Factors affecting the performance of 5' nuclease PCR assays for Listeria monocytogenes detection.

The design and operating parameters affecting the performance of 5' nuclease PCR (TaqMan) assays for the detection of Listeria monocytogenes was investigated. A system previously developed and based on the hlyA gene was used as a model [Appl. Environ. Microbiol. 61 (1995) 3724]. A series of fluorogenic probes labeled with a reporter and a quencher dye was synthesized to explore the effect of probe position and sequence content on the efficiency of probe hydrolysis. In addition, a series of PCR primer pairs that altered the distance between the upstream primer and the interceding probe was examined. The effects of various assay parameters were evaluated by measuring the ratio of the fluorescence intensity of the reporter dye over the quencher dye (deltaRQ). For a given probe sequence, the deltaRQ was typically lower if the 5' terminus was a G residue. Decreasing the probe concentration increased the deltaRQ, although this was at the expense of reproducibility in the assay readout. The distance between the upstream primer and the interceding probe has a significant effect on probe hydrolysis. Reducing the primer-probe distance from, for example, 127 to 4 nt increased the deltaRQ from 2.87 to 5.00. These general rules were used to develop a 5' nuclease PCR (TaqMan) assay with enhanced signal output, providing higher and more reproducible deltaRQ values for L. monocytogenes detection.

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP-NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4'-bipyridine (PPB) are described. The MBP domain of MBP-NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 x 10(4) and 7.1 x 10(4) M(-1) s(-1) were obtained for TNT and DNT, respectively. The apparent Michaelis-Menten constants (KM) for MBP-NR in solution and on the surface, using TNT as substrate, were determined to be 27 and 95 microM, respectively. The corresponding value for "wild-type" NR in solution containing TNT was 78 microM, which is very close to the value obtained for MBP-NR on the surface. The limits of detection for both TNT and DNT were estimated to be 2 microM, and the sensitivities were determined to be 205 and 222 nA/microM, respectively.

Novel immuno-FRET assay method for Bacillus spores and Escherichia coli O157:H7.

Novel immunofluorescence resonance energy transfer (immuno-FRET) assays for both Bacillus cereus spores and Escherichia coli O157:H7 are reported. Both assays involve the use of dual (QSY-7 and Oregon Green 514-antibody)-labeled spores or vegetative bacteria, such that Oregon Green 514-labeled antibodies are quenched by proximal QSY-7 molecules that are covalently bound to the dual (Oregon Green 514 and QSY-7)-labeled cells. Upon introduction of unlabeled bacteria or spores, in the respective assays, an increase in fluorescence is observed in proportion to the numbers of unlabeled cells. This is due to migration of Oregon Green 514-labeled antibody from the dual-labeled cells to the unlabeled target cells as verified by fluorescence microscopy. Optimization of the QSY-7 surface density led to a B. cereus spore detection sensitivity of approximately 1.0 x 10(5) to 2.5 x 10(5) spores per milliliter and 3.5 x 10(5) cells per milliliter for E. coli using a conventional cuvette-based spectrofluorometer.

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.

Time resolved collapse of a folding protein observed with small angle x-ray scattering.

High-intensity, "pink" beam from an undulator was used in conjunction with microfabricated rapid-fluid mixing devices to monitor the early events in protein folding with time resolved small angle x-ray scattering. This Letter describes recent work on the protein bovine beta-lactoglobulin where collapse from an expanded to a compact set of states was directly observed on the millisecond time scale. The role of chain collapse, one of the initial stages of protein folding, is not currently understood. The characterization of transient, compact states is vital in assessing the validity of theories and models of the folding process.

Molecular epidemiologic features and antimicrobial susceptibility profiles of various ribotypes of Pseudomonas aeruginosa isolated from humans and ruminants.

OBJECTIVES: To assess automated ribotyping for characterization of Pseudomonas aeruginosa isolates and to identify their type prevalence and geographic distribution. SAMPLE POPULATION: 39 human and 56 ruminant P aeruginosa isolates. PROCEDURES: Isolates were identified by use of bacteriologic techniques and automated Pvull-based ribotyping. Susceptibility to antimicrobials was tested in vitro. Data were analyzed for index of discrimination; prevalence ratio; geographic distribution of ribotypes found only in humans, only in cows, or only in goats (single-host ribotypes); and geographic distribution of ribotypes found in humans and ruminants (multihost ribotypes). RESULTS: All isolates were typeable (45 ribotypes, 35 single-host ribotypes). Ribotyping index of discrimination was 0.976. More isolates (45.3%) than expected yielded multihost ribotypes (22% of all ribotypes). Although 8.6% of single-host ribotypes were found in 4 or more isolates, 60% of multihost ribotypes were found in 4 or more isolates. Ninety percent of multihost ribotypes were isolated from different geographic areas, whereas 3.0% of single-host ribotypes were isolated from different geographic areas. All ruminant isolates were susceptible to gentamicin and polymyxin B. In contrast, antibiogram profiles differed for human isolates from different geographic areas. Susceptibility to antimicrobials differentiated 6 isolates not distinguished by ribotyping. CONCLUSIONS AND CLINICAL RELEVANCE: Automated ribotyping with Pvull discriminated more isolates than in vitro antimicrobial susceptibility. In combination, both tests provided more information than either test alone. Given the greater prevalence and geographic distribution of multihost ribotypes, immunocompromised humans and lactating ruminants may have a greater risk for disease if exposed to multihost P aeruginosa ribotypes, compared with single-host ribotypes.

