Transcriptional response of the model planctomycete Rhodopirellula baltica SH1T to changing environmental conditions

Background The marine model organism Rhodopirellula baltica SH1T was the first Planctomycete to have its genome completely sequenced. The genome analysis predicted a complex lifestyle and a variety of genetic opportunities to adapt to the marine environment. Its adaptation to environmental stressors was studied by transcriptional profiling using a whole genome microarray. Results Stress responses to salinity and temperature shifts were monitored in time series experiments. Chemostat cultures grown in mineral medium at 28°C were compared to cultures that were shifted to either elevated (37°C) or reduced (6°C) temperatures as well as high salinity (59.5‰) and observed over 300 min. Heat shock showed the induction of several known chaperone genes. Cold shock altered the expression of genes in lipid metabolism and stress proteins. High salinity resulted in the modulation of genes coding for compatible solutes, ion transporters and morphology. In summary, over 3000 of the 7325 genes were affected by temperature and/or salinity changes. Conclusion Transcriptional profiling confirmed that R. baltica is highly responsive to its environment. The distinct responses identified here have provided new insights into the complex adaptation machinery of this environmentally relevant marine bacterium. Our transcriptome study and previous proteome data suggest a set of genes of unknown functions that are most probably involved in the global stress response. This work lays the foundation for further bioinformatic and genetic studies which will lead to a comprehensive understanding of the biology of a marine Planctomycete.

knowledge about their functions is still sparse. The 'genomic revolution' [1] has opened the door to investigations targeting their genetic potential and activity on the molecular level.
A particularly interesting representative of the marine picoplankton community is Rhodopirellula baltica SH1 T , a free-living bacterium which was isolated from the water column of the Kiel Fjord (Baltic Sea) [2]. R. baltica belongs to the phylum Planctomycetes, a broadly distributed group of bacteria, whose members can be found in terrestrial, marine and freshwater habitats [3][4][5][6][7], but also in extreme environments like hot springs [8], marine sponges [9] and the hepatopancreas of crustaceans [10].
In terms of cell biology all Planctomycetes share several morphologically unique properties, such as a peptidoglycan-lacking proteinaceous cell wall [11,12], intracellular compartmentalization [13] and a mode of reproduction via budding. The latter results in a cell cycle that is characterized by motile and sessile morphotypes similar to Caulobacter crescentus [14][15][16][17]. A specific holdfast substance produced by sessile cells allows R. baltica to attach to macroscopic detrital aggregates (marine snow) [3,7].
At present, four planctomycete genomes are currently available [18]. Of these, the genome of R. baltica is the only one completely closed [16]. The genome was found to be 7,145,576 bases in size and codes for 7325 open reading frames (ORFs) plus 72 RNA genes. Originally, only 45% of the ORFs were assigned particular functions [16]. Thus, over 55% of all proteins in the genome remain functionally uncharacterized. These were referred to as 'hypothetical proteins' with or without the affix 'conserved' contingent on wider phylogenetic distribution [19]. A subset of these conserved hypothetical proteins is specific for Planctomycetes [18]. It seems likely that some of these genes code for the unique planctomycetal cellular characteristics and metabolic traits.
In summary, these studies confirmed the hypothesis of Glöckner et al. that R. baltica is a polysaccharide degrader [16]. It appears R. baltica is gaining carbon and energy from the decomposition of complex heteropolysaccharides originally produced by algae in the photic zone while slowly sedimenting with the marine snow.
Marine microorganisms like R. baltica are exposed to rapidly changing environmental conditions such as varying temperature, salinity, irradiance and oxygen concentra-tion. Typically, sudden changes of these environmental conditions induce a stress response in the exposed planktonic community characterized by a distinct change in their gene expression pattern. This stress response enables the organisms to protect vital processes and to adapt to the new condition. Such responses have been described for a set of organisms from different environments including Shewanella oneidensis [27,28], Pseudomonas aeruginosa [29], Desulfovibrio vulgaris Hildenborough [30], Xylella fastidiosa [31], Synechocystis sp. [32] and Yeast [33].
To gain insights into the stress responses of R. baltica with respect to salinity and temperature the first whole genome array for R. baltica -also the first Planctomycete microarray -was established and applied. The reported data will serve as a resource to expand our understanding of the physiological and transcriptional response of R. baltica to the wide range of changing environmental conditions a freeliving marine bacterium is exposed to.

