Endoribonuclease YbeY Is Essential for RNA Processing and Virulence in Pseudomonas aeruginosa

The increasing bacterial antibiotic resistance imposes a severe threat to human health. For the development of effective treatment and prevention strategies, it is critical to understand the mechanisms employed by bacteria to grow in the human body. Posttranscriptional regulation plays an important role in bacterial adaptation to environmental changes. RNases and small RNAs are key players in this regulation. In this study, we demonstrate critical roles of the RNase YbeY in the virulence of the pathogenic bacterium Pseudomonas aeruginosa. We further identify the small RNA ReaL as the direct target of YbeY and elucidate the YbeY-regulated pathway on the expression of bacterial virulence factors. Our results shed light on the complex regulatory network of P. aeruginosa and indicate that inference with the YbeY-mediated regulatory pathway might be a valid strategy for the development of a novel treatment strategy.

changes, bacteria control gene expression through transcriptional, posttranscriptional, and posttranslational mechanisms (2). Compared to transcriptional regulation that involves RNA synthesis, the posttranscriptional processing provides a way of regulation that saves time and energy as the mRNA translation and stability are regulated by RNases and small RNAs (sRNAs) (3)(4)(5). By binding to target mRNAs, sRNAs affect ribosome accessibility or RNase-mediated cleavage. Meanwhile, the processing and stabilities of sRNAs are under the control of RNases (6)(7)(8). Therefore, identification of the target sRNAs and mRNAs of RNases is essential for the elucidation of the RNasemediated regulatory pathways.
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes acute and chronic infections in human (9). Upon infection, phagocytes play an essential role in the host defense against pathogenic bacteria. One of the major bacterial killing mechanisms of phagocytes is the generation and release of reactive oxygen species (ROS) (10,11). P. aeruginosa harbors a variety of antioxidant enzymes, including catalases KatA and KatB and alkyl hydroperoxide reductase AphB, AhpC, and AhpF (12,13). KatA is a constitutively expressed catalase that plays a major role in the bacterial defense against oxidative stresses and virulence (14)(15)(16). Both KatA and KatB are regulated at the transcriptional level by a variety of factors, including OxyR and the stationary-phase sigma factor RpoS (17,18). Mutation of rpoS reduces the expression of these catalases, leading to increased susceptibility to oxidative stresses and attenuation of virulence in animal models (17,19,20).
RNases have been shown to play important roles in the regulation of virulence determinants in P. aeruginosa. Previously, we found that the polynucleotide phosphorylase (PNPase) controls the expression of the type III (T3SS) and type VI secretion systems and pyocin synthesis genes (21,22). We also found that the PNPase degrades sRNA P27, which directly controls the translation of the quorum sensing signal synthase RhlI (23). In P. aeruginosa, PNPase interacts with RNase E and RNA helicase DeaD to form an RNA degradosome that plays an important role in RNA processing (24). Both RNase E and DeaD are required for the expression of the T3SS genes (25)(26)(27).
YbeY is a highly conserved bacterial RNase that is involved in the maturation of 16S rRNA, ribosome quality control, regulation of sRNA, and stress responses (28)(29)(30)(31)(32)(33). In pathogenic bacteria, such as enterohemorrhagic Escherichia coli (EHEC), Vibrio cholerae, and Yersinia enterocolitica, YbeY has been shown to play important roles in bacterial virulence (29,34,35). However, the mechanisms by which YbeY affects bacterial virulence and stress response remain unclear. Among bacterial species, including P. aeruginosa, E. coli, and Staphylococcus aureus, the ybeY gene is colocalized with a ybeZ gene in the same operon (36)(37)(38). In E. coli, YbeY has been found to interact with YbeZ (37), indicating a functional connection between the two proteins. YbeZ contains an ATP binding and a nucleoside triphosphate hydrolase domain; however, its exact function remains unknown.
In this study, we demonstrate that the P. aeruginosa endoribonuclease YbeY is involved in the 16S rRNA maturation, ribosome assembly, and pathogenesis. We further identify the sRNA ReaL as the target of YebY and elucidate a YbeY-mediated regulatory pathway that controls the expression of rpoS and oxidative stress response genes. In addition, we elucidate a posttranscriptional regulatory mechanism of RpoS as well as a functional connection between YbeZ and YbeY.
