Generation and phenotyping of a collection of sRNA gene deletion mutants of the haloarchaeon Haloferax volcanii

The haloarchaeon Haloferax volcanii was shown to contain 145 intergenic and 45 antisense sRNAs. In a comprehensive approach to unravel various biological roles of haloarchaeal sRNAs in vivo, 27 sRNA genes were selected and deletion mutants were generated. The phenotypes of these mutants were compared to that of the parent strain under ten different conditions, i.e. growth on four different carbon sources, growth at three different salt concentrations, and application of four different stress conditions. In addition, cell morphologies in exponential and stationary phase were observed. Furthermore, swarming of 17 mutants was analyzed. 24 of the 27 mutants exhibited a difference from the parent strain under at least one condition, revealing that haloarchaeal sRNAs are involved in metabolic regulation, growth under extreme conditions, regulation of morphology and behavior, and stress adaptation. Notably, 7 deletion mutants showed a gain of function phenotype, which has not yet been described for any other prokaryotic sRNA gene deletion mutant. Comparison of the transcriptomes of one sRNA gene deletion mutant and the parent strain led to the identification of differentially expressed genes. Genes for flagellins and chemotaxis were up-regulated in the mutant, in accordance with its gain of function swarming phenotype. While the deletion mutant analysis underscored that haloarchaeal sRNAs are involved in many biological functions, the degree of conservation is extremely low. Only 3 of the 27 genes are conserved in more than 10 haloarchaeal species. 22 of the 27 genes are confined to H. volcanii, indicating a fast evolution of haloarchaeal sRNA genes

Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq)

Background: Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. Results: Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). Conclusion: This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated

Overview of sRNA gene deletion mutants and selected features.

<p>* published in Heyer <i>et al.</i>, 2012.</p

Scatter plot of the microarray analysis of the transcriptomes of parent strain and deletion mutant Δ63.

<p>Average signals of four biological replicates are shown. The experiment included a dye swap. The RNA was isolated after the exposures of the cultures to 1% (v/v) ethanol for 15 minutes. The solid line represents the diagonal (identical transcript levels in parent strain and mutant). The dotted lines represent a twofold difference between parent strain and mutant.</p

Schematic representation of all conditions under which sRNAs have important functions in <i>H. volcanii</i>.

<p>The importance of sRNAs was either deduced from phenotypic analysis of deletion mutants (this publication) or from the analyis of differential expression using Northern blot analysis and high throughput sequencing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090763#pone.0090763-Heyer1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090763#pone.0090763-Straub1" target="_blank">[33]</a>.</p

Conservation of <i>H. volcanii</i> sRNAs.

<p>* published in Heyer <i>et al.</i>, 2012.</p

Numbers of mutants with phenotypic difference to the parent strain during growth at three different NaCl concentrations.

<p>Numbers of mutants with phenotypic difference to the parent strain during growth at three different NaCl concentrations.</p

Growth curves of the parent strain (black) and selected mutants (in color) grown at extreme salt concentrations.

<p><b>A.</b> Parent strain and ΔH225.2R (red) grown at 1.2 M NaCl. <b>B.</b> Parent strain, Δ194 (purple), and Δ235 (red) grown at 2.1 M NaCl. <b>C.</b> Parent strain, Δ63 (orange), Δ194 (purple), Δ235 (red), and Δ288 (green) grown at 4.0 M NaCl.</p

Distribution of intergenic sRNA genes on the replicons of <i>H. volcanii</i>.

<p>Distribution of intergenic sRNA genes on the replicons of <i>H. volcanii</i>.</p