Introduction

The biosphere of the earth is dominated by permanently cold environments such as the Polar Regions, deep seas and high mountains. Many microorganisms inhabit these environments by adapting to low temperatures. Such cold-adapted microorganisms have been attracting a great deal of attention from both fundamental and application points of view (Cavicchioli et al. 2002; D’Amico et al. 2006). Cold-active enzymes from these microorganisms are expected to be useful as food-processing enzymes, additives to detergents, and tools for molecular biology (Cavicchioli et al. 2002). Applications of cold-adapted microorganisms include the construction of low-temperature protein expression systems by using these microorganisms as hosts to facilitate the overproduction of thermolabile proteins (Papa et al. 2007; Miyake et al. 2007). Extensive studies to understand the cold-adaptation mechanisms of these microorganisms are also being undertaken by many researchers (Cavicchioli 2006; D’Amico et al. 2006).

Recent studies of cold-adapted microorganisms revealed several strategies of these microorganisms to adapt to low temperatures, such as the production of cold-active enzymes (Feller and Gerday 2003; Siddiqui and Cavicchioli 2006), the modulation of the lipid composition to maintain the fluidity of the cell membrane (Chintalapati et al. 2004; Russell 1997), and the production of RNA chaperones to suppress the formation of undesired secondary structures of RNA (Hebraud and Potier 1999; Yamanaka et al. 1998). To obtain a better insight into microbial cold-adaptation mechanisms, genomic and proteomic approaches are being employed (D’Amico et al. 2006). Complete genomic DNA sequences are currently available for several cold-adapted microorganisms including Desulfotalea psychrophila LSv54 (Rabus et al. 2004), Photobacterium profundum SS9 (Vezzi et al. 2005), Colwellia psychrerythraea 34H (Methe et al. 2005), and Pseudoalteromonas haloplanktis TAC125 (Medigue et al. 2005). With the aid of these sequence data, it is possible to make a global identification of proteins produced under a particular growth condition. Because proteins that are inducibly produced at low temperatures are supposed to play important roles at low temperatures, the identification of cold-inducible proteins would provide clues for understanding the cold-adaptation mechanisms of microorganisms.

Shewanella livingstonensis Ac10, formerly called Shewanella sp. Ac10, is a cold-adapted bacterium isolated from Antarctic seawater (Kulakova et al. 1999). It was recently identified as a strain belonging to S. livingstonensis by Y. Nogi and C. Kato (Japan Agency for Marine-Earth Science and Technology) (unpublished). The strain grows well at low temperatures close to 0°C but does not grow at temperatures over 30°C (Kulakova et al. 1999). We have established transformation procedures, developed a heterologous protein expression system, and established targeted gene disruption methods for this bacterium (manuscript in preparation). In addition, we carried out whole genome sequencing of this bacterium and recently obtained the draft genome sequence. Shewanella species are widely distributed in various environments on the earth, and complete genomic DNA sequences of various Shewanella species have been determined [Genomes OnLine Database (http://www.genomesonline.org/)]. These include mesophilic Shewanella oneidensis MR-1 (Heidelberg et al. 2002) and piezophilic Shewanella violacea DSS12 (K. Nakasone, Kinki University, Higashihiroshima, Japan, personal communication). Comparative studies of these phylogenetically related strains that inhabit different environments would facilitate the understanding of the mechanisms of adaptation to various environments. In these respects, S. livingstonensis Ac10 is a fascinating model microorganism for the investigation of microbial cold-adaptation mechanisms.

In the present study, we performed proteomic studies of this bacterium to make a global identification of cold-inducible proteins and obtain clues to understand its cold-adaptation mechanism. We found that proteins involved in various cellular processes, such as the modulation of gene expression, motility, and membrane transport, were inducibly produced at low temperatures.

Materials and methods

Bacterial strain and culture conditions

Shewanella livingstonensis Ac10 isolated from Antarctic seawater was grown in 5 ml of Luria–Bertani (LB) medium (pH 7.0) for 48 h at 18°C, and then transferred to 300 ml of LB medium for further cultivation at 4°C and 18°C to the early stationary phase.