Results and Discussion
Overview 54 distinct, total RNA samples were analyzed by wholegenome microarray hybridization. Differential expression of 2372, 922 and 1127 genes was noted during heat shock, cold shock and salt stress, respectively, at one or more of the five time points when compared to reference samples (FIGURE 1i; ii &1iii). With only 45% of the genes in R. baltica's genome functionally annotated, it is not surprising that most of the differentially expressed genes were hypothetical or conserved hypothetical proteins. The complete list of the differentially expressed genes for each shift experiment and time point is available in the ADDI-TIONAL FILE 1.
Only 32% of the regulated genes in the heat and cold shock experiments could be assigned with a COG function (FIGURE 2i &2ii) while 37% were assignable in the salt stress experiment (FIGURE 2iii). This is in line with the 36% (2661 genes) of COG functional class designations in R. baltica. A striking feature of the expression profiles displayed is the stereotypical response of a large fraction of the genome to all three stress conditions. In summary, 152 genes are up-or down-regulated at any time point for all stressors. Of these 152 genes, 62 are induced and 90 are repressed (TABLE 1 and TABLE 2). 49% of the induced and 61% of the repressed genes were annotated as hypothetical proteins. The Venn diagrams shown in FIGURE 3 provide an overview of the specific and common genes of the three stress-specific responses. To identify co-regulated patterns of gene expression, we classified all differentially expressed genes of all three stress expressions into 30 k-means clusters based on their expression log ratio. To determine the necessary number of clusters a figure of merit was generated. 30 clusters were Number of regulated genes per stress experiment

Experimental design and array data quality assessment
The experimental conditions used were chosen to mimic the natural environment of R. baltica; however, stress conditions were constrained by the detection limit of the microarray technology used and, hence, were required to elicit a sufficiently pronounced response from the organism. In contrast to steady-state or single-time-point studies, time series experiments can show the dynamic of gene expression.
The negative, positive and stringency controls printed on the array gave no indications for unspecific hybridizations. Co-hybridizations of two cDNA samples prepared from the same total cellular RNA (self-self hybridization) suggested that genes with an expression log ratio value greater than 1.5 and smaller than -1.0 for heat and cold shock, respectively, could be regarded as differentially expressed. Salt stress log ratio values over 1.2 and below -1.0 were considered as significant.

Effect of stress on Rhodopirellula baltica
No growth was detectable during stress conditions nor were any obvious morphological changes by microscopic investigation. Under optimal conditions R. baltica has a doubling time of 10-12 hours [21], suggesting physiological effects are not measurable during the short stress period of, at maximum 5, hours.