Genetic evidence for a defective xylan degradation pathway in Lactococcus lactis.

Genetic and biochemical evidence for a defective xylan degradation pathway was found linked to the xylose operon in three lactococcal strains, Lactococcus lactis 210, L. lactis IO-1, and L. lactis NRRL B-4449. Immediately downstream of the xylulose kinase gene (xylB) (K. A. Erlandson, J.-H. Park, W. El Khal, H.-H. Kao, P. Basaran, S. Brydges, and C. A. Batt, Appl. Environ. Microbiol. 66:3974-3980, 1999) are two open reading frames encoding a mutarotase (xylM) and a xyloside transporter (xynT) and a partial open reading frame encoding a beta-xylosidase (xynB). These are functions previously unreported for lactococci or lactobacilli. The mutarotase activity of the putative xylM gene product was confirmed by overexpression of the L. lactis enzyme in Escherichia coli and purification of recombinant XylM. We hypothesize that the mutarotase links xylan degradation to xylose metabolism due to the anomeric preference of xylose isomerase. In addition, Northern hybridization experiments suggested that the xylM and xynTB genes are cotranscribed with the xylRAB genes, responsible for xylose metabolism. Although none of the three strains appeared to metabolize xylan or xylobiose, they exhibited xylosidase activity, and L. lactis IO-1 and L. lactis NRRL B-4449 had functional mutarotases.

Structural and kinetic characterization of early folding events in beta-lactoglobulin.

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.

High pressure NMR reveals a variety of fluctuating conformers in beta-lactoglobulin.

High pressure 1H/15N two-dimensional NMR spectroscopy has been used to study conformational fluctuation in bovine beta-lactoglobulin at pH 2.0 and 36 degrees C. Pressure dependencies of 1H and 15N chemical shifts and cross-peak intensities were analyzed at more than 80 independent atom sites between 30 and 2000 bar. Unusually large and non-linear chemical shift pressure dependencies are found for residues centering in the hydrophobic core region, suggesting the existence of low-lying excited native states (N') of the protein. Measurement of 1H/15N cross-peak intensities at individual amide sites as a function of pressure suggests that unfolding events occur independently in two sides of the beta-barrel, i.e. the hydrophobic core side (betaF-H) (producing I2) and the non-core side (betaB-E) (producing I1). At 1 bar the stability is higher for the core region (DeltaG0 = 6.5(+/-2.0) kcal/mol) than for the non-core region (4.6(+/-1.3) kcal/mol), but at high pressure the stability is reversed due to a larger DeltaV value of unfolding for the core region (90.0(+/-35.2) ml/mol) than that for the non-core region (57.4(+/-14.4) ml/mol), possibly due to an uneven distribution of cavities. The DeltaG0 profile along the amino acid sequence obtained from the pressure experiment is found to coincide well with that estimated from hydrogen exchange experiments. Altogether, the high pressure NMR experiment has revealed a variety of fluctuating conformers of beta-lactoglobulin, notably N, N', I1, I2 and the totally unfolded conformer U. Fluctuation of N to I1 and I2 conformers with open barrel structures could be a common design of lipocalin family proteins which bind various hydrophobic compounds in its barrel structure.

Dissolution of xylose metabolism in Lactococcus lactis.

Xylose metabolism, a variable phenotype in strains of Lactococcus lactis, was studied and evidence was obtained for the accumulation of mutations that inactivate the xyl operon. The xylose metabolism operon (xylRAB) was sequenced from three strains of lactococci. Fragments of 4.2, 4.2, and 5.4 kb that included the xyl locus were sequenced from L. lactis subsp. lactis B-4449 (formerly Lactobacillus xylosus), L. lactis subsp. lactis IO-1, and L. lactis subsp. lactis 210, respectively. The two environmental isolates, L. lactis B-4449 and L. lactis IO-1, produce active xylose isomerases and xylulokinases and can metabolize xylose. L. lactis 210, a dairy starter culture strain, has neither xylose isomerase nor xylulokinase activity and is Xyl(-). Xylose isomerase and xylulokinase activities are induced by xylose and repressed by glucose in the two Xyl(+) strains. Sequence comparisons revealed a number of point mutations in the xylA, xylB, and xylR genes in L. lactis 210, IO-1, and B-4449. None of these mutations, with the exception of a premature stop codon in xylB, are obviously lethal, since they lie outside of regions recognized as critical for activity. Nevertheless, either cumulatively or because of indirect affects on the structures of catalytic sites, these mutations render some strains of L. lactis unable to metabolize xylose.

