, affecting the level of hundreds of cellular transcripts indispensable for both environmental adaptation and virulence. The global impact of RhlE2 is mediated by its unique C-terminal extension, which supports the RNA unwinding activity of the N-terminal domain as well as an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery. Overall, our work reveals how the functional and molecular divergence between two homologous RNA helicases can contribute to bacterial fitness and pathogenesis.


26
DEAD-box RNA helicases are RNA-binding proteins that upon ATP binding and hydrolysis remodel 27 RNA structure and RNA-protein complexes (1). DEAD-box RNA helicases are found in all three 28 domains of life, as well as in some viruses, and they are identified on the basis of highly conserved 29 amino acid motifs within the catalytic core, "D-E-A-D" being one of those motifs (2,3). Besides the 30 conserved catalytic region, they possess variable N-and C-terminal extensions that can regulate the 31 core catalytic activity or coordinate interactions with RNA molecules or proteins (4-7). The Escherichia 32 coli DbpA RNA helicase, for example, contains an RNA-binding domain (RBD) at the C-terminus, 33 which is responsible for a specific and tight binding to hairpin 92 of 23S ribosomal RNA (8). 34 From bacteria to humans, DEAD-box RNA helicases are involved in maintaining a correct basal RNA 35 metabolism, by controlling mRNA translation, RNA degradation and ribosome biogenesis (1). In 36 bacteria, though, DEAD-box RNA helicases do not perform only housekeeping functions, but can also 37 regulate their environmental adaptation, host colonization and, in case of pathogens, infectious 38 processes (9-11). 39 Bacterial DEAD-box RNA helicases can be classified into to three major phylogenetic groups, that are 40 distinguished by the presence of DbpA-RBD: DbpA RBD-containing proteins, DbpA RBD-lacking 41 proteins and RhlE-like proteins (12). The RhlE-like RNA helicases, which are named from the E. coli 42 DEAD-box RNA helicase belonging to this group, seem to have originated from a duplication of a 43 DbpA-RBD lacking protein in a Proteobacterial ancestor and have successively expanded in this 44 phylum, where they can be present in several copies per genome (12). The role and molecular action of 45 RhlE-like proteins are not understood, and they vary across species. No phenotypic defect linked to 46 rhlE deletion has been described in E. coli, Pseudomonas syringae LZ4W and Mycobacterium 47 tuberculosis, while in Yersinia pseudotuberculosis and Caulobacter crescentus deletion of rhlE affects 48 cold acclimation (13)(14)(15)(16)(17)(18). In E. coli, RhlE seems to liaise with the SrmB and CsdA RNA helicases 49 during ribosome biogenesis, although the exact molecular action is not yet deciphered, and to interact 50 with the RNase E endonuclease for RNA degradation, even if the conditions in which the interaction 51 occurs in vivo are unclear (19,20). In P. syringae LZ4W, M. tuberculosis and C. crescentus, RhlE is 52 of a phenotype linked to a rhlE1 deletion even in absence of rhlE2 indicates that no buffering exists 134 among these two RNA helicases (Fig. 3A and 3B). 135 136 RNA sequencing analysis reveals the importance of RhlE2 for P. aeruginosa virulence 137 To examine further the role of RhlE2 in P. aeruginosa, we compared the transcriptome of rhlE2 deleted 138 strains in the wild type or in the ΔrhlE1 background, by performing RNA-sequencing. As experimental 139 condition, we choose to investigate the role of RhlE2 in swarming cells (Fig 4A). Swarming motility is 140 a coordinated movement of a bacterial population on semi-solid surfaces (0.5 to 0.7% agar), which is 141 mediated by flagella rotation and surfactants secretion (31). Importantly, P. aeruginosa swarming cells 142 possess a physiological state which is highly relevant to the bacterium pathogenicity as they express 143 virulence factors, they perform epithelial surfaces colonization and are associated to the establishment 144 of lungs infection through biofilm formation (32,33). Approximately 15% of the transcriptome was 145 significantly affected by rhlE2 mutation when compared to wild type PAO1 (N=836 genes, p-value 146 <0.05, FC>2), of which 4% (N=228) of transcripts were up-regulated and 11% (N=608) down-regulated 147 ( Fig 4B, Table S1). Differentially regulated genes were grouped into the corresponding KEGG 148 pathway: most significantly downregulated genes code for ribosomal proteins, metabolic pathways and 149 enzymes involved in the biosynthesis of secondary metabolites, while upregulated ones are linked to 150 xenobiotics biodegradation and metabolism (e.g. 4-fluorobenzoate, chlorocyclohexane and 151 chlorobenzene degradation, Table S2). Consistent with the phenotypic analysis, rhlE1 deletion had not 152 significant impact on the cellular transcript levels neither in the wild type nor in the rhlE2 mutant 153 background (Fig 4B). Next, we validated the transcriptome analysis by choosing a set of putative RhlE2 154 targets and examining their abundance by RT-qPCR in the ΔrhlE1, ΔrhlE2 and ΔrhlE1ΔrhlE2 mutants 155 as compared to the wild type, starting from cell grown under experimental conditions similar to those 156 used for the original RNA-sequencing analysis. The gene chosen have been previously shown to either 157 regulate swarming or being regulated in this condition, such as roeA, coding for a diguanylate cyclase 158 (34), lhpF, coding for the metabolic enzyme D-hydroxyproline dehydrogenase (35), the lasB elastase 159 (36) and the pvdF pyoverdine synthase (37) encoding genes, and finally the rhlA gene was chosen as 160 control (rhamnosyltransferase chain A) being unaffected by rhlE2 mutation, but previously shown 161 necessary for swarming motility (38). In agreement with the RNA-seq data, lasB and pvdF mRNA 162 levels were, respectively, 10.25 and 10.41-fold down-regulated in the ΔrhlE2 mutant when compared 163 to the wild type; while roeA and lhpF were, respectively, 3.98 and 8.94-fold up-regulated and rhlA 164 mRNA levels were unchanged (Fig 4C). To further validate the observation concerning regulation of 165 lasB expression, a transcriptional/translational lasB'-'lacZ fusion (39) was assayed for β-galactosidase 166 activity in the four strains, at different growth phases in NYB (Fig 4D). Expression of lasB is dependent 167 on cell-density and therefore maximal at stationary phase (40), the same trend was observed in all 168 mutants but in the ΔrhlE2 and ΔrhlE1ΔrhlE2 mutants the expression of the lasB'-'lacZ fusion was 169 reduced of ~2.5-fold in stationary phase, when compared to the wild type and ΔrhlE1 strains. 170 Besides lasB, one interesting observation concerning the rhlE2 mutant transcriptome was the significant 171 downregulation of various other genes encoding virulence factors (Table S1). To assess whether mRNA 172 levels regulation by RhlE2 also results in changes of virulence factor abundance, we measured in our 173 strains the extracellular levels of the LasB elastase and pyocyanin, a redox-active molecule inducing 174 oxidative stress in host cells (41). We found that the ΔrhlE2 and ΔrhlE1ΔrhlE2 mutants produced 2.6-175 and 3.6-fold less elastase and 2,1-and 2.4-fold less pyocyanin than the wild type, respectively, while 176 the rhlE1 deletion did not produce any significant difference (Fig 5A-B). 177 178