Proteome analysis

The cells grown at 4°C and 18°C were harvested by centrifugation and resuspended in 40 mM Tris–HCl (pH 7.0). The cell suspensions were sonicated and centrifuged. Proteins in the supernatants were used as soluble proteins for proteome analysis. Membrane proteins were extracted by using ReadyPrep Protein Extraction Kit (Membrane I) (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (300 μg) were loaded onto Immobiline DryStrip pH 4–7 (GE Healthcare UK Ltd, Buckinghamshire, UK), and isoelectric focusing was performed with PROTEAN IEF Cell (Bio-Rad Laboratories, Inc.) as recommended by the manufacturer. Treatment of the gel strips for the second-dimensional SDS-PAGE was carried out as described previously (Mineki et al. 2002). The second-dimensional SDS-PAGE was performed by using gradient gels with 10–20% acrylamide (PAG Large “Daiichi” 2D-10/20, Daiichi Pure Chemicals Co., Ltd, Tokyo, Japan). After fixation and staining with SYPRO Ruby (Invitrogen Corp., Carlsbad, CA, USA), the gels were scanned with an image analyzer, Typhoon 9400 (GE Healthcare UK Ltd). The expression pattern of proteins was analyzed using the image analysis software PDQuest ver. 7.0 (Bio-Rad Laboratories, Inc.). After scanning, the gels were restained with Negative Gel Stain MS Kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan).

Gel spots for cold-inducible proteins whose relative abundance at 4°C was more than twofold that at 18°C were excised, and the proteins were digested with sequencing-grade modified trypsin (Promega Corporation, Madison, WI, USA). Peptide mass fingerprinting (PMF) analysis was performed by the standard method with Autoflex II MALDI-TOF systems (Bruker Daltonics, Billerica, MA, USA). Spectrum acquisition was ensured in a reflector mode with the following parameters: 42% laser power with a mass range of 180–4,460 Da and 5.3% detector gain. Calibration was performed using Peptide Calibration Standard (Bruker Daltonics). A local version of Mascot (Matrix Science Ltd., London, UK) was used to identify the proteins.

Quantitative real-time RT-PCR analysis

Total RNA was extracted with RNeasy Kit (QIAGEN Inc., Valencia, CA, USA) from cells cultivated at 4°C and 18°C. The RNA pellets were dissolved in 0.1% diethyl pyrocarbonate-treated water and stored at −80°C until use. Quantitative real-time RT-PCR was performed with SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen Corp.) and the Mx3000P Multiple Quantitative RT-PCR system (Stratagene, La Jolla, CA, USA). The threshold cycle (Ct) value for each sample was normalized with the Ct value for 16S rRNA.

Results

Growth profile of S. livingstonensis Ac10

Shewanella livingstonensis Ac10 grew with a doubling time of 8.8 h at 4°C, whereas the doubling time was 2.5 h at 18°C. No growth was observed at temperatures over 30°C. The yield of the cells grown at 4°C (9.2 g/l) was comparable to that of the cells grown at 18°C (8.0 g/l).

Global identification of cold-inducible proteins

Shewanella livingstonensis Ac10 was grown at 4°C and 18°C to the early stationary phase, and soluble and membrane proteins were prepared for two-dimensional gel electrophoresis (2DE) to identify cold-inducible proteins. Figure 1a shows the gel images obtained for the soluble proteins. Image analysis of the gels revealed that 91 spots were cold-inducible (more than a twofold increase at 4°C) (Fig. 1a, indicated by arrowheads). Cold induction was reproducibly observed for 48 of them in at least three sets of experiments (Fig. 1a, numbered spots), and further analysis was performed for these spots. The gel images for the membrane proteins are shown in Fig. 1b. Thirty-four membrane proteins were inducibly produced at 4°C (Fig. 1b, indicated by arrowheads), and reproducibility was confirmed for five of them (Fig. 1b, numbered spots).

