**3. Results and discussion**

Consistent with the results of previous studies [4, 11, 30, 31], the total number of *E. coli* cells remained unchanged throughout the experimentation time under nutrient deprivation. The percentages of viable and culturable cells were calculated with respect to the total bacteria for each analyzed time (**Figure 1A** and **C**). Although the viable population did not show significant variations throughout the survival period, the culturable fraction declined progressively. Moreover, the loss of culturability of the cells incubated at 20°C occurred faster (already after 6 days of incubation). This result agrees with those obtained in previous works [4, 32] in which it was established that, in the absence of natural microbiota, the survival of *E. coli* reduces at higher temperatures.

Cellular dwarfing has been described as a typical response of bacteria exposed to adverse conditions. However, our work did not reveal any significant changes in the size of starved *E. coli* cells during 21 days (**Figure 1C** and **D**). For the main fraction of starved *E. coli* cells (63.5–73.5%), the cell length was preserved within the same range (>1.1–≤1.8 μm) during the incubation time. Similar results were obtained by Muela et al. [33], who found no changes in the cell size throughout the long-term survival of *E. coli* in sterile river water.

From survival assays carried out under starvation conditions, the samples for membrane subproteome analysis were collected at different incubation times: 0 (P0), 6 (P1), 12 (P2), and 21 days (P3). Despite the use of membrane fraction for mass spectrometry analysis, the PSORTb 3.0 program revealed that the resulting dataset potentially contained predicted cytosolic proteins (22%), including cytosolic subunits of ATP synthase or proteins that can conditionally be associated with the membrane (**Tables 1** and **2**). The fortuitous presence of cytoplasmic proteins in the membrane

**Figure 1.** *Escherichia coli* ATCC 27325 survival in the absence of nutrients at 4°C (A, C) and 20°C (B, D). (A and B) Variations in the percentages of viable ( ) and culturable ( ) bacteria obtained with respect to the total count at each period. (C and D) Variations in cell size distribution ( ≤ 1.12 μm; >1.12–≤1.77 μm; >1.77 μm). The data are mean values from three independent experiments with error bars representing the standard deviations calculated. Pannels (E and F), images of *E. coli* preparations stained with acridine orange (E) or Live/Dead BacLight™ kit (F) and examined through

**Percentage of cells (%)** 

*B* 

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

**Percentage of cells (%)** 

0 3 6 12 21

http://dx.doi.org/10.5772/67777

409

0 3 6 12 21

**Time (days)**

**Time (days)**

fractions was somewhat anticipated, as it was also observed in previous studies [24, 31].

epifluorescence microscopy.

**Percentage of cells (%)** 

*A* 

**Percentage of cells (%)** 

0 3 6 12 21

0 3 6 12 21

**Time (days)** 

*E F* 

**Time (days)** 

*C D* 

Thus, analysis of cell physiology and morphology revealed that, although *E. coli* cells remained active and maintained their integrity and size, starvation led to a decrease in the number of culturable cells. Moreover, these changes were temperature dependent. Similar behavior has been previously described for this bacterium [4], and it was attributed to the differences in metabolic activities of cells cultured at low and normal temperatures [34, 35].

Survival of *Escherichia coli* under Nutrient-Deprived Conditions: Effect on Cell Envelope Subproteome http://dx.doi.org/10.5772/67777 409

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

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 dif-

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

Consistent with the results of previous studies [4, 11, 30, 31], the total number of *E. coli* cells remained unchanged throughout the experimentation time under nutrient deprivation. The percentages of viable and culturable cells were calculated with respect to the total bacteria for each analyzed time (**Figure 1A** and **C**). Although the viable population did not show significant variations throughout the survival period, the culturable fraction declined progressively. Moreover, the loss of culturability of the cells incubated at 20°C occurred faster (already after 6 days of incubation). This result agrees with those obtained in previous works [4, 32] in which it was established that, in the absence of natural microbiota, the survival of *E. coli*