(RT-PCR) primers targeting the middle, 5= starting site, and 3= termination site of the 16S rRNA, representing the total, 5= immature, and 3= immature 16S rRNA, respectively. In both the logarithmic and stationary growth phases, the proportion of immature 16S rRNA in the ΔybeY mutant was higher than that in wild-type PA14, with the 5= immature Bacterial growth rates. Same numbers of cells of the indicated strains were inoculated in LB. The OD 600 was monitored every hour for 12 h. (B) Bacterial cells were cultured in LB to an OD 600 of 1, followed by RNA isolation. The 23S and 16S rRNA and the 16S rRNA precursor (17S) were separated by electrophoresis. (C) Top: Schematic diagram of the 16S rRNA precursor. Black arrows indicate the processing sites of RNase III. The red bars represent the regions amplified by real-time PCR. 16S-S, 16S-M, and 16S-T represent the 5= starting site, middle, and 3= termination site of the 16S rRNA, respectively. The bacteria were grown in LB to the OD 600 of 1 or 3. The bacterial total RNA was isolated, and the 5= and 3= immature ratios of the 16S rRNA were determined by real-time PCR. ***, P Ͻ 0.001 by Student's t test. (D) Bacteria were grown to an OD 600 of 1. The ribosome particles were subjected to sucrose gradient separation and quantified by UV absorbance at 260 nm.
Functions of YbeY in P. aeruginosa ® ratio much higher than the 3= immature ratio (Fig. 1C). We then examined the role of YbeY in ribosome assembly. Deletion of the ybeY gene reduced the proportion of assembled 70S ribosome while increasing the proportion of unassembled 30S and 50S ribosome components (Fig. 1D).
In previous studies, the ybeY mutant was not identified from Tn mutant libraries, indicating that ybeY might be an essential gene in P. aeruginosa (29,39). To examine whether ybeY is essential for P. aeruginosa, we deleted the gene in another two wild-type strains, PAO1 and PAK. Similar to the PA14 ΔybeY mutant, the ΔybeY mutants of PAO1 and PAK displayed reduced growth rate and increased proportion of 16S rRNA precursor (see Fig. S1 in the supplemental material). These results suggest that YbeY plays important roles in the rRNA processing and growth of P. aeruginosa.
To confirm that the P. aeruginosa ybeY (PA3982) gene is a true ortholog of the ybeY gene in E. coli, we complemented the PA14 ΔybeY mutant with an E. coli ybeY gene, which restored the bacterial growth rate and maturation of the 16S rRNA (Fig. S2A, B, and C). Consistent with previous studies, the E. coli ybeY mutant was more susceptible to heat shock and oxidative stresses (such as H 2 O 2 ) (29). Complementation of the E. coli ybeY mutant with the ybeY gene from PA14 restored the bacterial resistance to heat shock and H 2 O 2 ( Fig. S2D and E).
Previous studies in E. coli and Bacillus subtilis revealed that the amino acid residues R56 and H112 are critical for the function of YbeY (28,40,41). To determine the importance of these conserved amino acid residues in the P. aeruginosa YbeY, we replaced the R56 or H112 with alanine (A). Neither of the alleles restored the maturation of the 16S RNA and the growth rate of the ΔybeY mutant ( Fig. 2B and C), indicating the essentialities of these residues in the function of the P. aeruginosa YbeY.
YbeZ binds to YbeY and contributes to the processing of the 16S rRNA. In the P. aeruginosa genome, ybeY (PA3982) is predicted to be in the same operon with ybeZ (PA3981) and ybeX (PA3983). RT-PCR results confirmed the cotranscription of these three genes (Fig. S3). A previous study in E. coli revealed the interaction between YbeY and YbeZ (37), suggesting that YbeZ might function as a partner of YbeY. To test whether YbeY and YbeZ are functionally connected in P. aeruginosa, we performed a pulldown assay. Our results revealed active interaction between YbeY and YbeZ (Fig. 3A). Next, we examined the biological function of YbeZ. Mutation of ybeZ also reduced the bacterial growth rate (Fig. S4A) and maturation of the 16S rRNA, but to a lesser extent compared to the ybeY mutation ( Fig. 3B to D). For example, at the OD 600 of 1.0, the 16S rRNA 5= and 3= immature ratios of the ΔybeZ mutant were approximately 13-and 60-fold higher than those in the wild-type PA14, respectively, whereas the corresponding differences between the ΔybeY mutant and the wild-type PA14 were approximately 43-and 100-fold ( Fig. 3C and D). However, mutation of ybeZ did not affect the assembly of the ribosome (Fig. S4B). These results indicate that YbeY and YbeZ might form a complex that processes the 16S rRNA precursor, with YbeY playing a major role. Accordingly, we focused our following studies on the functions of YbeY.