Fig. 1
figure 1

Comparison of soluble (a) and membrane (b) proteins from S. livingstonensis Ac10 grown at 4°C (left) and 18°C (right). The gels were stained with SYPRO Ruby and analyzed with PDQuest ver. 7.0. The arrowheads indicate cold-inducible spots (91 and 34 spots for soluble and membrane proteins, respectively). Cold induction was reproducible for the numbered spots (48 and five spots for soluble and membrane proteins, respectively)

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The cold-inducible spots were excised from the gels and analyzed by PMF to identify the proteins. PMF analysis allowed the identification of 26 soluble proteins and two membrane proteins (Table 1). Twenty-two soluble proteins and three membrane proteins were not identified, probably due to the low abundance of the proteins or peptides extracted from the gels. The identified proteins can be classified into the following seven groups according to their sequence similarity to the proteins in the database: RNA synthesis and folding (group 1), protein synthesis and folding (group 2), membrane transport (group 3), motility (group 4), metabolism (group 5), other functions (group 6), and unknown function (group 7). The ratio of the spot intensity at 4°C to that at 18°C was more than 580 for CspA (Fig. 1a, no. 1111) and more than 610 for OmpA (Fig. 1b, no. 5401).

Table 1 Cold-inducible proteins of S. livingstonensis Ac10
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Analysis of the transcriptional levels of the genes coding for cold-inducible proteins

To determine whether the production of cold-inducible proteins is transcriptionally regulated, mRNAs for these proteins were quantified by real-time RT-PCR as described in Materials and methods. Because 2DE analysis showed that the spot intensities of TipA, DnaK, and ATP synthase β subunit (AtpD) were not significantly different at 4°C and 18°C (data not shown), mRNA coding for these proteins and 16S rRNA were used as controls. The results are summarized in Fig. 2.

Fig. 2
figure 2

Transcriptional levels of the genes coding for cold-inducible proteins. The ratios of the amounts of mRNA in the cells grown at 4°C and 18°C are indicated. All values were normalized with the ratio of the amounts of 16S rRNA in the cells grown at 4°C and 18°C. ND: No RT-PCR product was obtained for the cells grown at 4°C and 18°C. ∞: the RT-PCR product was obtained for the cells grown at 4°C but not for those grown at 18°C

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The amounts of mRNA for the following 14 genes were more than two times larger at 4°C than at 18°C: cspA, lysU, ompC, flgE, purD, deoC, nemA, ac10nuc, ac10C417081, ftsZ, ac10omp, ac10C130109, ac10C097028, and ac10C084124. In contrast, the cultivation of the cells at 4°C did not significantly increase the transcription level of 12 genes, rpoA, greA, tufB, efp, tig, ompA, ppx1, fixB, pdxJ, ac10cp, tsx, and ac10C346010, although the spot intensities of the proteins encoded by these genes were increased at low temperatures (Table 1).

Discussion

Regulation of the expression of cold-inducible proteins

We identified 28 cold-inducible proteins of S. livingstonensis Ac10 by proteomic analysis of the cells grown at 4°C and 18°C (Table 1). These proteins are supposed to participate in various cellular processes, such as the modulation of gene expression, motility, and membrane transport. Analysis of the expression of the genes by real-time RT-PCR revealed that 14 of them were cold-inducible at the level of transcription (>twofold) (Fig. 2). It is noticeable that the degree of increase in the mRNA amount was less significant than that of the increase in the protein amount for most of these genes: the only two exceptions were ompC and flgE. The degree of increase in the amount of mRNA for ompC was more significant than that of increase in its protein amount. The results suggest that the expression of these genes, except for flgE, is regulated at both the transcription and post-transcription levels. We found 12 cold-inducible proteins whose expression was not up-regulated at 4°C at the level of transcription. Thus the regulation of expression of the cold-inducible proteins of S. livingstonensis Ac10 depends on various mechanisms.

Cold-inducible proteins involved in RNA and protein synthesis

The amounts of three proteins involved in RNA synthesis and folding (RpoA, GreA, and CspA) increased at 4°C (Table 1). RpoA is a subunit of RNA polymerase, which plays important roles in the assembly of RNA polymerase, promoter recognition by site-specific protein–DNA interaction, and transcriptional activation (Ebright and Busby 1995). GreA induces the cleavage and removal of the 3′ proximal dinucleotide from the nascent RNA to control transcriptional fidelity (Hogan et al. 2002). CspA is an RNA chaperone, which is supposed to make the secondary structure of mRNA suitable for translation (Jiang et al. 1997). These proteins may be required for the efficient and accurate transcription and proper folding of RNA at low temperatures, where base pairing of nucleic acids is stable and undesired secondary structures of RNA tend to form.