Specific results of the shift experiments Heat shock
In their natural environment R. baltica cells can be regularly exposed to higher temperatures, for example, due to irradiation at the water surface. Therefore, R. baltica cells were rapidly shifted from 28°C to 37°C and observed over a period of 300 min in the first experiment. This is approximately 9°C above the optimal growth temperature reported by Schlesner et al. [2]. Employing a higher temperature is very likely to kill the cells. The time series reveals a quick response of R. baltica to sudden temperature up-shifts. In total 2372 genes are regulated out of which 1140 genes encode hypothetical proteins. 390 genes (5%) were regulated after 10 min. This number increased to 750 genes (10%) after 300 min (FIGURE 1i Taking a closer look at the response of R. baltica to thermal stress revealed the induction of many known heat shock proteins (Hsp): ClpB (RB6751), GroEL (RB8970), DnaJ (RB8972), GrpE (RB8974), Hsp20 (RB10279, RB10283), dnaK (RB9105), as well as the ATP-dependent protease ClpP (RB9103). Also up-regulated were the chaperonins Cpn10 (RB10627 and RB8969) and Cpn60 (RB8966) as well as the cell division protein FtsH (RB2966) (Cluster 4 in ADDITIONAL FILES 2). Previous proteomic studies found the proteins of these genes as well, except FtsH, DnaJ and Hsp20 [22,24].
The regulation of the heat shock response in R. baltica involves many transcriptional regulators. TetR (RB838) and GntR (RB1862, RB8695) showed an up-regulation, which affirms their important role in early heat shock response [34]. A gene encoding for GntR was also found in the environment on the planctomycete fosmid 3FN from a Namibian coast metagenome study [18]. In E. coli the induction of the majority of heat shock genes results from a rapid and transient increase in the cellular level of an alternative 32-kDa sigma factor (sigma32) encoded by rpoH along with the alternative sigma factors E and 54, encoded by rpoE (RB2302) and rpoN (RB6491), respectively [27]. Although, all genes are present in the R. baltica genome, they were not observed to be regulated, suggesting a significantly different response cascade.
R. baltica also showed an extracytoplasmic stress response. The gene coding for SecA (RB11690), belonging to the Sec system, was induced. This indicated an activation of protein translocation, most probably from the riboplasma to the paryphoplasm or medium. Proton channels were induced and motility was inhibited as the flagellar motor switch protein (FliG -RB12502) was down-regulated after 20 min. This was followed by the inhibition of the type 4 fimbrial assembly protein (pilC -RB11597) after 40 min.

Cold Shock
To investigate the response to cold shock, R. baltica cells were shifted from the optimal growth temperature 28°C [35] to 6°C and observed for a period of 300 min. 6°C was chosen for this study as this is a common temperature in the Baltic Sea. Sudden temperature chances occur naturally due to turbulences between water layers. Further, the temperature difference of 22°C is generally regarded as standard for cold shock studies with bacteria [27,36]. Compared to heat shock only one third (922) of the regulated genes were differentially expressed. Out of these 922 regulated proteins, 391 genes (42%) encode for  [28].
Transcriptional activity was regulated by the up-regulation of diverse RNA polymerase sigma factors, such as rpoD (RB6780) and sigK (RB1392). A homolog of rpoD (RB6780) was also found on the planctomycete fosmid 13FN [18]. 20 min after the exposure of R. baltica to cold stress conditions it started to express genes implicated in the modification of cytoplasmic membrane composition, fluidity as well as morphology. The alteration of the lipid composition in the cold has been previously reported in other microorganisms [37]. In R. baltica genes coding for cell envelope (RB6114 and RB6895), transport (RB4870), lipid metabolism (RB316) and 18 genes coding for membrane proteins were repressed after 20 min.
Furthermore, R. baltica repressed genes involved in sporulation oppB (RB12861) and O-antigen flippase (RB2503), flaA (RB4454) and pilus assembly (RB4061 and RB5478), leading to reduced motility and budding ability. Genes associated with amino acid biosynthesis, especially with synthesis and fate of glutamine (RB4269) and glutamate (RB5653) were also affected. The latter have been shown to be translated [22,24]. A glycosyltransferase (RB12831) and glycosidases (RB2988, RB2990 and RB2991) were upregulated at 300 min probably to aid in cell wall remodeling.
Although, incorrect protein folding at low temperature is less expected than at high temperatures, chaperons and proteases are required to deal with intracellular protein perturbations [28,38]. Here, this was observed in the induction of GroEL (RB8970) [22,24] and htrA-protease (RB12752). One of the most prominent responses of microorganisms to cold shock is the induction of cold shock proteins. However, the two annotated cold shock proteins of class I (CspA -RB4681 and Cspl -RB10009) [39,40] were not observed to be regulated. One may hypothesize that the stabilization of RNA in R. baltica employs a different protein compliment than observed in E. coli.