YbeY is required for the virulence of P. aeruginosa in a murine acute pneumonia model. To examine the role of YbeY in the virulence of P. aeruginosa, we utilized a murine acute pneumonia model. Mutation of ybeY reduced the bacterial load by approximately 600-fold, which was restored by the complementation with a wild-type ybeY gene (Fig. 4A).
To understand the mechanism of YbeY-mediated regulation on the bacterial virulence, we performed transcriptome analyses. Expression of 313 genes was altered by the mutation of ybeY ( Fig. 4B; Table S2). Of note, genes involved in the oxidative stress response were downregulated in the ΔybeY mutant, including the catalase genes katA and katB, the alkyl hydroperoxide reductase gene aphC, and the superoxide dismutase gene sodB ( Table 1). The catalases play critical roles during P. aeruginosa infection. KatA is a constitutive and housekeeping catalase produced by P. aeruginosa and plays a critical role in the bacterial tolerance to oxidative stresses and virulence (42,43), whereas the expression of katB is induced by oxidative stresses (15). Mutation of yebY reduced the mRNA levels of katA and katB in the presence and absence of H 2 O 2 , which were restored by complementation with the yebY gene ( Fig. 5A and B). Consistent with the gene expression pattern, the total catalase activity of the ΔybeY mutant was lower than that of the wild-type PA14, which was restored by overexpression of katA (Fig. 5C). The bacteria were cultured in LB to an OD 600 of 1, followed by RNA isolation. The 23S and 16S rRNA and the 16S rRNA precursor (17S) were separated by electrophoresis.
Functions of YbeY in P. aeruginosa ® In the acute pneumonia model, the neutrophil plays an important role in the host clearance of the invading P. aeruginosa (12). Generation of ROS is one of the major bacterium-killing mechanisms by the neutrophil (11,12). The downregulation of the oxidative response genes might render the ΔybeY mutant more susceptible to the neutrophil-mediated killing. Indeed, after incubation with neutrophils differentiated from HL60 cells (dHL60), the survival rate of the ΔybeY mutant was significantly lower than that of the wild-type strain (Fig. 5D). Supplementation of the ROS scavenger molecule N-acetylcysteine (NAC) or overexpression of katA restored the bacterial survival rate (Fig. 5D). In addition, the ΔybeY mutant was more susceptible to H 2 O 2 treatment and overexpression of the katA gene restored the bacterial survival rate (Fig. 5E). Furthermore, overexpression of katA in the ΔybeY mutant restored the bacterial load in the acute pneumonia model (Fig. 5F) without affecting the bacterial growth rate in LB (Fig. S5). These results indicate that the defective bacterial response to ROS contributes to the attenuated virulence of the ΔybeY mutant. YbeY controls the oxidative stress response genes through RpoS. In P. aeruginosa, the transcriptional regulator OxyR controls the expression of the catalase and the alkyl hydroperoxide reductase genes in response to oxidative stresses (44). In addition, the ATP-dependent helicase RecG facilitates the binding of OxyR to the promoters of target genes (45,46). In the ΔybeY mutant, the expression levels of oxyR, recG, and ahpB (alkyl hydroperoxide reductase) were similar to those in the wild-type PA14 in the presence and absence of H 2 O 2 (Fig. S6). The aphC mRNA level in the ΔybeY mutant was lower in the absence of H 2 O 2 but increased to a similar level as that in PA14 in the presence of H 2 O 2 . These results indicate that YbeY might not control the expression of katA/B through OxyR and RecG.
Besides OxyR and RecG, the alternative sigma factor RpoS had been demonstrated to affect the expression of katA/B and the bacterial tolerance to H 2 O 2 (18)(19)(20). Our RNA-seq result revealed downregulation of the rpoS gene in the ΔybeY mutant (Table 1), which was confirmed by real-time PCR (Fig. 6A). Overexpression of rpoS in the ΔybeY mutant restored the expression of katA (Fig. 6B) and the total catalase activity in the presence and absence of H 2 O 2 ( Fig. 6C) as well as the bacterial tolerance to H 2 O 2 (Fig. 6D). In addition, mutation of rpoS did not affect the expression of ybeY at either the logarithmic or stationary growth phase (Fig. S7). In combination, these results demonstrate that YbeY controls the expression of katA through RpoS.
