The Effect of Visible Light on Cell Envelope Subproteome during Vibrio harveyi Survival at 20 °C in Seawater

A number of Vibrio spp. belong to the well-studied model organisms used to understand the strategies developed by marine bacteria to cope with adverse conditions (starvation, suboptimal temperature, solar radiation, etc.) in their natural environments. Temperature and nutrient availability are considered to be the key factors that influence Vibrio harveyi physiology, morphology, and persistence in aquatic systems. In contrast to the well-studied effects of temperature and starvation on Vibrio survival, little is known about the impact of visible light able to cause photooxidative stress. Here we employ V. harveyi ATCC 14126T as a model organism to analyze and compare the survival patterns and changes in the protein composition of its cell envelope during the long-term permanence of this bacterium in seawater microcosm at 20 °C in the presence and absence of illumination with visible light. We found that V. harveyi exposure to visible light reduces cell culturability likely inducing the entry into the Viable but Non Culturable state (VBNC), whereas populations maintained in darkness remained culturable for at least 21 days. Despite these differences, the starved cells in both populations underwent morphological changes by reducing their size. Moreover, further proteomic analysis revealed a number of changes in the composition of cell envelope potentially accountable for the different adaptation pattern manifested in the absence and presence of visible light.


Introduction
Vibrio species are frequently used as models organisms to study the strategies developed by marine bacteria to cope with adverse and changing environments. A large number of studies have demonstrated that the survival of vibrios in the natural environment is largely determined by temperature, and some authors [1,2] have indicated that these bacteria represent an important and tangible barometer sensing the impact of climate change in marine ecosystems.
These effects are more profound during the summer seasons, which are also characterized by more intensive solar radiation. Several studies have reported the complex responses of aquatic bacteria exposed to photosynthetically active radiation (PAR; visible light) (400 to 700 nm). While it positively affects the physiology of autochthonous marine

Vibrio harveyi Strain and Inocula Preparation
A V. harveyi strain ATCC 14126 T was used throughout this study. For inocula preparation, cells were cultured aerobically in marine broth (MB, PanReac AppliChem, Barcelona, Spain) at 26 • C with shaking (120 rpm) until they reached the stationary phase. The cells were harvested by centrifugation (4000× g, 4 • C, 20 min), washed three times with sterile saline solution (1.94% NaCl, w/v) and were suspended afterwards in sterile saline solution.

Survival Experiments
All the glass flasks used for handling V. harveyi cultures were cleaned with H 2 SO 4 (96%, v/v) beforehand, rinsed with deionized water, and kept at 250 • C for 24 h to get rid of residual organic matter.
Erlenmeyer flasks containing 2 L filtered and autoclaved seawater, collected from Port of Armintza in the North of Spain (43 • 26 24 N and 2 • 54 24 W), were inoculated with stationary-phase V. harveyi cells to reach a density of 10 8 cells mL -1 and incubated at 20 • C with shaking (120 rpm) in darkness and exposed to photosynthetically active radiation (PAR) up to 21 days. Illumination was provided by five Sylvania Standard F25W/30 lamps emitting in the 400 to 700 range. The populations received a light intensity of 15.93 W m −2 . Periodically, samples were collected in triplicate for bacterial count, determination of cell size and extraction of membrane proteins.
All the experiments were performed three times. The values presented in datasets are the means of three experiments, and the standard deviations between replicates were less than 12%. The differences between the means were calculated by a one-way analysis of variances. Probabilities that were less than (or equal to) 0.05 were considered to be significant.

