Proteomic Insight into Functional Changes of Proteorhodopsin

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Journal of Proteome Research

Proteomic insight into functional changes of proteorhodopsincontaining bacterial species Psychroflexus torquis under different illumination and salinity levels Shi Feng1*, Shane M. Powell1, Richard Wilson2 & John P. Bowman1

1. Food Safety Centre, Tasmanian Institute of Agriculture, University of Tasmania, Sandy Bay, Hobart, Tasmania 7005, Australia. 2. Central Science Laboratory, University of Tasmania, Hobart, Tasmania, Australia. *Corresponding author: Email: [email protected]; Tel +61 03 6226 6380; Fax +61 03 6226 7444.

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ABSTRACT The

extremely

psychrophilic

proteorhodopsin-containing

bacterial

species

Psychroflexus torquis is considered a model sea-ice microorganism, which has adapted to an epiphytic lifestyle. So far not much is known about proteorhodopsinbased phototrophy and associated life strategies of sea ice bacteria although it has been previously shown that P. torquis can gain growth advantage from light using a proteorhodopsin proton pump the activity of which is influenced by environmental salinity. The comprehensive quantitative proteomic study performed here indicated that P. torquis responds to changing salinity and illumination conditions. Proteins in the electron transfer chain were down-regulated at a sub-optimal salinity level, TonBdependent transporters increased in abundance under supra-optimal salinity and decreased under sub-optimal salinity. In addition, several anaplerotic CO2 fixation proteins and three putative light sensing proteins that contain PAS and GAF domains became more abundant under illumination. Furthermore, central metabolic pathways (TCA and glycolysis) were also induced by both salinity stress and illumination. The data suggests that P. torquis responded to changes in both light energy and salinity to modulate membrane and central metabolic proteins which are involved in energy production as well as nutrient uptake and gliding motility processes that would be especially advantageous during the polar summer ice algal bloom. Key Words: Proteorhodopsin, Sea ice, Survival strategies, Proteomics

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INTRODUCTION Sea ice is a highly variable and dynamic environment covering a maximum 13% of the Earth’s surface area.1 Sea ice is dominated by extreme gradients of temperature and salinity. High salinity in sea-ice brine channels and pockets caused by extrusion of salt during ice formation is a major stressor for sea ice organisms, which may experience salinity up to three times higher than seawater.1 Nevertheless, sea-ice habitats are a major source of biomass in sub-polar and polar ocean regions1, 2 and support a diverse microbial community of phytoplankton, bacteria, archaea and viruses. The sea ice microbial community is highly metabolically active and sea ice bacteria have higher metabolic activity than some offshore temperate marine systems. This suggests the potential for substantial biological production that can be exported in polar regions.3,

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However, over the last decade, a severe loss of Arctic Ocean

multi-year sea ice has occurred, and the effect of continuous global warming may result in sea ice ecosystems disappearing in many regions. So far, most research has focused on characterizing the overall sea ice microbial community and the factors that influence them.5 Here, we present a quantitative proteomic study on the proteorhodopsin-containing sea ice dwelling bacterial species, Psychroflexus torquis, using a gel-free label-free based approach. P. torquis is an extreme psychrophile belonging to the family Flavobacteriaceae, phylum Bacteroidetes.6 Our genomic analysis of the P. torquis type strain revealed the presence of a proteorhodopsin (PR) gene and a cognate carotenoid monooxygenase involved in synthesis of its retinal co-factor.7 PR is a retinal binding transmembrane protein that is found in aquatic bacteria and are mainly characterized as light-driven proton pumps.8 Several recent studies demonstrated that some PR can function as sodium ion pumps.9, 10 However the sequence comparison

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analysis showed that PR harbored by P. torquis is phylogenetically closest to proton pumping proteorhodopsins rather than sodium ion pump-type PRs (Figure S1),9 thus suggest the PR in P. torquis is likely restricted to proton pumping. Proteorhodopsinbearing bacteria are widely distributed in marine ecosystems and also occur in sea ice microbial communities though the predominance in sea ice is so far unknown.3, 11 A significant amount of PR gene expression in sea-ice samples corresponding to the presence of metabolically active cells has been reported,3 which suggests that the PR gene is functional within sea ice. It is hypothesized that PR can provide light derived energy to bacteria under oligotrophic conditions. Another study showed that PR can function as an energy generating system replacing cellular respiration in E. coli.12 It was also shown that in Candidatus “Pelagibacter ubique” the ATP-dependent nutrient transporter activity increased in starved cells under light.13 However, the available data does not consistently show light stimulated growth in PR-containing bacteria.11 Thus, more studies are needed to reveal PR’s ecological functionality and significance. We recently reported14 that the growth of P. torquis is stimulated by light under osmotic pressure, and that the stimulatory effect and the PR protein abundance were regulated by salinity levels as well as by light intensity. This demonstrated the possibility that PR might be an adaptive strategy used by microorganisms under stress conditions associated with their specific econiche. However, the complete physiological role of PR-mediated proton transport in the overall cellular metabolism and bacterial growth remains unclear. To the best of our knowledge, no comprehensive proteomic studies that describe the physiology and metabolism of proteorhodopsin-containing sea ice dwelling bacteria have been performed. Our present proteome analysis describes the life style and responses of P. torquis to salinity and light providing insight into the role of PR in this microorganism in the

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sea-ice ecosystem.