Development of a fluorogenic probe-based PCR assay for detection of Bacillus cereus in nonfat dry milk.

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5'-to-3' exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

Is folding of beta-lactoglobulin non-hierarchic? Intermediate with native-like beta-sheet and non-native alpha-helix.

The refolding of beta-lactoglobulin, a beta-barrel protein consisting of beta strands betaA-betaI and one major helix, is unusual because non-native alpha-helices are formed at the beginning of the process. We studied the refolding kinetics of bovine beta-lactoglobulin A at pH 3 using the stopped-flow circular dichroism and manual H/(2)H exchange pulse labeling coupled with heteronuclear NMR. The protection pattern from the H/(2)H exchange of the native state indicated the presence of a stable hydrophobic core consisting of betaF, betaG and betaH strands. The protection pattern of the kinetic intermediate obtained about one second after initiating the reaction was compared with that of the native state. In this relatively late kinetic intermediate, which still contains some non-native helical structure, the disulfide-bonded beta-hairpin made up of betaG and betaH strands was formed, but the rest of the molecule was fluctuating, where the non-native alpha-helices may reside. Subsequently, the core beta-sheet extends, accompanied by a further alpha-helix to beta-sheet transition. Thus, the refolding of beta-lactoglobulin exhibits two elements: the critical role of the core beta-sheet is consistent with the hierarchic mechanism, whereas the alpha-helix to beta-sheet transition suggests the non-hierarchic mechanism.

A review of molecular recognition technologies for detection of biological threat agents.

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.

Solution structure and dynamics of bovine beta-lactoglobulin A.

Using heteronuclear NMR spectroscopy, we studied the solution structure and dynamics of bovine beta-lactoglobulin A at pH 2.0 and 45 degrees C, where the protein exists as a monomeric native state. The monomeric NMR structure, comprising an eight-stranded continuous antiparallel beta-barrel and one major alpha-helix, is similar to the X-ray dimeric structure obtained at pH 6.2, including betaI-strand that forms the dimer interface and loop EF that serves as a lid of the interior hydrophobic hole. [1H]-15N NOE revealed that betaF, betaG, and betaH strands buried under the major alpha-helix are rigid on a pico- to nanosecond time scale and also emphasized rapid fluctuations of loops and the N- and C-terminal regions.

Detection of viable Listeria monocytogenes with a 5' nuclease PCR assay.

A 5' nuclease assay has been developed to detect viable Listeria monocytogenes. The assay targets the hemolysin A (hlyA) transcript, which is found only in L. monocytogenes. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using Tth DNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribed hlyA mRNA. The viability of L. monocytogenes was reduced by heating at various temperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primers located in the most distal regions of the hlyA transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3' end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate.

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.

alpha-->beta transition of beta-lactoglobulin as evidenced by heteronuclear NMR.

Whereas bovine beta-lactoglobulin is a predominantly beta-sheet protein, it has a marked alpha-helical preference and can be considered to be a useful model of the alpha-->beta transition, a key issue for understanding the folding and biological function of a number of proteins. In order to understand the mechanism of the alpha-->beta transition, the backbone structures of the recombinant bovine beta-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol were characterized by 1H, 13C and 15N multidimensional NMR spectroscopy. Overall, the secondary structures in the native state were similar to those of the crystal structure. On the other hand, beta-lactoglobulin in the 2,2,2-trifluoroethanol state was composed of many alpha-helical segments. The presence of the persistent alpha-helices in the helical state and the core beta-sheet in the native state suggested that during folding native-like core beta-sheet and several non-native helices are formed first and the remaining beta-sheet is subsequently "induced" through interaction with the pre-existing beta-sheet.

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.

Lowering the pH optimum of D-xylose isomerase: the effect of mutations of the negatively charged residues.

Streptomyces rubiginosus D-xylose isomerase catalyzes the reversible isomerization of D-glucose to D-fructose. The isomerization reaction is maximized in the alkaline region of pH 8.5-8.8. The amino acid residues around two active site histidines (His-54 and His-220) and on the surface of the enzyme were mutated to improve the catalytic efficiency at neutral pH. The mutations have been made by removing the negatively charged residues based upon the sequence comparison of other D-xylose isomerases and the hypothesis proposed by Russell and Fersht (1987). The effects of these substitutions on kinetic parameter, pH dependence, and thermostability were characterized. The kcat values for D56N and E221A mutants on D-glucose are increased by 30-40% over that of the wild-type enzyme at pH 7.3 and the increased activities are maintained between pH 6 and 7.5. However, the surface mutants D65A, D81A, and D163N/E167Q only show 40-60% of the wild-type activity over the entire pH range. The pH activity profiles of the mutants are broader than that of the wild-type enzyme. The optimum pHs and the pKa values for all the mutants are lowered by 0.5-0.8 and 0.1-0.5 units, respectively. The small delta(deltaG) and high Tm values for all the mutants indicate that there is no significant change in the hydrogen bond network in the active site by mutations. These results indicate that D56N and E221A are possible candidates as good catalysts for High-Fructose Corn Syrup (HFCS) production.