Maite Orruño

Changes in Escherichia coli outer membrane subproteome under environmental conditions inducing the viable but nonculturable state.

Changes in the outer membrane subproteome of Escherichia coli along the transition to the viable but nonculturable state (VBNC) were studied. The VBNC state was triggered by exposure of E. coli cells to adverse conditions such as aquatic systems, starvation, suboptimal temperature, visible light irradiation and seawater. The subproteome, obtained according to Molloy et al., was analysed at the beginning of exposure (inoculum, phase 1), after a variable exposure time (95% of population culturable, phase 2) and when populations were mainly in the VBNC state (95% of cells VBNC, phase 3). Proteome changes were dependent on adverse conditions inducing the transition and were detected mainly in phase 2. The permanence of E. coli cells in seawater under illumination conditions entailed a dramatic rearrangement of the outer membrane subproteome involving 106 new spots, some of which could be identified by peptide fingerprinting. However, proteins exclusive to the VBNC state were not detected.

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T₉₀ values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.

Mechanisms involved in Escherichia coli and Serratia marcescens removal during activated sludge wastewater treatment

Wastewater treatment reduces environmental contamination by removing gross solids and mitigating the effects of pollution. Treatment also reduces the number of indicator organisms and pathogens. In this work, the fates of two coliform bacteria, Escherichia coli and Serratia marcescens, were analyzed in an activated sludge process to determine the main mechanisms involved in the reduction of pathogenic microorganisms during wastewater treatment. These bacteria, modified to express green fluorescent protein, were inoculated in an activated sludge unit and in batch systems containing wastewater. The results suggested that, among the different biological factors implied in bacterial removal, bacterivorous protozoa play a key role. Moreover, a representative number of bacteria persisted in the system as free‐living or embedded cells, but their distribution into liquid or solid fractions varied depending on the bacterium tested, questioning the real value of bacterial indicators for the control of wastewater treatment process. Additionally, viable but nonculturable cells constituted an important part of the bacterial population adhered to solid fractions, what can be derived from the competition relationships with native bacteria, present in high densities in this environment. These facts, taken together, emphasize the need for reliable quantitative and qualitative analysis tools for the evaluation of pathogenic microbial composition in sludge, which could represent an undefined risk to public health and ecosystem functions when considering its recycling.

Reprogramming of Vibrio harveyi gene expression during adaptation in cold seawater

The life and survival of the marine bacterium Vibrio harveyi during its adaptation in natural aquatic systems is highly influenced by the availability of nutrients and temperature. To learn about adaptation strategies evolved by this bacterium to cope with drastic temperature downshifts and nutrients depletion, we have studied the phenotypical and gene expression changes occurring in V. harveyi during its adaptation to cold seawater. We found that incubation in cold seawater up to 12 h did not cause any significant morphological changes in V. harveyi and had no effect on the number of viable and culturable cells. Microarray analysis revealed that the V. harveyi response to cold seawater leads to up- and downregulation of numerous genes controlling the central carbon metabolism, nucleotide and amino acid biosynthesis as well as DNA repair. In addition, expression of some genes controlling biosynthesis of lipids, molecular transport, and energy production was altered to likely affect the composition and properties of the V. harveyi cell envelope, thus implying the putative role of this compartment in adaptation to stress. Here, we discuss these results with regard to the putative adaptive responses likely triggered by V. harveyi to cope with environmental challenges in natural aquatic systems.

Survival strategies of Escherichia coli and Vibrio spp.: contribution of the viable but nonculturable phenotype to their stress-resistance and persistence in adverse environments

In their natural ecosystems, bacteria are continuously exposed to changing environmental factors including physicochemical parameters (e.g. temperature, pH, etc.), availability of nutrients as well as interaction(s) with other organisms. To increase their tolerance and survival under adverse conditions, bacteria trigger a number of adaptation mechanisms. One of the well-known adaptation responses of the non-spore-forming bacteria is the acquisition of the viable but non-culturable (VBNC) state. This phenotype is induced by different stress factors (e.g. low temperature) and is characterized by the temporal loss of culturability, which can potentially be restored. Moreover, this response can be combined with the bust and boom strategy, which implies the death of the main population of the stressed cells (or their entry into the VBNC state) upon stress, thus enabling the remaining cells (i.e. residual culturable population) to subsist at the expense of the dead or/and VBNC cells. In this review, we discuss the characteristics of the VBNC state, its biological significance and contribution to bacterial survival.

Survival of Escherichia coli under Nutrient-Deprived Conditions: Effect on Cell Envelope Subproteome

In the aquatic ecosystems, microorganisms are exposed to seasonal and circadian cycles. Abiotic factors (e.g. low temperature, nutrient deprivation) can cause morphological and physiological changes in bacteria, thereby facilitating cell survival. While representing the interface between the cells and external environment, the cell envelope plays a major role in bacterial response to stress and characterization of the changes it undergoes can help to understand the adaptation process. In this study, analysis of the morphological and physiological changes as well as variations in protein composition of the Escherichia coli cell envelope was carried out for populations maintained for 21 days under nutrient deprivation and suboptimal temperatures (4°C and 20°C). It was found that the absence of nutrients led to a temperature-dependent reduction of cell culturability but had no effect on cell viability and integrity. The concentration of membrane proteins playing the key roles in cellular transport, maintenance of cell structure or bioenergetics processes remained mainly unchanged. In contrast, the level of several proteins such as the elongation factor EFTu 1, components of Bam complex or proteins implicated in chemotaxis was altered, thus indicating that cells were readily responding and adapting to stress.