bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Role of
para-
Aminobenzoic Acid Biosynthesis in Folate Production by
Lactococcus lactis
Authors: Wegkamp Arno , van Oorschot Wietske , de Vos Willem M. , Smid Eddy J.
Published: February 2007

The
pab
genes for
para
-aminobenzoic acid (
p
ABA) biosynthesis in
Lactococcus lactis
were identified and characterized. In
L. lactis
NZ9000, only two of the three genes needed for
p
ABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (
pabC
) was initially annotated, but detailed analysis revealed that
pabC
was fused with the 3′ end of the gene coding for chorismate synthetase component II (
pabB
). Therefore, we hypothesize that all three enzyme activities needed for
p
ABA production are present in
L. lactis
, allowing for the production of
p
ABA. Indeed, the overexpression of the
p
ABA gene cluster in
L. lactis
resulted in elevated
p
ABA pools, demonstrating that the genes are involved in the biosynthesis of
p
ABA. Moreover, a
p
ABA knockout (KO) strain lacking
pabA
and
pabB
C
was constructed and shown to be unable to produce folate when cultivated in the absence of
p
ABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and
p
ABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of
L. lactis
. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway.
L. lactis
strain NZ7024, overexpressing both the folate and
p
ABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without
p
ABA. This is in sharp contrast to
L. lactis
strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the
p
ABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and
p
ABA biosynthesis pathway in the wild-type strain.
Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of
Escherichia coli
O157:H7
Authors: Ravva Subbarao V. , Korn Anna
Published: February 2007

The influence of nutrients in wastewater from dairy lagoons on the survival of
Escherichia coli
O157:H7 was monitored. Initially, the survival of
E. coli
O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of
E. coli
O157:H7 or
E. coli
ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of
E. coli
O157:H7 strains was monitored in 300 μl of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater.
E. coli
O157:H7 and
E. coli
ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of
E. coli
O157:H7 and
E. coli
ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of
E. coli
O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components,
E. coli
O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.
Molecular Basis of Fructose Utilization by the Wine YeastSaccharomyces cerevisiae: a MutatedHXT3Allele Enhances Fructose Fermentation
Authors: Guillaume Carole , Delobel Pierre , Sablayrolles Jean-Marie , Blondin Bruno
Published: February 2007

Fructose utilization by wine yeasts is critically important for the maintenance of a high fermentation rate at the end of alcoholic fermentation. ASaccharomyces cerevisiaewine yeast able to ferment grape must sugars to dryness was found to have a high fructose utilization capacity. We investigated the molecular basis of this enhanced fructose utilization capacity by studying the properties of several hexose transporter (HXT) genes. We found that this wine yeast harbored a mutatedHXT3allele. A functional analysis of this mutated allele was performed by examining expression in an hxt1-7Δ strain. Expression of the mutated allele alone was found to be sufficient for producing an increase in fructose utilization during fermentation similar to that observed in the commercial wine yeast. This work provides the first demonstration that the pattern of fructose utilization during wine fermentation can be altered by expression of a mutated hexose transporter in a wine yeast. We also found that the glycolytic flux could be increased by overexpression of the mutant transporter gene, with no effect on fructose utilization. Our data demonstrate that the Hxt3 hexose transporter plays a key role in determining the glucose/fructose utilization ratio during fermentation.
Genetic Engineering of
Zymobacter palmae
for Production of Ethanol from Xylose
Authors: Yanase Hideshi , Sato Dai , Yamamoto Keiko , Matsuda Saori , Yamamoto Sho , Okamoto Kenji
Published: February 2007

Its metabolic characteristics suggest that
Zymobacter palmae
gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered
Z. palmae
so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The
Escherichia coli
genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into
Z. palmae
, where their expression was driven by the
Zymomonas mobilis
glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant
Z. palmae
strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant
Z. palmae
, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using
Z. palmae
expressing
E. coli
xylose catabolic enzymes.
Oil Field Souring Control by Nitrate-Reducing
Sulfurospirillum
spp. That Outcompete Sulfate-Reducing Bacteria for Organic Electron Donors
Authors: Hubert Casey , Voordouw Gerrit
Published: February 2007

Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including
Sulfurospirillum
and
Thauera
spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile
Sulfurospirillum
spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of
Desulfovibrio
sp. strain Lac3, Lac6, or Lac15 and
Sulfurospirillum
sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.
Outbreak of an Acute Aflatoxicosis in Kenya in 2004: Identification of the Causal Agent
Authors: Probst Claudia , Njapau Henry , Cotty Peter J.
Published: February 2007

Maize contaminated with aflatoxins has been implicated in deadly epidemics in Kenya three times since 1981, but the fungi contaminating the maize with aflatoxins have not been characterized. Here we associate the S strain of
Aspergillus flavus
with lethal aflatoxicoses that took more than 125 lives in 2004.
Identification of
Lactobacillus sakei
Genes Induced during Meat Fermentation and Their Role in Survival and Growth
Authors: Hüfner Eric , Markieton Tobias , Chaillou Stéphane , Crutz-Le Coq Anne-Marie , Zagorec Monique , Hertel Christian
Published: February 2007

Lactobacillus sakei
is a lactic acid bacterium that is ubiquitous in the food environment and is one of the most important constituents of commercial meat starter cultures. In this study, in vivo expression technology (IVET) was applied to investigate gene expression of
L. sakei
23K during meat fermentation. The IVET vector used (pEH100) contained promoterless and transcriptionally fused reporter genes mediating β-glucuronidase activity and erythromycin resistance. A genomic library of
L. sakei
23K was established, and the clones were subjected to fermentation in a raw-sausage model. Fifteen in carne-induced fusions were identified. Several genes encoded proteins which are likely to contribute to stress-related functions. One of these genes was involved in acquisition of ammonia from amino acids, and the remaining either were part of functionally unrelated pathways or encoded hypothetical proteins. The construction and use of isogenic mutants in the sausage model suggested that four genes have an impact on the performance of
L. sakei
during raw-sausage fermentation. Inactivation of the heat shock regulator gene
ctsR
resulted in increased growth, whereas knockout of the genes
asnA2
, LSA1065, and LSA1194 resulted in attenuated performance compared to the wild-type strain. The results of our study are the first to provide an insight into the transcriptional response of
L. sakei
when growing in the meat environment. In addition, this study establishes a molecular basis which allows investigation of bacterial properties that are likely to contribute to the ecological performance of the organism and to influence the final outcome of sausage fermentation.
Tetracycline Resistance Mediated by
tet
(W),
tet
(M), and
tet
(O) Genes of
Bifidobacterium
Isolates from Humans
Authors: Aires J. , Doucet-Populaire F. , Butel M. J.
Published: February 2007

MICs of tetracyclines were determined for 86 human
Bifidobacterium
isolates and three environmental strains. The
tet
(O) gene was found to be absent in these isolates.
tet
(W) and
tet
(M) were found in 26 and 7%, respectively, of the
Bifidobacterium
isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of
tet
(W) and/or
tet
(M).
Prevalence and Genetic Properties ofSalmonella entericaSerovar Typhimurium Definitive Phage Type 104 Isolated fromRattus norvegicusandRattus rattusHouse Rats in Yokohama City, Japan
Authors: Yokoyama Eiji , Maruyama Soichi , Kabeya Hidenori , Hara Siro , Sata Shin , Kuroki Toshiro , Yamamoto Tomoko
Published: February 2007

Salmonella entericaserovar Typhimurium was isolated from the intestinal contents ofRattus rattusandRattus norvegicushouse rats captured at two buildings, designated buildings J and YS, in Yokohama City, Japan. From October 1997 to September 1998, 52 of 339 (15.3%) house rats were found to carrySalmonellaserovar Typhimurium definitive phage type 104 (DT104). In building J, 26 of 161 (16.1%) house rats carried DT104 over the 1-year study period, compared to 26 of 178 (14.6%) rats in building YS. The isolation rates of DT104 fromR. rattusandR. norvegicuswere similar in the two buildings. Most DT104 strains from building J (24 of 26) showed resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline and contained both the 1.0- and 1.2-kbp integrons, carrying genespse1,pasppflo-like,aadA2,sulI, andtet(G). All DT104 strains from building YS were resistant to ampicillin and sulfisoxazole, and had the 1.2-kbp integron carryingpse1andsulI. Cluster analysis of pulsed-field gel electrophoresis patterns of BlnI-digested DT104 DNAs showed that 22 of 26 DT104 strains from building J and 24 of 26 strains from building YS could be grouped into separate clusters each specific for the building origin. These results indicated that DT104 strains were prevalent in house rat colonies in each building and suggest that house rats may play an important role in the epidemiology of DT104.
Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of
Serratia marcescens
Authors: Thompson Stuart A. , Maani Elizabeth V. , Lindell Angela H. , King Catherine J. , McArthur J. Vaun
Published: February 2007