YbeY controls the expression of rpoS at the posttranscriptional level. We then explored the mechanism of YbeY-mediated regulation of rpoS. The transcription of rpoS was examined using a transcriptional fusion between the rpoS promoter and a pro-   moterless lacZ reporter gene (P rpoS -lacZ). ␤-Galactosidase assay revealed a reduced rpoS promoter activity in the ΔybeY mutant (Fig. 7A). A previous ChIP-seq analysis indicated a possible binding of RpoS to its own promoter, suggesting an autoregulation (47). To verify whether RpoS controls its own expression, we utilized an rpoS::Tn mutant from the PA14 transposon insertion mutant library (48). Mutation in rpoS reduced the LacZ expression from P rpoS -lacZ, which was restored by overexpression of an rpoS gene driven by an exogenous tac promoter (Fig. 7A). An EMSA demonstrated that RpoS indeed bound to its own promoter but not the coding sequence (Fig. 7B). Collectively, these results suggest an autoregulation of rpoS.
To elucidate the mechanism of YebY-mediated regulation of rpoS, we overexpressed the rpoS gene by a constitutively active tac promoter in the ΔybeY mutant, which restored the expression of the P rpoS -lacZ (Fig. 7A). These results raise the possibility that the lower rpoS promoter activity in the ΔybeY mutant might be due to a deficiency in the translation of the rpoS mRNA. To examine the translation of rpoS, we constructed a series of C-terminal Flag-tagged rpoS fusions (rpoS-Flag) driven by an exogenous arabinose-inducible promoter (P BAD ) with various lengths of the rpoS 5= UTR, resulting The bacteria were collected by centrifugation, and the cells were broken by sonication. The total intracellular catalase activity was measured using a catalase assay kit. (D) The indicated strains were grown in LB to an OD 600 of 1, and the cells were washed three times with PBS and then incubated with 10, 20, or 50 mM H 2 O 2 for 30 min. The bacteria were collected by centrifugation and resuspended with fresh LB, and the survival rates were determined by plating. ns, not significant; *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001 by Student's t test.
Functions of YbeY in P. aeruginosa ® in rpoS(F1)-FLAG, rpoS(F2)-FLAG, and rpoS(F3)-FLAG (Fig. 7C). The translation of rpoS was reduced in the ΔybeY mutant even when the 5= UTR was truncated to 25 nucleotides upstream of the start codon. However, when the 25-nucleotide sequence was replaced by an exogenous ribosome binding sequence from vector plasmid pET28a, the RpoS-FLAG protein levels were similar between wild-type PA14 and the ΔybeY mutant ( Fig. 7C and D). In combination, these results demonstrate that YbeY affects the translation of rpoS.
YbeY controls the rpoS translation through sRNA ReaL. In P. aeruginosa, two sRNAs, namely, RgsA and ReaL, repress the rpoS translation by targeting to the Ϫ25 to ϩ27 (relative to the start codon) and the Shine-Dalgarno (SD) regions (Ϫ13 to Ϫ6) of the rpoS mRNA, respectively (3,49,50). In the ΔybeY mutant, rgsA was downregulated whereas reaL was upregulated (Fig. 8A), indicating a possible involvement of ReaL in the repression of the rpoS translation. Indeed, deletion of reaL in the ΔybeY mutant restored the translation of the P BAD -driven rpoS-FLAG while the mRNA levels of the rpoS-FLAG were similar among the strains (Fig. 8B).
To further verify that the repression of rpoS is due to the upregulation of reaL, we mutated the predicted binding site of ReaL (3,49,50) in the rpoS(F1)-FLAG, rpoS(F2)-FLAG, and rpoS(F3)-FLAG (Fig. 8C), which resulted in similar expression levels of the RpoS-FLAG in wild-type PA14 and the ΔybeY mutant (Fig. 8C). Meanwhile, deletion of reaL in the ΔybeY mutant background restored the mRNA levels of katA and rpoS ( Fig. 8D and E). Overexpression of reaL in the double-knockout mutant reduced the rpoS mRNA level, whereas overexpression of ybeY resulted in a higher rpoS mRNA level than that in the wild-type PA14 (Fig. 8E). These results indicate that the high level of ReaL in the ΔybeY mutant contributes to the downregulation of rpoS and that YbeY might positively regulate rpoS through other mechanisms.
