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

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

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

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

Swarming velocity of the parent strains (black, grey) and 17 mutants.

<p>The mutant with a gain of function phenotype is shown in blue, the five mutants with a loss of function phenotype are shown in red. Average values of three replicates and their standard deviations are shown.</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

Overview of sRNA gene deletion mutants and selected features.

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

Swarm plate analysis of parent strain (H26) and deletion mutant Δ63 in the absence and presence of 1% (v/v) ethanol, as indicated.

<p>The pictures were taken at four different times after inoculation, as indicated.</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