Halobacterium sp. NRC-1 flourishes in environments that are not only hypersaline (4.3 M NaCl optimum) but also highly variable in total salinity (2.5–5.3 M NaCl) as a result of common evaporatic and dilution processes 1. Therefore, the cells must cope with high as well as dynamic concentrations of dissolved salts. The intracellular concentration of KCl is roughly equal (concentrations up to 5 M have been reported) to the external NaCl concentrations, and is used as the major compatible solute 17. Genome-wide, predicted proteins have a median pI of 4.9, with a concentration of negative charges on their surface, characteristics which permit effective competition for hydration and allow function in a cytoplasm with low water activity 318. High salt concentrations are also known to convert some DNA sequences, e.g. alternating (CG)-repeats, from the right-handed B to the left-handed Z form, facilitated by negative superhelical stress 1920. Organic compatible solutes commonly utilized in the response of bacterial and eukaryotic halophiles to osmotic stress, have also been reported in Halobacterium sp. NRC-1 2122.

The annotated genome of Halobacterium sp. NRC-1 provided an inventory of likely genes involved in maintaining the intracellular ionic conditions suitable for growth 1112. Genes coding for multiple active K⁺ transporters were found, including kdpABC, an ATP-driven K⁺ uptake system, and trkAH, low-affinity K⁺ transporters driven by the membrane potential. Genes coding for an active Na⁺ efflux system likely mediated by NhaC proteins were also present, corresponding to the unidirectional Na⁺-H⁺ antiporter activity described previously 23. Interestingly, several of these genes, including those coding for kdpABC, trkA (three copies), and nhaC (one copy) were found on the extrachromosomal pNRC200 replicon. However, a comprehensive study of the adaptation of Halobacterium sp. NRC-1 to various salinities was not previously reported.

In order to determine which genes were most responsive to high and low salinity, Halobacterium sp. NRC-1 cultures were grown at three salinities, low (2.9 M NaCl), optimal (4.3 M NaCl), and high (5.0 M NaCl), in rich media at 42°C (Figs. 1 &2). The high and low salinity conditions were selected to ensure that salts did not precipitate and cells did not lyse during culture, respectively. Growth at low salinity compared to optimal salinity displayed 143 up-regulated genes and 53 down-regulated genes by 1.5-fold or greater (Fig. 3). Growth at high salinity, where the NaCl concentration (5 M) was only slightly higher than the optimal salinity (4.3 M) displayed 32 up-regulated genes and 29 down-regulated genes by 1.5-fold or greater (Fig. 4).

Transcription of several potassium and sodium ion transporter genes was affected by salt conditions in NRC-1, mainly by low salt 2425. Among the putative potassium transporters, the trkA6 gene, was significantly up-regulated (2.0-fold) under low salt conditions (Table 1). This gene product may compensate for the loss of K⁺ under low salt conditions, via membrane leakiness, by uptake of more potassium ions. Among sodium transporters, only one (nhaC3, coded by pNRC200) gene was slightly down-regulated (1.3-fold) under low salt, while the others were unchanged. The lack of a more vigorous induction of the sodium-proton antiporter suggests that increases of Na⁺ concentration in cells may be tolerated during osmotic stress, possibly substituting for K⁺ intracellularly.

Expression of many other transporter genes was altered by growth in non-optimal salinities. The sfuB iron transporter-like permease protein was down-regulated (1.6-fold) under high salt (Table 2), and the phnC gene, a component of the phosphate/phosphonate transport system was down-regulated by 1.4-fold in high salt, suggesting that Halobacterium sp. NRC-1 responds to osmotic stress by reducing uptake of these species, which may potentially be toxic 26. The nosY gene, coding for an apparent ABC transporter component for nitrite/nitrate, was up-regulated under high salt (1.5-fold), although usage of nitrite or nitrate for respiration or as a nitrogen source has not been detected for NRC-1 13. Members of the two similarly organized gene clusters containing oligopeptide/dipeptide/Ni²⁺ transporter genes (appABCDF and dppABCDF, see Table 1), were responsive to salinity changes. Two genes in the app cluster were up-regulated between 1.5- and 1.9-fold under both high and low salt, while some genes in the dpp cluster were down-regulated 1.4- to 1.6-fold under low salt. In both cases, the permease genes, C and/or B, were more affected than the genes coding for the periplasmic and ATPase components. The increase in expression of these genes is consistent with an attempt by cells to minimize the loss of these ionic (or zwitterionic amino acids) species, whereas down-regulation may be related to growth rate or possible toxic effects. Interestingly, the proX and htr5 genes, which are proposed to code for compatible solute (possibly trimethylammonium) binding and transporter proteins (cosB and cosT, respectively, in the closely related Halobacterium salinarium 27) were unchanged in high or low salt concentrations in NRC-1.