MATERIALS AND METHODS Culture experiments The bacterial strain used in this study was Psychroflexus torquis ATCC 700755T. It was originally isolated from coastal sea ice from Prydz Bay, Vestfold Hills, Antarctica.6 Cells were grown on modified marine agar consisting of 5g of proteose peptone (Oxoid), 2g of yeast extract (Oxoid), 15g of agar, and the relevant amount of sea salt (Red Sea) in 1L of distilled water. To achieve four different salinities, 17.5g, 35g, similar to natural sea water salinity and considered the optimal salinity for growth in this study, 52.5g and 70g of sea salt were added per litter to achieve the target salinities. Considering that light intensity in sea ice is low, especially at the bottom of the ice1, for each salinity, cultures were exposed to three light conditions: high light (20-30 µmol photons m-2 s-1); low light (3-4 µmol photons m-2 s-1) and complete darkness (0 µmol photons m-2 s-1). In all experiments, light was provided from 30W fluorescent lamps. 100µL of working culture (6.0 log10 CFU/ml) was used for spread plates. Cultures were incubated under oxic conditions at 5 ºC for 3 weeks. Three biological replicates were performed for this study, each biological replicates contained two technical replicates. The biomass was harvested from agar plates, each harvest was made from three plates and the biomass from all three plates was combined together for protein extraction and digestion.

Protein extraction and post-treatment Harvested cells were lysed in 1 ml 50 mM Tris-HCl buffer (pH 7.0) by sonication in an ice bath, with 10s of sonication with a 10 s wait period, cycled 15 times until the

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opaque cell suspension became translucent. The suspensions were then centrifuged at 16,000 × g for 25 min at 4 °C. The supernatant protein was precipitated using trichloroacetic acid and the protein pellets were then treated with 0.2 M NaOH to improve subsequent solubilisation.15 The pellets were then solubilised using 100 µl 50 mM Tris-HCl buffer (pH 7.0) and protein concentration determined using the Bradford assay (Bio-Rad). Volumes of samples containing 50 µg of soluble protein extract were then reduced in a solution of 50 mM dithiothreitol, 100 mM ammonium bicarbonate for 1 h at room temperature. The samples were then alkylated with 200 mM iodoacetamide in 100 mM ammonium bicarbonate for 1h at room temperature. After alkylation, the reduction of proteins was repeated and they were digested in a buffer (50 mM ammonium bicarbonate, 1 mM calcium chloride) that contained sequencing grade modified trypsin (Promega), at a sample protein to trypsin ratio of 25:1, at 37 °C with gentle shaking overnight. Digestion was stopped by acidification with 10 µl of 10% (vol/vol) formic acid. The samples were then centrifuged for 5 min at 14,000 × g to remove any insoluble material, and an aliquot (100-200 µl) of peptides was transferred to HPLC vials for mass spectrometric analysis. Samples were prepared with three biological replicates and for each biological replicate, two technical replicates were performed.

NanoLC-Orbitrap Tandem Mass Spectrometry The separation of peptides utilized a Surveyor Plus HPLC system fitted in line with an LTQ-Orbitrap XLmass spectrometer (ThermoFisher Scientific). Aliquots of peptide samples were loaded at 0.05 mL/min onto a C18 capillary trapping column (Peptide CapTrap, Michrom BioResources) controlled by an Alliance 2690 separations module (Waters). Peptides were then separated on an analytical HPLC column packed with 5

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µm C18 media (PicoFrit Column, 75µm internal diameter, 15 µm i.d. pulled tip, 10 cm, New Objective) using four linear gradient segments controlled using a Surveyor MS Pump Plus (ThermoFisher Scientific) at 200 nl/min The solvent series included initially 5% acetonitrile in 0.2% formic acid (solvent A) shifting up to 90% acetonitrile in 0.2% formic acid (solvent B). The 4 stage process where solvent B gradually replaced solvent A comprised: 0–10% solvent B over 7.5 min; 10–25% solvent B over 50 min, 25–55% B over 20 min and then 55–100% solvent B over 5 min. This process was followed by re-equilibration of the column with solvent A for 15 min. The LTQ-Orbitrap XL was controlled using Xcalibur 2.0 software (ThermoFisher Scientific) and operated in data-dependent acquisition mode whereby the survey scan was acquired in the Orbitrap with a resolving power set to 60,000 (at 400 m/z). MS/MS spectra were concurrently acquired in the LTQ mass analyzer on the seven most intense ions from the FT survey scan. Charge state filtering, where unassigned and singly charged precursor ions were not selected for fragmentation, and dynamic exclusion (mass window 10ppm repeat count, 1; repeat duration, 30s; exclusion list size, 500), was used. Fragmentation conditions in the LTQ were 35% normalized collision energy, activation q of 0.25, 30 ms activation time, and minimum ion selection intensity of 500 counts.

Protein identification and bioinformatics The acquired MS/MS data were converted to .mzXML pleak list files from .RAW files using the msConvert command in Proteowizard. The MS/MS data was searched against the proteome of P. torquis ATCC 700755T (NCBI accession code CP003879, 3526 protein coding genes) employing X!Tandem running in the open source Computational Proteomics Analysis System environment.16 For searches parent ion

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tolerance of 20 ppm and fragment ion mass tolerances of 0.5 Da were used, and enzyme cleavage was set to trypsin, allowing for a maximum of two missed cleavages. Amino acid residue alterations were also accounted for including: Scarboxamidomethylation of cysteine residues specified as a fixed modification; and cyclisation of N-terminal glutamine to pyroglutamic acid, deamidation of asparagine, hydroxylation of proline and oxidation of methionine specified as variable modifications. Proteins identifications were assessed by the Peptide Prophet and Protein Prophet algorithms.17 Protein identifications were filtered by assigning a Protein Prophet and Peptide Prophet Probability > 0.9. This filtration constrains the protein false discovery rate to < 1%. Spectral counting was used to determine relative protein abundance taking into account the number of amino acid residues.18 In order to acquire the maximum coverage of proteins from each illumination and salinity treatment, spectra obtained from all three biological and two technical replicates samples were pooled together in this study