High salinity
As a marine organism, R. baltica must adjust to the haline stratification of the Baltic Sea [41,42]. While moving through the water column R. baltica cells are exposed to variable concentrations of dissolved salts. In general, an osmotic up-shift forces bacteria to change their physiology by activating or deactivating specific enzymes or transporters, in order to maintain osmotic balance [43].  ). Similar to other bacteria, R. baltica accumulated glutamate and trehalose as cytoplasmic osmoprotectants in response to osmotic stress [44]. Glutamate dehydrogenase (RB6930) showed an up-regulation after 10 min and was also present in the proteome [24]. Trehalose synthetase treS (RB519) was induced after 60 min. Cysteine, as a general protective component, was only needed in the first hour in elevated salt concentrations and was repressed afterwards (RB4386).
The accumulation of compatible solutes is a widely distributed mechanism used in coping with changing salinity concentrations [44,45]. In R. baltica 74 planctomycetes-Venn diagrams of specific and common stress response Figure 3 Venn diagrams of specific and common stress response. The diagram shows the distribution of stimulus-specific and common stress responses. All genes of all time points are represented in one diagram divided into repressed and induced genes.
group-specific genes are annotated as hypothetical proteins carrying a Domain of Unknown Function (DUF1559) [18]. This domain belongs to a new family of solute binding proteins (PF07596) [46] and was also found on the planctomycete fosmid 8FN [18]. Nine of these genes were up-regulated during the first hour of the cold and salt shock experiments. During the heat shock experiment, 16 of these genes were down-regulated. In vitro experiments have shown that some of these compatible solutes also possess general protein stabilization properties in addition to their osmoprotective property [47]. These homologous proteins do not play an integral role in the transport process per se, but probably serve as receptors that trigger or initiate translocation of solutes through membranes by binding external sites of the integral membrane proteins of the efflux system. In addition, some solute-binding proteins function in the initiation of sensory transduction pathways [46].
R. baltica up-regulated an efflux pump (RB7603) and a Na + /H + antiporter (RB1433) 300 min after salt shift. Both may play a role in the active export of salt ions out of the cells. Quinone oxidoreductase-like protein (RB10967), induced after 40 min, had been implicated in respirationcoupled Na + efflux as also shown in D. vulgaris [30]. Regulatory proteins like sigma-54 factor rpoN (RB6491), rpoA (RB12626) and rfaY (RB12251) were down-regulated. rpoN and rpoA were found to be translated [22,24]. R. baltica inhibited the genes for cell division (soj -RB2291) and chromosome segregation (SMC -RB6065) after 60 min salt stress, as well as diverse transferases (RB12080, RB8898, RB12690, RB2498, RB8222, RB9617) involved in the cell envelope modification. Interestingly, the pilin transport apparatus and the thin-pilus basal body (pilM -RB2860 and pilT -RB12773) were induced after one hour as were principle pilus associated adhesion (pilC -RB12781) and pilB (RB12774). Genes coding for biopolymer transport proteins (exbB -RB12053 and exbD -RB12055) were also induced. A homolog to exbD was annotated on the planctomycete fosmid 3FN [18]. It is known from studies of other organisms that genes encoding the flagellar and chemotaxis systems are up-regulated to move away from the stressful cations [30]. However, none of the flagellar genes were regulated and the genome does not harbor any essential chemotaxis genes except cheY [16]. Notably, the survival protein (SurE -RB10258) and two genes coding for the mechanosensitive ion channel (MscS -RB12279 and RB10255) were induced. The latter provides protection against hypo-osmotic shock, responding both to stretching of the cell membrane and to membrane depolarization [48]. Genes in Cluster 22 (ADDITIONAL FILE 2) seemed to be significantly affected by salt stress only.