Protein kinases are believed to be involved in the salt stress response cascade in eukaryotic cells 28 as well as some prokaryotic species 29. Among genes possibly involved in signaling in Halobacterium sp. NRC-1, the only signal transduction histidine kinase gene out of 13 in the genome that showed any significant change was hik5, for which expression under the high salt condition was slightly lowered (1.3-fold). In the large halotransducer (htr) family in NRC-1 only two of 17 htr genes, htr12 and htr13, were up-regulated under low salt (1.4- to 1.7-fold) 30. Knowledge of the true functions of these families of genes and their precise roles in signal transduction in Halobacterium sp. NRC-1 is quite limited at present.

Several transcription factors and regulators were significantly changed under salt stress conditions. Among these, one basal transcription factor gene out of 13 in NRC-1 12, tbpC (coded by pNRC200), was up-regulated 1.7-fold under low salt conditions. Also, the sirR gene, a putative transcriptional repressor, was significantly up-regulated under high salt (2.0-fold).

Interestingly, three stress genes were inducible under high and low salt conditions. Both sod genes, which encode superoxide dismutase, were salt-responsive. The sod2 transcript was up-regulated 1.4-fold under high salt and 2.0-fold under low salt, and sod1 was up-regulated 1.4-fold under high salt and 1.8-fold under low salt. Superoxide dismutase is responsible for limiting damage from reactive oxygen species, which are induced by extreme conditions, such as oxidative stress and excess irradiance 31 in many species. The gst gene, coding for glutathione S-transferase, another detoxification enzyme involved in cell protection from reactive oxygen species 32, was up-regulated (1.5-fold) under low salt conditions. These findings suggest that the removal of reactive oxygen species is of increased importance in osmotically stressed haloarchaea.

An earlier study 33 suggested that the small heat shock protein family coded by hsp genes may be involved in salt stress responses in the related haloarchaeon, Haloferax volcanii. We observed that two hsp genes in Halobacterium sp. NRC-1 were affected by salinity changes: hsp1 was up-regulated under low salt conditions (2.3-fold) and hsp5, coded by pNRC200, was down-regulated 1.4-fold under high salt conditions (as well as in the cold, see below). Two predicted stress genes, the cold shock gene cspD1 (1.4-fold up-regulated), and the heat shock gene dnaK (1.3-fold down-regulated), were regulated under low salt conditions, which suggests a wider role than temperature adaptation for these chaperones. Interestingly, a carboxypeptidase gene, cxp, which may function in protein turnover, was highly down-regulated in low salt (2.2-fold), and equally up-regulated in high salt.

Expression of a number of genes involved in cellular metabolism was significantly altered by salinity changes. Two highly regulated genes, carA and carB, coding for both subunits of carbamoyl-phosphate synthase, were up-regulated between 2.5- and 2.9-fold under high salt conditions and down-regulated 1.5- to 1.6-fold under low salt conditions. Any relation of these results to similar observations in Xenopus is unclear 34. One sugar kinase, product of the ush gene, was down-regulated (1.4-fold) at low salinity, and the malate dehydrogenase gene, mdhA, was up-regulated (1.4-fold) at high salt in NRC-1. The long-chain fatty acid-CoA ligase, coded by lfl3, was up-regulated 2.1-fold in high salt. However, we did not observe a similar change in other genes involved in lipid metabolism 35. The glutamine synthetase gene, glnA, was down-regulated (1.7-fold) under low salt conditions. The thioredoxin gene, trxA1, which is present on the inverted repeats of the pNRC replicons, was down-regulated under low salt conditions (1.4-fold). One of two aspartate aminotransferase genes, aspC2, was up-regulated under low salt (1.7-fold), and the two subunits of aspartate carbamoyltransferase, coded by pyrB and pyrI (both coded by pNRC200), were up-regulated under high salt (1.8-fold). A xanthine/uracil permease family protein, coded by xup, was down-regulated by 1.6-fold under low salt conditions, and the Na⁺-driven multidrug efflux pump, coded by the dfr gene, was down-regulated by 2.1-fold also under low salt. The cbiNQ genes, coding for part of the Co²⁺ transport system, were down-regulated 1.3- to 1.6-fold in low salt; however, this result may reflect the lower growth rate. Additional likely growth-rate dependent genes altered under low salt, included genes coding for the large ribosomal proteins, rpl2p, rpl22p, rpl23p, rpl29p (about 1.5-fold down-regulated) and the H⁺-transporting ATP synthase subunits, atpIKECFA (1.5 to 1.8-fold down-regulated). It is possible that the changes in gene expression of many of these salt-responsive metabolic genes in NRC-1 may be indirect, most likely as a result of growth rate changes.