Statistical Analysis of Mass Spectrometry Data The spectral count (SpC) output of the mass spectrometry data, filtered using Protein Prophet, was used to determine relative protein abundance. Fold changes in different protein expression levels were calculated based on the averaged SpC (for three biological replicates). The total spectral count normalization approach was used as described before.19 Significant differences in protein SpC were normalized and tested using beta-binomial distribution analysis implemented in R as previously described.19 CAP analysis20 of SpC data was carried out in Primer 6 (Primer-E, Plymouth, UK) with data transformed to the fourth root. The mass spectrometry proteomics data have been

deposited

to

the

ProteomeXchange

8


Consortium

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(http://www.proteomexchange.org) via the PRIDE partner repository21 with the dataset identifier PXD000673. Examination of Gliding Motility The gliding motility of P. torquis was examined under different salinity and illumination levels. Modified marine agar media with the different salinity levels described above were prepared and sterilized glass slides were imbedded to form a thin layer of agar on top of the slides. 10µL of working culture (6.0 log10 CFU/ml) was then inoculated on the glass slides and incubated under oxic conditions at 5 ºC for 3 weeks under the illumination conditions described above. Slides were subsequently excised and examined via light microscopy.

RESULTS AND DISCUSSION Growth conditions, proteome properties and broad changes in the proteome of Psychroflexus torquis This study used Psychroflexus torquis ATCC 700755T, which has a completely sequenced genome and contains 3572 predicted genes.7 It was originally isolated from Eastern Antarctic coastal sea ice but has a bipolar distribution.22 It is a slow growing organism, which takes more than 25 days to reach stationary phase in broth culture (Figure S2) and produces more biomass growing on agar than in liquid culture. The biomass for proteomic analysis was harvested from agar during late expoential phase. In addition to better cell yields the experiments were performed on agar plates because of the difficulties of separating cells from liquid culture. This is due to the propensity of P. torquis ATCC 700755T to produce prodigious amounts of exopolysaccharide resulting in cultures with substantially increased vsicosity which hindered sedimentation. Furthermore the EPS strongly reacts with protein detection

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analysis methods. More importanly, the influence of illumination is modeled better with an agar system than liquid media because culturing on agar provides evenly distributed illumination conditions. It also provides better oxic condition compared to broth culture and the solid surface for the biomass formation seems more suitable for studying the essentially epiphytic P. torquis. Analysis of the proteomes of cultures grown under a range of salinity and illumination conditions produced approximately 750,000 tandem mass spectra (Table S1 in the supplemental material). A total of 1406 proteins passed identification filtration criteria. TonB-dependent transporters, amino acid metabolism related proteins, central metabolism related proteins, membrane bioenergetics-associated proteins, and various uncharacterized proteins comprised the majority of high abundance proteins (Figure S3). The spectral count data of cells grown under different conditions were normalized and compared using canonical analysis of principal coordinates (CAP). A PERMANOVA test showed p (perm) = 0.0001 for the salinity factor and p (perm) = 0.0004 for the illumination factor. Furthermore, protein level volcano plots of log 2 ratio and the p value between different illumination levels are shown in Figure 1B and 1C to identify differentially expressed proteins. On average, 195 and 120 proteins (13.8% and 8.5% of 1406 detected proteins) among all salinity level changed significantly (P < 0.05 and fold change over 2.0 as the cutoff), at high and low illumination levels respectively. Of these 129 proteins were up-regulated and 91 proteins were down-regulated under high illumination conditions. In comparison 115 proteins were up-regulated and 58 proteins were down-regulated under low light conditions. Therefore, the volcano plots suggest that high levels of illumination induced a more significant effect on protein abundances (Figure 1B, 1C). It is worth noting that the abundance of only 60 proteins were significantly different when high

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and low illumination conditions were compared (Figure S4) indicating only subtle changes occur in the proteome expression pattern between high and low light conditions. Overall, sub-optimal salinity (17.5 g.L-1) levels and high light intensity among all factors were the most clearly influential factor among other salinity and light conditions tested.

Salinity and illumination have different effects on membrane bioenergetics Sea-ice ecosystems are hotspots for extremophiles, in which strong variations of salinity and light under partially sub-zero temperatures are experienced. Salinity may be a major stressor for sea-ice organisms since they can experience salinities ranging from three times that of seawater to hyposaline conditions when the ice thaws (1). Consistent with this hypersaline environment, P. torquis has a Na+-translocating NADH:quinone oxidoreductase (NQR) like other marine and halophilic bacteria.23-25 Our results showed that all NQR subunits were significantly down-regulated at a sea salt salinity of 17.5g·L-1 (sub-optimal for growth of P. torquis). As the salinity increased, the NQR level increased in abundance (Figure S5). P. torquis does not encode a bc1 complex (known as complex III) in the electron transport respiratory chain. Instead, like other aerobic members of phylum Bacteroidetes, P. torquis encodes components of the alternative complex III (ACIII) that is postulated to function as a menaquinol:cytochrome c oxidoreductase.24,26 However, based on structural analysis and the fact that no other proteins can replace its activity, ACIII is presumed to have proton pumping and electron transfer capabilities.27 Most proteins in the electron transfer chain were only reduced in abundance at low salinity (17.5g·L-1) levels, and did not show significant changes under the hypersaline conditions tested (Figure S5). Interestingly, the overall PR level

