Generation and Phenotyping of a Collection of sRNA Gene Deletion Mutants of the Haloarchaeon Haloferax volcanii

Abstract

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.

Small regulatory RNAs in Archaea

“…The efficient genetic system and the possibility of massive parallel cultivation together enabled the generation of a set of 27 deletion mutants of sRNA genes and their phenotyping under 11 different growth or stress conditions. In addition, cell morphology was checked and swarming was analyzed 54 . Interestingly, 24 of the 27 sRNA gene deletion mutants exhibited a phenotype under at least one of the tested conditions, unraveling the broad importance of sRNAs in haloarchaea for many biological functions.…”

Involvement of Enterococcus faecalis Small RNAs in Stress Response and Virulence

“…It has recently been demonstrated that several sRNA deletion mutants of the haloarchaeon Haloferax volcanii showed a gain-offunction phenotype (82). Taken together, it may be suggested that the biological role of some sRNAs can be to stabilize the homeostasis of cells, especially when they are confronted with the multiple environmental changes that occur in real life.…”

The Non-Coding Regulatory RNA Revolution in Archaea

“…While phenotypic characterization of sRNA deletion mutants, including 10 gain-of-function phenotypes out of 27 mutants tested, confirmed their roles in metabolic regulation, stress adaptation and complex behavior [12,36], their targets are still unknown with a few exceptions. Some of these exceptions comes from the study of M. mazei cultures grown under nitrogen starvation conditions where RNA-seq experiments revealed the differential expression of a number of sRNAs in response to nitrogen availability [23,37].…”

“…In H. volcanii, many intergenic sRNAs are differentially expressed in response to varying environmental conditions, including elevated temperature, osmotic stress, nutrient limitation, and 14 oxidative stress, [1,8,12,35,36]. While phenotypic characterization of sRNA deletion mutants, including 10 gain-of-function phenotypes out of 27 mutants tested, confirmed their roles in metabolic regulation, stress adaptation and complex behavior [12,36], their targets are still unknown with a few exceptions.…”