bluuebunny/crossref_metadata_2025_split huggingface dataset:
Production and Characterization of Antibodies against Each of the Three Subunits of the
Bacillus cereus
Nonhemolytic Enterotoxin Complex
Authors: Dietrich Richard , Moravek Maximilian , Bürk Christine , Granum Per Einar , Märtlbauer Erwin 
Published: December 2005

ABSTRACT

The nonhemolytic enterotoxin (Nhe) is one of the two three-component enterotoxins which are responsible for diarrheal food poisoning syndrome caused by
Bacillus cereus
. To facilitate the detection of this toxin, consisting of the subunits NheA, NheB, and NheC, a complete set of high-affinity antibodies against each of the three components was established and characterized. A rabbit antiserum specific for the C-terminal part (15 amino acids) of NheC was produced using a respective synthetic peptide coupled to a protein carrier for immunization. Using purified
B. cereus
exoprotein preparations as immunogens, one monoclonal antibody against NheA and several antibodies against NheB were obtained. No cross-reactivity with other proteins produced by different strains of
B. cereus
was observed. Antibodies against the NheB component were able to neutralize the cytotoxic activity (up to 98%) of Nhe. Based on indirect enzyme immunoassays, the antibodies developed in this study were successfully used in the characterization of the enterotoxic activity of several
B. cereus
strains. For the first time, it could be shown that strains carrying the
nhe
genes usually express the complete set of the three components, including NheC. However, the amount of toxin produced varies considerably between the different strains.
Engineering of Solvent-TolerantPseudomonas putidaS12 for Bioproduction of Phenol from Glucose
Authors: Wierckx Nick J. P. , Ballerstedt Hendrik , de Bont Jan A. M. , Wery Jan 
Published: December 2005

ABSTRACTEfficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strainPseudomonas putidaS12. Thetplgene fromPantoea agglomerans, encoding tyrosine phenol lyase, was introduced intoP. putidaS12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing thearoF-1gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolitesm-fluoro-dl-phenylalanine andm-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded aP. putidaS12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.
Mineralization of the Cyclic Nitramine Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by
Gordonia
and
Williamsia
spp
Authors: Thompson Karen T. , Crocker Fiona H. , Fredrickson Herbert L. 
Published: December 2005

ABSTRACT

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as
Williamsia
sp. KTR4 and
Gordonia
sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-
14
C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-
14
C]RDX to
14
CO
2
was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH
4
)
2
SO
4
greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.
Functional Analysis of Putative Adhesion Factors in
Lactobacillus acidophilus
NCFM
Authors: Buck B. Logan , Altermann Eric , Svingerud Tina , Klaenhammer Todd R. 
Published: December 2005

ABSTRACT

Lactobacilli are major inhabitants of the normal microflora of the gastrointestinal tract, and some select species have been used extensively as probiotic cultures. One potentially important property of these organisms is their ability to interact with epithelial cells in the intestinal tract, which may promote retention and host-bacterial communication. However, the mechanisms by which they attach to intestinal epithelial cells are unknown. The objective of this study was to investigate cell surface proteins in
Lactobacillus acidophilus
that may promote attachment to intestinal tissues. Using genome sequence data, predicted open reading frames were searched against known protein and protein motif databases to identify four proteins potentially involved in adhesion to epithelial cells. Homologous recombination was used to construct isogenic mutations in genes encoding a mucin-binding protein, a fibronectin-binding protein, a surface layer protein, and two streptococcal R28 homologs. The abilities of the mutants to adhere to intestinal epithelial cells were then evaluated in vitro. Each strain was screened on Caco-2 cells, which differentiate and express markers characteristic of normal small-intestine cells. A significant decrease in adhesion was observed in the fibronectin-binding protein mutant (76%) and the mucin-binding protein mutant (65%). A surface layer protein mutant also showed reduction in adhesion ability (84%), but the effect of this mutation is likely due to the loss of multiple surface proteins that may be embedded in the S-layer. This study demonstrated that multiple cell surface proteins in
L. acidophilus
NCFM can individually contribute to the organism's ability to attach to intestinal cells in vitro.
In Vitro Determination of Prebiotic Properties of Oligosaccharides Derived from an Orange Juice Manufacturing By-Product Stream
Authors: Manderson K. , Pinart M. , Tuohy K. M. , Grace W. E. , Hotchkiss A. T. , Widmer W. , Yadhav M. P. , Gibson G. R. , Rastall R. A. 
Published: December 2005