Cellular dwarfing has been described as a typical response of bacteria exposed to adverse conditions. However, our work did not reveal any significant changes in the size of starved *E. coli* cells during 21 days (**Figure 1C** and **D**). For the main fraction of starved *E. coli* cells (63.5–73.5%), the cell length was preserved within the same range (>1.1–≤1.8 μm) during the incubation time. Similar results were obtained by Muela et al. [33], who found no changes in

Thus, analysis of cell physiology and morphology revealed that, although *E. coli* cells remained active and maintained their integrity and size, starvation led to a decrease in the number of culturable cells. Moreover, these changes were temperature dependent. Similar behavior has been previously described for this bacterium [4], and it was attributed to the differences in

the cell size throughout the long-term survival of *E. coli* in sterile river water.

metabolic activities of cells cultured at low and normal temperatures [34, 35].

the program using enolase peptides as an internal standard [28].

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

protein, (v) stress response, or (vi) have miscellaneous functions.

ferentially affected by survival conditions.

**3. Results and discussion**

reduces at higher temperatures.

**Figure 1.** *Escherichia coli* ATCC 27325 survival in the absence of nutrients at 4°C (A, C) and 20°C (B, D). (A and B) Variations in the percentages of viable ( ) and culturable ( ) bacteria obtained with respect to the total count at each period. (C and D) Variations in cell size distribution ( ≤ 1.12 μm; >1.12–≤1.77 μm; >1.77 μm). The data are mean values from three independent experiments with error bars representing the standard deviations calculated. Pannels (E and F), images of *E. coli* preparations stained with acridine orange (E) or Live/Dead BacLight™ kit (F) and examined through epifluorescence microscopy.

From survival assays carried out under starvation conditions, the samples for membrane subproteome analysis were collected at different incubation times: 0 (P0), 6 (P1), 12 (P2), and 21 days (P3). Despite the use of membrane fraction for mass spectrometry analysis, the PSORTb 3.0 program revealed that the resulting dataset potentially contained predicted cytosolic proteins (22%), including cytosolic subunits of ATP synthase or proteins that can conditionally be associated with the membrane (**Tables 1** and **2**). The fortuitous presence of cytoplasmic proteins in the membrane fractions was somewhat anticipated, as it was also observed in previous studies [24, 31].


a OM, outer membrane; CM, cytoplasmic membrane; Cyt, cytosolic protein; ?, unknown.

**Table 1.** Membrane proteins that did not show significant changes in their level after 0, 6, 12, and 21 days of *E. coli* starvation in saline solution (NaCl 0.9%).

**Category**

**Protein** 

**Locationa**

**Protein name**

**4°C** **P1/P0**

**P3/P1**

**P1/P0**

**P2/P1**

**P3/P2**

**20°C**

**accession** 

**number**

Cell structure

YBJP\_ECOLI ?

OSME\_ECOLI

YIDC\_ECOLI

BAMA\_ECOLI

BAMB\_ECOLI

Transport

PTW3C\_ECOLI

 CM

 OM

 OM

CM

Membrane protein

NC

ND

NC

NC

ND

insertase YidC

Outer membrane protein

ND

ND

ND

ND

ND

assembly factor BamA

Outer membrane protein

ND

ND

ND

ND

ND

assembly factor BamB

PTS system

NC

ND

NC

NC

NC

N-acetylglucosamine-

specific

EIICBA component

Protein translocase

NC

NC

NC

ND

ND

subunit SecD

PTS system mannitol

NC

ND

NC

ND

ND

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

specific EIICBA

component

SECD\_ECOLI

PTM3C\_ECOLI

Bioenergetics

NUOCD\_

Cyt

NADH-quinone

NC

ND

NC

NC

NC

oxidoreductase subunits

C/D

NADH dehydrogenase

ATP-dependent zinc

NC

NC

NC

0.57

NC

http://dx.doi.org/10.5772/67777

metalloprotease FtsH

ND

ND

ND

ND

ND

ECOLI

DHNA\_ECOLI

Synthesis,

FTSH\_ECOLI

CM

degradation

and turnover of

proteins

Stress response

BFR\_ECOLI

Cyt

Bacterioferritin

NC

NC

ND

ND

ND

411

 CM

 CM

CM

 ?