Cell Counting and Estimation of Bacterial Size
The total number of bacteria (TNB) was determined according to the procedure described by Hobbie et al. [31]. Viable bacteria, estimated as bacteria with intact cytoplasmic membranes (MEMB+), were counted with Live/Dead BacLight TM kit (Thermo Fisher Scientific Inc., Madrid, Spain) as described by Joux et al. [32]. The number of culturable bacteria (CFU) was determined by spreading cell suspensions on marine agar (MA, PanReac AppliChem, Barcelona, Spain) followed by incubation for 24 h at 26 • C and cell counting.
The length variations of V. harveyi cells during their survival at 20 • C were estimated via image analysis of epifluorescence preparations [33] by using an image analysis system, which included a video camera of high resolution (Hamamatsu 2400, Hamamatsu Photonics, Hamamatsu City, Japan). Digitized images of microscopic fields were analyzed by Scion Image 1.62 a software. In total, 200 cells were measured in each sample. The mean value (x) and the corresponding standard deviation (SD), which defined the size of the cells in the initial inoculate, were used to establish three arbitrary ranges of cell size (≤x-SD, >x-SD and ≤ x+SD, >x+SD) subsequently used to present the time-dependent changes of cell size in V. harveyi populations [5].

Isolation of Membrane Proteins by Using Sodium Carbonate Extraction
To extract membrane proteins, cells were harvested by centrifugation (8000× g, 40 min, 4 • C) and the pellet obtained was suspended in 10 mL of Tris-buffered saline (TBS, pH 8). The cells were collected by centrifugation again (8000× g, 20 min, 4 • C), the cell pellet was briefly washed with TBS, which was decanted and the cell pellet was suspended in 10 mL of TBS followed by addition of 250 µL of Protease Inhibitor Cocktail (Sigma-Aldrich, Madrid, Spain) per g of cell pellet and 90 µL of 2 mM of phenylmethylsulfonyl fluoride (PSMF, PanReac AppliChem, Barcelona, Spain), the suspensions were frozen in liquid nitrogen and stored at −80 • C.
Cells were disrupted by intermittent sonication (SONICS VibraCell TM VCX130 Ultrasonic Cell Disruptor, Sonics & Materials Inc., Newtown, CT, USA) using a 6-mm-diameter probe (65% amplitude setting, 30 s on/45 s off cycles for 3 min in total). Unbroken cells and cellular debris were removed by centrifugation at 6000× g for 20 min at 4 • C. The supernatant fractions were stored on ice while the pellets were suspended in 10 mL of TBS and sonicated under the same conditions as specified above. This procedure was repeated at least three times.
The supernatants obtained from the same samples were combined, diluted (1:1) with 0.2 M sodium carbonate solution, incubated on ice for 1 h with gentle shaking and ultracentrifuged at 115,000× g for 1 h at 4 • C. The supernatants were discarded and protein pellets containing membrane proteins were suspended in 1 mL of TBS.