ABSTRACT

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and
Eubacterium rectale
numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the
Eubacterium rectale
numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the
Eubacterium rectale
population was found, and butyrate levels increased, which is of potential benefit to the host.
Ecological Behavior ofLactobacillus reuteri100-23 Is Affected by Mutation of theluxSGene
Authors: Tannock Gerald W. , Ghazally Salina , Walter Jens , Loach Diane , Brooks Heather , Cook Gregory , Surette Michael , Simmers Cameron , Bremer Phil , Dal Bello Fabio , Hertel Christian 
Published: December 2005

ABSTRACTTheluxSgene ofLactobacillus reuteri100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for theluxSgene inL. reuteri100-23C, aluxSmutant was constructed by insertional mutagenesis. TheluxSmutant did not produce autoinducers AI-2 or AI-3. Complementation of theluxSmutation by a plasmid construct containingluxSrestored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in theluxSmutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of theluxSmutant was, however, 65% of that of wild-type cells. Biofilms formed by theluxSmutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of theluxSmutant, when in competition withL. reuteristrain 100-93 in the mouse cecum, was reduced compared to that of axylAmutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes ofL. reuteri100-23C, the consequences of which are niche specific.
Phospholipid Furan Fatty Acids and Ubiquinone-8: Lipid Biomarkers That May Protect
Dehalococcoides
Strains from Free Radicals
Authors: White David C. , Geyer Roland , Peacock Aaron D. , Hedrick David B. , Koenigsberg Stephen S. , Sung Youlboong , He Jianzhong , Löffler Frank E. 
Published: December 2005

ABSTRACT

Dehalococcoides
species have a highly restricted lifestyle and are only known to derive energy from reductive dehalogenation reactions. The lipid fraction of two
Dehalococcoides
isolates, strains BAV1 and FL2, and a tetrachloroethene-to-ethene-dechlorinating
Dehalococcoides
-containing consortium were analyzed for neutral lipids and phospholipid fatty acids. Unusual phospholipid modifications, including the replacement of unsaturated fatty acids with furan fatty acids, were detected in both
Dehalococcoides
isolates and the mixed culture. The following three furan fatty acids are reported as present in bacterial phospholipids for the first time: 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6), 9-(5-butyl-2-furyl)-nonanoate (Fu17:2ω5), and 8-(5-pentyl-2-furyl)-octanoate (Fu17:2ω6). The neutral lipids of the
Dehalococcoides
cultures contained unusually large amounts of benzoquinones (i.e., ubiquinones [UQ]), which is unusual for anaerobes. In particular, the UQ-8 content of
Dehalococcoides
was 5- to 20-fold greater than that generated in aerobically grown
Escherichia coli
cultures relative to the phospholipid fatty acid content. Naphthoquinone isoprenologues (MK), which are often found in anaerobically grown bacteria and archaea, were also detected.
Dehalococcoides
shows a difference in isoprenologue pattern between UQ-8 and MK-5 that is atypical of other bacteria capable of producing both quinone types. The difference in UQ-8 and MK-5 isoprenologue patterns strongly suggests a special function for UQ in
Dehalococcoides
, and
Dehalococcoides
may utilize structural modifications in its lipid armamentarium to protect against free radicals that are generated in the process of reductive dechlorination.
Genetic Variability of the Heme Uptake System among Different Strains of the Fish Pathogen
Vibrio anguillarum
: Identification of a New Heme Receptor
Authors: Mouriño Susana , Rodríguez-Ares Isabel , Osorio Carlos R. , Lemos Manuel L. 
Published: December 2005

ABSTRACT

The ability to utilize heme compounds as iron sources was investigated in
Vibrio anguillarum
strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes
huvABCD
, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all
huvABCD
genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene
huvA
. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a
V. anguillarum
serotype O3 strain lacking
huvA
. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in
Vibrionaceae
, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the
V. anguillarum
strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in
Escherichia coli
and
V. anguillarum
mutants. The
huvS
and
huvA
sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.
Twin-Arginine Translocation of Active Human Tissue Plasminogen Activator in
Escherichia coli
Authors: Kim Jae-Young , Fogarty Elizabeth A. , Lu Franklin J. , Zhu Hui , Wheelock Geoffrey D. , Henderson Lee A. , DeLisa Matthew P. 
Published: December 2005