Uncharacterized YbjP

Osmotically inducible

1.93

NC

NC

ND

ND

lipoprotein E

NCb

NC

0.59c

NC

NC


**Category Protein accession number Locationa Protein name**

SLP\_ECOLI OM Slp

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

YDGA\_ECOLI CM YdgA

OMPC\_ECOLI OM OmpC OMPW\_ECOLI OM OmpW OMPX\_ECOLI OM OmpX TOLC\_ECOLI OM TolC

Bioenergetics ATPA\_ECOLI Cyt ATP synthase, subunit alpha

Stress responses YQJD\_ECOLI ? Uncharacterized protein YqjD ELAB\_ECOLI ? ElaB

OM, outer membrane; CM, cytoplasmic membrane; Cyt, cytosolic protein; ?, unknown.

Others MIND\_ECOLI CM Septum site-determining protein MinD

ATPB\_ECOLI; ATPL\_ECOLI

CYDA\_ECOLI; CYDB\_ECOLI

FRDB\_ECOLI; FRDA\_ECOLI

DHSA\_ECOLI DHSB\_ECOLI

starvation in saline solution (NaCl 0.9%).

Synthesis, degradation, and turnover of proteins

a

Transport OMPA\_ECOLI OM OmpA

Cell structure LPP\_ECOLI OM Major outer membrane lipoprotein Lpp

PAL\_ECOLI OM Peptidoglycan-associated lipoprotein

METQ\_ECOLI CM D-Methionine-binding lipoprotein MetQ DACC\_ECOLI CM D-Alanyl-D-alanine carboxypeptidase DacC

SLYB\_ECOLI OM Outer membrane protein SlyB

GLPT\_ECOLI CM Glycerol 3 phosphate transporter

PTND\_ECOLI CM Mannose permease IID component COPA\_ECOLI CM Copper-exporting P-type ATPase A ACRA\_ECOLI CM Multidrug efflux pump subunit AcrA

YHII\_ECOLI CM Uncharacterized protein YhiI

PTNAB\_ECOLI Cyt PTS system mannose-specific EIIAB component

DCUA\_ECOLI CM Anaerobic C4-dicarboxylate transporter DcuA

NARG\_ECOLI CM Respiratory nitrate reductase 1 alpha chain

HFLK\_ECOLI Cyt Modulator of FtsH protease HflK HFLC\_ECOLI CM Modulator of FtsH protease HflC

**Table 1.** Membrane proteins that did not show significant changes in their level after 0, 6, 12, and 21 days of *E. coli*

CM ATP synthase, subunits beta and c

iron-sulfur subunit

CM Cytochrome bd-I ubiquinol oxidase, subunits 1 and 2

CM Succinate dehydrogenase flavoprotein subunit and

CM Fumarate reductase iron-sulfur subunit and flavoprotein subunit


**Table 2.** Membrane proteins that exhibited significant changes in their level at 6 (P1), 12 (P2), and 21 days (P3) of *E. coli* starvation in sterile saline solution (NaCl 0.9%, w/v). A large group of proteins (**Table 1**) did not show any significant upregulation (>1.5-fold) or downregulation (<0.6-fold) during the survival experiments. This group included proteins related to the maintenance of cell structure (some lipoproteins, YdgA, and other) and/or the transport (porins such as OmpA, OmpW, OmpC, and TolC) (**Table 1**). Noteworthy, some of these proteins (namely, Lpp lipoprotein and OmpA and OmpC porins) belong to the group of the most abundant polypeptides detected in all samples. The above data suggest a role for these proteins in the maintenance of cell integrity observed here and in previous studies [12, 15, 16, 36, 37] upon *E. coli* exposure to adverse conditions. While some of them (e.g., lipoproteins) may be critical for maintaining the lipid bilayer, others (e.g., OmpA and OmpW) are likely involved in sustaining the integrity of the outer membrane [38–40]. No changes in protein level were also observed for different proteases implicated in synthesis, degradation, and turnover of membrane proteins (HflK and HflC) (**Table 1**). It seems that their presence is critical for preservation of cell stability as these proteases might degrade damaged or unnecessary proteins that could potentially accumulate in the lipid bilayer, thus restricting membrane permeability [41, 42], which is one of the fundamental functions of biological membranes [41]. This idea is supported by the results of staining with the Live/Dead BacLight™ kit used to differentiate *live* and *dead* cells (**Figure 1A** and **B**), demonstrating that the membranes of the