Common stress response
R. baltica showed a common stress response to all three tested environmental factors. Several known general stress genes were induced, such as genes coding for the manganese-containing catalase (RB10727), which is also present in the proteome [21,22,24]. Ferritin and Dps (RB4433) or pyridoxamine 5'-phosphate oxidase (RB4438) belong to a general stress cluster (RB4432-4438) and were initially described by Hieu et al. [24]. Thioredoxin (RB10378) could serve as an electron donor for the up-regulated methionine sulfoxide reductase gene (msrB -RB2268) [49,50]. The genes could be regulated via rpoN found on the proposed upstream sigma 54-dependent promoter (RB10378) [51].
Perhaps to cope with reactive oxygen species (ROS), typically present under stressful conditions [50], the nitrogen fixation protein (nifU -RB3596) was induced. NifU is involved in the biosynthesis and repair of ROS scavenging iron-sulfur clusters. Finally, the peptidase M50 (RB6092) may have been induced to regulate stress response, sporulation, cell division, and cell differentiation [52].
Interestingly, the machinery for the rearrangement and interchange of genetic material was induced under all three stressful conditions. It seems to play an important role in the organism's long-term adaptation. R. baltica harbors 81 non-randomly distributed transposases in its genome. Notably, under heat stress three times more transposase genes were up-regulated than under cold stress and twice as many as under salt stress. Shared induction shows five IS3/IS911, three ISXo8, two putative transposases (RB170, RB5888, RB11749, RB11802, RB12940, RB2186, RB9907, RB12239, RB934 and RB7389), and one integrase (RB11750). Rearranging the genome to select the most efficient gene combination has been described as a common way to adapt quickly to extreme environments [34]. Relaxed DNA may also be required to get better access to the gene regions for increased expression. Here, DNA relaxation is suggested by the repression of histone-like DNA-binding protein (RB6276).
In line with an alternative global sensing and regulation system initially proposed by Glöckner et al. [16], a common pattern concerning sensing and regulation response was detected. R. baltica contains 37 genes belonging to the extracytoplasmic function (ECF) subfamily of sigma 70 [53]. The genes RB138, RB13241 and RB10049 are up-regulated under all three stress conditions. Studholme et al. [46] suggests that ECF-factor RB10049 is the regulator for the conserved hypothetical protein RB10051. The conserved domain belongs to a new group of proteins that share novel domains referred to as planctomycete-specific (PSD) or planctomycete-specific cytochrome C (PSC). RB10051 contains the PSD1 (DUF1553 -PF07587) and PSC2 (DUF1549 -PF07583) domains, suggesting a function in redox reactions [46]. Each domain is represented 41 times in the whole genome of R. baltica [18].
Additionally, at 300 min the ECF-sigma factor RB138 was up-regulated together with serine/threonine protein kinase (RB140). Protein kinases are believed to be involved in stress response [37,54]. The serine/threonine protein kinase (RB12942) and two histidine-kinases (RB4511 and RB10330) were up-regulated during heat shock. Whereas, under cold shock only one serine/threonine kinase (RB8505) was induced. Under salt stress a histidine-kinase (RB13122) and three two-component systems (RB5780, RB12952 and RB13118) were induced.
Finally, the ECF-sigma factor RB1790 was up-regulated, but only under high salinity conditions. In summary, the results confirmed that ECF sigma factors, as well as twocomponent systems, are heavily involved in stress sensing and regulation of R. baltica. The importance of these genes in the natural environment is asserted by the presence of a homolog to RB12952 on the planctomycete fosmid 6N14 [18].
The down-regulation of genes associated with the ribosomal machinery (55%) was observed. During heat shock and high salinity these genes were permanently repressed, whereas under cold shock they were only repressed within the first hour. Of the 51 ribosomal proteins in the whole genome, 18 genes encoding proteins of the small-and large subunit (RB1233, RB12821, RB12824, RB12839, RB7117, RB7837 -RB7841, RB7849, RB7850, RB7852, RB7854, RB7856, RB7857, RB7859 and RB7899) were repressed. Additionally, a set of genes involved in RNA metabolism, protein synthesis, as well as R. baltica's only translation elongation factor (EF-Tu -RB7894) were repressed. The genes for the conserved hypothetical protein RB12818 and the hypothetical protein RB12837 were co-regulated which suggests an association with the translation machinery. The repression of the ribosomal genes, along with a large set of genes involved in RNA metabolism, protein synthesis, cell growth (Cluster 1 ADDI-TIONAL FILE 2), has been reported as a general feature of the environmental stress responses (ESR) [33]. It has been assumed that they are acting as stress sensors [55]. This coincides nicely with the induction of the ribosomal proteins at 300 min under cold shock conditions. Recovery and ongoing adaptation of R. baltica was further supported by the up-regulation of the ribosomal-binding fac-tor rbfA (RB5503), which is, aside from csdA, required for optimal growth at low temperatures [56].

