Haloarcula hispanica JCM8911 was obtained from the Japan Collection of Microorganisms (JCM, Tsukuba, Ibaraki, Japan). The strain was grown in Medium 307, which contained 2 g casamino acid (BD, Franklin Lakes, NJ, USA), 2 g Bacto yeast extract (BD), 1 g sodium glutamate, 3 g trisodium citrate, 10 g MgSO₄⋅7H₂O, 1 g CaCl₂⋅2H₂O, 1 g KCl, 200 g NaCl, 0.36 mg FeCl₂⋅4H₂O, and 0.36 mg MnCl₂⋅4H₂O per liter of distilled water. After the pH of the medium was adjusted to 7.0, the medium was sterilized by filtration with a polyethersulfone membrane filter (pore size, 0.22 μm; Thermo Fisher Scientific, Waltham, MA, USA) and autoclaving at 121°C for 20 min. Exactly 20 ml of the medium was injected into autoclaved 60-ml screw-cap test tubes and inoculated with cells of H. hispanica in exponential growth phase in pre-culture, and the cultures were incubated in the darkness with shaking at 180 rpm at 37°C. The cultures were centrifuged at 6230 × g for 3 min. The pelleted cells were stored at -25°C until DNA extraction.

In order to make standards for qPCR described below, we performed cloning and sequencing of 16S rRNA genes of H. hispanica. Bulk DNA was extracted from H. hispanica cells grown in Medium 307 with modifications (Tchinda et al., 2016). Briefly, the pelleted cells were lysed with lysozyme and proteinase K solution. Then the genomic DNA was extracted with successive phenol:chloroform:isoamyl alcohol and chloroform:isoamyl alcohol steps and precipitated with ethanol. Next, 16S rRNA genes were amplified from the bulk DNA using the archaea-specific primer set 8aF/1512uR (Table 1). PCR products were purified with a MicroSpin S-400 HR column (GE Healthcare, Little Chalfont, UK) and cloned using the Zero Blunt TOPO PCR Cloning kit (Life Technologies, Carlsbad, CA, USA). The PCR products were ligated into vector pCR4Blunt-TOPO (Life Technologies). Escherichia coli TOP10 cells (Life Technologies) were transformed with the ligated plasmid to construct a clone library. Insert DNA from selected recombinant colonies was sequenced by the dideoxy cycle-sequencing method using a Model 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). The most similar 16S rRNA gene sequence was determined by the BLAST program (Altschul et al., 1990). The 16S rRNA gene sequences obtained in this study were deposited in the DDBJ/EMBL/GenBank database under accession numbers LC085245, LC085246, and LC085247.

Because thermophilic and hyperthermophilic archaea have greater P_GC values for 16S rRNA genes compared with psychrophilic and mesophilic archaea (Galtier and Lobry, 1997; Khachane et al., 2005; Kimura et al., 2006, 2007, 2010), Kimura et al. (2013) proposed linear regression equations to infer T_min, T_opt, and T_max of cultured and not-yet cultured archaea based on P_GC of 16S rRNA genes. We therefore used these equations to estimate growth temperatures of H. hispanica based on P_GC value of each 16S rRNA gene.

Kimura et al. (2013) used partial 16S rRNA gene sequences (ca. 800 bp) between the archaea-specific primers 109aF and 915aR in order to estimate their growth temperatures (Table 1). Thus, we manually selected the internal sequences from the 16S rRNA gene sequences determined in this study. P_GC values for the internal sequences were calculated using Genetyx-Mac ver. 17.0.6 (Genetyx, Tokyo, Japan). T_min, T_opt, and T_max were calculated based on P_GC of the respective sequence using Kimura’s equations.

Haloarcula hispanica cells in exponential growth phase in pre-culture were inoculated into 60-ml screw-capped tubes containing 20 ml of Medium 307. The cultures were incubated in the dark with shaking at 180 rpm at 25, 30, 35, 40, 45, and 50°C. The optical density at 660 nm (OD₆₆₀) of the culture was monitored using a Spectronic 200 spectrophotometer (Thermo Fisher Scientific) with sterilized medium as the negative control. When the cultures reached the early exponential growth phase (OD₆₆₀ = 0.25–0.50), the cultures were centrifuged at 6230 × g for 3 min. The pelleted cells were mixed with 100 μl RNAlater (Life Technologies) and stored at –85°C until RNA extraction.