Protein Identification and Quantification
Analysis of protein samples containing membrane proteins was performed in the Proteomics Core Facility-SGIKER at the University of the Basque Country, using the protocol previously described by González-Fernández et al. [34]. Briefly, 50 µg of total protein were precipitated by using a 2-D Clean-Up kit (GE Healthcare, Bilbao, Spain) according to the manufacturer's instructions. The pellet was suspended in 0.2% RapiGest solution (Waters Corporation, Cerdayola del Vallès, Spain), heated (85 • C, 15 min), reduced with DL-dithiothreitol (DTT, 5 mM), alkylated with iodoacetamide (15 mM) and digested with trypsin (Roche Diagnostics, Leganés, Spain; 2 µg per sample) overnight at 37 • C. RapiGest was inactivated by the addition of HCl at a final concentration of 0.5% and incubation at 37 • C for 40 min. Samples were centrifuged at 16,000× g for 10 min, the supernatant was collected and MassPREP Enolase Digestion Standard (Waters Corporation, Cerdayola del Vallès, Spain) was added as an internal standard for protein absolute quantification. Data independent acquisition analyses were performed in a NanoAcquity UPLC System coupled to a SYNAPT HDMS (Waters Corporation, Cerdayola del Vallès, Spain). A final amount of 0.5 µg (containing tryptic peptides and 100 fmol of MassPREP Enolase Digestion Standard) were loaded onto a Symmetry 300 C18, 180 µm × 20 mm precolumn (Waters Corporation). The precolumn was connected to a BEH130 C18 column (75 µm × 200 mm, 1.7 µm [Waters Corporation, Cerdayola del Vallès, Spain]) and peptides were eluted with a 120 min linear gradient (3 to 40%) of acetonitrile (v/v) followed by a 15 min linear gradient (40 to 60%) of acetonitrile (v/v). Mass spectra (MS) were acquired using a data independent acquisition mode (MSE) described by Silva et al. [35]. Briefly, 1 s alternate MS acquisitions were performed at low (6 eV) and high (12-35 eV ramping) collision energies and the radio frequency (RF) offset was adjusted so that the MS data were acquired from m/z 350 to 1990. [Glu1]-fibrinopeptide B (Sigma-Aldrich) at a concentration of 100 fmol/µL sprayed through the NanoLockSpray source and sampled every 30 s. The obtained spectra were processed with ProteinLynx Global Server v2.4 Build RC7 (Waters Corporation) using the doubly protonated monoisotopic ion of [Glu1]-fibrinopeptide B for mass correction. Protein identification was carried out by using the embedded database search algorithm of the program [36] and a Vibrio harveyi UniProt Knowledgebase (UniProtKB) (version 2020_06, 4950 sequences). For protein identification, the following parameters were adopted: carbamidomethylation of C as a fixed modification; N-terminal acetylation, N and Q deamidation, and M oxidation, as variable modifications; 1 missed cleavage and default automatic precursor and fragment error tolerance. A maximum false positive rate of 5% was allowed. Absolute protein quantification based on peak area intensity of peptide precursors was automatically calculated by ProteinLynx Global Server using Enolase peptides as an internal standard [37]. A total of 648 proteins were confirmed by finding at least three protein-derived peptides in the tryptic digest, 327 proteins were detected in at least two biological replicates and were subsequently used for absolute quantification. Individual absolute quantification values were normalized versus the total protein amount present in the sample. Proteins with a significant (p < 0.05, t-test) increase (>1.5 fold) or decrease (<0.5 fold) in their relative abundance with respect to the initial time (P0) were considered to be differentially affected by survival conditions. UniProt (http://www.uniprot.org/) and KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) databases were used to verify the name and possible function of the proteins (accessed on 1 November 2020). The subcellular localization of many polypeptides annotated as membrane-associated proteins with known functions was further scrutinized by searching for the cognate membrane-binding domains with the PSORTb 3.0 program [38].

Analysis of V. harveyi Persistence at 20 • C
The variations in integrity, viability, culturability, and cell size distribution of V. harveyi populations maintained at 20 • C under nutrient scarcity (i.e., incubation in seawater microcosms) are shown in Figure 1. The numbers of total (TNB) and viable (MEMB+) bacteria remained practically unchanged throughout the experimentation time regardless of PAR irradiation. However, the number of culturable cells (CFU) declined approximately 0.53 and 1.83 log after 21 d of incubation in the absence and presence of illumination, respectively ( Figure 1A,B). The significant loss of culturability for population exposed to PAR, along with the preservation of cell viability, indicated that the major part of the population (98.51%) had likely acquired the VBNC phenotype at the end of the incubation time.
The size of the starved V. harveyi cells varied along the survival process, in fact, the cells reduced considerably their length during incubation, from a medium length of 1.93 µm at the beginning of the experiments to 0.97 or 0.92 µm after 21 d of incubation in the absence or presence of illumination, respectively. These phenotypical changes led to the appearance of cells with the coccoid-like morphology associated with the VBNC state in Vibrio species [39]. The length reduction was more profound when the experiments were carried out under illumination. The fraction of shorter cells (length ≤ 0.91 µm) increased nearly 3.6 times during exposure to visible light and about 2.7 times in darkness when compared to the initial values ( Figure 1C,D), ultimately reaching 43.5 and 57.5%, respectively. Moreover, the cells with a length exceeding 1.74 µm were not found after 21 days of incubation under both conditions. The size of the starved V. harveyi cells varied along the survival process, in fact, the cells reduced considerably their length during incubation, from a medium length of 1.93 µ m at the beginning of the experiments to 0.97 or 0.92 µ m after 21 d of incubation in the absence or presence of illumination, respectively. These phenotypical changes led to the appearance of cells with the coccoid-like morphology associated with the VBNC state in Vibrio species [39]. The length reduction was more profound when the experiments were carried out under illumination. The fraction of shorter cells (length ≤ 0.91 μm) increased nearly 3.6 times during exposure to visible light and about 2.7 times in darkness when compared to the initial values ( Figure 1C,D), ultimately reaching 43.5 and 57.5%, respectively. Moreover, the cells with a length exceeding 1.74 µ m were not found after 21 days of incubation under both conditions.