Hypothetical proteins
Approximately 50% of the regulated genes observed have no known function in each of the three environmental stress experiments. Some of these share a similar expression profile (ADDITIONAL FILE 2). We propose that some of these genes are involved in cell morphology changes, stress sensing and regulation. The low number of known transcriptional regulators (2.4%) in the genome of R. baltica [53], coupled with the fact that most of the essential pathways encoded are not organized in operon structures [16] support the hypothesis of novel global regulation mechanisms. Hypothetical proteins that carry regulatory domains, like the FHA domain in RB1789 or a putative transcriptional regulatory domain in RB9999 are strong candidates. RB11766 might regulate the gene next to it, which is a so called giant gene (RB11769) [57]. This giant gene encodes a novel peptide motif that is most likely involved in cell morphology changes [46]. The importance of the hypothetical proteins RB11505, RB10954, RB10956 and RB10958 was further supported by their presence on the proteome gels of Hieu et al. as well as Gade et al. [21,22,24]. The latter three of these genes were claimed to be among the most abundant proteins in R. baltica cultures grown on mineral medium.

Planctomycete special feature: Genes encoding sulfatases
The genome of R. baltica contains no less than 110 sulfatases. It is assumed that they are involved in the recycling of carbon from complex sulfated heteropolysaccharides. Although the mineral medium does not contain any sulfated polysaccharides, we found 11 sulphatase genes were up-or down-regulated (TABLE 3) during the different stress experiments. These included one choline sulphatase (RB1205), seven arylsulfatases (RB13148, RB1477, RB3403, RB406, RB5146, RB684 and RB9498), two sulphatase genes without specificity (RB3956, RB5294), and one alkylsulfatase (RB11502). Furthermore, during life cycle experiments (unpublished data) we found evidence that certain sulfatases are only regulated in specific growth stages, which could indicate their involvement in the remodeling of the distinct morphological features of R. baltica. Sulfatase genes RB1477, RB5294, RB9498 and RB11502 were induced. We propose that RB9498 and RB11502 have an extracellular function and may be involved in the formation of an extrapolymeric substance.
Six sulfatase genes (RB406, RB684, RB1205, RB3403, RB5146 and RB13145) were repressed after 300 min of heat shock. They may have been involved in the rearrangement of the cell wall formation, which comprises a protein sacculus with disulfide bonds [12]. In summary, these results show the diverse roles that sulfatases may have and, furthermore, that only a variety of different experimental approaches will increase our knowledge of these roles.