Resistances to tetracycline and mercury were identified in an environmental strain of
Serratia marcescens
isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in
Escherichia coli
. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [
tetA
(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of
Acinetobacter
spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the
tetA
(41) gene was a divergently transcribed gene [
tetR
(41)] predicted to encode a tetracycline-responsive repressor protein. The
tetA
(41)-
tetR
(41) intergenic region contained putative operators for TetR(41) binding. The
tetA
(41) and
tetR
(41) promoters were analyzed using
lacZ
fusions, which showed that the expression of both the
tetA
(41) and
tetR
(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this
S. marcescens
strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the
S. marcescens
chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to
E. coli
, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.
Diversity, Activity, and Abundance of Sulfate-Reducing Bacteria in Saline and Hypersaline Soda Lakes
Authors: Foti Mirjam , Sorokin Dimitry Y. , Lomans Bart , Mussman Marc , Zacharova Elena E. , Pimenov Nikolay V. , Kuenen J. Gijs , Muyzer Gerard
Published: February 2007

Soda lakes are naturally occurring highly alkaline and saline environments. Although the sulfur cycle is one of the most active element cycles in these lakes, little is known about the sulfate-reducing bacteria (SRB). In this study we investigated the diversity, activity, and abundance of SRB in sediment samples and enrichment cultures from a range of (hyper)saline soda lakes of the Kulunda Steppe in southeastern Siberia in Russia. For this purpose, a polyphasic approach was used, including denaturing gradient gel electrophoresis of
dsr
gene fragments, sulfate reduction rate measurements, serial dilutions, and quantitative real-time PCR (qPCR). Comparative sequence analysis revealed the presence of several novel clusters of SRB, mostly affiliated with members of the order
Desulfovibrionales
and family
Desulfobacteraceae
. We detected sulfate reducers and observed substantial sulfate reducing rates (between 12 and 423 μmol/dm
3
day
−1
) for most lakes, even at a salinity of 475 g/liter. Enrichments were obtained at salt saturating conditions (4 M Na
+
), using H
2
or volatile fatty acids as electron donors, and an extremely halophilic SRB, strain ASO3-1, was isolated. Furthermore, a high
dsr
gene copy number of 10
8
cells per ml was detected in a hypersaline lake by qPCR. Our results indicate the presence of diverse and active SRB communities in these extreme ecosystems.
Gene Expression and Biochemical Analysis of Cheese-Ripening Yeasts: Focus on Catabolism ofl-Methionine, Lactate, and Lactose
Authors: Cholet Orianne , Hénaut Alain , Casaregola Serge , Bonnarme Pascal
Published: February 2007

DNA microarrays of 86 genes from the yeastsDebaryomyces hansenii,Kluyveromyces marxianus, andYarrowia lipolyticawere developed to determine which genes were expressed in a medium mimicking a cheese-ripening environment. These genes were selected for potential involvement in lactose/lactate catabolism and the biosynthesis of sulfur-flavored compounds. Hybridization conditions to follow specifically the expression of homologous genes belonging to different species were set up. The microarray was first validated on pure cultures of each yeast; no interspecies cross-hybridization was observed. Expression patterns of targeted genes were studied in pure cultures of each yeast, as well as in coculture, and compared to biochemical data. As expected, a high expression of theLACgenes ofK. marxianuswas observed. This is a yeast that efficiently degrades lactose. Several lactate dehydrogenase-encoding genes were also expressed essentially inD. hanseniiandK. marxianus, which are two efficient deacidifying yeasts in cheese ripening. A set of genes possibly involved inl-methionine catabolism was also used on the array.Y. lipolytica, which efficiently assimilatesl-methionine, also exhibited a high expression of theSaccharomyces cerevisiaeorthologsBAT2andARO8, which are involved in thel-methionine degradation pathway. Our data provide the first evidence that the use of a multispecies microarray could be a powerful tool to investigate targeted metabolism and possible metabolic interactions between species within microbial cocultures.
Increased Prevalence of Ubiquitous Ascomycetes in an Acropoid Coral (
Acropora formosa
) Exhibiting Symptoms of Brown Band Syndrome and Skeletal Eroding Band Disease
Authors: Yarden Oded , Ainsworth Tracy D. , Roff George , Leggat William , Fine Maoz , Hoegh-Guldberg Ove
Published: February 2007