Changes of Membrane Subproteome during Permanence at 20 °C
From survival assays carried out in darkness or upon exposure to visible light, the samples were collected at different incubation times: immediately after inoculation (P0), 6 days (P1), and 21 days (P2). Proteins detected in at least two biological replicates, whose biological functions were previously defined or could be inferred by homology, were selected for further analysis. The dataset of proteins contained a high number of predicted cytosolic proteins (35.5%). Some of them belonged to cytosolic subunits of membrane protein complexes or were annotated as proteins that can transiently be associated with the membrane [5]. The above properties may explain the presence of these "cytosolic" proteins in the membrane fraction. After determining the composition of the membrane subproteomes, the identified proteins were sorted according to their biological functions and grouped to form the following categories of proteins involved in: (i) maintenance of cell structure, (ii) transport, (iii) bioenergetics, (iv) signal transduction, (v) protein synthesis, degradation and turnover, or other (vi) miscellaneous functions. rveyi cells varied along the survival process, in fact, the length during incubation, from a medium length of 1.93 iments to 0.97 or 0.92 μm after 21 d of incubation in the ion, respectively. These phenotypical changes led to the oid-like morphology associated with the VBNC state in uction was more profound when the experiments were he fraction of shorter cells (length ≤ 0.91 μm) increased e to visible light and about 2.7 times in darkness when igure 1C,D), ultimately reaching 43.5 and 57.5%, respeclength exceeding 1.74 μm were not found after 21 days ons.
ome during Permanence at 20 °C d out in darkness or upon exposure to visible light, the nt incubation times: immediately after inoculation (P0), teins detected in at least two biological replicates, whose sly defined or could be inferred by homology, were seataset of proteins contained a high number of predicted f them belonged to cytosolic subunits of membrane prod as proteins that can transiently be associated with the rties may explain the presence of these "cytosolic" profter determining the composition of the membrane subs were sorted according to their biological functions and ategories of proteins involved in: (i) maintenance of cell nergetics, (iv) signal transduction, (v) protein synthesis, er (vi) miscellaneous functions. The size of the starved V. harveyi cells varied along the survival process, in fact cells reduced considerably their length during incubation, from a medium length of μm at the beginning of the experiments to 0.97 or 0.92 μm after 21 d of incubation in absence or presence of illumination, respectively. These phenotypical changes led to appearance of cells with the coccoid-like morphology associated with the VBNC sta Vibrio species [39]. The length reduction was more profound when the experiments w carried out under illumination. The fraction of shorter cells (length ≤ 0.91 μm) incre nearly 3.6 times during exposure to visible light and about 2.7 times in darkness w compared to the initial values ( Figure 1C,D), ultimately reaching 43.5 and 57.5%, res tively. Moreover, the cells with a length exceeding 1.74 μm were not found after 21 of incubation under both conditions.

Changes of Membrane Subproteome during Permanence at 20 °C
From survival assays carried out in darkness or upon exposure to visible light samples were collected at different incubation times: immediately after inoculation 6 days (P1), and 21 days (P2). Proteins detected in at least two biological replicates, w biological functions were previously defined or could be inferred by homology, wer lected for further analysis. The dataset of proteins contained a high number of predi cytosolic proteins (35.5%). Some of them belonged to cytosolic subunits of membrane tein complexes or were annotated as proteins that can transiently be associated with membrane [5]. The above properties may explain the presence of these "cytosolic" teins in the membrane fraction. After determining the composition of the membrane proteomes, the identified proteins were sorted according to their biological functions grouped to form the following categories of proteins involved in: (i) maintenance of structure, (ii) transport, (iii) bioenergetics, (iv) signal transduction, (v) protein synth degradation and turnover, or other (vi) miscellaneous functions.