Optimum growth for Halobacterium sp. NRC-1 occurs at 42°C, but measurable growth is observed down to a temperature of 15°C, with very slow growth at temperatures as low as 10°C 1136. The growth temperature optimum and minimum for NRC-1 are typical of most haloarchaea, but significantly higher than for some cold-adapted species, e.g. Halorubrum lacusprofundi, where the temperature minimum for growth has been reported down to -2°C 3637.

In order to determine which genes in Halobacterium sp. NRC-1 are responsive to reduced temperatures, cultures were grown at low (15°C) and optimal (42°C) temperatures (Fig. 5). Growth at 15°C resulted in up-regulation of 151 genes and down-regulation of 287 genes by 1.5-fold or greater (Fig. 6). Interestingly, 29% of the up-regulated and 37% of the down-regulated genes are of unknown function, suggesting the involvement of novel genes in adaptation of Halobacterium sp. NRC-1 to cold temperature.

An interesting class of responsive genes was those involved in membrane lipid metabolism. The gene coding for the first step in synthesis of polar lipids, sn-1-glycerol phosphate dehydrogenase (gldA), which is coded by pNRC200, was down regulated 2.7-fold (Table 3), suggesting that polar lipid biosynthesis is altered in Halobacterium sp. NRC-1 cells growing at low temperature. There is also an up-regulation of genes encoding dehydrogenases, such as acd4 (1.5-fold) coding for acyl-CoA dehydrogenase, and genes coding for increased turnover of polar lipids, e.g. lfl3 (2.2-fold), coding for a long-chain fatty acid-CoA ligase, and aca (1.4-fold), coding an acetoacetyl-CoA thiolase. These results are consistent with Halobacterium sp. NRC-1 altering the composition of its lipids in the cold, as has been previously reported in other microorganisms 3839404142.

The genomes of several haloarchaea have shown the presence of multiple cold shock genes, the products of which may bind to single-stranded DNA and RNA, functioning as RNA chaperones, and facilitating the initiation of translation under low temperatures 364344. As expected, expression of both cold shock genes, cspD1 and cspD2, was altered in NRC-1 cells during growth at 15°C, with the former up-regulated 2.3-fold and the latter up-regulated 3.1-fold. In addition, three heat shock genes, dnaK, grpE and hsp5, were down-regulated from 1.5- to 4.8-fold in the cold. Most of these changes, some of which were among the highest fold-changes observed in our investigations, were reversed when cells were heat shocked (see below).

A striking observation was that gas vesicle gene expression and content were increased when Halobacterium sp. NRC-1 was grown in the cold. In particular, the rightward transcribed genes of the buoyant gas vesicle gene cluster (gvpACNO coded on both pNRC replicons), were significantly up-regulated, showing increases from 1.8- to 8.0-fold in the cold. The first two genes of the leftward operon, gvpDE, which encode putative regulators for gas vesicle biosynthesis, were also up-regulated (3- and 3.4-fold). Microscopic examination of cells yielded results that were consistent with the microarray data, i.e. an increase in gas vesicle content observable in cells grown at lower temperatures (data not shown).