The prevalence of coral-associated fungi was four times higher in diseased
Acropora formosa
colonies than in healthy colonies. Since taxonomically related fungal species were isolated from diseased and healthy colonies, we suggest that their association with coral may be constitutive but that their abundance is dependent on coral health.
Unusual Codon Bias in Vinyl Chloride Reductase Genes of
Dehalococcoides
Species
Authors: McMurdie Paul J. , Behrens Sebastian F. , Holmes Susan , Spormann Alfred M.
Published: February 2007

Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes
vcrA
and
bvcA
showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available
Dehalococcoides
genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other
Dehalococcoides
genes.
New Insights into Microbial Oxidation of Antimony and Arsenic
Authors: Lehr Corinne R. , Kashyap Des R. , McDermott Timothy R.
Published: February 2007

Sb(III) oxidation was documented in anAgrobacterium tumefaciensisolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the orderCyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.
Amariner-Based Transposition System forListeria monocytogenes
Authors: Cao Min , Bitar Alan Pavinski , Marquis Hélène
Published: February 2007

In this study, we developed a newmariner-based transposition system forListeria monocytogenes. Themariner-based system has a high rate of transposition and a low rate of plasmid retention, and transposition is very random, making it an ideal tool for high-throughput transposon mutagenesis inL. monocytogenes.
Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria
Authors: Müller Rainer , Gröger Gerhard , Hiller Karl-Anton , Schmalz Gottfried , Ruhl Stefan
Published: February 2007

For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated.
Streptococcus gordonii
,
Streptococcus mitis
, and
Staphylococcus aureus
were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.
Persistence and Decontamination of
Bacillus atrophaeus
subsp.
globigii
Spores on Corroded Iron in a Model Drinking Water System
Authors: Szabo Jeffrey G. , Rice Eugene W. , Bishop Paul L.
Published: February 2007

Persistence of
Bacillus atrophaeus
subsp.
globigii
spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10
6
CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log
10
reduction of adhered
B. atrophaeus
subsp.
globigii
, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation.
B. atrophaeus
subsp.
globigii
spores injected in the presence of a typical distribution system chlorine residual (∼0.75 mg/liter) resulted in a steady reduction of adhered
B. atrophaeus
subsp.
globigii
over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log
10
reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where
B. atrophaeus
subsp.
globigii
spores had adhered.
B. atrophaeus
subsp.
globigii
spores are capable of persisting for an extended time in the presence of high levels of free chlorine.
The
Dehalococcoides
Population in Sediment-Free Mixed Cultures Metabolically Dechlorinates the Commercial Polychlorinated Biphenyl Mixture Aroclor 1260
Authors: Bedard Donna L. , Ritalahti Kirsti M. , Löffler Frank E.
Published: February 2007

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the
Dehalococcoides
group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the
Dehalococcoides
cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 × 10
6
± 0.42 × 10
6
to 1.67 × 10
8
± 0.04 × 10
8
cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No
Dehalococcoides
growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 × 10
8
± 0.04 × 10
8
Dehalococcoides
cells per μmol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e.,
Desulfitobacterium
,
Dehalobacter
,
Desulfuromonas
,
Sulfurospirillum
,
Anaeromyxobacter
,
Geobacter
, and
o
-17/DF-1-type
Chloroflexi
organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the
Dehalococcoides
population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of
Dehalococcoides
organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.
Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments
Authors: La Duc Myron T. , Dekas Anne , Osman Shariff , Moissl Christine , Newcombe David , Venkateswaran Kasthuri
Published: February 2007

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 106cells/m2. However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of theBacillaceaefamily were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (OceanobacillusandExiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.
The 29-Kilodalton Thiol-Dependent Peroxidase of
Entamoeba histolytica
Is a Factor Involved in Pathogenesis and Survival of the Parasite during Oxidative Stress
Authors: Sen Abhik , Chatterjee Nabendu Sekhar , Akbar M. Ali , Nandi Nilay , Das Pradeep
Published: February 2007