Cells were thawed on ice, and the RNAlater was removed and discarded. Total RNA was extracted from the cells using the mirVana miRNA Isolation kit (Ambion, Austin, TX, USA). Contaminating genomic DNA in the extracted RNA samples was removed using the TURBO DNA-Free kit (Life Technologies). Total RNA was purified with the RNeasy MinElute Cleanup kit (Qiagen, Hilden, Germany). The quality and concentration of RNA were verified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and a NanoVue Plus spectrophotometer (GE Healthcare). Single-strand complementary DNA (cDNA) was synthesized from the purified total RNA using the SuperScript III first strand synthesis system (Life Technologies) as following manufacturer’s protocol. The cDNA was purified using QIAquick PCR Purification kit (Qiagen). The purified cDNA was stored at -25°C until qPCR analysis.

The specific primer sets rrsAf/rrsAr for rrsA and rrsBCf/rrsBCr for rrsBC were designed using Primer Express 2.0 software ver. 2.0 (Applied Biosystems) (Table 1). To test the specificity of these primer sets, qPCR was performed with PCR products of rrsA, rrsB, and rrsC that were amplified from the clones in the 16S rRNA gene-clone library described above. The PCR products were purified with QIAquick PCR purification kit (Qiagen). The qPCR was performed on an ABI Prism 7300 Real Time PCR System (Applied Biosystems) with PowerUP SYBR Green master mix (Life Technologies). Since the sequences of rrsB and rrsC are nearly identical, it was not possible to design primer sets for specifically amplifying rrsB and rrsC, independently.

Next, rrsA and rrsBC in the cDNA were quantified by qPCR using the ABI Prism 7300 Real Time PCR System. Each PCR mixture contained 2 μl of diluted cDNA template, 2 μl of each designed primer set (each 300 nM), 10 μl of PowerUP SYBR Green PCR master mix (Life Technologies), and 4 μl of nuclease-free water (Ambion). The PCR conditions included an initial step of 50°C for 2 min and 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 1 min. The standard curves were prepared from diluted PCR products (1/10, 1/100, 1/1000, 1/10000) of rrsA, rrsB, and rrsC that were amplified from the clones in the 16S rRNA gene-clone library described above. The PCR reactions were performed in triplicate for technical repeats and four individuals for biological repeats.

To assess any advantage to H. hispanica of having multiple distinct 16S rRNA genes, we disrupted the three rRNA operons by using a mutation method (See Supplementary Materials and Methods for details). Briefly, an rRNA operon in wild-type H. hispanica was replaced with novobiocin resistance gene to construct rRNA operon single-mutant strains (HA2, HB2, or HC2) that lack rrnA, rrnB, or rrnC, respectively (Supplementary Table 1 and Figure 1). Furthermore, rrnB in HC2, rrnA in HC2 (or rrnC in HA2), and rrnB in HA2 were replaced with mevinolin resistance genes to construct the rRNA operon double-mutant strains HCB2, HCA2 (or HAC2), or HAB2 that contain only rrnA, rrnB, or rrnC, respectively (Supplementary Table 1 and Figure 2). Disruption of operons rrnA, rrnB, and rrnC in these mutant strains was confirmed by PCR amplification using primer sets, AVF/ACR, BVF/BCR, or CVF/CCR (Supplementary Table 2). The rRNA operon double-mutant strains harboring only rrnB (HCA2 and HAC2) could not be constructed despite repeated attempts (Supplementary Table 1).

The rRNA operon single- and double-mutant strains and wild-type strain were inoculated into Medium 307 and incubated in the dark with shaking at 180 rpm at 25, 30, 35, 40, 45, and 50°C. OD₆₆₀ of the cultures was monitored using a Spectronic 200 spectrophotometer (Thermo Fisher Scientific), and growth curves were drawn based on the values. The culture experiments were performed in quadruplicate or quintuplicate. The culture experiment at 50°C (actual T_max) was carried out twice to confirm the growth of the double-mutant and wild-type strains.

