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The effect of NaCl concentration during sporulation and subsequent growth on the distributions of times associated with different stages of the lag phase of individual spores of nonproteolytic Clostridium botulinum strain Eklund 17B is determined. The results on distribution of lag times for individual spores have helped in understanding the mechanisms that can be used in improving the predictions of growth in food and refining risk assessments.

The successive generation times for single cells of Escherichia coli K-12 were measured and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. The results show that the differences in the lag time duration was reported for greater inoculum sizes, but those cells were severely stressed by starvation or heating or growth conditions were very close to the growth-no growth boundary.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. The role of a homologue of the negative transcriptional regulatory protein HspR, which in other organisms participates in the control of the heat-shock response, was investigated. Following inactivation of hspR in C. jejuni, members of the HspR regulon were identified by DNA microarray transcript profiling. In agreement with the predicted role of HspR as a negative regulator of genes involved in the heat-shock response, it was observed that the transcript amounts of 13 genes were increased in the hspR mutant, including the chaperone genes dnaK, grpE and clpB, and a gene encoding the heat-shock regulator HrcA. Proteomic analysis also revealed increased synthesis of the heat-shock proteins DnaK, GrpE, GroEL and GroES in the absence of HspR. The altered expression of chaperones was accompanied by heat sensitivity, as the hspR mutant was unable to form colonies at 44 [degrees]C. Surprisingly, transcriptome analysis also revealed a group of 17 genes with lower transcript levels in the hspR mutant. Of these, eight were predicted to be involved in the formation of the flagella apparatus, and the decreased expression is likely to be responsible for the reduced motility and ability to autoagglutinate that was observed for hspR mutant cells. Electron micrographs showed that mutant cells were spiral-shaped and carried intact flagella, but were elongated compared to wild-type cells. The inactivation of hspR also reduced the ability of Campylobacter to adhere to and invade human epithelial INT-407 cells in vitro, possibly as a consequence of the reduced motility or lower expression of the flagellar export apparatus in hspR mutant cells. It was concluded that, in C. jejuni, HspR influences the expression of several genes that are likely to have an impact on the ability of the bacterium to successfully survive in food products and subsequently infect the consumer.

Campylobacter jejuni is a zoonotic pathogen and the most common cause of bacterial foodborne diarrhoeal illness worldwide. To establish intestinal colonization prior to either a commensal or pathogenic interaction with the host, C. jejuni will encounter iron-limited niches where there is likely to be intense competition from the host and normal microbiota for iron. To gain a better understanding of iron homeostasis and the role of ferric uptake regulator (Fur) in iron acquisition in C. jejuni, a proteomic and transcriptome analysis of wild-type and fur mutant strains in iron-rich and iron-limited growth conditions was carried out. All of the proposed iron-transport systems for haemin, ferric iron and enterochelin, as well as the putative iron-transport genes p19, Cj1658, Cj0177, Cj0178 and cfrA, were expressed at higher levels in the wild-type strain under iron limitation and in the fur mutant in iron-rich conditions, suggesting that they were regulated by Fur. Genes encoding a previously uncharacterized ABC transport system (Cj1660-Cj1663) also appeared to be Fur regulated, supporting a role for these genes in iron uptake. Several promoters containing consensus Fur boxes that were identified in a previous bioinformatics search appeared not to be regulated by iron or Fur, indicating that the Fur box consensus needs experimental refinement. Binding of purified Fur to the promoters upstream of the p19, CfrA and CeuB operons was verified using an electrophoretic mobility shift assay (EMSA). These results also implicated Fur as having a role in the regulation of several genes, including fumarate hydratase, that showed decreased expression in response to iron limitation. The known PerR promoters were also derepressed in the C. jejuni Fur mutant, suggesting that they might be co-regulated in response to iron and peroxide stress. These results provide new insights into the effects of iron on metabolism and oxidative stress response as well as the regulatory role of Fur. Present address: Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Abbreviation: EMSA, electrophoretic mobility shift assay. The raw microarray data are available as a supplement to the online version of this paper (at http://mic.sgmjournals.org), which also has links to Supplementary Tables A-C (available with the online version of the paper only).

Byline: Roberto Carcoba (1), Carmen Pin (2), Ana Rodriguez (1) Keywords: Lactococcus lactis; Chemostat cultures; Lactose metabolism; Dilution rate; pH Abstract: Lactococcus lactis subsp. lactis IPLA 947 is a starter strain isolated from a Spanish farmhouse acid-coagulated cheese. To use this strain as starter culture in Afuega'l Pitu cheese manufacture, anaerobic lactose-limited chemostat cultures were used to optimise the active biomass production in a simple medium (BRFS). Growth, lactose consumption and metabolite production were measured at different pH values (5.5, 6.0 and 6.5) and dilution rates (0.06 to 0.96 h.sup.-1). The influence of both variables on the quoted parameters was described by means of mathematical models. These provided a satisfactory representation of the experimental data. The bacterial response surface showed a significant increase in the biomass when the pH increased and the dilution rate decreased. As dilution rate increased, biomass yield increased but only at the lowest pH. The lactose consumption and the ratio of metabolic end products were also influenced by both variables. As dilution rate decreased, the residual lactose decreased and increased amounts of metabolites other than lactate (acetate, formate, ethanol) were produced. The minimum lactose consumption was obtained at pH 5.5 and 0.96 h.sup.-1 dilution rate. A significant rise of acetate and formate levels occurred as pH increased. A maximum lactate level, along with an acetate, formate and ethanol production close to the minimum, was predicted by mathematical modelling at 0.5173 h.sup.-1 dilution rate and pH 6.054. Author Affiliation: (1) Instituto de Productos Lacteos de Asturias (IPLA-CSIC), Apdo. 85, 33300, Villaviciosa, Asturias, Spain (2) Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, UK Article History: Registration Date: 01/06/2004 Received Date: 28/01/2004 Online Date: 02/07/2004

Research has been conducted on Brochothrix thermosphacta glucose metabolism. The role of atmosphere composition in this metabolism has been investigated and the decision whether the metabolism is dominantly anaerobic or anaerobic for a given atmosphere composition has been made.

