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C A Batt

Publications and source records attributed to C A Batt.

At least 37 records · Page 2Linked to original sources
PubMed1998

Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris.

beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and functional expression in lactobacillus plantarum 80 of xylT, encoding the D-xylose-H+ symporter of Lactobacillus brevis.

A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Amino Acid Sequence↗
PubMed1997

High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties.

Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.

Amino Acid Sequence↗
PubMed1997

Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.

Cheese↗
PubMed1997

Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential.

A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers. PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles. The combination of these virulence gene alleles and ribotype patterns separated L. monocytogenes into three distinct lineages. While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently. These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed. The clinical history of the L. monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages. Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III. Animal isolates were found in all three lineages. We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay. Strains from lineage III also seem to form larger plaques than strains from lineage II. A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L. monocytogenes subspecies. None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

Alleles↗
PubMed1997

Molecular diagnostics for dairy-borne pathogens.

Advances in diagnostic assays based on nucleic acids will revolutionize the ability of the industry to maintain the safety of dairy foods. Two complementary assay formats are explored, one of which permits the rapid detection of bacterial pathogens and the other the identification of reservoirs of these pathogens. The first format is an assay based on the polymerase chain reaction that employs homogeneous detection (TaqMan polymerase chain reaction detection; Perkin Elmer, Applied Biosystems Division, Foster City, CA) of the target sequence. This assay has been applied to the detection of Listeria monocytogenes. A primary problem with current assays that are based on polymerase chain reaction is the complexity of sample handling and the quantification of the initial target number. This fluorogenic assay takes advantage of the endogenous 5',3'-endonuclease activity in Taq DNA polymerase. Approximately 100 samples can be analyzed in 2 to 3 h with a sensitivity of < 50 cells and a dynamic range of > 1000-fold. The TaqMan polymerase chain reaction detection assay is a robust format that is readily applicable to a wide array of other pathogens found in foods and in the environment. The second format is an instrument for automated ribosomal RNA analysis (Riboprinter; DuPont, Wilmington, DE) that can be used to locate the reservoirs harboring the bacterial pathogen. Use of this typing method it has been shown that, although a number of different ribotypes can be isolated from a single environmental sample, only a selected number of these strains apparently have the ability to cause disease. The future of food microbiology lies in the development and integration of molecular methods that can be automated into a testing regimen that extends from the farm to finished products.

Bacterial Infections↗
PubMed1997

Diversity of Streptococcus agalactiae and Staphylococcus aureus ribotypes recovered from New York dairy herds.

OBJECTIVES: To develop a reference database for characterization of bovine Staphylococcus aureus and Streptococcus agalactiae strains by automated ribotyping and to use it to assess the discriminatory power of this typing procedure and the geographic distribution of Sta aureus and Str agalactiae strains in New York state dairy herds. SAMPLE POPULATION: 22 commercial dairy herds. PROCEDURE: Isolates of Sta aureus and Str agalactiae from bovine milk were identified by standard bacteriologic procedures, then typed by automated ribotyping. Antimicrobial susceptibility of isolates was tested in vitro. Two indicators made from the data were percentage of farms with multiple ribotypes and percentage of single ribotypes found in several geographic regions. Standard bacteriologic diagnosis, automated ribotyping, and determination of antibiograms (Kirby-Bauer method) also were done. RESULTS: Of 50 Sta aureus and 44 Str agalactiae isolates from composite milk samples of 12 and 10 herds, respectively, 18 and 14 ribotypes, respectively, were identified. The discriminatory power of automated ribotyping was approximately 0.96 (Hunter-Gaston's formula). A higher percentage of herds with Sta aureus had multiple ribotypes. The most common Sta aureus ribotypes tended to have broader geographic distribution. Some Sta aureus ribotypes were significantly associated with antibiotic resistance profiles. CONCLUSIONS: Automated ribotyping appears to characterize bovine strains of bacteria associated with intramammary infections with a high discriminatory index. Potential applications include identification of strains that appear to have broad geographic distribution suggesting interfarm transfer, discrimination between recurrent versus new intramammary infections (ie, for control of Str agalactiae and Sta aureus), and evaluation of antibiotic therapy.

Animals↗
PubMed1997

Investigation of a listeriosis epizootic in sheep in New York state.

OBJECTIVE: To investigate potential sources of an epizootic of listerial encephalitis, using molecular diagnostic and typing methods. SAMPLE POPULATION: A flock of about 655 sheep. PROCEDURE: An epizootiologic investigation was performed. Clinical, feed, and environmental samples were tested for Listeria monocytogenes, using polymerase chain reaction and culture methods; recovered isolates were "fingerprinted," using an automated ribotyping system. RESULTS: Listeria monocytogenes was recovered from brain specimens of 7 sheep with clinical signs of listerial encephalitis. All clinical isolates had fingerprints identical to those of isolates from farm equipment used to transport silage. Corn silage, which was not fed to the sheep, also contained L monocytogenes of the same pattern type as defined by ribotyping. Listeria monocytogenes was not isolated from the stored haylage designated for feeding the sheep (the cut-off point for isolation being < 10(2) colony-forming units/g). CONCLUSIONS: Corn silage was implicated as the source of a listeriosis epizootic. It appears to have cross-contaminated the haylage destined for the sheep during handling with a front-end loader. Suspension of silage feeding coincided with cessation of listeriosis cases. CLINICAL RELEVANCE: Use of advanced molecular techniques can help to identify the sources and restrict the scope of an epizootic. In epizootics, a single L monocytogenes strain can lead to infection of multiple animals, with rapid progression of the disease.