RhlE2 affects P. aeruginosa virulence in Galleria mellonella 179
Galleria mellonella has become an important model to study bacterial pathogenesis (42). We used this 180 system to confirm that RhlE2 has indeed an important role during P. aeruginosa pathogenesis in vivo. 181 In agreement with data disclosed above, the ΔrhlE2 and ΔrhlE1ΔrhlE2 mutants displayed a significant 182 attenuation of virulence in the G. mellonella infection model. Indeed, at 24 hours post-infection the 183 mortality of larvae injected with either ΔrhlE2 or ΔrhlE1ΔrhlE2 reached less than 40%, while the 184 mortality rate of larvae challenged with wild type or ΔrhlE1 strain was 80% (Fig 5C). Altogether, these 185 results indicate that RhlE2 controls, directly and/or indirectly, the expression of virulence or virulence-186 related genes and is necessary for P. aeruginosa pathogenicity in vivo. 187

RhlE1 and RhlE2 display an RNA-dependent ATPase activity in vitro 189
To fuel their molecular actions, RNA helicases possess an ATP hydrolytic activity that is dependent on 190 RNA binding (2). To confirm this prediction, based on RhlE1 and RhlE2 sequence conservation, we 191 assess their enzymatic activity in vitro. The proteins were produced in E. coli as  tagged fusion and purified from a soluble extract by adsorption to nickel-