Previously, increases in the supercoiling of DNA have been reported upon cold shock in E. coli 4546; congruently, the levels of certain DNA topoisomerases change during cold shock 4748. Surprisingly, the Halobacterium sp. NRC-1 gyrB gene was found to be down-regulated 1.4-fold in the cold, and we did not observe significant changes in gyrA, top6AB, or topA. Interestingly, the archaeal histone gene (hpyA) was down-regulated (1.5-fold), while the actin (mbl) and tubulin-like (ftsZ2) genes were up-regulated (1.7- and 1.6-fold respectively) in the cold, suggesting reduced need for genomic compaction but increased requirement for intracellular organization in the cold.

At reduced temperatures, the solubility of gases and the stability of toxic reactive oxygen species may increase 4950. However, we did not observe a corresponding increase in either superoxide dismutase (sod1 and sod2) gene or the glutathione S-transferase (gst) gene. Further, the peptidyl-prolyl cis-trans isomerase (slyD) and the peptidyl-prolyl isomerase (ppiA) genes were not up regulated. These results suggest that at low temperatures, NRC-1 does not enhance the use of these gene products to remove oxygen radicals or interact with hydrophobic patches and aid in the folding of proteins 51.

A variety of important genes necessary for metabolism and cellular functions were altered during growth at 15°C, likely as a result of decreased growth rate. Genes coding for the H⁺-transporting ATP synthase (atpIKEBD), several of the 30 and 50S ribosomal proteins (rps and rpl genes), transport (secE, gspE2), purine/pyrimidine metabolism (purSQ, apt, cmk, pyrBI), and chemotaxis (cheY) were all down-regulated (1.4 to 3.0-fold). For genes of carbohydrate metabolism, fbaA, a sugar aldolase, and pmu2, a sugar kinase, were about 1.7-fold down-regulated. Among genes related to peptide metabolism, e.g. the dipeptide transporter coded by the dpp gene, dppA, was up-regulated (1.6-fold), as were two aminopeptidase genes, pepQ2 (3.4-fold) and yuxL (2.1-fold). These findings suggest an effort to increase the amino acid pool available to cells in the cold; however, decreased growth rate would result from inhibition of ATP production, protein synthesis and export, DNA metabolism, and taxis.

Halobacterium sp. NRC-1 is known to be slightly thermotolerant, exhibiting relatively normal growth up to 48°C. As the temperature is increased from the growth optimum of 42°C, the growth rate declines and temperatures above 50°C prevent growth and provoke photobleaching. After treatment of cells at 49°C, normal growth resumes after shifting back to more moderate temperatures. Pretreatment of NRC-1 cells at 49°C for 1 hour, and then shifting to 56°C, increased survival 2.5-fold compared to cells directly shifted from 42 to 56°C, indicating a classic heat shock effect (Fig. 7) 52.

In order to determine which genes are responsive to heat shock, Halobacterium sp. NRC-1 cultures which were shifted for one hour from 42 to 49°C were compared to cultures remaining at 42°C. Heat shock at 49°C resulted in up-regulation of 64 genes and down-regulation of 43 genes by 1.5-fold or greater (Fig. 8). Genes of unknown function constituted 34% of the up-regulated and 28% of the down-regulated genes.

Of the four genes coding small heat shock proteins belonging to the Hsp20/α-crystallin family in Halobacterium sp. NRC-1, which are known to protect against irreversible aggregation of cellular proteins and assist in protein refolding during stress conditions 53, hsp5 was the most highly affected, being up-regulated 3.0-fold after heat shock (Table 4). Among genes encoding the dnaK/dnaJ/grpE Hsp70 family chaperones involved in disaggregation and reactivation of proteins 54, only the dnaK gene was found to be highly up-regulated, 1.8-fold after heat shock. The cctA gene, coding the chaperonin-containing t-complex polypeptide (Cct) thermosome family was up-regulated 8.7-fold 53. This family has been hypothesized to be involved in general environmental stress 555657; however, in this transcriptomic study, we only observed an increase in transcript under heat shock in NRC-1. In addition, members of the AAA⁺ ATPase class have been found to be up-regulated during heat shock and aid in the refolding of proteins 5859. Two members of this class of genes, cdc48b (5.6-fold) and cdc48d (3.4-fold), the latter of which is coded by pNRC200, were significantly up-regulated during heat shock.