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decreased significantly at 17.5g·L-1 compared to 35g·L-1 (4.5 fold change on average), and was also reduced at 52.5g·L-1 and 70g·L-1 by 1.3 fold and 2 fold respectively (Figure S5). This could be due to energy limitation under stressful conditions. Thus, the microorganism may potentially devote more effort to other life strategies such as stress defense, nutrient uptake, adhesion and gliding. At salinities of 17.5g L-1 and 35g L-1 NQR subunits became more abundant under illumination, whereas, at other salinities, NQR levels remained unaffected by illumination. The effect of illumination on ACIII and complex IV was weak (Figure 2, Table S2 in the supplemental material), however, the effect on the F1F0 ATP synthase complex was stronger. Two of its major subunits, AtpD and AtpE, were significantly down-regulated under light at 17.5g·L-1 (0.7 fold and 0.4 fold respectively, p value < 0.01). Both AtpB and AtpC were stimulated by light in 17.5g·L-1 and 52.5g·L-1 sea salt, but exhibited a relatively small change in 70g·L-1 (Table S2). Under optimal salinity conditions (35g·L-1), the abundance of the ATPase complex was reduced under illumination. In particular, the gamma and delta subunits, which are involved in rotating the F1-ATPase, were greatly reduced (Table S2 in the supplemental material). The results suggest that under illumination at optimal salinity, although the electron transport chain retained a higher potential for proton pumping compared to dark conditions, ATP production by ATPase was limited. Overall, ATP synthase abundance was influenced by both light and salinity. This response may be the result of different proton gradient levels produced by PR and other proton transporters, and also by cellular energy requirements, which may explain reduced ATPase subunit protein abundances under illumination at optimal salinity. The accumulated data implies that PR-photophysiology can potentially regulate Na+-translocating NQR to adapt to different salinity conditions. Under specific levels of light intensity and salinity, NQR

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and PR proton transport activity likely becomes additive, contributing to growth stimulation.13

Hypersalinity and illumination leads to increased transporter protein abundances Functional analysis of the proteome showed that amongst all functionally allied protein sets, TonB-dependent transporters (TBDTs) were the most abundant proteins (Figure S3). TBDTs are known to utilize the proton motive force to transport nutrients including iron, copper, vitamin B12, and complex carbohydrates across the outer membrane. They may also be involved in degradation and uptake of the products of hydrolytic enzymes.28,

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It has been proposed that marine flavobacteria rely on

proteins and carbohydrate polymers for growth and thus contribute to marine secondary production.23 Since sea-ice phytoplankton are the primary producers and P. torquis has an epiphytic relationship with them,6 it likely uses TBDTs to take up nutrients and substrates directly derived from algal products. These high abundance TBDTs include several SusC-like carbohydrate uptake and SusD-like outer membrane proteins that putatively bind polysaccharides, similar to what has been surmised in the related species “Gramella forsetii” which also has many TBDT systems.30 These highly abundant transporters were observed to increase in abundance under supraoptimal salinity conditions and decrease under sub-optimal salinity (Figure 3A, Table S2). Moreover, TBDTs and some putative ABC-type nutrient transporters exhibit lower abundance in cells grown with 17.5g·L-1 sea salt under illumination, but became more abundant or remained unchanged under the combination of illuminated, hypersaline conditions. Increases in transporter abundance under supra-optimal salinity would assist cells under conditions where there is less availability of labile

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growth substrates due to lower levels of algal biomass. The reduced abundance of transporters under sub-optimal salinity levels, could be a response to the situation in thawing during which nutrient availability spikes co-occur.1, 31 Reduced salinity also corresponded to a lower growth yield as described in Feng et al (2013). Thus if increased growth substrates were presented, there could be less need for transporters owing to slower biomass synthesis rates. The high abundance of TBDTs is also consistent with the concept that enhanced mass transfer of nutrients helps overcome salinity and low temperature stresses.32 Proteomic data also revealed that several other transporters were significantly more abundant in cultures exposed to light. In particular, 33% of detected efflux transporters were more abundant under the combination of low salinity and illumination (Table S2, Figure 3B). Based on the protein classification, these efflux systems may remove toxic compounds from the cell33,34 though at this stage the function of these transporters is speculative. Efflux activities, however could prove useful in adapting to the sea-ice ecosystem, which has an active microbial loop in constrained spaces (such as brine channels) and thus is a highly competitive system. It is known higher temperatures and continuous light during the late spring and summer in polar regions leads to sea ice melting and intensification of microbial loop activity.5,35 When competing for nutrients sea-ice algae have been shown to secrete compounds that inhibit the growth of bacteria temporarily.36 This apparent light-associated increase in overall efflux transporters could potentially provide P. torquis with a survival/growth advantage by transporting noxious compounds out of the cell; however, further analysis is required to determine the exact roles of these proteins and whether such changes can be reproduced in a more natural setting.

Glycolysis and TCA cycle enzyme abundance is stimulated under salt stress and 14


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illuminated conditions The main energy generating metabolic pathways of P. torquis were identified as the glycolysis pathway and the tricarboxylic acid (TCA) cycle. In response to different salinity levels, several enzymes in glycolysis that are involved in energy production became more abundant. In particular, fructose-bisphosphate aldolase (EC 4.1.2.13), pyruvate kinase (EC 2.7.1.40) and enolase (EC 2.4.2.11) were induced under both sub-

and

supra-optimal

salinities

(Figure

S6).