YbeY directly degrades ReaL. To explore the mechanism of the upregulation of ReaL, we constructed a transcriptional fusion of the reaL promoter with a promoterless lacZ gene (P reaL -lacZ). The LacZ levels were similar in wild-type PA14 and the ΔybeY mutant (Fig. 9A), indicating a similar transcriptional level of the sRNA. We then examined the stability of ReaL. Following blockage of the RNA synthesis by rifampin, the ReaL level dropped quickly in wild-type PA14, whereas a slower reduction was observed in the ΔybeY mutant (Fig. 9B). Meanwhile, the mRNA level of RpsL (a ribosomal subunit) dropped at a similar rate in the two strains (Fig. 9C). These results suggest that the higher level of ReaL in the ΔybeY mutant is likely due to its increased stability.
To address whether ReaL is a direct target of YbeY, we tested whether ReaL can interact with YbeY by EMSA in the presence of an RNase inhibitor. As shown in Fig. 9D, YbeY retarded the migration of ReaL, indicating a direct interaction. Next, we investigated whether YbeY can degrade ReaL directly. As shown in Fig. 9E and F, the presence of Mn 2ϩ significantly increased the degradation of the ReaL RNA by YbeY, which is consistent with previous reports that YbeY is a metal-dependent endoribonuclease (1,6,10). Furthermore, mutation of the conserved residues R56 and H112 or both of them in YbeY reduced the degradation efficacy ( Fig. 9E and F).
Downregulation of rpoS by ReaL plays a major role in the attenuated virulence of the ⌬ybeY mutant. Next, we examined whether the upregulation of ReaL contributes to the defective oxidative response and attenuated virulence of the ΔybeY mutant. Deletion of reaL in the ΔybeY mutant restored the total catalase activity (Fig. 10A) as well as the bacterial tolerance to H 2 O 2 (Fig. 10B), whereas deletion of reaL in the wild-type PA14 did not affect these phenotypes. In addition, overexpression of rpoS or deletion of reaL in the ΔybeY mutant restored the bacterial survival rate after incubation with dHL60 cells (Fig. 10C). We further examined the bacterial virulence in the acute pneumonia model. Overexpression of rpoS or deletion of reaL in the ΔybeY mutant restored the bacterial loads in the lungs (Fig. 10D), whereas deletion of reaL in the wild-type PA14 did not affect the bacterial loads in the lungs (date not shown). Deletion of reaL in the ΔybeY mutant did not affect the bacterial growth rate, while overexpression of RpoS slightly increased the growth rate (Fig. S5). In combination, these results indicate that the ReaL-mediated downregulation of RpoS and subsequent defective oxidative stress response contribute to the attenuated virulence of the ΔybeY mutant.
Mutation of ybeZ results in similar phenotypes as those of the ⌬ybeY mutant. As we found that YbeZ binds to YbeY and is involved in the maturation of the 16S rRNA, it is likely that YbeZ plays similar roles as YbeY. Indeed, mutation of ybeZ resulted in upregulation of reaL and downregulation of rpoS and katA (Fig. 11A). Consistently, the ΔybeZ mutant was more susceptible to H 2 O 2 and displayed attenuated virulence in the acute pneumonia model (Fig. 11B and C). These results suggest that YbeY and YbeZ are likely to function together in the regulation of reaL, rpoS, and oxidative stress response genes.