Research was conducted to examine a novel numerical approach in estimating bacterial growth parameters using detection times generated by different initial counts. Detection times were subjected to a transformation involving the maximum specific growth rate and the ratios between the different inoculum sizes and the constant detectable level of counts. The more accurate the cell number at the detection level, the better the estimate will be for the variance of the lag times of the individual cells.

Abstract Background Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge. Results A computing tool (ArrayLeaRNA) has been developed for gene expression analysis. It implements a Bayesian approach which is based on the Gumbel distribution and uses printed genomic DNA control features for normalization and for estimation of the parameters of the Bayesian model and prior knowledge from predicted operon structure. The method is compared with two other approaches: the classical LOWESS normalization followed by a two fold cut-off criterion and the OpWise method (Price, et al. 2006. BMC Bioinformatics. 7, 19), a published Bayesian approach also using predicted operon structure. The three methods were compared on experimental datasets with prior knowledge of gene expression. With ArrayLeaRNA, data normalization is carried out according to the genomic features which reflect the results of equally transcribed genes; also the statistical significance of the difference in expression is based on the variability of the equally transcribed genes. The operon information helps the classification of genes with low confidence measurements. ArrayLeaRNA is implemented in Visual Basic and freely available as an Excel add-in at http://www.ifr.ac.uk/safety/ArrayLeaRNA/ Conclusion We have introduced a novel Bayesian model and demonstrated that it is a robust method for analysing microarray expression profiles. ArrayLeaRNA showed a considerable improvement in data normalization, in the estimation of the experimental variability intrinsic to each hybridization and in the establishment of a clear boundary between non-changing and differentially expressed genes. The method is applicable to data derived from hybridizations of labelled cDNA samples as well as from hybridizations of labelled cDNA with genomic DNA and can be used for the analysis of datasets where differentially regulated genes predominate.

Drug-induced gastrointestinal toxicities (DI-GITs) are among the most common adverse events in clinical trials. High prevalence of DI-GIT has persisted among new drugs due in part to the lack of robust experimental tools to allow early detection or to guide optimization of safer molecules. Developing in vitro assays for the leading GI toxicities (nausea, vomiting, diarrhoea, constipation, and abdominal pain) will likely involve recapitulating complex physiological properties that require contributions from diverse cell/tissue types including epithelial, immune, microbiome, nerve, and muscle. While this stipulation may be beyond traditional 2D monocultures of intestinal cell lines, emerging 3D GI microtissues capture interactions between diverse cell and tissue types. These interactions give rise to microphysiologies fundamental to gut biology. For GI microtissues, organoid technology was the breakthrough that introduced intestinal stem cells with the capability of differentiating into each of the epithelial cell types and that self-organize into a multi-cellular tissue proxy with villus- and crypt-like domains. Recently, GI microtissues generated using miniaturized devices with microfluidic flow and cyclic peristaltic strain were shown to induce Caco2 cells to spontaneously differentiate into each of the principle intestinal epithelial cell types. Second generation models comprised of epithelial organoids or microtissues co-cultured with non-epithelial cell types can successfully reproduce cross-'tissue' functional interactions broadening the potential of these models to accurately study drug-induced toxicities. A new paradigm in which in vitro assays become an early part of GI safety assessment could be realized if microphysiological systems (MPS) are developed in alignment with drug-discovery needs. Herein, approaches for assessing GI toxicity of pharmaceuticals are reviewed and gaps are compared with capabilities of emerging GI microtissues (e.g., organoids, organ-on-a-chip, transwell systems) in order to provide perspective on the assay features needed for MPS models to be adopted for DI-GIT assessment. [ABSTRACT FROM AUTHOR]

The dynamic model for the growth of a bacterial population was applied to model the lag period and exponential growth of Listeria monocytogenes under conditions of fluctuating temperature and water activity values. Observations for several scenarios in which exponential growth was halted by a sudden change in the temperature and/or water activity values were in good agreement with predictions.

The effects of C[O.sub.2] on gene expression and neurotoxin formation by proteolytic Clostridium botulinum type A1 strain ATCC 3502 are analyzed during the growth cycle. Microarray analysis has enabled the identification of coding sequences whose expression profiles have matched with those of the neurotoxin cluster and a DNA microarray is used for studying the transcriptome.

A study was conducted to assess the suitability of real-time PCR for the quantification of Listeria monocytogenes under different stress conditions and no-stress conditions in a culture model. The differences observed between the data obtained from low-stress conditions plate count and real-time PCR data are related to the stress imposed on L. monocytogenes and changed during growth.

The development of a stochastic model is reported to simulate the growth of the population resulting from the single-cell division times in Escherichia coli. Results suggest that greater the variability of the single-cell interdivision intervals, the shorter are both the lag time and the doubling time of the population.

A novel attempt is made to determine the distributions of times associated with the various stages of germination and subsequent growth from spores and the relationships between these stages. As the variability in postgermination stages is relatively large, the first spore to germinate will not necessarily be the first spore to produce actively dividing cells and start neurotoxin production.