Glyceraldehyde-3-phosphate

dehydrogenase (EC 1.2.1.12), which is involved in the reaction that produces two NADH and one H+ molecules, was more abundant under sub-optimal salinity levels. Phosphoglycerate kinase (EC 2.7.2.3), which catalyzes ATP generation (to balance consumption of ATP in the pathway), was only induced under supra-optimal salinity levels. In contrast triosephosphate isomerase (EC 5.3.1.1) was less abundant under both sub- and supra-optimal salinity conditions. This may lead to reduced conversion of glyceraldehyde-3-phosphate to glycerone-phosphate, and to an increase in NADH production efficiency. Both pyruvate carboxylase (EC 6.4.1.1) and pyruvate dehydrogenase complex were more abundant at lower salinity, but were not affected by the high salinity levels (Figure S6, Table S2 in the supplemental material) suggesting a different emphasis in the synthesis of acetyl-CoA between the salinity extremes. The citrate synthase complex, which introduces acetyl-CoA into the TCA cycle, is slightly inhibited. However, the TCA cycle enzymes that are involved in ATP and NADH generation steps all became more abundant (Figure S6). Previous growth experiments14 indicated that both sub- and supra- salinity levels are stressful for P. torquis, with lower growth yields obtained. Because cells were grown in a nutrient rich medium our findings suggest that changes the abundance of proteins involved in glycolysis and the TCA cycle are indicative of general responses to salinity stress. The

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expression pattern of central metabolic pathways indicates a need to generate more energy to overcome the salinity stress. Since light mediates proton pumping via PR and energizes the electron transport chain, some energy requirements imposed by low or high salt stress are alleviated. Since PR provides light derived ATP to supplement cell energy usage, the effect of illumination on central metabolic pathways was smaller than the effect of different salinity levels (Figure 4, Figure S6, Table S2). Nevertheless, illumination significantly increased the level of the energy generation related enzymes glyceraldehyde-3phosphate dehydrogenase (EC 1.2.1.12), triosephosphate isomerase (EC 5.3.1.1) and phosphoglycerate kinase (EC2.7.2.3) in glycolysis; and fumarate hydratase (EC 4.2.1.2) in the TCA cycle under all salinity levels tested (Figure 4). As well, oxoglutarate dehydrogenase SucB (EC 2.3.1.61), which catalyses the production of NADH, significantly increased 2.5-fold at 17.5g·L-1 under illumination. The increasing amount of NADH could potentially stimulate membrane bioenergetic processes. TBDTs rely on the proton motive force to import nutrients especially carbohydrates. Therefore, more carbon uptake is expected under illumination resulting in the observed stimulation of various glycolysis and TCA cycle enzymes. It is presumed that efficient harnessing of light energy can assist biological processes that involve nutrient and energy strategies in P. torquis. This could be the basis for prior observations of growth stimulation by light under optimal salinity conditions.14

Certain anaplerotic CO2 fixation enzymes are more abundant under illuminated conditions Several proteins that are involved in anaplerotic CO2 fixation37 could be quantified, including pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase

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(EC 4.1.1.49), malate dehydrogenase (1.1.1.38) and adenylosuccinate lyase (EC 4.3.2.2), which can act to replenish the TCA cycle. Pyruvate carboxylase that generates oxaloacetate from HCO3- and pyruvate22 exhibited no salinity-dependent changes under illumination while its abundance was increased under sub-optimal salinity conditions (Table S2in the supplemental material). Phosphoenolpyruvate carboxykinase and adenylosuccinate lyase, which generate oxaloacetate and fumarate, increased 1.3-3.7 fold under illumination (Figure 4, Table S2 in the supplemental material). In a previous study it was shown that Polaribacter sp. MED152 fixed more HCO3- under illuminated conditions.21 A subsequent report on light-stimulated growth of Dokdonia sp. MED134, however showed significant down-regulation of pyruvate carboxylase expression under illuminated conditions.38 These differences could be due to strain variation as well as poor correlations between changes in mRNA transcript and protein abundance.39 In general, PR proton pumping is an independent process from organic matter respiration, however provision of supplementary ATP and reductant due to more active electron transport could enhance carbon flow through the TCA cycle, thus requiring increased uptake of CO2.

Putative light sensing proteins are stimulated under illuminated conditions The genomic analysis of P. torquis revealed that it harbors genes coding for the enzymes responsible for the production of carotenoids (crtEBIY) and retinal (blh) which are chromophores for PR.7 We observed an extremely low level of these proteins in our proteomic survey except for phytoene desaturase which was presented in relatively constant amounts under different growth conditions. Previous studies also demonstrated the presence of both carotenoids and retinal in P. torquis and their levels were affected by light intensity and salinity.14 We assume that these proteins

