bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiple Microfermentor Battery: a Versatile Tool for Use with Automated Parallel Cultures of Microorganisms Producing Recombinant Proteins and for Optimization of Cultivation Protocols
Authors: Frachon Emmanuel , Bondet Vincent , Munier-Lehmann Hélène , Bellalou Jacques 
Published: August 2006

ABSTRACT

A multiple microfermentor battery was designed for high-throughput recombinant protein production in
Escherichia coli
. This novel system comprises eight aerated glass reactors with a working volume of 80 ml and a moving external optical sensor for measuring optical densities at 600 nm (OD
600
) ranging from 0.05 to 100 online. Each reactor can be fitted with miniature probes to monitor temperature, dissolved oxygen (DO), and pH. Independent temperature regulation for each vessel is obtained with heating/cooling Peltier devices. Data from pH, DO, and turbidity sensors are collected on a FieldPoint (National Instruments) I/O interface and are processed and recorded by a LabVIEW program on a personal computer, which enables feedback control of the culture parameters. A high-density medium formulation was designed, which enabled us to grow
E. coli
to OD
600
up to 100 in batch cultures with oxygen-enriched aeration. Accordingly, the biomass and the amount of recombinant protein produced in a 70-ml culture were at least equivalent to the biomass and the amount of recombinant protein obtained in a Fernbach flask with 1 liter of conventional medium. Thus, the microfermentor battery appears to be well suited for automated parallel cultures and process optimization, such as that needed for structural genomics projects.
Comparative, Collaborative, and On-Site Validation of a TaqMan PCR Method as a Tool for Certified Production of Fresh,
Campylobacter
-Free Chickens
Authors: Krause M. , Josefsen M. H. , Lund M. , Jacobsen N. R. , Brorsen L. , Moos M. , Stockmarr A. , Hoorfar J. 
Published: August 2006

ABSTRACT

Certified
Campylobacter
-free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled “
Campylobacter
free.” This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols on naturally contaminated samples (99 shoe covers, 101 cloacal swabs, 102 neck skins from abattoirs, and 100 retail neck skins). Culturing included enrichment in both Bolton and Preston broths followed by isolation on Preston agar and mCCDA. In one or both culture protocols, 169 samples were identified as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of
Campylobacter jejuni
. Valid results were obtained from six of the participating laboratories. Accuracy for high levels was 100% for neck skin and cloacal swab samples. For low levels, accuracy was 100% and 92% for neck skin and cloacal swab samples, respectively; however, detection in shoe cover samples failed. A second collaborative trial, with an optimized DNA extraction procedure, gave 100% accuracy results for all three spiking levels. Finally, on-site validation at the abattoir on a flock basis was performed on 400 samples. Real-time PCR correctly identified 10 of 20 flocks as positive; thus, the method fulfilled the NordVal validation criteria and has since been implemented at a major abattoir.
Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions
Authors: Reischer Georg H. , Kasper David C. , Steinborn Ralf , Mach Robert L. , Farnleitner Andreas H. 
Published: August 2006

ABSTRACT

A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum
Bacteroidetes
. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 10
9
marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 10
5
marker equivalents per liter).
Identification of a Gene Cluster for Biosynthesis of Mannosylerythritol Lipids in the Basidiomycetous Fungus
Ustilago maydis
Authors: Hewald Sandra , Linne Uwe , Scherer Mario , Marahiel Mohamed A. , Kämper Jörg , Bölker Michael 
Published: August 2006