Changes of Membrane Subproteome during Permanence at 20 °C
From survival assays carried out in darkness or upon exposure to visibl samples were collected at different incubation times: immediately after inocul 6 days (P1), and 21 days (P2). Proteins detected in at least two biological replica biological functions were previously defined or could be inferred by homolog lected for further analysis. The dataset of proteins contained a high number of cytosolic proteins (35.5%). Some of them belonged to cytosolic subunits of mem tein complexes or were annotated as proteins that can transiently be associate membrane [5]. The above properties may explain the presence of these "cyto teins in the membrane fraction. After determining the composition of the mem proteomes, the identified proteins were sorted according to their biological fun grouped to form the following categories of proteins involved in: (i) maintena structure, (ii) transport, (iii) bioenergetics, (iv) signal transduction, (v) protein degradation and turnover, or other (vi) miscellaneous functions.

Changes of Membrane Subproteome during Permanence at 20 • C
From survival assays carried out in darkness or upon exposure to visible light, the samples were collected at different incubation times: immediately after inoculation (P0), 6 days (P1), and 21 days (P2). Proteins detected in at least two biological replicates, whose biological functions were previously defined or could be inferred by homology, were selected for further analysis. The dataset of proteins contained a high number of predicted cytosolic proteins (35.5%). Some of them belonged to cytosolic subunits of membrane protein complexes or were annotated as proteins that can transiently be associated with the membrane [5]. The above properties may explain the presence of these "cytosolic" proteins in the membrane fraction. After determining the composition of the membrane subproteomes, the identified proteins were sorted according to their biological functions and grouped to form the following categories of proteins involved in: (i) maintenance of cell structure, (ii) transport, (iii) bioenergetics, (iv) signal transduction, (v) protein synthesis, degradation and turnover, or other (vi) miscellaneous functions.
The proteins that did not show any significant variation in abundance (i.e., they were not upregulated [>1.5-fold] or downregulated [<0.5-fold]) during the survival experiments are listed in Table 1.
This group includes proteins involved in (i) maintaining the structure of cell envelope (e.g., lipoproteins [D0XEL2_VIBH1, D0XD95_VIBH1], components of the β-barrel assembly machinery [BAM] complex, membrane protein insertase YidC and rod shape-determining protein MreB); (ii) transmembrane transport (ion transporters as OmpU or D0XAK6_VIBH1 porin, vitamin B12 transporter BtuB, maltose operon periplasmic protein [MalM] and others) and in protein translocation (YajC, SecA, and SecD subunits) and secretion (Type II secretion system core protein G, a TolC family protein or multidrug resistance protein MdtA); as well as (iii) proteins whose function is related to bioenergetics (namely, different subunits of ATP synthase, cytochrome b or subunits of Na(+)-translocating NADH-quinone reductase); (v) protein biogenesis (HflC and HflK proteins); and (vi) translation (elongation factors EF-Tu and EF-G). Table 1. Membrane proteins of V. harveyi ATCC 14126 T whose level did not show a significant change (>1.5 or <0.5 fold change) after 6 days (P1), and 21 days (P2) of starvation in seawater at 20 • C with respect to initial values (P0). In addition to proteins listed in Table 1, there was a group of proteins whose level was affected by experimental conditions (Table 2). In other words, we found that the level of numerous proteins was altered after 21 d of starvation at 20 • C both in the absence and presence of PAR irradiation.

