**2. Materials and methods**

To prepare experimental samples, *E. coli* ATCC 27325 cells were cultured aerobically in Tryptone soy broth (TSB) at 37°C with shaking (120 rpm) until they reached the stationary growth phase (24 h). The cells were harvested by centrifugation (4000 g, 4°C, 20 min) and washed three times with sterile saline solution (0.9% NaCl, w/v). All the glass flasks used for handling *E. coli* cultures were cleaned with H2 SO4 (96%, v/v) beforehand, rinsed with deionized water, and kept at 250°C for 24 h to get rid of residual organic compounds.

Cells were inoculated in Erlenmeyer flasks containing sterile saline solution (absence of nutrients) to obtain a final density of 10<sup>8</sup> cells ml−1 and further incubated for 21 days at 4 and 20°C (suboptimal temperatures) with shaking (120 rpm) in darkness.

Subsamples were collected at the beginning of the experiments and after 3, 6, 12, and 21 days of nutrient deprivation to determine the number of total, viable, and culturable cells and to estimate the size of the cells (see below). Subsamples were also collected at 0 (P0), 6 (P1), 12 (P2), and 21 days (P3) to extract membrane proteins further analyzed by mass spectroscopy (see below).

conditions. Moreover, during transfer from their natural environments to other ecosystems, bacteria can change their status from autochthonous to allochthonous one. This scenario is very typical for enteric bacteria, which are regularly transferred from their hosts to aquatic systems, a process accompanied by changes in the temperature and nutrient content of their

*Escherichia coli* (allochthonous, copiotroph, mesophile bacterium) is an indicator of fecal pollution and, therefore, its detection, quantification, as well as assessment of its ability to survive in aquatic environments are important subjects of the study and monitoring pursued

Many bacteria, including *E. coli*, can cope with adverse conditions and successfully thrive in new environments by adjusting their physiology and metabolism. *E. coli* survival in a hostile aquatic environment depends on both biotic and abiotic factors [1–3]. Several abiotic factors including suboptimal temperature [4, 5], limitation of nutrients [6–8], and exposure to solar radiation [9–11] can lead to the loss of culturability. Barcina and Arana [12], Lothigius et al. [13], and others have demonstrated that, under these conditions, cells still remain physiologically active and intact. Transition from culturable to non-culturable state is known to involve con-

To learn more about *E. coli* adaptation in aquatic systems, we undertook the present study to focus on adaptation changes affecting the composition of cell envelope and appearance of *E. coli* cells. While representing the interface between the cytoplasm and external environment, the cell envelope plays a major role in how bacteria sense and respond to stress [17] during its adaptation to changing environments [18, 19]. These functions of the cell envelope prompted us to characterize the morphological and physiological changes undertaken by *E. coli* populations during their permanence at suboptimal temperatures and under limitation of nutrients. Moreover, we have analyzed the variations in the subproteome of cell envelopes

To prepare experimental samples, *E. coli* ATCC 27325 cells were cultured aerobically in Tryptone soy broth (TSB) at 37°C with shaking (120 rpm) until they reached the stationary growth phase (24 h). The cells were harvested by centrifugation (4000 g, 4°C, 20 min) and washed three times with sterile saline solution (0.9% NaCl, w/v). All the glass flasks used for

SO4

Cells were inoculated in Erlenmeyer flasks containing sterile saline solution (absence of nutri-

Subsamples were collected at the beginning of the experiments and after 3, 6, 12, and 21 days of nutrient deprivation to determine the number of total, viable, and culturable cells and to

ized water, and kept at 250°C for 24 h to get rid of residual organic compounds.

(suboptimal temperatures) with shaking (120 rpm) in darkness.

(96%, v/v) beforehand, rinsed with deion-

cells ml−1 and further incubated for 21 days at 4 and 20°C

by both public administrations and the scientific community.

406 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

siderable changes in the biochemical content of the cells [14–16].

accompanying this survival process.

handling *E. coli* cultures were cleaned with H2

ents) to obtain a final density of 10<sup>8</sup>

**2. Materials and methods**

habitats.

The results from survival experiments are presented as the means of three independent experiments, with coefficient of variation between replicates less than 12%. The one-way analysis of variance (ANOVA) was used to determine the differences between the means. Probabilities less than (or equal to) 0.05 were considered significant.