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

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413

We also observed that the level of numerous proteins implicated in bioenergetics (namely, different subunits of ATP synthase, cytochromes, and reductases) and transport (porins, mannose permease, components of PTS systems, or glycerol 3 phosphate transporter) was nearly the same in the control sample (P0) and samples (P3) mainly containing non-culturable bacteria (**Figure 1** and **Table 1**). Despite the constant presence of these proteins in cell envelope, several studies suggested that starving cells likely preserve a minimal level of metabolic activities. For instance, Ozkanca and Flint [43] indicated that respiration rates greatly decreased to almost undetectable levels in *E. coli* cells exposed to starvation during their incubation in sterile lake water. Likewise, Barcina et al. [44] detected a decrease of glucose uptake for populations maintained in freshwater. Thus, the constant presence of the energy- and sugar metabolism-related proteins seems to indicate that starving cells still stay alarmed and prepared to quickly respond to favorable environmental conditions. Indeed, analysis of glucose uptake by the starving cells revealed a quick response and, as a result, an increase in the respiration rate [44]. Consistently, several authors have demonstrated the function of the electron transport chains in non-culturable bacteria by showing their ability to reduce intracellularly tetrazolium salts [31, 45, 46]. Moreover, in a previous work, Arana et al. [30] found that some *E. coli* cells could release nutrients (mainly monomeric carbohydrates and amino acids) into the surrounding medium under stress conditions. The released nutrients are taken up by other cells, thus could aid in the survival of remaining culturable cells and, therefore,

Other constantly present proteins include YqjD and its paralogous protein ElaB, known to be abundant in the stationary growth phase. These proteins seem to be involved in inhibition of ribosomal activity and in localization of ribosomes on the inner membrane during the stationary phase of growth. In cells exposed to some stress conditions (e.g., starvation), both ribosomal biogenesis and protein synthesis are known to be suppressed. Thus, the negative

starved cells remain intact and preserve their selective permeability.

ensure the persistence of the species in adverse environments.

A large group of proteins (**Table 1**) did not show any significant upregulation (>1.5-fold) or downregulation (<0.6-fold) during the survival experiments. This group included proteins related to the maintenance of cell structure (some lipoproteins, YdgA, and other) and/or the transport (porins such as OmpA, OmpW, OmpC, and TolC) (**Table 1**). Noteworthy, some of these proteins (namely, Lpp lipoprotein and OmpA and OmpC porins) belong to the group of the most abundant polypeptides detected in all samples. The above data suggest a role for these proteins in the maintenance of cell integrity observed here and in previous studies [12, 15, 16, 36, 37] upon *E. coli* exposure to adverse conditions. While some of them (e.g., lipoproteins) may be critical for maintaining the lipid bilayer, others (e.g., OmpA and OmpW) are likely involved in sustaining the integrity of the outer membrane [38–40]. No changes in protein level were also observed for different proteases implicated in synthesis, degradation, and turnover of membrane proteins (HflK and HflC) (**Table 1**). It seems that their presence is critical for preservation of cell stability as these proteases might degrade damaged or unnecessary proteins that could potentially accumulate in the lipid bilayer, thus restricting membrane permeability [41, 42], which is one of the fundamental functions of biological membranes [41]. This idea is supported by the results of staining with the Live/Dead BacLight™ kit used to differentiate *live* and *dead* cells (**Figure 1A** and **B**), demonstrating that the membranes of the starved cells remain intact and preserve their selective permeability.