The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of
Entamoeba histolytica
exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of
eh29
gene expression by antisense RNA technology has shown approximately 55% inhibition in
eh29
expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of
E. histolytica
has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis.
Purification of Components of the Translation Elongation Factor Complex of
Plasmodium falciparum
by Tandem Affinity Purification
Authors: Takebe Sachiko , Witola William Harold , Schimanski Bernd , Günzl Arthur , Ben Mamoun Choukri
Published: February 2007

Plasmodium falciparum
is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in
P. falciparum
. Transgenic parasites were generated which express the translation elongation factor PfEF-1β harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1β-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1β, the G-protein PfEF-1α, and two new proteins that we named PfEF-1γ and PfEF-1δ based on their homology to other eukaryotic γ and δ translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in
P. falciparum
, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1β. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.
Gamma Interferon Responses of CD4 and CD8 T-Cell Subsets Are Quantitatively Different and Independent of Each Other during PulmonaryMycobacterium bovisBCG Infection
Authors: Ngai Patricia , McCormick Sarah , Small Cherrie , Zhang Xizhong , Zganiacz Anna , Aoki Naoko , Xing Zhou
Published: February 2007

Gamma interferon (IFN-γ) is a key cytokine in host defense against intracellular mycobacterial infection. It has been believed that both CD4 and CD8 T cells are the primary sources of IFN-γ. However, the relative contributions of CD4 and CD8 T-cell subsets to IFN-γ production and the relationship between CD4 and CD8 T-cell activation have not been examined. By using a model of pulmonary mycobacterial infection and various immunodetection assays, we found that CD4 T cells mounted a much stronger IFN-γ response than CD8 T cells at various times after mycobacterial infection, and this pronounced IFN-γ production by CD4 T cells was attributed to both greater numbers of antigen-specific CD4 T cells and a greater IFN-γ secretion capacity of these cells. By using major histocompatibility complex class II-deficient or CD4-deficient mice, we found that the lack of CD4 T cells did not negatively affect primary or secondary CD8 T-cell IFN-γ responses. The CD8 T cells activated in the absence of CD4 T cells were capable of immune protection against secondary mycobacterial challenge. Our results suggest that, whereas both CD4 and CD8 T cells are capable of IFN-γ production, the former represent a much greater cellular source of IFN-γ. Moreover, during mycobacterial infection, CD8 T-cell IFN-γ responses and activation are independent of CD4 T-cell activation.
Enterococcus faecalis
Mutations Affecting Virulence in the
Caenorhabditis elegans
Model Host
Authors: Maadani Arash , Fox Kristina A. , Mylonakis Elftherios , Garsin Danielle A.
Published: February 2007

Enterococcus faecalis
transposon insertion mutants were screened for attenuated killing of the nematode model host
Caenorhabditis elegans
. The genes disrupted in the attenuated mutants encode a variety of factors including transcriptional regulators, transporters, and damage control and repair systems. Five of nine mutants tested were attenuated in a mouse peritonitis model.
Chlamydial Hsp60-2 Is Iron Responsive in
Chlamydia trachomatis
Serovar E-Infected Human Endometrial Epithelial Cells In Vitro
Authors: LaRue Richard W. , Dill Brian D. , Giles David K. , Whittimore Judy D. , Raulston Jane E.
Published: February 2007

Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in
Chlamydia trachomatis
serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.
Identification of a Prepore Large-Complex Stage in the Mechanism of Action of
Clostridium perfringens
Enterotoxin
Authors: Smedley James G. , Uzal Francisco A. , McClane Bruce A.
Published: February 2007

Clostridium perfringens
enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPE-containing complexes in eukaryotic cell membranes. Formation of these ∼155- and ∼200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca
2+
influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the β-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the ∼155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore ∼155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.
Primary Infection of C57BL/6 Mice withPlasmodium yoeliiInduces a Heterogeneous Response of NKT Cells
Authors: Soulard Valérie , Roland Jacques , Sellier Christèle , Gruner Anne Charlotte , Leite-de-Moraes Maria , Franetich Jean-François , Rénia Laurent , Cazenave Pierre-André , Pied Sylviane
Published: February 2007