DISCUSSION
YbeY is a highly conserved bacterial RNase that is involved in the 16S rRNA maturation, ribosome assembly, virulence, and stress responses (28)(29)(30)(31)(32)(33). In previous studies, the ybeY mutant was not identified from P. aeruginosa transposon mutant libraries. Thus, ybeY was predicted to be an essential gene (29,39). In this study, we were able to delete ybeY in the backgrounds of PA14, PAO1, and PAK. However, it took at least 2 days for the ΔybeY mutant to form visible colonies on the LB plate. We suspect that the growth defect might explain why the ybeY mutant was not identified from the Tn mutant population. Mutation of ybeY resulted in defective processing of the 16S rRNA, defective response to oxidative stresses, and attenuated virulence in the murine acute pneumonia model. In addition, we found that the ybeY mutant was more susceptible to heat shock (45°C, data not shown).   (29,51,52). The YbeY purified from S. meliloti displays a metal-dependent endoribonuclease activity that cleaves both ssRNA and double-stranded RNA (dsRNA) substrates (33). A previous bacterial two-hybrid analysis revealed that YbeY interacts with the ribosomal protein S11, Era, Der, SpoT, and YbeZ in E. coli (37). The interaction between YbeY and S11 is required for the maturation of the 16S rRNA (37). Overexpression of the GTPase gene era in a ΔybeY mutant improves the 16S rRNA maturation and 70S ribosome assembly (53). SpoT plays an important role in the bacterial stringent response by controlling the homeostasis of the alarmone molecule (p)ppGpp (54). Of note, (p)ppGpp controls the bacterial growth rate by suppressing the rRNA production (55). Further studies are warranted to explore whether YbeY affects the (p)ppGpp level and thus the bacterial stringent response. The GTPase Der (double Era-like GTPase) contains two GTP-binding domains. Studies in E. coli demonstrated its essentiality in the biogenesis of the 50S ribosomal subunit (56). In bacteria, the colocalization of ybeZ and ybeY in an operon is highly conserved (36)(37)(38). Here, we demonstrated the interaction between YbeY and YbeZ in P. aeruginosa and found that mutation of ybeZ resulted in similar phenotypes as the ybeY mutant. Further studies are needed to understand the exact function of YbeZ.
Here, we found that YbeY affects the expression of RpoS. A comparison of the transcription profiles between the ybeY mutant and an rpoS mutant revealed that 49 genes displayed similar expression patterns in the two mutants compared to the wild-type strain (see Table S2 in the supplemental material) (57). For example, previous studies revealed that the P. aeruginosa lectin PA-IL coding gene lecA and the twocomponent response regulator gene pprB are under the direct regulation of RpoS (58,59). Mutation of rpoS resulted in downregulation of lecA, pprB, and genes regulated by the PprB, including the tad locus (PA4297-PA4305) and the type IVb pilin gene flp (59). Mutation of ybeY also reduced the expression of those genes (Table S2). However, 7 genes were oppositely expressed in the ybeY mutant and the rpoS mutant (Table S2), and many other genes displayed different expression patterns in the two mutants. These results indicate that YbeY controls global gene expression through multiple pathways.
In P. aeruginosa, RpoS controls the expression of the catalase genes katA/B (18); however, no RpoS binding sequence has been identified in the promoter regions of the two genes, indicating an indirect regulation (47). Overexpression of rpoS in the ΔybeY mutant restored the expression levels of katA (Fig. 6) and sodB (data not shown). However, overexpression of rpoS only restores the expression level of katB in the ΔybeY mutant in the absence of H 2 O 2 , but not in the presence of H 2 O 2 (data not shown). The RpoS-mediated regulatory pathways on katA, katB, and sodB warrant further studies.
Upon invading host, the bacteria encounter phagocytes that kill the bacteria mainly through ROS, phagocytosis, antimicrobial peptides, and hydrolases (12,60,61). Bacteria survive the phagocyte-generated ROS by producing superoxide dismutase and catalases (13,62). Our in vitro infection assay revealed that the ybeY mutant was more susceptible to neutrophils. Neutralization of ROS by NAC restored the bacterial survival rate, indicating that the hypersusceptibility to neutrophil is mainly due to defective detoxification of the ROS. Therefore, the downregulation of katA might partially contribute to the attenuated virulence of the ybeY mutant. Meanwhile, RpoS controls multiple stress response genes and affects the quorum sensing systems (57). The Overall, our study reveals an important physiological role of YbeY in the RNA processing in P. aeruginosa. Further studies are required to understand how the expression and activity of YbeY are regulated, particularly under environmental stresses.

MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterial strains, plasmids, and primers used in this study are listed in Table S1 in the supplemental material. Bacteria were cultured in the L-broth (63) medium at 37°C with agitation at 200 rpm. Antibiotics were used at the following concentrations: for E. coli, gentamicin 10 g ml Ϫ1 , tetracycline 10 g ml Ϫ1 , kanamycin 50 g ml Ϫ1 , ampicillin 100 g ml Ϫ1 ; for P. aeruginosa, tetracycline 50 g ml Ϫ1 , gentamicin 50 g ml Ϫ1 , rifampin 100 g ml Ϫ1 , carbenicillin 150 g ml Ϫ1 .