Conclusion
This work presents the first transcriptome study of the environmental stress response of a marine, free-living Planctomycete. Although R. baltica is an unusual organism in many aspects, its stress responses to heat and cold shock as well to changing salinity were in line with earlier results reported for other model organisms. Heat shock induced a set of chaperons, likely to protect cellular proteins from denaturation and breakdown. Growth in the cold may be followed by the induction of genes altering lipid metabolism. Salinity shifts resulted in the activation of planctomycete-specific groups of genes including genes involved in morphological change and an extracytoplasmic stress response. All stressors triggered the down-regulation of the ribosomal machinery, the up-regulation of transposases and the induction of several ECF-sigma factors and two-component systems. This supports the hypothesis that R. baltica is regulating its gene activity on a global rather than operon level. Aside from well characterized stress response genes, about 2000 genes of unknown function, constituting 30% of the genes predicted in the genome, were affected. This, combined with proteome studies and the presence of some of the genes in fosmid libraries, provides a strong indication that the vast number of genes with unknown function play a vital role in the organism's environmental response. The regulation of 11 sulfatases during stressful conditions suggests that these genes are heavily involved in the core cellular function of R. baltica. The data presented lead to the conclusion that R. baltica's rich repertoire of genes is combined with a fine tuned regulation mechanism to best respond to the changing conditions of its habitat. Nevertheless, data analysis has just started and further investigations concerning the genes involved in the life-cycle, the stress response pathways, promoter regions and network analysis are already ongoing or planned for the near future.

Bacterial growth conditions
For all experiments Rhodopirellula baltica SH1 T cells were grown as chemostat cultures in a mineral medium containing 10 mM glucose as the sole carbon source and 1 mM ammonium chloride as a nitrogen source at 28°C [20]. Chemostat (Ø 13.5 cm × 25 cm, 1 l, Schott, modified by Ochs, Bovenden) parameters used were: pH 7.4, average dilution rate 0.75 ml/min and pO 2 around 100%. The cultures had an OD 600 nm of 0.5 -0.6 (corresponding to log phase). The cells were harvested after 5 dwell times.

Sample collection, cell lyses, RNA Isolation and cDNA synthesis
After harvesting the R. baltica cultures, an aliquot was collected to serve as the time-zero reference. The culture broth was collected in 500 ml tubes and swirled briefly in an ethanol-dry ice bath to rapidly cool the cultures and prevent shifts in the RNA profile. Subsequently, the broth was centrifuged at 6000 rpm for 20 min at 4°C (Beckman Coulter™ AvantiTM626 J-20XP, JA10 Rotor). The pellets were re-suspended in 0.1 M Tris-HCL and then re-centrifuged. Cell pellets were shock-frozen in liquid nitrogen and stored at -80°C. Total RNA was isolated using the protocol of the TRI Reagent ® Kit by Ambion (Austin, USA).
The purity and quality of the extracted total RNA was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Palto Alto, USA) and gel electrophoresis. cDNA synthesis was performed using the SuperScript direct cDNA labeling kit by Invitrogen (Karlsruhe, Germany) according to the manufacturer's instructions with random hexamers and unlabeled dCTP/dUTP, followed by a three hour reverse transcription incubation step at 46°C. The RT reaction was halted by incubation for 3 min at 95°C. To hydrolyze the RNA, 0.1 M NaOH was added, incubated at 65°C for 15 min and neutralized with 0.1 M HCL. The Concentrations of RNA and cDNA were measured, and incorporation of the dyes Alexa 546 and Alexa 647 were checked using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, USA).

Experimental design and sample preparation
In three independent hybridizations conducted for each experiment and time point, the expression profiles of cells that had undergone stress were compared with those of cells at time zero. That is, the array analysis of each Alexa 647 labeled sample was compared with those of Alexa 546 labeled time-zero samples. The data shown are based on the analysis of all three replicates performed for each of the conditions. Samples for expression profiling and microscopic analysis were collected at 10, 20, 40, 60 and 300 min in all three stress experiments.

Heat shock from 28°C to 37°C
Cells grown continuously at 28°C were collected by centrifugation. An aliquot was removed for RNA extraction and taken as the time zero reference for the heat, cold and salt stress experiments. Aliquots were re-suspended in an equal volume of 37°C medium and returned to 37°C for cultivation.

