximum pH that allows growth (pHmax) for 34 strains of lactobacilli. High alkali tolerance was exhibited by strains of
Lactobacillus casei
,
L. paracasei
subsp.
tolerans
,
L. paracasei
subsp.
paracasei
,
L. curvatus
,
L. pentosus
, and
L. plantarum
that originated from plant material, with pHmax values between 8.5 and 8.9. Among these,
L. casei
NRIC 1917 and
L. paracasei
subsp.
tolerans
NRIC 1940 showed the highest pHmax, at 8.9. Digestive tract isolates of
L. gasseri
,
L. johnsonii
,
L. reuteri
,
L. salivarius
subsp.
salicinius
, and
L. salivarius
subsp.
salivarius
exhibited moderate alkali tolerance, with pHmax values between 8.1 and 8.5. Dairy isolates of
L. delbrueckii
subsp.
bulgaricus
,
L. delbrueckii
subsp.
lactis
, and
L. helveticus
exhibited no alkali tolerance, with pHmax values between 6.7 and 7.1. Measurement of the internal pH of representative strains revealed the formation of transmembrane proton gradients (ΔpH) in a reversed direction (i.e., acidic interior) at alkaline external-pH ranges, regardless of their degrees of alkali tolerance. Thus, the reversed ΔpH did not determine alkali tolerance diversity. However, the ΔpH contributed to alkali tolerance, as the pHmax values of several strains decreased with the addition of nigericin, which dissipates ΔpH. Although neutral external-pH values resulted in the highest glycolysis activity in the presence of nigericin regardless of alkali tolerance, substantial glucose utilization was still detected in the alkali-tolerant strains, even in a pH range of between 8.0 and 8.5, at which the remaining strains lost most activity. Therefore, the alkali tolerance of glycolysis reactions contributes greatly to the determination of alkali tolerance diversity.
Preliminary Evidence that American Bullfrogs (
Rana catesbeiana
) Are Suitable Hosts for
Escherichia coli
O157:H7
Authors: Gray Matthew J. , Rajeev Sreekumari , Miller Debra L. , Schmutzer A. Chandler , Burton Elizabeth C. , Rogers Emily D. , Hickling Graham J.
Published: April 2007

We orally inoculated
Rana catesbeiana
tadpoles (
n
= 23) and metamorphs (
n
= 24) to test their suitability as hosts for
Escherichia coli
O157:H7. Tadpoles were housed in flowthrough aquaria and did not become infected. Metamorphs were housed in stagnant aquaria, and 54% tested positive through 14 days postinoculation, suggesting that they are suitable hosts for
E. coli
O157:H7.
Recurrent and Sporadic
Listeria monocytogenes
Contamination in Alheiras Represents Considerable Diversity, Including Virulence-Attenuated Isolates
Authors: Felício M. T. S. , Hogg T. , Gibbs P. , Teixeira P. , Wiedmann M.
Published: April 2007

Microbiological characterization of alheiras, traditional smoked meat sausages produced in northern Portugal, had previously shown that more than 60% of the lots analyzed were contaminated with
Listeria monocytogenes
at levels higher than 100 CFU/g. In order to better understand
L. monocytogenes
contamination patterns in alheiras, we characterized 128
L. monocytogenes
isolates from alheiras using a variety of subtyping techniques (i.e., molecular serotyping; arsenic, cadmium, and tetracycline resistance typing; and pulsed-field gel electrophoresis [PFGE]). Subtyping of isolates from products collected on two separate dates provided evidence for the persistence of specific
L. monocytogenes
PFGE types in the production and distribution chains of alheiras from four different processors. A subset of 21 isolates was further characterized using ribotyping and Caco-2 cell invasion assays to evaluate the pathogenic potential of
L. monocytogenes
present in alheiras. Caco-2 invasion assays revealed seven isolates with invasion efficiencies that were less than 20% of that of the control strain 10403S. All seven isolates had premature stop codons in
inlA
that represented three distinct mutations, which had previously been observed in isolates from the United States or France. Our findings indicate the need for a comprehensive approach to control
L. monocytogenes
in alheiras, including strategies to reduce persistence. The presence of considerable diversity in invasion phenotypes among
L. monocytogenes
strains present in alheiras, including the presence of subtypes likely to be virulence attenuated, may provide an opportunity to initially focus control strategies on the subtypes most likely to cause human disease.
Diversity of Environmental
Mycobacterium
Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach
Authors: Gomila Margarita , Ramirez Antonio , Lalucat Jorge
Published: April 2007