Chromosomal gene mutations were generated as described previously (64). To construct a ybeY deletion mutant in P. aeruginosa, a 945-bp fragment and a 1,306-bp fragment that are upstream and downstream of the ybeY coding region, respectively, were amplified by PCR using PA14 chromosome as the template and primers listed in Table S1. The fragments were cloned into the plasmid pEX18TC. The resulting plasmid was transferred into E. coli S17 and then transferred into P. aeruginosa by conjunction. Single crossover mutants were selected on 50 g ml Ϫ1 tetracycline and 50 g ml Ϫ1 kanamycin (to kill the E. coli donor strain), and double crossover mutants were selected by growth on LB plates containing 7.5% sucrose. The ybeY deletion mutant was screened by PCR with the primers YbeY-L and YbeY-R (Table S1). In the ybeY deletion mutant, a 421-bp fragment was deleted from the 483-bp coding region of the ybeY gene.
Transcriptome sequencing and analysis. Bacteria were cultured in LB at 37°C to the stationary phase (OD 600 ϭ 3). Total RNA was extracted by the RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). Sequencing and analysis services were performed by the Suzhou Genewiz as previously described (65).
Real-time PCR. Bacterial cells were cultured under indicated conditions. Total bacterial RNA was extracted by an RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). cDNAs were synthesized using random primers and reverse transcriptase (TaKaRa, Dalian, China). Real-time PCR was performed with the SYBR II Green supermix (TaKaRa, Dalian, China). The ribosomal gene rpsL or PA1805 was used as the internal control (66). rRNA analysis. Bacteria were cultured in LB at 37°C until late logarithmic phase (OD 600 ϭ 1). Total RNA was isolated with the RNA prep Pure Cell/Bacteria kit (Tiangen Biotec, Beijing, China). One microgram of the total RNA was mixed with an RNA loading buffer (TaKaRa, Dalian, China) and incubated at 65°C for 10 min, followed by incubation on ice for 10 min. Then the 16S and 23S rRNAs were separated by electrophoresis on a gel made of 0.9% Synergel (BioWorld, USA) and 0.7% agarose in TAE as described previously (67).
Ribosome separation by sucrose gradients. Separation of P. aeruginosa ribosomal particles was performed as previously described (41) with minor modifications. Bacteria were grown in LB at 37°C to an OD 600 of 1. One liter of the bacterial cells was collected and resuspended in 40 ml precooled Buffer A (10 mM Tris-HCl, 10 mM MgCl 2 , 100 mM NH 4 Cl, 6 mM ␤-mercaptoethanol, pH 7.5) with 10 g/ml DNase I. The bacterial cells were lysed by a French press (600 MPa), and the cell debris was removed by centrifugation at 12,000 ϫ g for 30 min at 4°C. One milliliter of the supernatant was loaded onto a 10% to 40% sucrose gradient in buffer A, followed by centrifugation at 36,000 rpm for 3 h at 4°C in an SW41 rotor (Beckman). The stratified sucrose gradient solutions were collected, and the RNA contents were quantified by UV absorbance at 260 nm.
Pulldown assay. The ybeY and ybeZ coding regions were amplified by PCR using PA14 chromosome DNA as the template, and the primers are listed in Table S1. A 6ϫHis tag coding sequence was included in the downstream primer of the ybeY gene (Table S1), thus resulting in a C-terminal 6ϫHis-tagged ybeY (ybeY-His). The amplified ybeY gene was cloned into the plasmid pMMB67EH. The ybeZ gene was cloned into the plasmid pET41a, resulting in a translational fusion with the gst gene at the C terminus. E. coli strain BL21 carrying the gst gene or ybeZ-gst fusion gene was cultured at 37°C to an OD 600 of 0.4 to 0.6. Expression of the YbeZ-GST was induced by 1 mM IPTG for 4 h, and then the bacteria were collected and resuspended in a lysis buffer (1 M Na 2 HPO 4 , 1 M NaH 2 PO 4 , 0.3 M NaCl, pH 8.0), followed by sonication. The cell debris was removed by centrifugation at 12,000 ϫ g for 20 min at 4°C. The cell lysate containing GST or YbeZ-GST protein was incubated with the GST tag purification resin (Beyotime Biotechnology, Shanghai, China) for 2 h at 4°C and then washed three times with the lysis buffer. PA14 carrying the ybeY-His fusion gene was grown in LB to an OD 600 of 0.6, followed by induction by 1 mM IPTG for 4 h. The bacteria were resuspended in the lysis buffer and lysed by sonication. The cell lysate was then incubated with the GST or YbeZ-GST bound resin at 4°C for 2 h. The resin was washed three times with the lysis buffer. The bound proteins were then eluted with 60 mM reduced glutathione (GSH) in an elution buffer provided by the purification kit (Beyotime Biotechnology, Shanghai, China) and subjected to Western blot analysis.
