Data_Sheet_1_The PL6-Family Plasmids of Haloquadratum Are Virus-Related.DOCX

<p>Plasmids PL6A and PL6B are both carried by the C23^T strain of the square archaeon Haloquadratum walsbyi, and are closely related (76% nucleotide identity), circular, about 6 kb in size, and display the same gene synteny. They are unrelated to other known plasmids and all of the predicted proteins are cryptic in function. Here we describe two additional PL6-related plasmids, pBAJ9-6 and pLT53-7, each carried by distinct isolates of Haloquadratum walsbyi that were recovered from hypersaline waters in Australia. A third PL6-like plasmid, pLTMV-6, was assembled from metavirome data from Lake Tyrell, a salt-lake in Victoria, Australia. Comparison of all five plasmids revealed a distinct plasmid family with strong conservation of gene content and synteny, an average size of 6.2 kb (range 5.8–7.0 kb) and pairwise similarities between 61–79%. One protein (F3) was closely similar to a protein carried by betapleolipoviruses while another (R6) was similar to a predicted AAA-ATPase of His 1 halovirus (His1V_gp16). Plasmid pLT53-7 carried a gene for a FkbM family methyltransferase that was not present in any of the other plasmids. Comparative analysis of all PL6-like plasmids provided better resolution of conserved sequences and coding regions, confirmed the strong link to haloviruses, and showed that their sequences are highly conserved among examples from Haloquadratum isolates and metagenomic data that collectively cover geographically distant locations, indicating that these genetic elements are widespread.</p

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09 janvier 19211921/01/09 (N529,FASC78)

Translational efficiencies of a consecutive SD mutant series under standard conditions.

<p>For the comparative analysis of translational efficiencies H. volcanii cultures of pPK10-pPK18 and pNP10 negative control (without hdrA) were grown to mid-exponential growth phase under standard conditions (2.1 M NaCl, 42°C). A. Schematic overview of the reporter gene constructs pPK10-pPK18. The SD sequence (green and underlined) was mutated as indicated. B. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s002" target="_blank">Table S2</a>. C. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3).</p

Bioinformatic analysis of the <i>H. volcanii</i> genome.

<p>A. Sequence logo of 5′ regions of 3025 putative monocistronic genes or proximal genes in operons with an intergenic distance at least 40 nt to the adjacent gene. B. Sequence logo of 5′ regions of 791 putative distal genes in operons with an intergenic distance of less than 10 nt to the adjacent gene.</p

Occurrence of SD motifs upstream of distal genes in operons.

<p>108 nt upstream of all genes with a distance of less than 10 nt to the preceding were retrieved from the genome of <i>H. volcanii</i>. The sequences were searched for the occurrence of motifs with a 5–8 nt match to the SD motif GGAGGUGA. A. the occurrence of motifs with matches of at least 6 nt (purple curve), at least 7 nt (blue curve), and 8 nt (red curve) are shown. B. smoothed curves (3 nt average) are shown for the occurrence of exactly 5 nt (green curve), exactly 6 nt (purple curve), exactly 7 nt (blue curve), and exactly 8 nt (red curve).</p

Translational efficiencies of the consecutive SD mutant series at reduced growth rates.

<p>For the comparative analysis of translational efficiencies <i>H. volcanii</i> cultures of pPK10-pPK18 and pNP10 negative control (NC) were grown to mid-exponential growth phase at the reduced temperature of 30°C (A, B) or with acetate as carbon and energy source (C, D). A. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s003" target="_blank">Table S3</a>. B. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3). C. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s004" target="_blank">Table S4</a>. D. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3).</p

Translational efficiencies of different transcripts with and without an SD sequence.

<p>For the comparative analysis of translational efficiencies <i>H. volcanii</i> cultures of pPK19-pPK22 were grown to mid-exponential growth phase under standard conditions. A. Schematic overview of the reporter gene constructs pPK19-pPK22. The native SD motif of the <i>sod</i> gene (green and underlined) was replaced by three randomly generated unrelated sequences in the mutants pPK20-pPK22. A second leaderless translation start site AUG was introduced at the 5′-end of the 51 nt elongated <i>sod</i> 5′-UTR (blue). B. One representative example of three independent experiments is shown for a Western blot analysis (upper panel), a Northern blot analysis (middle panel), and the 16S rRNA of an agarose gel used for normalization. The protein and transcript levels were analyzed as described in Experimental Procedures. The results are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094979#pone.0094979.s005" target="_blank">Table S5</a>. C. Graphic representation of the normalized average translational efficiencies and their standard deviations (n = 3). Results of proteins originating from the leaderless start codon are shown in blue, results of proteins originating from the <i>sod</i> start codon are shown in green.</p

Spacing between the 8 nt SD motif to the start codon of the downstream gene.

<p>In 31 cases the extended 8 nt SD motif was found to be localized at the 3′-end near the stop codon. The sequences around the SD motif were retrieved from the genome sequence of <i>H. volcanii</i> and the gene identifier (HVO numbers) and sequences are shown. It turned out that all 31 genes were proximal genes in operons and were closely followed by a downstream gene. The SD motif and the start codon of the downstream gene are shown in red, and the stop codon of the upstream gene is shown in bold.</p

Model Construction and Analysis of Respiration in <i>Halobacterium salinarum</i>

<div><p>The archaeon <i>Halobacterium salinarum</i> can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of <i>H. salinarum</i>.</p></div

The building blocks of a cell obtained by harmonizing the different data gathered from literature [25, 26].

<p>A 1 ml OD cell suspension contains 1.81 <i>μ</i>L cell pellet and 1.36 × 10⁹ halobacterial cells. (A) The 1.81 <i>μ</i>L cell pellet consists of 0.47 <i>μ</i>L cellular organic material, 0.45 <i>μ</i>L cellular basal salt and 0.89 <i>μ</i>L inter-cellular basal salt. The total cell volume (1.36 <i>μ</i>L from the sum of organic material and cellular basal salt) contains 0.80 <i>μ</i>L water. (B) Using a buoyant density of 1.2 mg/mL for the cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151839#pone.0151839.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151839#pone.0151839.ref026" target="_blank">26</a>], the 1.36 <i>μ</i>L cell volume is 1.63 <i>μ</i>g. This consists of 0.80 <i>μ</i>g water and other components. (C) Assuming that a cell pellet contains 1.36 × 10⁹ halobacterial cells, then an individual cell has a mass of 1.2 pg. (D) The components by volume of a cell is given. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151839#pone.0151839.s001" target="_blank">S1 File</a> for more details. Units: <i>μ</i>L = micro liters, pg = pico grams, fL = femto liters.</p