We also observed that the level of numerous proteins implicated in bioenergetics (namely, different subunits of ATP synthase, cytochromes, and reductases) and transport (porins, mannose permease, components of PTS systems, or glycerol 3 phosphate transporter) was nearly the same in the control sample (P0) and samples (P3) mainly containing non-culturable bacteria (**Figure 1** and **Table 1**). Despite the constant presence of these proteins in cell envelope, several studies suggested that starving cells likely preserve a minimal level of metabolic activities. For instance, Ozkanca and Flint [43] indicated that respiration rates greatly decreased to almost undetectable levels in *E. coli* cells exposed to starvation during their incubation in sterile lake water. Likewise, Barcina et al. [44] detected a decrease of glucose uptake for populations maintained in freshwater. Thus, the constant presence of the energy- and sugar metabolism-related proteins seems to indicate that starving cells still stay alarmed and prepared to quickly respond to favorable environmental conditions. Indeed, analysis of glucose uptake by the starving cells revealed a quick response and, as a result, an increase in the respiration rate [44]. Consistently, several authors have demonstrated the function of the electron transport chains in non-culturable bacteria by showing their ability to reduce intracellularly tetrazolium salts [31, 45, 46]. Moreover, in a previous work, Arana et al. [30] found that some *E. coli* cells could release nutrients (mainly monomeric carbohydrates and amino acids) into the surrounding medium under stress conditions. The released nutrients are taken up by other cells, thus could aid in the survival of remaining culturable cells and, therefore, ensure the persistence of the species in adverse environments.

Other constantly present proteins include YqjD and its paralogous protein ElaB, known to be abundant in the stationary growth phase. These proteins seem to be involved in inhibition of ribosomal activity and in localization of ribosomes on the inner membrane during the stationary phase of growth. In cells exposed to some stress conditions (e.g., starvation), both ribosomal biogenesis and protein synthesis are known to be suppressed. Thus, the negative

**Category**

**Protein** 

**Locationa**

**Protein name**

**4°C** **P1/P0**

**P3/P1**

**P1/P0**

**P2/P1**

**P3/P2**

**20°C**

**accession** 

**number**

Others

EFTU1\_ECOLI

HEMX\_ECOLI

HEMY\_ECOLI

QMCA\_ECOLI

PPID\_ECOLI FLIC\_ECOLI MCP1\_ECOLI

 CM

Ex

Flagellin Methyl accepting

ND

ND

ND

ND

ND

chemotaxis protein I

aOM, outer membrane; CM, cytoplasmic membrane; Cyt, cytosolic; ?, unknown; Ex, extracellular.

bNC, no significant changes with respect to the previous sample; ND, not detected.

**Table 2.**

cValues higher than 1.5 indicate significant increases, and values lower than 0.6 indicate significant decreases of protein level with respect to the previous time.

Membrane proteins that exhibited significant changes in their level at 6 (P1), 12 (P2), and 21 days (P3) of *E. coli* starvation in sterile saline solution (NaCl 0.9%, w/v).

NC

ND

NC

NC

ND

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

CM

Peptidyl-prolyl *cis*-trans

NC

NC

NC

ND

ND

isomerase D

 ?

 CM

 CM

 Cyt

Elongation factor Tu 1

Putative

uroporphyrinogen-III C

methyltransferase

Protein HemY Protein QmcA

NC

NC

NC

ND

ND

NC

ND

NC

NC

NC

2.81 NC

NC

0.59

NC

NC

NC

4.22

NC

NC

regulation of these processes by YqjD and ElaB could be important for bacterial adaptation and survival in harsh environments [47].

