Showing papers by "Douglas B. Kell published in 2002"

Rapid and quantitative detection of the microbial spoilage of meat by fourier transform infrared spectroscopy and machine learning.

Abstract: Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable “fingerprints.” Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial leastsquares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10 7 bacteria·g 1 the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.

A family of autocrine growth factors in Mycobacterium tuberculosis.

Abstract: Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells We show here that the five cognate proteins from M tuberculosis have very similar characteristics and properties to those of Rpf They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling The five M tuberculosis proteins show cross-species activity against M luteus, Mycobacterium smegmatis and M bovis (BCG) Actively growing cells of M bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections

Formation and resuscitation of "non-culturable" cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase.

Abstract: After growth of Rhodococcus rhodochrous in Sauton’s medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3–4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ‘non-culturable’ cells of the ‘Academia’ strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton’s medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4–5 months post-inoculation, of a homogeneous population of ostensibly ‘non-culturable’ cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 105 organisms ml−1, and this value was further increased by one log using supernatant from an actively growing culture. Populations of ‘non-culturable’ cells of Mycobacterium tuberculosis were also obtained by the filtration of ‘clumpy’ cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ‘non-culturable’ cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ‘non-culturable’ bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

The rpf gene of Micrococcus luteus encodes an essential secreted growth factor

Abstract: Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non-productively at the cell surface, as a truncated form of the protein, encompassing only the 'Rpf domain' is fully active. The C-terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti-Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid-located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf-like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.

Metabolic profiling using direct infusion electrospray ionisation mass spectrometry for the characterisation of olive oils

Abstract: There is a continuing need for improved methods for assessing the adulteration of foodstuffs. We report some highly encouraging data, where we have developed direct infusion electrospray ionisation mass spectrometry (ESI-MS) together with chemometrics as a novel, rapid (1 min per sample) and powerful technique to elucidate key metabolite differences in vegetable and nut oils. Principal components analysis of these ESI-MS spectra show that the reproducibility of this approach is high and that olive oil can be discriminated from oils which are commonly used as adulterants. These adulterants include refined hazelnut oil, which is particularly challenging given its chemical similarity to olive oils.

Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor

Abstract: The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their annotated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites.

Schemes of flux control in a model of Saccharomyces cerevisiae glycolysis

Abstract: We used parameter scanning to emulate changes to the limiting rate for steps in a fitted model of glucose-derepressed yeast glycolysis. Three flux-control regimes were observed, two of which were under the dominant control of hexose transport, in accordance with various experimental studies and other model predictions. A third control regime in which phosphofructokinase exerted dominant glycolytic flux control was also found, but it appeared to be physiologically unreachable by this model, and all realistically obtainable flux control regimes featured hexose transport as a step involving high flux control.

Monitoring of complex industrial bioprocesses for metabolite concentrations using modern spectroscopies and machine learning: Application to gibberellic acid production

Abstract: Two rapid vibrational spectroscopic approaches (diffuse reflectance-absorbance Fourier transform infrared [FT-IR] and dispersive Raman spectroscopy), and one mass spectrometric method based on in vacuo Curie-point pyrolysis (PyMS), were investigated in this study. A diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi producing the natural product gibberellic acid, were analyzed directly without a priori chromatographic separation. Partial least squares regression (PLSR) and artificial neural networks (ANNs) were applied to all of the information-rich spectra obtained by each of the methods to obtain quantitative information on the gibberellic acid titer. These estimates were of good precision, and the typical root-mean-square error for predictions of concentrations in an independent test set was <10% over a very wide titer range from 0 to 4925 ppm. However, although PLSR and ANNs are very powerful techniques they are often described as "black box" methods because the information they use to construct the calibration model is largely inaccessible. Therefore, a variety of novel evolutionary computation-based methods, including genetic algorithms and genetic programming, were used to produce models that allowed the determination of those input variables that contributed most to the models formed, and to observe that these models were predominantly based on the concentration of gibberellic acid itself. This is the first time that these three modern analytical spectroscopies, in combination with advanced chemometric data analysis, have been compared for their ability to analyze a real commercial bioprocess. The results demonstrate unequivocally that all methods provide very rapid and accurate estimates of the progress of industrial fermentations, and indicate that, of the three methods studied, Raman spectroscopy is the ideal bioprocess monitoring method because it can be adapted for on-line analysis.