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formed complexes with carotenoid pigments,40 which were lost during protein extraction, or they are expressed at low levels. P. torquis contains several regulatory systems that potentially sense light and invoke regulatory responses. These include several proteins with PAS and GAF domains known to be common components of phytochromes. PAS domains are reported to respond to cellular energy levels, oxygen levels, redox potential and light, whereas GAF domains often work as phototransducers.22,41,42 Another gene related to light sensing containing the BLUF domain that specifically responds to blue light was also found.43 Like other PR containing marine bacteria the genome also encodes deoxyribodipyrimidine photolyases and DASH family cryptochrome-like coding genes.23, 24 In addition to light sensors, several histidine kinase and two-component system sensors serve as a basic environmental stimulation response coupling mechanism that helps organisms to sense and respond to the environment.44 In this study, most proteins in the signal transduction group were only detected at low abundances. Nevertheless, our data is sufficiently sensitive to reveal PAS and GAF domain protein levels are always higher when cells were grown exposed to light, additionally these signal transduction systems were also influenced by salinity. In general, these proteins are less abundant under optimal salinity levels (Figure 5). BLUF domain, cryptochromes and deoxyribodipyrimidine photolyase proteins were detected only at low to very low abundance levels under all conditions suggesting that these proteins do not couple with the responses linked to activities associated with the green-light absorbing PR that P. torquis contains. Some secondary transduction enzymes including two-component histidine kinases showed increasing expression levels under illumination and have the same trend of response to salinity as the PAS and GAF domain-containing proteins (Figure 5). These sensory systems may

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therefore also be involved in illumination-influenced regulation of gene expression.45

P. torquis photo-inhibition under high light levels is linked to oxidative stress and lower transporter abundance Illumination experiments were conducted at two levels (20-30 µmol photons m-2 s-1 and 3-4 µmol photons m-2 s-1). Previous studies have reported that high light levels can inhibit bacterial activity and this is also the case for P. torquis.14, 46 The consensus is that strong and continual light exposure creates photooxidative stress; however, this stress is unlikely to be derived from UV irradiation-related damage since the light source used had low UV energy emittance levels and the petri dish cover on cultures would have further impeded UV light exposure. The proteome data suggests that under higher levels of light, the protein abundance changes in the central metabolic pathway and membrane bioenergetics pathway are generally more significant (Table S2). In addition, the antioxidant enzyme peroxiredoxin was induced by light, implying that P. torquis has a mechanism that responds to photooxidative stress. Nevertheless, some TBDTs were less abundant under high illumination levels (Figure 3B). This reduction could partly explain light inhibition of growth since chemoheterotrophic P. torquis relies on uptake of nutrients via TBDTs.

Illumination and sub-optimal salinity promotes the abundance of adhesion proteins P. torquis harbors several types of putative adhesion proteins such as fasciclin repeat domain-containing proteins and putative ice-binding adhesion-like proteins.7 These are potentially associated with adhesion to algal and ice surfaces aiding the colonization of living phytoplankton cells and recruitment into forming sea-ice.47 We

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found two fasciclin domain containing proteins that exhibited 20-fold and 6-fold increases under illumination at sub-optimal salinity, and a Por secretion system protein48 that was 15-fold more abundant under the same conditions, yet they all have smaller scales of stimulation at higher salinities (Table S2). This finding provides a hypothesis that adhesion proteins are involved in maintaining phytoplankton colonization at the time of sea-ice thaw and formation.5 These associations are likely to be especially important during and after the sea-ice microbial community is dispersed into the water column.

Dark conditions promote gliding whilst illumination suppresses gliding Previous studies have shown that after transferring PR into E. coli illumination can increase the swimming motility of E. coli.12 In our proteomic data four proteins critical for efficient gliding motility (GldJ, GldL, GldM and GldN)49 were abundant and could be readily measured. Illumination did not affect the abundance of these proteins at the optimal growth salinity, but illumination suppressed the level of GldJ and GldN by 0.3-fold and 0.7-fold respectively at sub-optimal salinity and stimulated GldJ’s abundance 4-fold at the highest salinity. GldM was increased under illumination 2.5-fold at sub-optimal salinity but did not change at other salinity levels (Table S2 in the supplemental material), which indicates that gliding of P. torquis could be dependent on external stimulation. Our direct observation of P. torquis’ gliding motility on agar showed obvious increasing motility under dark at salinities of 17.5g·L-1, 35g·L-1 and 70g·L-1. However, the motility was largely suppressed by illumination at 17.5g·L-1 and 35g·L-1 while some gliding ability was retained at 52.5g·L-1 and 70g·L-1. The overall effect of salinity on gilding motility is smaller than the effect of light (Figure S7) indicating overexpression of GldM may inhibit gliding

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and GldJ and GldN are potentially important for gliding in P. torquis. Thus, darkness may induce the motility of P. torquis potentially aiding nutrient uptake during the polar winter when algal primary production is negligible. It is reasonable to expect there would be greater gliding motility in the natural sea ice environment: the experiment was conducted on nutrient rich agar whereas in natural sea ice nutrient levels are lower.

Exopolysaccharides (EPS) formation is salinity regulated EPS is one of the major outputs of bacterial secondary production in sea ice communities and contributes to the overall carbon cycle in the Antarctic environment.50 EPS is important for sea ice inhabitation providing a structural network for microbial associations and acting as nutrient ligands for growth at low temperature and high salinity.48 However, there are few reports on the mechanism of the effect of EPS on bacterial tolerance to high salinity, such as that found in the brine channels of sea ice. In this study we found the production of EPS in P. torquis was clearly salinity regulated. Compared to optimal salinity both low and high levels of salinity induced the enzymes involved in EPS biosynthesis as well as some lipopolysaccharide biosynthesis proteins (Table S2 in the supplemental material). A recent study has shown that EPS from a sea ice diatom can alter the physical properties of sea ice resulting in the retention of more salt in the sea ice.51 Secretion of EPS by P. torquis could theoretically act to exclude salt to achieve optimal salinity in the direct vicinity of cells. This could explain why EPS production and the associated enzymes were slightly increased under hypersaline conditions.