As shown in **Figure 1**, *E. coli* response to starvation was temperature dependent. We identified several proteins whose level was differently affected by temperature. Namely, the protein translocase subunit SecD and bacterioferritin became undetectable in starved cells maintained at 20°C, whereas PTS system N-acetylglucosamine-specific EIICBA component was gradually lost in populations maintained at 4°C (**Table 2**). Unlike above examples, the temperature-dependent regulation of osmotically inducible lipoprotein E (OSME\_ECOLI) and ATP-dependent zinc metalloprotease FtsH did not reveal a clear

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

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415

Interestingly, the levels of three proteins were below detection at the starting point (P0), but their level was increased afterward. This group of proteins includes the structural protein MreB (MREB\_ECOLI), putative porin NmpC, and the cytosolic universal stress protein F (USPF\_ECOLI). MreB is a homolog of eukaryotic actin, which has been found to be associated with the membrane in several bacteria [60–62]. Shih et al. [63] stated that the MreB system is required for establishment of the rod shape of cells. MreB proteins form actin-like cables lying beneath the inner cell membrane. The cables are required to guide longitudinal cell wall synthesis. Chiu et al. [64] demonstrated that, in non-culturable *Vibrio parahaemolyticus* populations appeared upon starvation, MreB protein was located near the cytoplasmic membrane. Moreover, these authors reported a reduction in cellular size associated with the increase in the expression of the *mreB* gene. However, Ben Abdallah et al. [65] and Parada et al. [24] described the decline in the expression of the *mreB* gene and MreB protein content, respectively, in *Vibrio* species undergoing morphological changes in response to stress. Kruse et al. [66] demonstrated that a decrease of MreB concentration leads to merodiploid spherical and inflated *E. coli* cells prone to cell lysis. Moreover, Defeu Soufo et al. [67] demonstrated a relationship between MreB and EFTu for *E. coli* and *Bacillus subtilis* cells, and they suggested that EFTu could affect the cellular morphology through interaction with MreB. EFTu improves the ability of MreB to form filaments functioning as a basal structure. Our work suggests that both proteins could interact, as both of them are present during the cell

Thus, the present study showed that, although incubation in the absence of nutrients reduced cell culturability in a temperature-dependent manner, the cells still remained active and preserved their integrity and size. In addition, proteome analysis of the cell's envelope revealed that the concentration of membrane proteins playing the key roles in cellular transport, maintenance of cell structure, as well as bioenergetic processes remained almost unchanged, indicating their crucial roles in *E. coli* survival under nutrient-limiting conditions. Moreover, some of the proteins critical for preservation of cell stability and membrane permeability (such as the modulators of FtsH protease, HflK, and HflC) appeared to be steadily present in the populations of mainly non-culturable cells. We also found the continuous increase in the level of elongation factor EFTU1 along the survival process, thus suggesting its essential role in the adaptation process. Interestingly, the level of some proteins (e.g., bacterioferritin) was differently affected by temperature (see above). Finally, the observed depletion of the key components of the Bam complex, insertase YidC, and/or proteins implicated in chemotaxis suggested their redundancy for preserving cell integrity and therefore allowed to save energy

regulation pattern.

response to stress.

during *E. coli* adaptation and survival.

**Table 2** shows the membrane proteins (accounted for 17–19% of the total analyzed polypeptides) that became less or more abundant upon starvation of *E. coli* in sterile saline solution. Some of these proteins underwent variations dependent on nutrient status and/or incubation temperature. For example, the level of two proteins (namely, BamA and BamB) belonging to the outer membrane complex Bam (additionally containing BamB, BamC, BamD, and BamE [48, 49]) as well as the membrane protein insertase YidC sharply declined and became undetectable in the starved cells (**Table 2**). Since the β-barrel assembly machinery (BAM) is essential for maintaining the bacterial cell envelope and is involved in OMP recognition, folding, and assembly [48, 50, 51], its depletion with BamA, one of the key components of the *E. coli* Bam complex, after 3 days of incubation under starvation conditions could indicate the reduction in the production and/or active assembly of proteins in the outer membrane. Volokhina et al. [48] suggested that loss of activity of this protein promotes accumulation of proteins in the outer membrane that cannot be inserted therein. This accumulation could be lethal for the bacterium since aggregates would be formed in the periplasmic space. Moreover, this could lead to the incorporation of these OMPs into inner bacterial membranes, which would dissipate the proton-motive force and kill bacteria [52]. However, in this study, we have not detected dead cells (**Figure 1**). This fact could indicate that the Bam complex might become redundant in the nondividing *E. coli* cells due to reduction of production and maturation of OMPs in bacterial cells exposed to starvation.