To calculate the maximum growth rates at each temperature, we determined a cell number factor to convert from OD₆₆₀ value to cell density. Briefly, wild-type H. hispanica was grown in Medium 307, and the cultures were diluted with sterilized medium. After OD₆₆₀ of the cultures was measured using Spectronic 200 spectrophotometer (Thermo Fisher Scientific), the cells in the cultures were fixed in formaldehyde (final concentration 7%) for 16 h at 4°C as described previously (Antón et al., 1999). The cultures were filtered using pre-blackened polycarbonate filters (pore size, 0.2 μm; diameter, 25 mm) (Millipore, Billerica, MA, USA). The cells collected on the filters were stained with SYBR Green I (1:100 dilution) (Life Technologies). The cells were observed under a model BX51 epifluorescence microscope equipped with a U-MNIB3 fluorescence filter (Olympus, Tokyo, Japan), and 50 microscopic fields were counted for each sample. A cell number factor of 2.1 × 10⁹ cells ml^-1 per OD₆₆₀ determined in this study was used to determine cell density in the cultures (Supplementary Figure 3). Growth rate (μ) was calculated between the individual incubation periods (t₁ and t₂) with an assumption of exponential growth; i.e., μ (h^-1) = (ln N_t2 – ln N_t1)/(t₂ -t₁), where N_t1 and N_t2 are the cell densities. On the basis of the growth rate, T_min, T_opt, and T_max of the strains were determined.

A total of 16 clones were randomly selected from a clone library of H. hispanica strain JCM8911, and the sequences of 16S rRNA genes were determined (1440 bp). Three types of 16S rRNA genes were identified and matched rrsA, rrsB, and rrsC in the genome sequence as determined by Liu et al. (2011). The sequences of rrsB and rrsC were 99.6% identical, whereas the sequences of rrsA and rrsBC were 94.6–94.9% identical. These results confirm previous reports of intragenomic polymorphism of 16S rRNA genes in Haloarcula (e.g., Cui et al., 2009).

The sequence regions between the archaea-specific primers 109aF and 915aR (795 bp) were selected from the 16S rRNA gene sequences. The P_GC of the internal sequences of rrsA, rrsB, and rrsC were 58.9, 56.5, and 56.4%, respectively (Table 2). The offset between the P_GC of rrsA and rrsBC was ∼2.5%. Table 2 summarizes the estimated growth temperatures based on these P_GC values. The estimated T_min, T_opt, and T_max based on the P_GC of rrsA were 32.6 ± 16.7, 51.6 ± 11.8, and 59.7 ± 13.1°C, which are much higher than those calculated from the P_GC of rrsB and rrsC. The offsets between the estimated growth temperatures based on P_GC of rrsA and rrsBC was >10°C. These findings may indicate that harboring 16S rRNA genes with relatively high- and low-P_GC values allows H. hispanica to maintain rapid growth over a wide temperature range.

To check the selectivity of the specific primers designed in this study, qPCR was performed with PCR products of rrsA, rrsB, and rrsC that were amplified from the clones in the 16S rRNA gene-clone library. The rrsA-specific primer set, rrsAf/rrsAr, provided the proper products from only diluted PCR products of rrsA as templates at annealing temperature of 58°C, whereas the rrsBC-specific primer set, rrsBCf/rrsBCr, provided the proper products from only diluted PCR products of rrsB and rrsC at annealing temperature of 58°C. These results indicated that these primer sets were sufficiently selective to detect and quantify rrsA and rrsBC, respectively.

Our survey of 16S RNA gene expression demonstrated that rrsA and rrsBC expression varied with temperatures. In particular, the expression ratio of rrsA to rrsBC (rrsA:rrsBC) increased with culture temperature (Figure 1). The ratios at 45 and 50°C exceeded 1.0, which were significantly greater than those in the 25–35°C range (P < 0.05 by Student’s t-test). On the other hand, the ratios at 25, 30, and 35°C were below 1.0. Especially, the ratios at 25 and 30°C were 0.56, which means that total expression of rrsB and rrsC was almost as twice as that of rrsA at the low temperatures. Our results suggest that transcription of high-P_GC 16S rRNA gene rrsA and low-P_GC 16S rRNA genes rrsBC may be regulated in response to culture temperature.

We constructed rRNA operon single-mutant strains (HA2, HB2, and HC2) that lack rrnA, rrnB, or rrnC, respectively (Supplementary Table 1). We further constructed rRNA operon double-mutant strains, namely HCB2 harboring only rrnA and HAB2 harboring only rrnC (Supplementary Table 1). Operon deletion was confirmed by PCR with specific primer sets and by electrophoresis of the PCR products (Supplementary Figure 4).