Some researchers have reported an increase in DNA repair proteins during heat shock responses 6061. In our experiments, about 5% of the up-regulated genes were involved with DNA metabolism; however, only one DNA repair gene, srl2, a SMC/Rad50-like ATPase found on pNRC200 was slightly up-regulated (1.5-fold). Among other genes coding DNA binding proteins, we also observed an increase in dpsA (1.9-fold), a stress inducible DNA binding protein, consistent with an earlier proteomic study 52.

Many genes seem to be coordinately regulated when Halobacterium sp. NRC-1 is exposed to the cold and heat, including twenty-five genes that were inversely regulated under the two conditions. Although many of these genes are of unknown function, several genes known to be involved in heat shock (hsp5, dnaK, cctA), as well as two putative regulators (gvpE1 and boa2) were identified. In addition, ten genes were regulated in the same direction under both conditions, including tfbG (2.4-fold up-regulated), one of seven TFB genes in NRC-1.

Halobacterium sp. NRC-1 cultures were grown in standard CM⁺ medium with shaking at 220 rpm on a New Brunswick Scientific platform shaker. For growth in the cold, cultures were aerobically grown at 15°C. For heat shock experiments, cells were grown at 42°C followed by incubation at 49°C for one hour and 56°C for three or four hours. For salt experiments, cells were grown at 42°C with either 2.9, 4.3, or 5.0 M NaCl. Pre-cultures grown under the same conditions used in the growth curve were used as inocula. For the heat killing profile, Halobacterium sp. NRC-1 cultures used were grown under standard laboratory conditions (aerobically at 42°C in CM⁺ medium). Each growth curve and heat killing profile was based on the average of three cultures. For each microarray experiment, three cultures were grown in the same manner as those used for the growth curves and RNA was pooled from all three cultures for cDNA synthesis and hybridization. Cultures grown at high and low salinities and 15°C were harvested at early exponential phase (OD₆₀₀ = 0.19 to 0.23) for microarray analysis. Cultures used for the heat shock microarrays were grown to early exponential phase and then incubated at 49°C for 1 hour. Before harvesting the cells to collect RNA, cultures were swirled briefly in an ethanol-dry ice bath to rapidly cool the cultures and "freeze" the RNA profile. Total RNA was isolated using the Agilent total RNA isolation mini kit (Agilent Technologies, Palo Alto, CA) and then treated with RNase-free DNase I. cDNA was prepared with equal amounts of RNA from control and experimental samples and then fluorescently labeled with Cy3-dCTP and Cy5-dCTP. Concentrations of RNA and cDNA were measured using a Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies, Wilmington, DE). Incorporation of the Cy3 and Cy5 labels was checked via gel electrophoresis and scanning on a GE Typhoon fluorescence scanner (GE Healthcare, Piscataway, NJ). Washing and hybridization of the arrays was performed as recommended by Agilent and as previously described 5. Slides were scanned for Cy-3 and Cy-5 signals with an Agilent DNA-microarray scanner. Probe signals were extracted with the Agilent Feature Extraction Software and analyzed using the statistical methods described below. Oligonucleotide arrays, in situ synthesized using ink-jet technology, were used for transcriptome analysis of Halobacterium sp. NRC-1. Oligomer (60-mer) probes were previously designed for 2474 ORFs utilizing the program OligoPicker 16. Two replicate microarrays were performed for the high and low salinity and 15°C growth experiments, and three replicate microarrays were performed for the heat shock experiment. Data shown are based on the analysis of all arrays performed for each of the given conditions.

The Agilent Feature Extraction program was used for image analysis and processing of the microarray image file. Background signals were subtracted from raw signals using the area either on or around the features. Dye biases created by differences in the red and green channel signals caused by different efficiencies in labeling, emission and detection were also estimated and corrected. Signals from each channel were normalized using the LOWESS algorithm to remove intensity-dependent effects within the calculated values. Further statistical analyses were performed as described below.