Conclusions

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In this study, we characterized the proteome expression pattern associated with different growth rates and biomass yields observed in P. torquis when exposed to various salinity and illumination levels. Our analysis revealed a number of proteins that changed in response to salinity and illumination. Several proteins were upregulated under different light levels and under salinity stress (both sub and supraoptimal salinity), these included proteins associated with predicted light sensors energy generation, adhesion, and transporters. We propose that the inferred protein abundance data can be related to the capability of P. torquis to adapt within a highly dynamic sea-ice ecosystem leading to an apparent interaction between the effects of salinity and exposure to light. Several studies had suggested that PR is involved in light-enhanced growth of a few bacterial species.14,52,53 However, in P. torquis PR protein abundance does not fully correlate with its growth rate and yield which indicates that PR-photrophy may be a more complicated process. The protein expression patterns of P. torquis show that it can use light to obtain an advantage in carrying out colonization of phytoplankton and to take up more nutrients by significantly up-regulating transporters, efflux pumps and adhesion proteins, as well as optimizing energy production to stay within or even modify the sea-ice habitat. Our results also support a hypothesis proposed in a recent study which suggested that PR may be involved in some non-ATP forming functions, such as environmental sensing that may contribute to bacterial survival.50 Analysis of the proteome of P. torquis provides insights into its life strategies and survival and provides a basis for the study of other sea-ice associated microorganisms.

ASSOCIATED CONTENT Supporting Information

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Supporting Information data Figure S1: Phylogenetic tree and sequence comparison of microbial rhodopsins’ amino acid sequences. Figure S2: Growth curve of P. torquis in marine broth. Figure S3: Spectral abundance of P. torquis proteins observed in sets organized on the basis of functional class. Figure S4: Volcano plot of 1406 proteins from high light compared to low light conditions. Figure S5: Proteomic response of the electron transport chain proteins under different salinities when compared to optimal salinity conditions. Figure S6: Diagram of central metabolic pathway change under different salinities when compare to the optimal salinity level. Figure S7: Gliding motility assay of P. torquis under different salinity and illumination levels. Table S1: Spectral count and number of proteins identified by LC-MS/MS from all biological replicates. Table S2: A functional/regulation comparative analysis of the proteomic data including P. torquis proteome response to different levels of illumination compare to dark under different salinities and its proteome response to sub-/supra-optimal salinity compare with optimal salinity regardless the presence of light. The Supporting Information is available free of charge via http://pubs,acs,org/ Acknowledgments The work was supported by a grant from the Department of Sustainability, Environment, Water, Population and Communities and the Australian Antarctic Division (project no. 3127). We thank Dr. Jay Kocharunchitt for his assistance with statistical analysis. We also want to thank Professor Tom McMeekin for critical discussion related to the manuscript.

Conflict of Interest: The authors declare no conflict of interest.

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Titles and Legends to Figures Figure 1 CAP analysis plot (A) comparing the proteomes of P. torquis grown under different levels of salinity and light conditions. Filtered spectra data was fourth-root transformed. Cultures were grown under high light (20-30 µmol photons m-2 s-1), low light (3-4 µmol photons m-2 s-1) and dark conditions (0 µmol photons m-2 s-1) in salinities of 17.5g·L-1 (circles), 35g·L-1 (diamonds), 52.5g·L-1 (triangles) and 70g·L-1 (squares). Volcano plot of 1406 proteins from high light compared to dark (B) and low light compared to dark (C) conditions, where the log ratio of protein fold change is plotted against negative log p value of the beta-binomial distribution analysis from six replicates. Red line represent cut-off for p-value=0.05; blue line represent cut-off for fold change=±2.

Figure 2 Proteomic response of electron transfer chain protein complexes in P. torquis under high light and low light conditions. The change in abundance is compared between high light (20-30 µmol photons m-2 s-1) and dark (as control: 0 µmol photons m-2 s-1) conditions and also between low light (3-4 µmol photons m-2 s1

) and dark (as control) conditions at different salinity levels. Each bar represents the

total abundance of a complex to its cognate complex labelled below it. Asterisks indicate p value of the fold change is less than 0.05. Figure 3 Heat map generated from proteomic data for some TonB-dependent transporters and efflux transporters. Hierarchical clustering was performed based on the spectral counts. (A) represents the response to different salinities. The optimal salinity was used as control and spectral counts from all three light conditions for each salinity level were pooled together and compared to the optimal salinity. (B) represents response of proteins to different illumination levels compared to dark

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conditions at corresponding salinity levels. Figure 4 Diagram of central metabolic pathway changes that occur in P. torquis under illumination. The fold-change was determined using the dark incubation of each salinity level as the control. Each salinity level is shown separately. Within the metabolic map, red lines indicate an enzyme with greater abundance under light, green lines indicate lower abundance under light, and blue lines indicate an unchanged abundance level. The thickness of the lines implies the degree of abundance change. The size of the EC enzyme code provides an indication of the overall abundance of the enzyme. Figure 5 Comparison of total abundance of light sensing proteins between different salinity and light conditions, and the changes of each light sensing protein under different salinity of light levels. Illumination intensity is: high light (20-30 µmol photons m-2 s-1); low light (3-4 µmol photons m-2 s-1) and complete darkness (0 µmol photons m-2 s-1).