NKT cells are a population of innate-like lymphocytes that display effector functions and immunoregulatory properties. We characterized the NKT cell response induced in C57BL/6 mice during a primary infection withPlasmodium yoeliisporozoites. We observed a heterogeneous NKT cell response that differed between liver and spleen. Hepatic NKT cells found in infected livers consisted mainly of CD1d-dependent CD4+and double-negative (DN) NKT cells, whereas CD1d-independent NKT cells exhibiting a TCRhighCD4highphenotype were prominent among splenic NKT cells during the infection. Hepatic and splenic NKT cells isolated from infected mice were activated and secreted mainly gamma interferon and tumor necrosis factor alpha in response to stimulation. Finally,P. yoelii-activated hepatic DN NKT cells inhibited the parasite's liver stage in a CD1d-dependent manner in vitro. However, experiments using B6.CD1d-deficient mice showed that CD1d and CD1d-restricted NKT cells are not necessary to control the parasite's development in vivo during neither the preerythrocytic stage nor the erythrocytic stage. Thus, our results show that a primaryP. yoeliiinfection induces a heterogeneous and organ-specific response of NKT cells and that CD1d-dependent NKT cells play a minor role in the control of the development ofPlasmodiumin vivo in our model.
The
Escherichia coli
Enterotoxin STb Permeabilizes Piglet Jejunal Brush Border Membrane Vesicles
Authors: Gonçalves Carina , Vachon Vincent , Schwartz Jean-Louis , Dubreuil J. Daniel
Published: February 2007

The membrane-permeabilizing ability of the
Escherichia coli
enterotoxin STb was evaluated using brush border membrane vesicles isolated from piglet jejunum and a membrane-potential-sensitive fluorescent probe, 3,3′-dipropylthiadicarbocyanine iodide. A strong membrane potential was generated by the efflux of K
+
ions from the vesicles in the presence of the potassium ionophore valinomycin. Under these conditions, preincubation of the vesicles with STb efficiently depolarized the membrane in a dose-dependent and saturable manner. This activity was independent of pH, however, at least between pH 5.5 and 8.0. On the other hand, in the absence of valinomycin, STb had no significant influence on the measured fluorescence levels, indicating that it was unable to modify the ionic selectivity of the intact membrane. In agreement with the fact that the integrity of the disulfide bridges of STb is known to be essential for its biological activity, a reduced and alkylated form of the toxin was unable to depolarize the membrane in the presence of valinomycin. Furthermore, two previously described poorly active STb mutants, M42S and K22A-K23A, showed no membrane-permeabilizing capacity. These results demonstrate for the first time that STb can permeabilize its target membrane and suggest that it does so by forming nonspecific pores.
Gamma Interferon Suppresses Erythropoiesis via Interleukin-15
Authors: Mullarky Isis K. , Szaba Frank M. , Kummer Lawrence W. , Wilhelm Lindsey B. , Parent Michelle A. , Johnson Lawrence L. , Smiley Stephen T.
Published: February 2007

Impaired erythropoiesis causes anemia during genetic disorders, chronic disease, and infection. In studies of the underlying mechanisms researchers have increasingly focused on gamma interferon (IFN-γ). Here, we identified a previously unrecognized role for interleukin-15 (IL-15) in red blood cell homeostasis and demonstrated that IFN-γ and signal transducer and activator of transcription protein 1-dependent pathways up-regulate expression of IL-15 in vivo. These findings identified new therapeutic targets for anemia.
Priming with an Adenovirus 35-Circumsporozoite Protein (CS) Vaccine followed by RTS,S/AS01B Boosting Significantly Improves Immunogenicity toPlasmodium falciparumCS Compared to That with Either Malaria Vaccine Alone
Authors: Stewart V. Ann , McGrath Shannon M. , Dubois Patrice M. , Pau Maria G. , Mettens Pascal , Shott Joseph , Cobb Michelle , Burge J. Robert , Larson David , Ware Lisa A. , Demoitie Marie-Ange , Weverling Gerrit Jan , Bayat Babak , Custers Jerome H. H. V. , Dubois Marie-Claude , Cohen Joe , Goudsmit Jaap , Heppner D. Gray
Published: February 2007