ABSTRACT

Many microorganisms produce surface-active substances that enhance the availability of water-insoluble substrates. Although many of these biosurfactants have interesting potential applications, very little is known about their biosynthesis. The basidiomycetous fungus
Ustilago maydis
secretes large amounts of mannosylerythritol lipids (MELs) under conditions of nitrogen starvation. We recently described a putative glycosyltransferase, Emt1, which is essential for MEL biosynthesis and whose expression is strongly induced by nitrogen limitation. We used DNA microarray analysis to identify additional genes involved in MEL biosynthesis. Here we show that
emt1
is part of a gene cluster which comprises five open reading frames. Three of the newly identified proteins, Mac1, Mac2, and Mat1, contain short sequence motifs characteristic for acyl- and acetyltransferases. Mutational analysis revealed that Mac1 and Mac2 are essential for MEL production, which suggests that they are involved in the acylation of mannosylerythritol. Deletion of
mat1
resulted in the secretion of completely deacetylated MELs, as determined by mass spectrometry. We overexpressed Mat1 in
Escherichia coli
and demonstrated that this enzyme acts as an acetyl coenzyme A-dependent acetyltransferase. Remarkably, Mat1 displays relaxed regioselectivity and is able to acetylate mannosylerythritol at both the C-4 and C-6 hydroxyl groups. Based on these results, we propose a biosynthesis pathway for the generation of mannosylerythritol lipids in
U. maydis
.
Resolution of Distinct Membrane-Bound Enzymes fromEnterobacter cloacaeSLD1a-1 That Are Responsible for Selective Reduction of Nitrate and Selenate Oxyanions
Authors: Ridley Helen , Watts Carys A. , Richardson David J. , Butler Clive S. 
Published: August 2006

ABSTRACTEnterobacter cloacaeSLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett.228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of ab-type cytochrome in the active complex. The apparentKmfor selenate was determined to be ∼2 mM, with an observedVmaxof 500 nmol SeO42−min−1mg−1(kcat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.
Characterization of the β-Ketoadipate Pathway inSinorhizobium meliloti
Authors: MacLean Allyson M. , MacPherson Gordon , Aneja Punita , Finan Turlough M. 
Published: August 2006

ABSTRACTAromatic compounds represent an important source of energy for soil-dwelling organisms. The β-ketoadipate pathway is a key metabolic pathway involved in the catabolism of the aromatic compounds protocatechuate and catechol, and here we show through enzymatic analysis and mutant analysis that genes required for growth and catabolism of protocatechuate in the soil-dwelling bacteriumSinorhizobium melilotiare organized on the pSymB megaplasmid in two transcriptional units designatedpcaDCHGBandpcaIJF. ThepcaDpromoter was mapped by primer extension, and expression from this promoter is demonstrated to be regulated by the LysR-type protein PcaQ. β-Ketoadipate succinyl-coenzyme A (CoA) transferase activity inS. melilotiwas shown to be encoded by SMb20587 and SMb20588, and these genes have been renamedpcaIandpcaJ, respectively. These genes are organized in an operon with a putative β-ketoadipyl-CoA thiolase gene (pcaF), and expression of thepcaIJFoperon is shown to be regulated by an IclR-type transcriptional regulator, SMb20586, which we have namedpcaR. We show thatpcaRtranscription is negatively autoregulated and that PcaR is a positive regulator ofpcaIJFexpression and is required for growth ofS. melilotion protocatechuate as the carbon source. The characterization of the protocatechuate catabolic pathway inS. melilotioffers an opportunity for comparison with related species, includingAgrobacterium tumefaciens. Differences observed betweenS. melilotiandA. tumefaciens pcaIJoffer the first evidence ofpcagenes that may have been acquired after speciation in these closely related species.
Molecular Epidemiologic Investigation of
Campylobacter coli
in Swine Production Systems, Using Multilocus Sequence Typing
Authors: Thakur Siddhartha , Morrow W. E. Morgan , Funk Julie A. , Bahnson Peter B. , Gebreyes Wondwossen A. 
Published: August 2006

ABSTRACT

Multilocus sequence typing of 151
Campylobacter coli
isolates from swine reared in conventional (
n
= 74) and antimicrobial-free (
n
= 77) production systems revealed high genotypic diversity. Sequence type (ST) 1413 was predominant and observed among ciprofloxacin-resistant strains. We identified a
C. coli
ST 828 clonal complex consisting of isolates from both production systems.
Improvement of Lactic Acid Production in
Saccharomyces cerevisiae
by Cell Sorting for High Intracellular pH
Authors: Valli Minoska , Sauer Michael , Branduardi Paola , Borth Nicole , Porro Danilo , Mattanovich Diethard 
Published: August 2006