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1,
gyrB
,
hsp65
,
recA
,
rpoB
, and
sodA
). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.
Transcriptome Analysis in Response to Heat Shock and Cadmium in the Aquatic Fungus
Blastocladiella emersonii
Authors: Georg Raphaela C. , Gomes Suely L.
Published: April 2007

The global transcriptional response of the chytridiomycete
Blastocladiella emersonii
to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in
B. emersonii
. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the
B. emersonii
transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of
B. emersonii
cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up- and 60 downregulated genes during heat shock and 189 up- and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress,
B. emersonii
cells induced genes encoding six different glutathione
S
-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus.
Carbohydrate Metabolism in theToxoplasma gondiiApicoplast: Localization of Three Glycolytic Isoenzymes, the Single Pyruvate Dehydrogenase Complex, and a Plastid Phosphate Translocator
Authors: Fleige Tobias , Fischer Karsten , Ferguson David J. P. , Gross Uwe , Bohne Wolfgang
Published: April 2007

Many apicomplexan parasites, such asToxoplasma gondiiandPlasmodiumspecies, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites’ viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism ofT. gondiito either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in theT. gondiigenome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. TheT. gondiigenome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.
Transcriptional Profiling of Cross Pathway Control in
Neurospora crassa
and Comparative Analysis of the Gcn4 and CPC1 Regulons
Authors: Tian Chaoguang , Kasuga Takao , Sachs Matthew S. , Glass N. Louise
Published: April 2007

Identifying and characterizing transcriptional regulatory networks is important for guiding experimental tests on gene function. The characterization of regulatory networks allows comparisons among both closely and distantly related species, providing insight into network evolution, which is predicted to correlate with the adaptation of different species to particular environmental niches. One of the most intensely studied regulatory factors in the yeast
Saccharomyces cerevisiae
is the bZIP transcription factor Gcn4p. Gcn4p is essential for a global transcriptional response when
S. cerevisiae
experiences amino acid starvation. In the filamentous ascomycete
Neurospora crassa
, the ortholog of
GCN4
is called the
c
ross
p
athway
c
ontrol-1 (
cpc-1
) gene; it is required for the ability of
N. crassa
to induce a number of amino acid biosynthetic genes in response to amino acid starvation. Here, we deciphered the CPC1 regulon by profiling transcription in wild-type and
cpc-1
mutant strains with full-genome
N. crassa
70-mer oligonucleotide microarrays. We observed that at least 443 genes were direct or indirect CPC1 targets; these included 67 amino acid biosynthetic genes, 16 tRNA synthetase genes, and 13 vitamin-related genes. Comparison among the
N. crassa
CPC1 transcriptional profiling data set and the Gcn4/CaGcn4 data sets from
S. cerevisiae
and
Candida albicans
revealed a conserved regulon of 32 genes, 10 of which are predicted to be directly regulated by Gcn4p/CPC1. The 32-gene conserved regulon comprises mostly amino acid biosynthetic genes. The comparison of regulatory networks in species with clear orthology among genes sheds light on how gene interaction networks evolve.
Cryptococcus neoformans
Mates on Pigeon Guano: Implications for the Realized Ecological Niche and Globalization
Authors: Nielsen Kirsten , De Obaldia Anna L. , Heitman Joseph
Published: April 2007

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of
Cryptococcus neoformans
and
Cryptococcus gattii
on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches.
C. neoformans
is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (
grubii
[serotype A] and
neoformans
[serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast,
C. gattii
often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced.
C. neoformans
exhibited prolific mating on pigeon guano, whereas
C. gattii
did not. The observations that
C. neoformans
completes the life cycle on pigeon guano but that
C. gattii
does not indicates that pigeon guano could represent the realized ecological niche for
C. neoformans
. Because
C. gattii
grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for
C. gattii
. Based on these studies, we hypothesize that an ancestral
Cryptococcus
strain gained the ability to sexually reproduce in pigeon guano and then swept the globe.
Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon
Pyrococcus furiosus
: Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase
Authors: Schut Gerrit J. , Bridger Stephanie L. , Adams Michael W. W.
Published: April 2007

The hyperthermophilic archaeon
Pyrococcus furiosus
uses carbohydrates as a carbon source and produces acetate, CO
2
, and H
2
as end products. When S
0
is added to a growing culture, within 10 min the rate of H
2
production rapidly decreases and H
2
S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S
0
reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (
M
r
, 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S
0
addition. This primary response to S
0
also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H
2
-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S
0
addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S
0
. A secondary response to S
0
is observed 30 min after S
0
addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S
0
-reducing system involving NSR and MBX has been found so far only in the heterotrophic
Thermococcales
and is in contrast to the cytochrome- and quinone-based S
0
-reducing system in autotrophic archaea and bacteria.