Cold shock from 28°C to 6°C
Cells grown continuously at 28°C were collected by centrifugation, re-suspended in an equal volume of 6°C medium and returned to 6°C for cultivation.

Signal detection and data analysis
Slides were scanned at a resolution of 5 μm using a ScanArray Express Microarray scanner (Perkin Elmer, Wellesley, USA) with varied laser power and photomultiplier tube (PMT sensitivity) for each slide. The accompanying image analysis software, ScanArray Express Version 4.0, was used for automatic spot detection and signal quantification of both fluorophores. Raw data were automatically processed using the microarray data analysis software tool MADA http://www.megx.net/mada, developed in-house. Firstly, the spot intensities were corrected for local background (mean spot intensity minus mean spot background intensity). Signals were only assessed as positive if mean spot pixel intensity was higher than the mean local background intensity plus twice the standard deviation of the mean local background pixel intensity. Each gene is spotted in three replicates. Spot replicates with poor quality were removed from the data set according to MADA's outlier test results. This test first computes the standard deviation of all replicates. Secondly, one replicate is omitted and the standard deviation is recalculated; if the deviation differs more than 50% from the previous deviation, the omitted replicate is regarded as an outlier. This procedure is repeated for all replicates Expression is described through the ratio and intensity, where R is the fluorescence log ratio of the experiment time point relative to the control condition (e.g. R = log2 (result of channel 10 min/result of channel control/refer-ence)) and I is the log mean fluorescence intensity (e.g. I = log10 (result of channel 10 min × result of channel control/reference)).
Each data point represents a regulation factor (ratio) in a logarithmic scale for one gene calculated from the positive replicates for a particular probe coming from two RNA pools (reference and sample). Normalization was carried out by LOWESS fitting on an R-versus-I plot with a smoothing factor of 0.5. Each time point of the time-series experiment was hybridized independently three times. The expression data (ratio) of the three hybridizations were combined to one expression data point (ratio) by averaging and the standard deviation of the average value was calculated. Only ratios with a standard deviation less than 25% were regarded as genes that are regulated. Differentially expressed genes were detected by a fixed threshold cut off method (i.e. a two-fold increase or decrease) based on the results of self-self hybridization. Using the same biological sample, the reference (untreated sample) is labeled twice, once with Alexa 546 and once with Alexa 647, and the variability between the two sets of measurements is calculated to estimate the experimental noise. Ideally, there should not be any variability and all expression points should have a ratio close to zero. In reality, however, this is never the case and thresholds based on the distribution of these data along the y-axis were defined for the further experiments.
Consequently, R. baltica genes detected with intensities resulting in ratios above or below these thresholds can beregarded as up-or down-regulated.

Cluster analysis
Differentially expressed genes present in the complete time course profile (10, 20, 40, 60 und 300 min) for all three experiments were clustered using the k-means clustering approach (Euclidean distance metric, k = 30 clusters and 49 (max. 500) iterations) [58] with the software tool Multiexperiment Viewer MeV Version 4.0.2 from the TM4 microarray software suite [59]. Briefly, the clustering algorithm arranges genes into a given number of clusters, k, according to similarity in their expression profiles across the entire array experiments, such that genes with similar expression patterns are clustered together. The data are displayed in tabular format where each row of colored boxes stores the variation in transcript abundance for each given gene and each column stores the variation in transcript levels of every gene in a given mRNA sample, as detected on one array. The variations in transcript abundance for each gene are depicted by means of a color scale, in which shades of red represents increases and shades of green represent decrease in mRNA levels, relative to the unstressed culture, and the saturation of the color corresponds to the magnitude of the differences. Black colora-tion indicates no change in transcript level while grey represents missing data.
FOG contributed background information and was involved in writing and finishing the manuscript.
All authors read and approved the final manuscript.