RNA polymerase is composed of RpoA, RpoB, RpoC and a sigma factor. Since we did not identify the spots for RNA polymerase subunits other than RpoA in this study, it is not clear whether the amounts of these proteins are increased at low temperatures. Inducible nature of the subunits other than RpoA will be studied to reveal whether the amount of functional RNA polymerase is increased at low temperatures.

CspA was identified as a major cold-shock protein in Escherichia coli (Yamanaka et al. 1998). CspA is supposed to play a central role in the cold-shock response in E. coli. In S. livingstonensis Ac10, however, CspA was produced in the stationary phase, suggesting that it is required for normal growth at low temperatures.

Tig of S. livingstonensis Ac10 was inducibly produced at 4°C (Table 1). It is a ribosome-associated chaperone with peptidyl-prolyl cis–trans isomerase (PPIase) activity and facilitates proper folding of newly synthesized proteins (Kramer et al. 2004). Cold induction of Tig may contribute to efficient translation at low temperatures. The protein consists of three domains, an N-terminal domain mediating association with ribosomes, a central substrate-binding domain with homology to FKBP proteins showing PPIase activity, and a C-terminal domain of unknown function. Suzuki et al. reported that an FKBP family member protein with PPIase activity (FKBP22) is inducibly produced at low temperatures in Shewanella sp. SIB1 (Suzuki et al. 2004). Although Tig of S. livingstonensis Ac10 is supposed to have PPIase activity, it is different from FKBP22 in that Tig has N-terminal and C-terminal domains that are not found in FKBP22. S. livingstonensis Ac10 has two copies of the genes coding for FKBP22 homologs, FkpA_C295011 and FkpA_C367122, showing 90 and 43% identities to FKBP22, respectively, but their expression was not confirmed in the present study.

Tig was first discovered in E. coli as a protein triggering the translocation of the precursor of pro-OmpA into a membrane vesicle (Crooke and Wickner 1987). pro-OmpA is a precursor of a major outer membrane protein, OmpA. We found that OmpA of S. livingstonensis Ac10 was inducibly produced at 4°C (Table 1). Cold-inducible Tig is possibly involved in the production of OmpA at low temperatures.

Translation elongation factors play a key role in protein synthesis on ribosomes and assist in the elongation of nascent polypeptide chains (Weijland et al. 1992). At low temperatures, protein synthesis is slowed because the activities of proteins involved in protein synthesis are suppressed. We found that putative elongation factors, TufB and Efp, which are supposed to act as carriers of aminoacyl-tRNAs, were inducibly produced at 4°C (Table 1). These proteins may enhance global protein synthesis at low temperatures.

Lysyl-tRNA synthetase, LysU (Freist and Gauss 1995), was identified as a cold-inducible protein (Table 1). Thus S. livingstonensis Ac10 possibly enhances the insertion of lysine into proteins at low temperatures. Many cold-adapted enzymes have a high Lys/Arg ratio in order to increase protein flexibility (Siddiqui and Cavicchioli 2006). LysU may contribute to the production of such lysine-rich cold-adapted enzymes.

Cold-inducible proteins involved in membrane transport

Two putative outer membrane porin homologs, OmpA and OmpC, were inducibly produced at 4°C (Table 1). Homologs of these proteins from other bacteria form channels for hydrophilic solutes and play important roles in the uptake of nutrients (Nikaido 2003). It is well known that bacteria regulate the production of their outer membrane porin depending on their environments. For example, Shewanella frigidimarina NCIMB400R inducibly produces an outer membrane porin, IfcO, under the control of the iron-responsive element under anaerobic conditions (Reyes-Ramirez et al. 2003). A deep-sea bacterium, Photobacterium profundum SS9, inducibly produces OmpL under high hydrostatic pressure conditions (Welch and Bartlett 1996). Thus these bacteria modulate the permeability of their outer membranes to adapt to their environments. Cold induction of OmpA and OmpC of S. livingstonensis Ac10 may counteract the low diffusion rate of solutes at low temperatures and enables the efficient uptake of nutrients.