The total number of bacteria was determined according to the procedure described by Hobbie et al. [20]. Namely, aliquots of cell suspensions from survival assays were filtered throughout 0.22 μm pore size black polycarbonate filters, stained with acridine orange (0.01%, w/v), and examined through epifluorescence microscopy. Viable bacteria, estimated as bacteria with intact cytoplasmic membranes (MEMB+), were counted with Live/Dead BacLight™ kit (Invitrogen) as described by Joux et al. [21]. The bacteria with intact (green fluorescence, MEMB+) and permeabilized (red fluorescence) cytoplasmic membranes were enumerated separately. The number of culturable bacteria was determined by the spread plate method on Tryptone soy agar (TSA) followed by their incubation for 24 h at 37°C.

The length variations of *E. coli* cells during their survival at 4 and 20°C were estimated through image analysis of epifluorescence preparations [22] by using an image analysis system, which included a high-resolution video camera (Hamamatsu 2400). Images of microscopic fields with enough bacteria were selected to be digitized and analyzed by Scion Image 1.62ᵃ software. For each subsample, 200 bacteria were measured. The values of mean size (x = 1.45 μm) and standard deviation (SD = 0.32) of initial population were used to establish three size categories (≤ x−SD, > x−SD – ≤ x + SD, > x + SD). Therefore, according to their length, the cells fell into one of the following size ranges: ≤ 1.12 μm, >1.12–≤1.77 μm, or >1.77 μm.

Membrane protein preparations were obtained according to the method described by Molloy et al. [23] with minor variations [24]. Subsequent analysis of these proteins was performed by the Proteomics Core Facility-SGIKER at the University of the Basque Country, using the protocol previously described by Gonzalez-Fernandez et al. [25]. Briefly, after protein precipitation by using a 2D Clean-Up Kit (GE Healthcare), the pellet was suspended in RapiGest solution (0.2%) (Waters Corporation) and heated at 85°C for 15 min. Then, the preheated suspension was reduced with DTT (5 mM), alkylated with iodoacetamide (15 mM), and digested with trypsin (2 μg per sample) overnight. MassPREP Enolase Digestion Standard (Waters Corporation) was added to the supernatants collected after centrifugation (16,000 g, 10 min) of HCl-treated samples (inactivation of RapiGest) and was used as a standard for protein absolute quantification. A nanoACQUITY UPLC System coupled to a SYNAPT HDMS (Waters Corporation) was used for data-independent acquisition analyses. Subsamples with tryptic peptides and MassPREP Enolase Digestion Standard were loaded onto a Symmetry300 C18, 180 μm × 20 mm precolumn (Waters Corporation) connected to a BEH130 C18 column (75 μm × 200 mm, 1.7 μm [Waters Corporation]). Peptides were eluted with a linear gradient of acetonitrile (120 min from 3 to 40% and 15 min from 40 to 60% [v/v]). Mass spectra were acquired using a data-independent acquisition mode (MSE) [26] as previously described by Gonzalez-Fernandez et al. [25] and processed with ProteinLynx Global SERVER v2.4 Build RC7 (Waters Corporation). Protein identification was carried out using the database search algorithm of the program [27] and the parameters specified by Parada et al. [24]. The absolute protein quantification based on peak area intensity of peptide precursors was calculated by the program using enolase peptides as an internal standard [28].

Among proteins confirmed by the presence of at least three protein-derived peptides in the tryptic digests, those detected in two or three of the biological replicates were considered for further analysis. Quantification values of individual proteins were normalized *versus* the total protein in the samples. Only those proteins showing a 1.5-fold increase or a 0.6-fold decrease in their relative abundance (with respect to the previous sampling time) were considered differentially affected by survival conditions.

UniProt and KEGG databases were used to verify the identity and function of proteins. For the prediction of the bacterial protein subcellular localization, the PSORTb 3.0 program [29] was used. According to their main biological functions specified in UniProt database, selected proteins were further grouped to form the categories of proteins that (i) play structural roles involved in (ii) transport, (iii) bioenergetics, (iv) synthesis, degradation, and turnover of protein, (v) stress response, or (vi) have miscellaneous functions.