Animals↗
PubMed1996

Equine motor neuron disease is not linked to Cu/Zn superoxide dismutase mutations: sequence analysis of the equine Cu/Zn superoxide dismutase cDNA.

The cDNA encoding the equine copper/zinc superoxide dismutase (SOD1) was cloned from leukocyte total RNA from healthy horses and its nucleotide (nt) sequence was determined. We further sequenced the SOD1 gene from 16 horses diagnosed with equine motor neuron disease (EMND) and eight unrelated, clinically normal horses to determine if this disease, similar to amyotrophic lateral sclerosis (ALS) in humans, is linked to SOD1 mutations. The 465-bp SOD1 coding region in the horse encodes 153 amino acid (aa) residues. Equine SOD1 exhibited 81.8 and 79.9% sequence identity to the human homolog at the nt and aa levels, respectively, with only five distinct aa in the two loops that constitute the active site of the enzyme. None of the human SOD1 mutations found in the familial form of ALS were detected in SOD1 of the 16 affected horses. Although DNA sequence analysis identified three potential polymorphisms in equine SOD1, these were silent and were found in both normal and EMND-afflicted horses. At this time, there is no conclusive evidence for EMND linkage to SOD1 mutations.

Amino Acid Sequence↗
PubMed1996

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Allergens↗
PubMed1996

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider.

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10 g of ground beef when samples were enriched in modified EC broth for 7 h at 37 degrees C. The minimum detectable number of cells for the apple cider samples was calculated to be approximately 0.5 cfu ml-1. The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.

Animals↗
PubMed1996

A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef.

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'-->3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, delta RQ values increased from approximately 1.5 to 3.0. The delta RQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 +/- 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I-producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.

Animals↗
PubMed1996

Ribotype diversity of Listeria monocytogenes strains associated with outbreaks of listeriosis in ruminants.

Ribotyping is a molecular method for the characterization, identification, and typing of bacterial isolates that has value in epidemiological studies. To demonstrate the utility of this technique for typing of Listeria monocytogenes, four outbreaks of epizootic listeriosis in ruminants were investigated through coordinated detection and characterization methods utilizing classical microbiology and nucleic acid-based techniques. L. monocytogenes strains isolated from clinical samples and the silage consumed by the affected animals were ribotyped to establish the causal relationship between feed and the disease outbreak. For all but one outbreak, we were able to isolate L. monocytogenes strains represented by the same ribotype from both clinical and silage samples. Additional L. monocytogenes strains with ribotypes different from those of the respective clinical samples were isolated from all silage samples. This indicates that a diverse population of L. monocytogenes strains exists in farm environments, of which some may be more likely than others to cause disease.

Abortion, Veterinary↗
PubMed1995

Probing the roles of active site residues in D-xylose isomerase.

The roles of active site residues His54, Phe94, Lys183, and His220 in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate.

Aldose-Ketose Isomerases↗
PubMed1995

Applications of DNA amplification techniques in veterinary diagnostics.

An overview of the principles of the polymerase chain reaction, ligase chain reaction, self-sustained sequence replication and Q beta replicase is given. The application of these methods for the diagnosis of veterinary infectious and hereditary diseases as well as for other diagnostic purposes is discussed and comprehensive tables of reported assays are provided. Specific areas where these DNA-based amplification methods provide substantial advantages over traditional approaches are also highlighted. With regard to PCR-based assays for the detection of viral pathogens, this article is an update of a previous review by Belák and Ballagi-Pordány (1993).

Animals↗
PubMed1995

Effect of cellular physiology on PCR amplification efficiency.

Culture conditions, and other variables that modulate a cell's physiology, can bias a polymerase chain reaction (PCR) amplification against generating a representative population profile. Two Pseudomonas putida nahR alleles were constructed in pUC19 that differ solely in a 31-bp internal segment whose sequence has been inverted. After PCR amplification, the products could be distinguished on the basis of a change in a unique restriction site. When an Escherichia coli strain carrying one nahR allele is submitted to different growth conditions, the consequences of such variations on the relative PCR amplification of whole cells can be ascertained through coamplification with a strain carrying the other allele and subsequent restriction analysis. Cells in stationary phase displayed improved amplifiability while cells grown at 42 degrees C were equally amplifiable as compared to cells grown at 37 degrees C. However, sublethal levels of tetracycline or growth in minimal medium made the PCR target in these cells relatively less amplifiable. When cells are completely lysed and the plasmid DNA is purified beforehand, the coamplification bias is eliminated. These results suggest that mixed populations containing cells in different physiological states may not be representatively amplified by PCR unless a DNA extraction step is included.

Alleles↗
PubMed1995

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Animals↗