Cold-inducible proteins involved in cell motility

The amounts of hook-related proteins of flagella, FlgE and FlgL (Homma et al. 1990), were increased at 4°C (Table 1). We found the consensus sequence of σ 54-dependent promoters and the gene coding for the σ 54-dependent transcriptional activator protein (Wosten 1998) in the upstream and downstream regions, respectively, of the gene cluster containing flgE. These factors possibly regulate the expression of flgE. Cold induction of FlgE and FlgL suggests that S. livingstonensis Ac10 forms different types of flagella depending on the cultivation temperature or produces flagella only at low temperatures to modulate its motility. To support this speculation, we found that the cells were more motile at 4°C than at 18°C. The physiological significance of higher motility at low temperatures remains to be clarified.

Cold-inducible FtsZ

FtsZ, which forms the division septum, is an essential protein for cell division highly conserved among prokaryotes (Addinall and Holland 2002). Most microorganisms have one copy of FtsZ, whereas S. livingstonensis Ac10 has two copies of the genes coding for FtsZ homologs, FtsZC26 and FtsZC19. FtsZC26 shows much higher similarity to other bacterial FtsZ than FtsZC19. We found that FtsZC26 was inducibly produced at 4°C (Table 1). The result raised the possibility that this bacterium changes its morphology depending on its growth temperature by altering the amount of the cell division protein. To support this speculation, we found that the cells were significantly shorter at 4°C than at 18°C.

Comparison of proteomes of various cold-adapted microorganisms

Proteomic studies have been conducted for several cold-adapted microorganisms such as Methanococcoides burtonii (Goodchild et al. 2004, 2005), Bacillus psychrosaccharolyticus (Seo et al. 2004), Psychrobacter cryohalolentis K5 (Bakermans et al. 2007), and Psychrobacter articus 273-4 (Zheng et al. 2007). It was found that different cold-adapted microorganisms produce different cold-inducible proteins. Thus different cold-adapted microorganisms are supposed to use different strategies to cope with cold environments. Nevertheless, some of those strategies appear to be common in several cold-adapted microorganisms. Cold induction of an RNA chaperone, CspA, is observed in many bacteria, including Arthrobacter globiformis SI55 (Berger et al. 1997), P. cryohalolentis K5 (Bakermans et al. 2007), and S. livingstonensis Ac10. PPIase, which facilitates protein folding, is inducibly produced at low temperatures in several cold-adapted microorganisms, such as Shewanella sp. SIB1 (Suzuki et al. 2004), M. burtonii (Goodchild et al. 2004), P. articus 273-4 (Zheng et al. 2007), and S. livingstonensis Ac10, although the type of cold-inducible PPIase is different in different cold-adapted microorganisms. It is likely that isomerization of peptidyl-prolyl bonds for proper protein folding is a crucial process for many microorganisms to survive at low temperatures. The modulation of RNA polymerase is also supposed to be important for the cells to grow at low temperatures because subunits of these complexes of several cold-adapted microorganisms, RNA polymerase subunit E from M. burtonii (Goodchild et al. 2004) and RNA polymerase α subunit from P. articus 273-4 (Zheng et al. 2007) and S. livingstonensis Ac10, are up-regulated at low temperatures. More distinctive features of proteomes of cold-adapted microorganisms will be clarified by future proteomic studies of other cold-adapted microorganisms, which will facilitate our understanding of their cold-adaptation mechanisms.

Cold adaptation is similar to pressure adaptation in several respects (Welch et al. 1993; Ishii et al. 2005). For example, both low temperature and high pressure decrease membrane fluidity, and the cells must modify the membrane component to maintain the membrane fluidity upon exposure to these environments. It has been reported that many cold shock proteins are inducibly synthesized under high-pressure conditions. Thus, characterization of cold-inducible proteins may also be helpful to understand mechanism of cellular adaptation to high pressure.