The wild-type H. hispanica was able to grow at temperature ranging from 25 to 50°C, with optimum growth at 45°C (Table 2). The fastest growth was 0.16 h^-1 at the T_opt of 45°C (Supplementary Figure 5). The growth characteristics of the wild-type strain at various temperatures were almost the same as those of the rRNA operon single-mutant strains (data not shown). In the culture experiments using the double-mutant strains, the wild-type grew faster than HCB2 and HAB2 at all tested temperatures (Figure 2). HCB2 and HAB2 were able to grow within the same temperature range (i.e., 25–50°C) as the wild-type strain (Supplementary Figure 5). The HCB2 that harbors only rrnA containing high-P_GC rrsA grew optimally at 45°C. On the other hand, optimum growth of HAB2, which harbors only rrnC containing low-P_GC rrsC, was slightly shifted to low temperature of 40°C.

At 25°C, the wild-type H. hispanica grew significantly faster than HAB2 and HCB2 (Figure 2A). Condon et al. (1995) used Escherichia coli with multiple rRNA operons inactivated by antibiotic cassettes to demonstrate that the number of rRNA operons in the genome affects cell proliferation rate. Yano et al. (2013) found that the existence of multiple rRNA operons underlies the high growth rate of Bacillus subtilis. Another study reported that the copy number of rRNA operons on the genomes is correlated with growth rate of the prokaryotes under optimal culture condition (Vieira-Silva and Rocha, 2010; Roller et al., 2016). Our results support these studies and suggest that the number of rRNA operons influences cell proliferation rate at low temperatures close to actual T_min (25°C).

At 30°C, HCB2 had slightly higher growth rate than HAB2 (Figure 2B), and this difference increased in cultures at 35 and 40°C (Figures 2C,D). At 45 and 50°C, the growth curves for HCB2 were almost identical with those of the wild-type strain (Figures 2E,F). These findings suggest that the rRNAs transcribed from rrnA, including the high-P_GC 16S rRNA gene rrsA, result in more rapid growth of H. hispanica at high temperatures of 45 and 50°C. The expression survey in this study also showed that expression of rrsA was higher than those of rrsBC at 45 and 50°C (Figure 1), which also supports that rRNAs transcribed from rrnA including high-P_GC 16S rRNA gene rrsA may be important for growth under the high temperature conditions.

Haloarcula hispanica has been identified in a number of solar salterns and desert salt lakes (e.g., Arahal et al., 1996; Pašić et al., 2005; Tapilatu et al., 2010; Mani et al., 2012). These hyper-saline habitats generally have large daily temperature fluctuations, i.e., temperature can vary by >10°C (Post, 1977; Wieland et al., 2005; Sima et al., 2013; Andrade et al., 2015). Because of this habitat feature, it is predicted that H. hispanica would express the rRNA operon with high-P_GC 16S rRNA gene to grow faster in the daytime, when environmental temperatures rise to around T_max (50°C). On the other hand, H. hispanica would express all of the three rRNA operons to grow in the nighttime and/or early morning, when environmental temperatures drop to around T_min (25°C).

Fifteen Haloarcula strains have been isolated from hyper-saline environments worldwide (e.g., Juez et al., 1986; Ihara et al., 1997; Oren et al., 1999; Yang et al., 2007). Except for H. aidinensis, 14 Haloarcula strains so far examined harbor several different 16S rRNA gene sequences in the genome (Supplementary Table 3). Thus, intragenomic 16S rRNA gene heterogeneity seems to be common feature in the genus Haloarcula. We further confirmed that 9 of the 14 Haloarcula strains for which sequence was available show a >2.0% difference in P_GC among the 16S rRNA genes. The estimated growth temperatures based on P_GC values of the respective 16S rRNA genes suggested >10°C differences as well as H. hispanica (Supplementary Table 3). Additionally, previous study using a Haloarcula strain suggested that the sequences of putative promoter regions were obviously different among upstream regions of rRNA operons (Dennis et al., 1998; Dennis, 1999; López-López et al., 2007). These findings suggest that Haloarcula strains may regulate the expression of these 16S rRNA genes in response to culture temperature conditions, and this can be tested in future studies.