Metabolomics and Machine Learning: Explanatory Analysis of Complex Metabolome Data Using Genetic Programming to Produce Simple, Robust Rules

Abstract: Kell, D. B. (2002). Metabolomics and machine learning: explanatory analysis of complex metabolome data using genetic programming to produce simple, robust rules. Molecular Biology Reports, 29, (1-2), 237-241.

Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20 kDa) proteins.

Abstract: Vaidyanathan, S., Winder, C. L., Wade, S. C., Kell, D. B., Goodacre, R. (2002). Sample preparation in matrix-assisted laser desorption/ionization mass spectrometry of whole bacterial cells and the detection of high mass (>20 kDa) proteins. Rapid Communications in Mass Spectrometry, 16, (13), 1276-1286 Sponsorship: BBSRC

Differentiation of Phytophthora infestans sporangia from other airborne biological particles by flow cytometry

Abstract: The ability of two different flow cytometers, the Microcyte (Optoflow) and the PAS-III (Partec), to differentiate sporangia of the late-blight pathogen Phytophthora infestans from other potential airborne particles was compared. With the PAS-III, light scatter and intrinsic fluorescence parameters could be used to differentiate sporangia from conidia of Alternaria or Botrytis spp., rust urediniospores, and pollen of grasses and plantain. Differentiation between P. infestans sporangia and powdery mildew conidia was not possible by these two methods but, when combined with analytical rules evolved by genetic programming methods, could be achieved after staining with the fluorescent brightener Calcofluor white M2R. The potential application of these techniques to the prediction of late-blight epiphytotics in the field is discussed. Current methods for predicting the occurrence of late-blight of potato (Phytophthora infestans) rely on climatic modeling to identify conditions conducive to pathogen reproduction and thus to disease spread (5). Such methods can provide considerable savings to growers, as well as environmental benefits. For United Kingdom potato production, calendar spraying is normal practice, with sprays commencing when the crop canopy has closed and continuing at 7- to 10-day intervals until crop desiccation or harvest. Use of forecasting models can reduce the number of required sprays by allowing a delay in the first fungicide application and/or an extension of the interval between subsequent sprays (e.g., references 25 and 28).

On Fitness Distributions and Expected Fitness Gain of Mutation Rates in Parallel Evolutionary Algorithms

Abstract: Setting the mutation rate for an evolutionary algorithm (EA) is confounded by many issues. Here we investigate mutation rates mainly in the context of large-population-parallelism. We justify the notion that high rates achieve better results, using underlying theory which notices that parallelization favourably alters the fitness distribution of a mutation operator. We derive an expression which sets out how this is changed in terms of the level of parallelization, and derive further expressions that allow us to adapt the mutation rate in a principled way by exploiting online-sampled landscape information. The adaptation technique (called RAGE - Rate Adaptation with Gain Expectation) shows promising preliminary results. Our motivation is the field of Directed Evolution (DE), which uses large-scale parallel EAs for limited numbers of generations to evolve novel proteins. RAGE is highly suitable for DE, and is applicable to large-scale parallel EAs in general.

Metabolic control analysis and engineering of the yeast sterol biosynthetic pathway.

Abstract: Groeneveld, P., Rolley, N., Kell, D. B., Kelly, S. L., Kelly, D. E. (2002). Metabolic control analysis and engineering of the yeast sterol biosynthetic pathway. Molecular Biology Reports, 29, (1-2), 27-29

Rapidhigh-throughput microbial characterization by metabolite and protein profiling of whole cells using soft-ionization mass spectrometry.

Abstract: The development of techniques for the rapid characterization of microorganisms is important in assessing food and environmental safety, in medical diagnosis, and in the screening of biotechnol. relevant metabolites. Soft ionization mass spectrometry of whole cells, with minimal sample prepn., offers the potential for rapid, high-throughput microbial characterization based on specific biomarkers, protein or metabolite profiles. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are two complementary soft-ionization mass spectrometry (MS) methods that we are developing for the anal. of whole microbial cells and which produce predominantly metabolite and protein profiles. This presentation will give an overview on the characterization of the spectral information, optimization of exptl. conditions and the application of flow injection ESI-MS and MALDI-MS for the discrimination of microbial strains using chemometric techniques.