ABSTRACT

Yeast strains expressing heterologous
l
-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pH
i
) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of
Saccharomyces cerevisiae
, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pH
i
. During batch culture, we observed in both strains a reduction of the mean pH
i
and the appearance of a subpopulation of cells with low pH
i
. Simultaneous analysis of pH
i
and viability proved that the cells with low pH
i
were dead. Based on the observation that the better lactic-acid-producing strain had a higher pH
i
and that the cells with low pH
i
were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pH
i
range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pH
i
range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.
Isolation, Characterization, and Ecology of Cold-Active, Chemolithotrophic, Sulfur-Oxidizing Bacteria from Perennially Ice-Covered Lake Fryxell, Antarctica
Authors: Sattley W. Matthew , Madigan Michael T. 
Published: August 2006

ABSTRACT

Novel strains of obligately chemolithoautotrophic, sulfur-oxidizing bacteria have been isolated from various depths of Lake Fryxell, Antarctica. Physiological, morphological, and phylogenetic analyses showed these strains to be related to mesophilic
Thiobacillus
species, such as
T. thioparus
. However, the psychrotolerant Antarctic isolates showed an adaptation to cold temperatures and thus should be active in the nearly freezing waters of the lake. Enumeration by most-probable-number analysis in an oxic, thiosulfate-containing medium revealed that the sulfur-oxidizing chemolithotroph population peaks precisely at the oxycline (9.5 m), although viable cells exist well into the anoxic, sulfidic waters of the lake. The sulfur-oxidizing bacteria described here likely play a key role in the biogeochemical cycling of carbon and sulfur in Lake Fryxell.
Unlocking
Streptomyces
spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering
Authors: van Wezel Gilles P. , Krabben Preben , Traag Bjørn A. , Keijser Bart J. F. , Kerste Rob , Vijgenboom Erik , Heijnen Josef J. , Kraal Barend 
Published: August 2006

ABSTRACT

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene
ssgA
. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of
Streptomyces coelicolor
and
Streptomyces lividans
. Product formation was also stimulated, with a twofold increase in yield of enzyme production by
S. lividans
. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.
Temporal Expression of Respiratory Genes in an Enrichment Culture Containing
Dehalococcoides ethenogenes
Authors: Rahm Brian G. , Morris Robert M. , Richardson Ruth E. 
Published: August 2006

ABSTRACT

Multiple reductive dehalogenase (RDase), hydrogenase (H
2
ase), and other respiration-associated (RA) oxidoreductase genes have been identified in cultured representatives of
Dehalococcoides
. Although their products are likely to play key roles in the environmentally important process of reductive dechlorination, very little information is available about their regulation and specific functions. Here we show increased expression and temporal variability in the expression of five RDase genes and in the expression of genes for a putative formate dehydrogenase (Fdh) and two H
2
ases, including a periplasmic [Ni/Fe] H
2
ase (Hup) and a cytoplasmic [Fe] H
2
ase (Vhu). mRNA transcripts extracted from tetrachloroethene-dechlorinating mixed cultures corresponding to Fdh, the H
2
ase Hup, and the RDase targets TceA and DET0162 were expressed most highly, with average levels 34 (± 7.5)-, 23 (± 6.7)-, 16 (± 3.3)-, and 13 (± 3.3)-fold higher, respectively, than that for RNA polymerase (RpoB). H
2
ase and RA transcripts reached their respective expression maxima within the first 2 h after feeding. RDase transcripts, however, were most highly expressed after 3 h and exhibited greater temporal variability than other transcripts. Comparison with
D. ethenogenes
strain 195 pure culture expression levels indicated that RDase DET1545 was more highly expressed in mixed cultures, where, on average, its transcript level was sixfold higher than that of RpoB. While the specific functions of several of these gene products remain elusive, the high expression levels and temporal variability reported here suggest that these groups of enzymes are metabolically important for the respiration of chlorinated ethenes in mixed cultures containing
Dehalococcoides
.
Virus-Bacterium Interactions in Water and Sediment of West African Inland Aquatic Systems
Authors: Bettarel Yvan , Bouvy Marc , Dumont Claire , Sime-Ngando Télesphore 
Published: August 2006