Protein Accession Number
Namely, some components of phosphotransferase systems (PTS) (D0X8N6_VIBH1, D0XD90_VIBH1), TatA protein translocase, cytochrome c5, so-called methyl-accepting chemotaxis proteins (D0XEK4_VIBH1, D0XEY1_VIBH1, D0X9J1_VIBH1, D0XEC5_VIBH1, D0XHW4_VIBH1, D0XGG1_VIBH1, D0X5R4_VIBH1, D0X9F5_VIBH1, and D0XCQ6_VIBH1) and flagellin became undetectable (Table 2), whereas the level of YhcB readily declined after 21 d. On the contrary, only a few proteins (e.g., mechanosensitive ion channel MscS, bacterioferritin and catalase-peroxidase, see Table 2) undetectable in the initial inoculates (time P0) were detected after long-term starvation at 20 • C under both conditions. These proteins were detected earlier (6 d) in populations maintained under illumination. Table 2. Changes in the level of membrane proteins of V. harveyi ATCC 14126 T subjected to starvation in seawater at 20 • C. The data are presented for the initial population (time P0) and populations analyzed after starvation of V. harveyi for 6 (P1) and 21 (P2) days in the absence (−) and presence (+) of illumination.  In addition, there was a group of membrane proteins differentially affected by incubation in the presence vs. absence of illumination. For instance, several proteins related to bioenergetic (e.g., cytochrome c oxidase subunit CcoO, cytochrome c4, ubiquinolcytochrome c reductase iron sulfur subunit, ubiquinol-cytochrome c reductase cytochrome c1) and others (penicillin-binding protein activator LpoA and proteases, D0XAK5_VIBH1 and ATP-dependent Zn protease) were downregulated only in populations that were exposed to PAR.

Protein Accession
Vice versa, several proteins, in particular those related to transport of phosphate and glucose, one isoform of ATP synthase subunit beta, OmpK, OmpA-like protein D0X6J9_VIBH1, and ATP-dependent zinc metalloprotease FtsH, were downregulated (or undetectable) only in the populations maintained in darkness. The upregulation of several proteins was likewise light-dependent. Namely, while some proteins (ATP synthase subunit c, subunit I of cytochrome d ubiquinol oxidase or NAD (P) transhydrogenase subunit beta) were upregulated in darkness, the same polypeptides were undetectable in the populations exposed to PAR. Similarly, illumination of starved V. harveyi cells for 21 days led to an increase in the level of some structural and transport-related proteins including the outer membrane protein Slp, YcfL peptidoglycan-associated lipoprotein D0XFJ5_VIBH1, general secretion pathway protein D, outer membrane protein TolC and agglutination protein D0XI94_VIBH1.