The RTS,S/AS02A protein-based vaccine consistently demonstrates significant protection against infection withPlasmodium falciparummalaria and also against clinical malaria and severe disease in children in areas of endemicity. Here we demonstrate with rhesus macaques that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding circumsporozoite protein (CS) (Ad35.CS), followed by boosting with RTS,S in an improved MPL- and QS21-based adjuvant formulation, AS01B, maintains antibody responses and dramatically increases levels of T cells producing gamma interferon and other Th1 cytokines in response to CS peptides. The increased T-cell responses induced by the combination of Ad35.CS and RTS,S/AS01B are sustained for at least 6 months postvaccination and may translate to improved and more durable protection againstP. falciparuminfection in humans.
Broad Up-Regulation of Innate Defense Factors during Acute Cholera
Authors: Flach Carl-Fredrik , Qadri Firdausi , Bhuiyan Taufiqur R. , Alam Nur H. , Jennische Eva , Lönnroth Ivar , Holmgren Jan
Published: February 2007

We used a whole-genome microarray screening system (Affymetrix human GeneChips covering 47,000 different transcripts) to examine the gene expression in duodenal mucosa during acute cholera. Biopsies were taken from the duodenal mucosa of seven cholera patients 2 and 30 days after the onset of diarrhea, and the gene expression patterns in the acute- and convalescent-phase samples were compared pairwise. Of about 21,000 transcripts expressed in the intestinal epithelium, 29 were defined as transcripts that were up-regulated and 33 were defined as transcripts that were down-regulated during acute cholera. The majority of the up-regulated genes characterized were found to have an established or possible role in the innate defense against infections; these genes included the LPLUNC1, LF, VCC1, TCN1, CD55, SERPINA3, MMP1, MMP3, IL1B, LCN2, SOCS3, GDF15, SLPI, CXCL13, and MUC1 genes. The results of confirmative PCR correlated well with the microarray data. An immunohistochemical analysis revealed increased expression of lactoferrin in lamina propria cells and increased expression of CD55 in epithelial cells, whereas increased expression of the SERPINA3 protein (α1-antichymotrypsin) was detected in both lamina propria and epithelial cells during acute cholera. The expression pattern of CD55 and SERPINA3 in cholera toxin (CT)-stimulated Caco-2 cells was the same as the pattern found in the intestinal mucosa during acute cholera, indicating that the activation of the CD55 and SERPINA3 genes in intestinal epithelium was induced by CT. In conclusion, during acute cholera infection, innate defense mechanisms are switched on to an extent not described previously. Both direct effects of CT on the epithelial cells and changes in the lamina propria cells contribute to this up-regulation.
Gingival Epithelial Cell Transcriptional Responses to Commensal and Opportunistic Oral Microbial Species
Authors: Hasegawa Yoshiaki , Mans Jeffrey J. , Mao Song , Lopez M. Cecilia , Baker Henry V. , Handfield Martin , Lamont Richard J.
Published: February 2007

Transcriptional profiling and ontology tools were utilized to define the biological pathways of gingival epithelial cells modulated by coculture with the oral commensal
Streptococcus gordonii
and the opportunistic commensal
Fusobacterium nucleatum
. Overall,
F. nucleatum
and
S. gordonii
perturbed the gingival epithelial cell transcriptome much less significantly than the oral pathogens
Porphyromonas gingivalis
and
Aggregatibacter actinomycetemcomitans
perturbed the transcriptome, indicating that there was a greater degree of host adaptation by the commensal species (M. Handfield, J. J. Mans, G. Zheng, M. C. Lopez, S. Mao, A. Progulske-Fox, G. Narasimhan, H. V. Baker, and R. J. Lamont, Cell. Microbiol.
7:
811-823, 2005). The biological pathways significantly impacted by
F. nucleatum
and
S. gordonii
included the mitogen-activated protein kinase (MAPK) and Toll-like receptor signaling pathways. Differential regulation of GADD45 and DUSP4, key components of the MAPK pathway, was confirmed at the protein level by Western blotting. Modulation of the MAPK pathway is likely to affect host cell proliferation and differentiation. In addition, both the MAPK and Toll-like receptor pathways ultimately converge on cytokine gene expression. An enzyme-linked immunosorbent assay of secreted interleukin-6 (IL-6) and IL-8 demonstrated that
F. nucleatum
induced production of these cytokines, whereas
S. gordonii
inhibited secretion from the epithelial cells. Stimulation of secretion of proinflammatory cytokines from epithelial cells may reflect the invasive phenotype of
F. nucleatum
and contribute to the greater pathogenic potential of
F. nucleatum
than of
S. gordonii
.