ABSTRACT
The ecology of virioplankton in tropical aquatic ecosystems is poorly documented, and in particular, there are no references concerning African continental waters in the literature. In this study, we examined virus-bacterium interactions in the pelagic and benthic zones of seven contrasting shallow inland waters in Senegal, including one hypersaline lake. SYBR Gold-stained samples revealed that in the surface layers of the sites, the numbers of viruses were in the same range as the numbers of viruses reported previously for productive temperate systems. Despite high bacterial production rates, the percentages of visibly infected cells (as determined by transmission electron microscopy) were similar to the lowest percentages (range, 0.3 to 1.1%; mean, 0.5%) found previously at pelagic freshwater or marine sites, presumably because of the local environmental and climatic conditions. Since the percentages of lysogenic bacteria were consistently less than 8% for pelagic and benthic samples, lysogeny did not appear to be a dominant strategy for virus propagation at these sites. In the benthic samples, viruses were highly concentrated, but paradoxically, no bacteria were visibly infected. This suggests that sediment provides good conditions for virus preservation but ironically is an unfavorable environment for proliferation. In addition, given the comparable size distributions of viruses in the water and sediment samples, our results support the paradigm that aquatic viruses are ubiquitous and may have moved between the two compartments of the shallow systems examined. Overall, this study provides additional information about the relevance of viruses in tropical areas and indicates that the intensity of virus-bacterium interactions in benthic habitats may lower than the intensity in the adjacent bodies of water.
Molecular Biological Detection of Anaerobic Gut Fungi (
Neocallimastigales
) from Landfill Sites
Authors: Lockhart Robert J. , Van Dyke Michele I. , Beadle Ian R. , Humphreys Paul , McCarthy Alan J. 
Published: August 2006

ABSTRACT

Oligonucleotide primers were designed for the 18S rRNA genes of members of the
Neocallimastigales
and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the
Neocallimastigales
outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.
Salt, Alone or in Combination with Sucrose, Can Improve the Survival of
Escherichia coli
O157 (SERL 2) in Model Acidic Sauces
Authors: Chapman B. , Jensen N. , Ross T. , Cole M. 
Published: August 2006

ABSTRACT

The commercial production of microbiologically safe and stable sauces containing acetic acid is guided by the Comité des Industries des Mayonnaises et Sauces Condimentaires de la Communauté Économique Européenne's (CIMSCEE) code. The CIMSCEE safety value is calculated using a linear regression equation combining weighted contributions of pH and aqueous-phase concentrations of undissociated acetic acid, NaCl, and sugars. By implication, the CIMSCEE safety equation predicts that increasing concentrations of hurdles will always increase inactivation of the target pathogen. In this study, the time to achieve a 3-log
10
reduction of an acid-resistant, acid-adapted, Shiga toxin-producing
Escherichia coli
(STEC) O157 isolate was determined experimentally for 81 formulations at various pHs and acetic acid, NaCl, and sucrose concentrations in a broth model. The combinations were intended to simulate the aqueous phase of acidic sauces and dressings. Experimental data were fitted to the log logistic model to estimate the time to 3-log
10
reduction (
t
3D
). Comparison of fitted
t
3D
estimates with CIMSCEE values showed agreement in predicting safety (as defined by CIMSCEE) for the majority of formulations. However, CIMSCEE safety predictions were “fail dangerous” for 13 of 81 formulations. Among these formulations and others, the observed
E. coli t
3D
initially increased and then decreased with increasing osmolalities (NaCl and sucrose). Relative protection increased with exposure time where the protective effect of NaCl predominated. While commercial acidic sauces are not considered high-risk vehicles for STEC, interactions among hurdles that decrease their combined effectiveness are deserving of further investigation because they may reveal mechanisms of broader relevance in the inactivation of pathogens in foods.
Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes
Authors: Kubota Kengo , Ohashi Akiyoshi , Imachi Hiroyuki , Harada Hideki 
Published: August 2006

ABSTRACT
Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.