The illuminant intensity, log₂(x) value, and standard deviation of the log₂(x) value were calculated for the normalized red and green probe values for each gene in each microarray. The illuminant intensity was calculated through the logarithm of the geometric mean of Cy5 and Cy3 processed signal intensities by first calculating:probesignal=12log⁡2(Cy5*Cy3) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqWGWbaCcqWGYbGCcqWGVbWBcqWGIbGycqWGLbqzcqWGZbWCcqWGPbqAcqWGNbWzcqWGUbGBcqWGHbqycqWGSbaBcqGH9aqpdaWcaaqaaiabigdaXaqaaiabikdaYaaacyGGSbaBcqGGVbWBcqGGNbWzdaWgaaWcbaGaeGOmaidabeaakiabcIcaOiabdoeadjabdMha5jabiwda1iabcQcaQiabdoeadjabdMha5jabiodaZiabcMcaPaaa@4D87@. Then for a finite-sized sample of size N, we calculated the intensity using the arithmetic mean: illuminant intensity=1/N∑i=1nprobesignali MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqqGPbqAcqqGSbaBcqqGSbaBcqqG1bqDcqqGTbqBcqqGPbqAcqqGUbGBcqqGHbqycqqGUbGBcqqG0baDcqqGGaaicqqGPbqAcqqGUbGBcqqG0baDcqqGLbqzcqqGUbGBcqqGZbWCcqqGPbqAcqqG0baDcqqG5bqEcqGH9aqpcqaIXaqmcqGGVaWlcqqGobGtdaaeWbqaaiabdchaWjabdkhaYjabd+gaVjabdkgaIjabdwgaLjabdohaZjabdMgaPjabdEgaNjabd6gaUjabdggaHjabdYgaSnaaBaaaleaacqWGPbqAaeqaaaqaaiabdMgaPjabg2da9iabigdaXaqaaiabd6gaUbqdcqGHris5aaaa@6342@. Biased estimators for sample means of log₂(x) ratios were calculated by the arithmetic mean of log₂(x) ratios for a set of N probes for a gene; where r_i is the processed Cy5 illuminant intensity level and g_i is the processed Cy3 illuminate intensity value for the ith probe: log⁡2¯=1/N∑i=1nlog⁡2(ri,gi) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaqdaaqaaiGbcYgaSjabc+gaVjabcEgaNnaaBaaaleaacqaIYaGmaeqaaaaakiabg2da9iabigdaXiabc+caViabb6eaonaaqahabaGagiiBaWMaei4Ba8Maei4zaC2cdaWgaaqaaiabikdaYaqabaGccqGGOaakcqWGYbGCdaWgaaWcbaGaemyAaKgabeaaaeaacqWGPbqAcqGH9aqpcqaIXaqmaeaacqWGUbGBa0GaeyyeIuoakiabcYcaSiabdEgaNnaaBaaaleaacqWGPbqAaeqaaOGaeiykaKcaaa@4AAD@62. Standard deviations for sample means of log₂(x) ratios were then calculated: (1/N∑i=1n(log⁡2(ri,gi)−log⁡2¯)2)1/2 MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaadaqadaqaaiabigdaXiabc+caViabd6eaonaaqahabaGaeiikaGIagiiBaWMaei4Ba8Maei4zaC2aaSbaaSqaaiabikdaYaqabaGccqGGOaakcqWGYbGCdaWgaaWcbaGaemyAaKMaeiilaWcabeaakiabdEgaNnaaBaaaleaacqWGPbqAaeqaaOGaeiykaKIaeyOeI0Yaa0aaaeaacyGGSbaBcqGGVbWBcqGGNbWzdaWgaaWcbaGaeGOmaidabeaaaaGccqGGPaqkdaahaaWcbeqaaiabikdaYaaaaeaacqWGPbqAcqGH9aqpcqaIXaqmaeaacqWGUbGBa0GaeyyeIuoaaOGaayjkaiaawMcaamaaCaaaleqabaGaeGymaeJaei4la8IaeGOmaidaaaaa@51EF@. Changes in transcript levels were considered significant if they were changed more than 1.3- to 1.5-fold using a linear transform function, with the upper figure used to calculate total numbers of altered genes under specific conditions. See Tables 1, 2, 3, 4 for a listing of notable regulated genes, their fold change, log₂(x) value, and standard deviation under the high and low salinity and temperature conditions tested.

All microarray data were stored in our HaloArray database 63 mirrored on two Linux servers. The systems use Apache server software, MySQL databases, and custom Perl script-based webtools designed for access and data analysis. This database is a part of our comprehensive HaloWeb database 64, which includes our genome sequence and annotation data, and is indexed in the Thompson ISI Web of Knowledge and Current Web Contents.