Discussion
The life style and persistence of microorganisms in natural aquatic systems are greatly dependent on diverse abiotic and biotic stress factors (e.g., suboptimal salinity and pH, temperature up-and downshifts, nutrient availability, solar radiation, predation, etc.). A number of studies, which were aimed at addressing the effect of these environmental factors, previously employed V. harveyi as a model organism. Although the individual impact of some stress factors on V. harveyi is well characterized [40,41], little is known about their joined action.
Here we studied the combined effects of nutrient limitation, temperature, and visible light on V. harveyi adaptation in seawater microcosms. A typical and well-documented survival response of several Vibrio species under nutrient limitation is the acquisition of the VBNC state [4,8,9,42,43], which is more frequently observed at low temperatures rather than at temperatures ranging from 13 to 22 • C [9,26,44,45]. Consistently, we found that, unlike in the experiments carried out at 4 • C [5], V. harveyi strain ATCC 14126 T populations did not acquire the VBNC state after at least three weeks of incubation in seawater (nutrient scarcity, darkness) at 20 • C. Thus, our data indicate that, similar to the key role of temperature in adaptation of V. parahaemolyticus [46] or V. vulnificus [10,47], it also determines V. harveyi survival responses. Moreover, the persistence of V. harveyi populations was accompanied by a progressive reduction in cell size. This observation supports the idea that the initial response of V. harveyi to starvation leads to morphological changes rather than an immediate transition to the VBNC state [5,26].
In addition to nutrient availability and temperature, exposure to visible light is another important stress factor known for its contribution to Vibrio growth and survival [27][28][29]. To compare the long-term adaptation of V. harveyi in the absence and presence of illumination, experiments were also carried out upon exposure of V. harveyi populations to visible light. Our data demonstrate that illumination with visible light not only accelerates the bacterial size reduction but also decreases cell culturability, thus suggesting the cell entry into the VBNC state. However, the effect of visible light on transition to the VBNC state might be less profound than that in other bacteria, in which the acquisition of this phenotype can occur within a few days (e.g., in Escherichia coli [48,49] or Enterococcus faecalis [48,50]) or even hours (e.g., in Pseudomonas aeruginosa [51]).
As exposure to visible light can provoke oxidative stress, the prolong persistence of Vibrio spp. population could be due to their ability to activate protective mechanisms mitigating the damaging effects of light. In fact, Rees et al. [52] speculated on the protective role of bacterial luminescence against oxidative stress. Consistently, other authors have indicated that bacterial bioluminescence may play an important role in detoxification of reactive oxygen species [53,54] and in stimulating DNA repair [55,56]. Another important response to oxidative stress involves catalase overproduction. It has been described for different Vibrio species subjected to abiotic stress [57][58][59]. In addition, recent studies described overexpression of other enzymes conferring protection against the toxic effects of H 2 O 2 and reactive oxygen species during V. harveyi permanence in seawater at different temperatures [24,26]. Therefore, our observation that catalase peroxidase becomes detectable in populations exposed to visible light earlier (i.e., at time P1) than in those lacking illumination could indicate a role of this enzyme in sustaining V. harveyi resistance to visible light.
V. harveyi survival is determined by the capacity of this bacterium to respond to changing environment by reprogramming gene expression, thus affecting the entire proteome. Owing to the essential role of cell envelope in bacterial adaptation to stress, the second part of this study was focused on determining the stress-related changes in V. harveyi membrane subproteome and discussing their possible contribution to cell resistance to stress. Analysis of cell envelope subproteome revealed that the level of some membrane proteins with key roles in maintenance of cellular structure, transport, and bioenergetic processes remained unchanged until 21 d (Table 1). Moreover, some of those proteins (i.e., OmpW, maltose operon periplasmic protein, vitamin B12 transporter BtuB, several ATP synthase subunits, Na(+)-translocating NADH quinone reductase subunits or Hflk protein) were also maintained in VBNC populations induced during exposure to cold temperature [5]. Therefore, these proteins appear to constitute a pool of proteins inherently present in the viable (culturable or nonculturable) cells under starvation. Additionally, the level of many structural proteins detected in this study was preserved or even increased under stress. They include lipoproteins, Bam factors and other outer membrane proteins (e.g., OmpW porin [60]) apparently essential for maintaining the integrity of the outer cell membrane throughout the survival process.
MreB (Table 1) is another protein whose level remained nearly the same during the survival process. This protein plays an important role in cell shape maintenance and division [61]. While sequence analysis predicts that it is a cytoplasmic protein, there is some evidence that suggests the transient association of this protein with the membrane [62]. Moreover, Chiu et al. [63] demonstrated that the MreB protein could be detected close to the membrane in starved cells. Although previous work had shown that MreB became undetectable in the membrane fraction during the first 12 h of permanence at 4 • C [5], we did not see any significant time-dependent changes in MreB levels for populations incubated at 20 • C in the presence or absence of illumination (present study). These results suggest that MreB association with the membrane (and therefore its cellular localization) in the starved cells are likely dependent on temperature.