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A

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ATP synthase

70g∙L-1

3.5

*


3.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


Figure 2

PR

High Light Low Light

2.5 1.5 0.5 -0.5

-1.5 -2.5

-2.5 Complex I (NQR)

ACIII

Complex IV

ACS Paragon Plus Environment Complex I

ATP synthase

PR

ACIII

Complex IV

ATP synthase

PR

A 17.5 g·L-1 52.5g·L-1 70g·L-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


B 17.5L 17.5DL 35L 35DL 52.5L 52.5DL 70L 70DL




Page 36 of 38

Figure 4

H+

PR

glyceraldehyde-3-phosphate

ATP synthase

H+ ADP

PPi EC 2.7.4.1 PPPi

Sedoheptulose-7phosphate

3-phosphoglycerate

Glycerone-phosphate

EC 2.7.1.11

EC 5.1.3.1

EC 2.7.2.3

EC 5.3.1.1

EC 2.2.1.1

Erythrose-4-phospahte

1,3-bisphosphoglycerate

fructose-1,6-bisphosphate

EC 2.2.1.1

D-Xylulose 5-phosphate

EC 3.6.1.1

Glycolysis EC 1.2.1.12

EC 4.1.2.13

Pentose Phosphate

ATP

EC 5.4.2.1

2-phosphoglycerate

fructose-6-phosphate

EC 4.2.1.11

EC 2.2.1.2 EC 5.3.1.9

Phosphoenolpyruvate

glucose-6-phosphate

EC 2.7.1.40

EC 4.1.1.49

Complex Ⅳ

H+

Ribulose-5-phosphate

EC 6.4.1.1 CO2

Pyruvate

HCO3-

z

Malate pyruvate

EC 1.1.1.38

SuccinateNAD+

Complex Ⅱ

Fumarate

Alternative Complex Ⅲ

H+

EC 1.2.4.1 EC 2.3.1.12 EC 1.8.1.4 Acetyl-CoA

Citrate

Oxaloacetate

EC 1.1.1.37

EC 2.3.3.1 EC 4.2.1.3

EC 4.2.1.2

Adenylosuccinate

TCA Cycle

Fumarate

Isocitrate

EC 4.3.2.2

EC 1.3.99.1

Sub-optimal Salinity Na+

Complex Ⅰ

EC 6.2.1.5

supra-optimal Salinity H+

NADH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Na+

Succinat e

HCO3-

Na+

TBDT


Succinyl-CoA

EC 1.2.4.2 EC 2.3.1.61 EC 1.8.1.4

EC 1.1.1.42

2-Oxoglutarate



High Light

25

50

25

50

20

40

20

40

15

30

15

30

10

20

10

20

5

10

5

10

0

0

0

17.5g∙L-1

35g∙L-1

52.5g∙L-1

Spectral Count

30

70g∙L-1

30

60

0 17.5g∙L-1

35g∙L-1

52.5g∙L-1

70g∙L-1

Salinity Level

Salinity Level

Spectral Count

Spectral Count

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Low Light

60

30

60

Dark

25

50

Bacteriophytochrome-like sensor, PAS domains (YP_006867167.1)

20

40

Bacteriophytochrome, GAF domain (YP_006869231.1)

15

30

10

20

Bacteriophytochrome-like sensor, PAS and GAF domain (YP_006869331.1) Bacteriophytochrome-like sensor, multiple PAS domains (YP_006867168.1)

5

10 Total SpC of PAS+GAF domains

0

0 17.5g.L-1

35g.L-1

52.5g.L-1

70g.L-1

Salinity Level


Total SpC of two-component regulators


Graphical Abstract

H+

PR

glyceraldehyde-3-phosphate

ATP synthase

H+ ADP

PPi EC 2.7.4.1 PPPi

Sedoheptulose-7phosphate

3-phosphoglycerate

Glycerone-phosphate

EC 2.7.1.11

EC 5.1.3.1

EC 2.7.2.3

EC 5.3.1.1

EC 2.2.1.1

Erythrose-4-phospahte

1,3-bisphosphoglycerate

fructose-1,6-bisphosphate

EC 2.2.1.1

D-Xylulose 5-phosphate

EC 3.6.1.1

Glycolysis EC 1.2.1.12

EC 4.1.2.13

Pentose Phosphate

ATP

EC 5.4.2.1

2-phosphoglycerate

fructose-6-phosphate

EC 4.2.1.11

EC 2.2.1.2 EC 5.3.1.9

Phosphoenolpyruvate

glucose-6-phosphate

EC 2.7.1.40

EC 4.1.1.49

Complex Ⅳ

H+

Ribulose-5-phosphate

EC 6.4.1.1 CO2

Pyruvate

HCO3-

z

Malate pyruvate

EC 1.1.1.38

SuccinateNAD+

Complex Ⅱ

Fumarate

Alternative Complex Ⅲ

H+

EC 1.2.4.1 EC 2.3.1.12 EC 1.8.1.4 Acetyl-CoA

Citrate

Oxaloacetate

EC 1.1.1.37

EC 2.3.3.1 EC 4.2.1.3

EC 4.2.1.2

Adenylosuccinate

TCA Cycle

Fumarate

Isocitrate

EC 4.3.2.2

EC 1.3.99.1

Sub-optimal Salinity Na+

Complex Ⅰ

EC 6.2.1.5

supra-optimal Salinity H+

NADH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Page 38 of 38

Na+

Succinat e

HCO3-

Na+

TBDT


Succinyl-CoA

EC 1.2.4.2 EC 2.3.1.61 EC 1.8.1.4

EC 1.1.1.42

2-Oxoglutarate

Proteomic Insight into Functional Changes of Proteorhodopsin

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