Similar to BamA and BamB, the membrane protein insertase YidC also was not detectable after 21 days of starvation. This protein has been proposed to mediate the transfer of transmembrane segments of hydrophilic polypeptide chains from the Sec-translocon into the lipid bilayer and can assist folding of inner membrane proteins [53] including ATP synthase subunit c [17]. This finding together with the data obtained for BamA and BamB (see above) suggests that limitation of nutrients leads to the overall reduction of cell envelope biogenesis.

Other proteins that became undetectable in starved cells were the methyl-accepting chemotaxis protein I and the flagellin FLIC\_ECOLI. Chen and Chen [54] demonstrated that under starvation, *Vibrio vulnificus* populations exhibited reduced motility. Lemke et al. [55] and Chandrangsu et al. [56] concluded that DksA (protein required for the regulation of certain promoters) and the alarmone ppGpp inhibit expression of the flagellar cascade during cells' entry into the stationary phase or during their starvation. This mechanism could prevent unnecessary waste of energy on synthesis of macromolecular complexes and generation of proton-motive force used to rotate the flagella apparatus [57] and, therefore, would free more energy to sustain the survival process.

Unlike chemotaxis protein I and the flagellin FLIC\_ECOLI, the elongation factor Tu 1 became one of the most abundant proteins in populations maintained 6 days at 20°C. This elongation factor is known as a cytoplasmic chaperone implicated in protein synthesis, growth regulation, and stress responses [58, 59]. The high level of this protein in starving cells is consistent with the data presented by Muela et al. [31]. They observed an increase in the level of EFTu in *E. coli* populations under starvation conditions and its subsequent localization on the membrane.

As shown in **Figure 1**, *E. coli* response to starvation was temperature dependent. We identified several proteins whose level was differently affected by temperature. Namely, the protein translocase subunit SecD and bacterioferritin became undetectable in starved cells maintained at 20°C, whereas PTS system N-acetylglucosamine-specific EIICBA component was gradually lost in populations maintained at 4°C (**Table 2**). Unlike above examples, the temperature-dependent regulation of osmotically inducible lipoprotein E (OSME\_ECOLI) and ATP-dependent zinc metalloprotease FtsH did not reveal a clear regulation pattern.

regulation of these processes by YqjD and ElaB could be important for bacterial adaptation

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

**Table 2** shows the membrane proteins (accounted for 17–19% of the total analyzed polypeptides) that became less or more abundant upon starvation of *E. coli* in sterile saline solution. Some of these proteins underwent variations dependent on nutrient status and/or incubation temperature. For example, the level of two proteins (namely, BamA and BamB) belonging to the outer membrane complex Bam (additionally containing BamB, BamC, BamD, and BamE [48, 49]) as well as the membrane protein insertase YidC sharply declined and became undetectable in the starved cells (**Table 2**). Since the β-barrel assembly machinery (BAM) is essential for maintaining the bacterial cell envelope and is involved in OMP recognition, folding, and assembly [48, 50, 51], its depletion with BamA, one of the key components of the *E. coli* Bam complex, after 3 days of incubation under starvation conditions could indicate the reduction in the production and/or active assembly of proteins in the outer membrane. Volokhina et al. [48] suggested that loss of activity of this protein promotes accumulation of proteins in the outer membrane that cannot be inserted therein. This accumulation could be lethal for the bacterium since aggregates would be formed in the periplasmic space. Moreover, this could lead to the incorporation of these OMPs into inner bacterial membranes, which would dissipate the proton-motive force and kill bacteria [52]. However, in this study, we have not detected dead cells (**Figure 1**). This fact could indicate that the Bam complex might become redundant in the nondividing *E. coli* cells due to reduction of production and matura-