The permanent presence of other membrane proteins (Table 1) is likely linked to their essential functions (e.g., protein transport) during starvation. Indeed, our data revealed the constant presence of proteins (D0X124_VIBH1, D0X520_VIBH1) that were components of Types I and II secretion systems. Moreover, the concentration of some components (D0X523_VIBH1, D0X7A6_VIBH1, D0XI94_VIBH1) has even increased in the illuminated populations after 21 days. Concerning the Sec-mediated transport in V. harveyi, the abundance of SecA, SecD, and YajC proteins also remained unaltered along survival, while TatA became already undetectable after 6 days of starvation, thereby suggesting that nutrient limitation favored Tat-independent secretion. Unlike the mechanisms affecting protein secretion in V. harveyi, Brucella suis response to starvation appears to limit the Sec-dependent transport possibly to reduce the overall metabolic activity and energy consumption [64]. Similarly, Campylobacter jejuni persistence in tap water at different temperatures [65] has been reported to support the Tat-dependent (rather than Sec-dependent) transport.
Elongation factors are essential bacterial proteins, and EF-TU has been described as a cytoplasmic chaperone [42,66] involved in protein synthesis and other cellular processes [67,68]. The higher abundance of EF-TU in the subproteomes of the stressed cells [5,18] and its upregulation upon exposure to stress [5,69] could imply the involvement of EF-TU in cell adaptation. Nevertheless, the lack of significant variations in the level of these proteins in populations examined in the present study does not support the general role of these protein factors in bacterial adaptation to stress.
Besides the continuous presence of many proteins apparently essential for both the normal growth and cell survival (Table 1), there was a group of polypeptides differentially affected by starvation (Table 2). Variations in their levels could be attributable to stress adaptation induced by nutrient deprivation, and subsequent energy and carbon depletion. In particular, we found that multiple methyl-accepting chemotaxis proteins became rapidly undetectable in V. harveyi populations maintained in seawater at 20 • C regardless of expo-sure to visible light, thus mimicking V. harveyi response to starvation at 4 • C [5]. Similarly, the level of flagellin declined after 21 days. These results agree with previous observations obtained with starved Vibrio S14 cells by Malmcrona-Friberg et al. [70]. They found that most cells lost motility under starvation and suggested that the chemosensory system could be shutdown already after first 24 h of starvation. Likewise, our findings are also consistent with the results of Stretton et al. [71], who showed the detachment of flagellum during the first days of starvation and argued that due to high energy cost of synthesis, assembly, and function of flagellum, the transition to the non-motile (but metabolically active) state would be more beneficial for cells under starvation. In a similar study, Chen and Chen [72] likewise demonstrated that V. vulnificus motility diminished along the time of permanence under nutrient scarcity conditions. Moreover, some authors [73][74][75] revealed that starvation not only leads to the loss of motility but also increases cell adhesion. Therefore, the loss of chemotactic activity and motility observed in our study for V. harveyi populations under starvation could be an important strategy enabling to save energy and ensure cell survival under stress.
Dissolved iron concentrations in open ocean surface waters typically stay below 0.2 nM [76], thus establishing iron-limiting conditions for marine organisms. Several authors [77,78] indicated that the control of iron homeostasis and responses to oxidative stress are interdependent. In other words, iron is not only an essential element for bacterial growth but it is also a toxic metal able to promote the formation of reactive oxygen species (ROS). They cause oxidative stress, consequently elevating the level of catalase peroxidase. In previous work, the success of V. harveyi permanence in seawater microcosms at 4 • C was linked to iron homeostasis involving bacterioferritin during the entry into the VBNC [5]. Regarding the populations exposed to starvation at 20 • C (present study), bacterioferritin, which was undetectable at the beginning of experiments, becoming expressed even though no culturability loss was detected.
A similar expression pattern was observed for the mechanosensitive ion channel protein MscS. It seems likely that in addition to its role in coping with osmotic stress, this protein is also involved in cell wall repairing to protect against sustained stress [79] stimulated by visible light.
Taken together, our results demonstrate that V. harveyi adaptation to starvation at 20 • C induces morphological changes leading to cell size reduction and acquisition of the coccoid-like morphology, apparently triggering the acquisition of the VBNC state by the cell exposed to visible light. This finding suggests that the exposure to visible light along with variations in temperature, salinity, and others, might promote the V. harveyi entry to the VBNC state in aquatic systems. Moreover, several studies have proposed that the VBNC cells can potentially preserve their capacity to elicit infections [6,39,80]. Further analysis of cell envelope subproteome revealed that a number of membrane proteins playing the key roles in maintenance of major cell envelope functions constitute a pool of proteins continuously present in viable (culturable and nonculturable) V. harveyi ATCC14126 T populations exposed to stress. The presence of these proteins enables to sustain the key functions of the membranes, such as selective permeability and transport. In contrast, nutrient depletion leads to the loss of proteins involved in cell mobility and chemotaxis. In addition, starvation could potentially affect iron homeostasis largely dependent in the stressed populations on bacterioferritin. Likewise, as exposure to visible light potentially increases oxidative stress, there was a continuous presence of catalase and peroxidase proteins in the long starved cells. Taken together, our proteomic data indicate that adjustments in the cell envelope subproteome were more profound in the case of populations exposed to visible light.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.