In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MFO is maximal at a growth temperature of 17.5°C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MFO, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.Pseudomonas fluorescens is well known as a major psychrotrophic contaminant of raw milk stored in refrigerated tanks (16). Many studies of this bacterium have been concerned with the production of deleterious extracellular enzymes, such as thermostable proteases (8,15,27). Among the numerous observations concerning these enzymes, it has been repeatedly shown that most strains maximally produce proteases at a temperature (15 to 20°C) well below the optimal growth temperature (25 to 30°C) (13,19,25). However, no studies have yet dealt with the mechanism of regulation of protease production with regard to temperature.At this stage two main questions can be raised regarding the elucidation of this mechanism. The first one relates to the specificity of this temperature effect, i.e., whether it is restricted to the production of proteases or extended to the production of other enzymes. To this end, the activities of extracellular proteases as a function of growth temperature were compared with those of several periplasmic phosphatases. The exported enzymes all showed the same regulation by temperature even though they are clearly differentially regulated by other growth conditions. Thus, it was important to determine whether this temperature effect might involve protein export through the cytoplasmic membrane. If so, any foreign exported protein should be submitted to the same effect. The expression of a gene from the mesophilic species Escherichia coli, under the control of its own promoter, was studied in P. fluorescens at different growth temperatures. In this case, a temperature effect similar to that observed with the native enzymes was not demonstrated.The second question is whether the temperature itself is the direct cause of the regulation or an indirect effector acting through the growth rate variation; such an indirect effect has indeed been demonstrated for several activities or proteins in mesophilic bacteria (5). To answer this question, the activities of the two acidic phosphatases were assayed in cells grown in a chemostat at two different temperatures and * Corresponding author. several dilution rates. The ...

Background Enterotoxigenic Escherichia coli (ETEC) substantially contributes to the burden of diarrheal illnesses in developing countries. With the use of complementary in vitro models of the human digestive environment, TNO gastrointestinal model (TIM-1), and Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we provided the first detailed report on the spatial-temporal modulation of ETEC H10407 survival, virulence, and its interplay with gut microbiota. These systems integrate the main physicochemical parameters of the human upper digestion (TIM-1) and simulate the ileum vs ascending colon microbial communities and luminal vs mucosal microenvironments, captured from six fecal donors (M-SHIME). Results A loss of ETEC viability was noticed upon gastric digestion, while a growth renewal was found at the end of jejunal and ileal digestion. The remarkable ETEC mucosal attachment helped to maintain luminal concentrations above 6 log10 mL−1 in the ileum and ascending colon up to 5 days post-infection. Seven ETEC virulence genes were monitored. Most of them were switched on in the stomach and switched off in the TIM-1 ileal effluents and in a late post-infectious stage in the M-SHIME ascending colon. No heat-labile enterotoxin production was measured in the stomach in contrast to the ileum and ascending colon. Using 16S rRNA gene-based amplicon sequencing, ETEC infection modulated the microbial community structure of the ileum mucus and ascending colon lumen. Conclusions This study provides a better understanding of the interplay between ETEC and gastrointestinal cues and may serve to complete knowledge on ETEC pathogenesis and inspire novel prophylactic strategies for diarrheal diseases.

Differences in digestive physicochemical parameters may partially explain why children are more susceptible to EHEC infection than adults. Such data are essential for a full understanding of EHEC pathogenesis and would help in designing novel therapeutic approaches.

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Fluorescence spectroscopy is an emerging tool for the analysis of biomolecules from complex matrices. We explored the potentialities of the method for the pseudomonad taxonomic purpose at the genus and species level. Emission spectra of three intrinsic fluorophores (namely, NADH, tryptophan, and the complex of aromatic amino acids and nucleic acid) were collected from whole bacterial cells. Their comparisons were performed through principal component analysis and factorial discriminant analysis. Reference strains from the Xanthomonas, Stenotrophomonas, Burkholderia, and Pseudomonas genera were well separated, with sensitivity and selectivity higher than 90%. At the species level, P. lundensis, P. taetrolens, P. fragi, P. chlororaphis, and P. stutzeri were also well separated, in a distant group, from P. putida, P. pseudoalcaligenes, and P. fluorescens. These results are in agreement with the generally admitted rRNA and DNA bacterial homology grouping but they also provide additional information about strain relatedness. In the case of environmental isolates, the method allows good discrimination, even for strains for which ambiguity still remained after PCR and API 20NE identification. Rapid, easy to perform, and low cost, fluorescence spectroscopy provides substantial information on cell components. Statistical analysis of collected data allows in-depth comparison of strains. Our results strongly support the view that fluorescence spectroscopy fingerprinting can be used as a powerful tool in a polyphasic approach to pseudomonad taxonomy.