Similar to BamA and BamB, the membrane protein insertase YidC also was not detectable after 21 days of starvation. This protein has been proposed to mediate the transfer of transmembrane segments of hydrophilic polypeptide chains from the Sec-translocon into the lipid bilayer and can assist folding of inner membrane proteins [53] including ATP synthase subunit c [17]. This finding together with the data obtained for BamA and BamB (see above) suggests that limitation of nutrients leads to the overall reduction of cell envelope biogenesis. Other proteins that became undetectable in starved cells were the methyl-accepting chemotaxis protein I and the flagellin FLIC\_ECOLI. Chen and Chen [54] demonstrated that under starvation, *Vibrio vulnificus* populations exhibited reduced motility. Lemke et al. [55] and Chandrangsu et al. [56] concluded that DksA (protein required for the regulation of certain promoters) and the alarmone ppGpp inhibit expression of the flagellar cascade during cells' entry into the stationary phase or during their starvation. This mechanism could prevent unnecessary waste of energy on synthesis of macromolecular complexes and generation of proton-motive force used to rotate the flagella apparatus [57] and, therefore, would free more

Unlike chemotaxis protein I and the flagellin FLIC\_ECOLI, the elongation factor Tu 1 became one of the most abundant proteins in populations maintained 6 days at 20°C. This elongation factor is known as a cytoplasmic chaperone implicated in protein synthesis, growth regulation, and stress responses [58, 59]. The high level of this protein in starving cells is consistent with the data presented by Muela et al. [31]. They observed an increase in the level of EFTu in *E. coli* populations under starvation conditions and its subsequent localization on the membrane.

and survival in harsh environments [47].

tion of OMPs in bacterial cells exposed to starvation.

energy to sustain the survival process.

Interestingly, the levels of three proteins were below detection at the starting point (P0), but their level was increased afterward. This group of proteins includes the structural protein MreB (MREB\_ECOLI), putative porin NmpC, and the cytosolic universal stress protein F (USPF\_ECOLI). MreB is a homolog of eukaryotic actin, which has been found to be associated with the membrane in several bacteria [60–62]. Shih et al. [63] stated that the MreB system is required for establishment of the rod shape of cells. MreB proteins form actin-like cables lying beneath the inner cell membrane. The cables are required to guide longitudinal cell wall synthesis. Chiu et al. [64] demonstrated that, in non-culturable *Vibrio parahaemolyticus* populations appeared upon starvation, MreB protein was located near the cytoplasmic membrane. Moreover, these authors reported a reduction in cellular size associated with the increase in the expression of the *mreB* gene. However, Ben Abdallah et al. [65] and Parada et al. [24] described the decline in the expression of the *mreB* gene and MreB protein content, respectively, in *Vibrio* species undergoing morphological changes in response to stress. Kruse et al. [66] demonstrated that a decrease of MreB concentration leads to merodiploid spherical and inflated *E. coli* cells prone to cell lysis. Moreover, Defeu Soufo et al. [67] demonstrated a relationship between MreB and EFTu for *E. coli* and *Bacillus subtilis* cells, and they suggested that EFTu could affect the cellular morphology through interaction with MreB. EFTu improves the ability of MreB to form filaments functioning as a basal structure. Our work suggests that both proteins could interact, as both of them are present during the cell response to stress.

Thus, the present study showed that, although incubation in the absence of nutrients reduced cell culturability in a temperature-dependent manner, the cells still remained active and preserved their integrity and size. In addition, proteome analysis of the cell's envelope revealed that the concentration of membrane proteins playing the key roles in cellular transport, maintenance of cell structure, as well as bioenergetic processes remained almost unchanged, indicating their crucial roles in *E. coli* survival under nutrient-limiting conditions. Moreover, some of the proteins critical for preservation of cell stability and membrane permeability (such as the modulators of FtsH protease, HflK, and HflC) appeared to be steadily present in the populations of mainly non-culturable cells. We also found the continuous increase in the level of elongation factor EFTU1 along the survival process, thus suggesting its essential role in the adaptation process. Interestingly, the level of some proteins (e.g., bacterioferritin) was differently affected by temperature (see above). Finally, the observed depletion of the key components of the Bam complex, insertase YidC, and/or proteins implicated in chemotaxis suggested their redundancy for preserving cell integrity and therefore allowed to save energy during *E. coli* adaptation and survival.