ABSTRACT

When eukaryotic proteins with multiple disulfide bonds are expressed at high levels in
Escherichia coli
, the efficiency of thiol oxidation and isomerization is typically not sufficient to yield soluble products with native structures. Even when such proteins are secreted into the oxidizing periplasm or expressed in the cytoplasm of cells carrying mutations in the major intracellular disulfide bond reduction systems (e.g.,
trxB gor
mutants), correct folding can be problematic unless a folding modulator is simultaneously coexpressed. In the present study we explored whether the bacterial twin-arginine translocation (Tat) pathway could serve as an alternative expression system for obtaining appreciable levels of recombinant proteins which exhibit complex patterns of disulfide bond formation, such as full-length human tissue plasminogen activator (tPA) (17 disulfides) and a truncated but enzymatically active version of tPA containing nine disulfides (vtPA). Remarkably, targeting of both tPA and vtPA to the Tat pathway resulted in active protein in the periplasmic space. We show here that export by the Tat translocator is dependent upon oxidative protein folding in the cytoplasm of
trxB gor
cells prior to transport. Whereas previous efforts to produce high levels of active tPA or vtPA in
E. coli
required coexpression of the disulfide bond isomerase DsbC, we observed that Tat-targeted vtPA and tPA reach a native conformation without thiol-disulfide oxidoreductase coexpression. These results demonstrate that the Tat system may have inherent and unexpected benefits compared with existing expression strategies, making it a viable alternative for biotechnology applications that hinge on protein expression and secretion.
Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes
Authors: Coolen Marco J. L. , Post Eduard , Davis Catherine C. , Forney Larry J. 
Published: December 2005

ABSTRACT

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of
Lactobacillus
. In contrast, the communities of two other women included numerically abundant populations that differed from
Lactobacillus
strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.
Community Analysis of a Mercury Hot Spring Supports Occurrence of Domain-Specific Forms of Mercuric Reductase
Authors: Simbahan Jessica , Kurth Elizabeth , Schelert James , Dillman Amanda , Moriyama Etsuko , Jovanovich Stevan , Blum Paul 
Published: December 2005

ABSTRACT

Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercury-rich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg
2+
, necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial
merA
sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal
merA
in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.
FlhA InfluencesBacillus thuringiensisPlcR-Regulated Gene Transcription, Protein Production, and Virulence
Authors: Bouillaut Laurent , Ramarao Nalini , Buisson Christophe , Gilois Nathalie , Gohar Michel , Lereclus Didier , Nielsen-LeRoux Christina 
Published: December 2005

ABSTRACTBacillus thuringiensisandBacillus cereusare closely related.B. thuringiensisis well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins.B. cereusappears to be an emerging opportunistic human pathogen.B. thuringiensisandB. cereusproduce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation ofplcRdecreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis407 Cry−ΔflhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in theflhAmutant. Moreover, we quantified the effect of FlhA onplcAandhblCgene transcription. The results show that theflhAmutation results in a significant reduction ofplcAandhblCtranscription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, theflhAmutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence againstGalleria mellonellalarvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.
Improvement of Catalytic Properties of
Escherichia coli
Penicillin G Acylase Immobilized on Glyoxyl Agarose by Addition of a Six-Amino-Acid Tag
Authors: Scaramozzino Francesca , Estruch Ilona , Rossolillo Paola , Terreni Marco , Albertini Alessandra M. 
Published: December 2005

ABSTRACT
A tag of three lysines alternating with three glycines was added to the C-terminal end of the β chain of penicillin G acylase (PGA). This modification improved the immobilization efficiency of PGA on glyoxyl agarose and the catalytic properties of the PGA derivative, although it impaired the posttranslational steps of overexpressed protein maturation.
Legionella
Confirmation Using Real-Time PCR and SYTO9 Is an Alternative to Current Methodology
Authors: Giglio Steven , Monis Paul T. , Saint Christopher P. 
Published: December 2005

ABSTRACT

The currently accepted culture techniques for the detection of
Legionella
spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.
Differential Real-Time PCR Assay for Enumeration of Lactic Acid Bacteria in Wine
Authors: Neeley Ezekiel T. , Phister Trevor G. , Mills David A. 
Published: December 2005

ABSTRACT

Oenococcus oeni
is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.