␤-Galactosidase assay. Bacterial cells were grown in LB at 37°C until the OD 600 reached 1. An 0.5-ml portion of the bacterial culture was collected and resuspended in 1.5 ml Z buffer (50 mM Functions of YbeY in P. aeruginosa ® ␤-mercaptoethanol, 60 mM Na 2 HPO 4 , 60 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , pH 7.0). The ␤-galactosidase activity was determined as previously described (68). H 2 O 2 susceptibility assay. Bacteria were cultured to an OD 600 of 1 and then washed three times with PBS and resuspended in PBS. The bacteria were treated with 10, 20, or 50 mM H 2 O 2 at 37°C for 30 min. The live bacterial numbers were determined by serial dilution and plating. The survival rate was calculated by comparing the live bacterial number after the H 2 O 2 treatment with that before the treatment.
Catalase activity assay. Bacteria were cultured to an OD 600 of 1 and then incubated with or without 2 mM H 2 O 2 for 30 min. The bacteria were collected by centrifugation, and the cells were broken by sonication. The total intracellular catalase activity was measured using a catalase assay kit (Beyotime, Shanghai, China). The total bacterial protein concentrations were quantified by a BCA analysis (Beyotime, Shanghai, China) for calibration.
HL60 differentiation and killing assay. HL60 cells were cultured in RPMI 1640 medium (HyClone, USA), with streptomycin (100 mg/ml), penicillin G (100 U/ml), and 10% (vol/vol) thermally inactivated fetal bovine serum (Gibco, Australia) at 37°C with 5% CO 2 . The HL60 cells were diluted to 4.5 ϫ 10 5 cells/ml and cultured in the differentiation medium (RPMI 1640 medium, 20% [vol/vol] heat-inactivated fetal bovine serum, 1.3% dimethyl sulfoxide [Sigma, USA]) for 6 to 7 days (20). The differentiated HL60 cells (designated dHL60) were diluted to 1 ϫ 10 7 cells/ml in warm HBSS, and 100 l cell suspension was added to each well of a 96-well plate. Then the cells were infected with bacteria at an MOI of 10 and incubated at 37°C for 3 h. The live bacterial numbers were determined by serial dilution and plating. The survival rate was calculated by comparing the numbers of live bacterial cells after incubation with or without the dHL60 cells.
RNA stability analysis. P. aeruginosa strains were cultured in LB at 37°C to an OD 600 of 1. The bacterial cultures were then treated with 100 g/ml rifampin to stop the transcription. At the indicated time points, the bacterial cells were collected and mixed with the same number of E. coli cells expressing a gfp gene. Total RNA was purified, and the levels of reaL and rpsL were determined by real-time PCR. The gfp mRNA levels were used as the internal control for normalization.
In vitro transcription and RNA gel mobility shift assay. The in vitro transcription of sRNA was performed as previously described (22). The sRNA ReaL was synthesized using the Riboprobe System-T7 (Promega) from PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1 according to the manufacturer's instructions. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of purified YbeY or GFP in the binding buffer (10 mM Tris-HCl [pH 7.5], 5 mM MgCl 2 , 50 mM KCl, 10% glycerol, 1 U recombinant RNase inhibitor [TaKaRa]) and incubated on ice for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. The electrophoresis was performed at 100 V for 150 min in 0.5ϫ TBE buffer on ice. The RNA bands were visualized after staining with Gel-red (Biotium) for 10 min.
In vitro RNA degradation assay. The sRNA ReaL was synthesized using a Riboprobe System-T7 (Promega) from the PCR product amplified from PA14 chromosomal DNA with the primers listed in Table S1. The RNA was purified by isopropanol precipitation and refolded by heating at 90°C for 10 min and then cooling down naturally at room temperature for 30 min. One hundred nanograms of the purified RNA was mixed with indicated amounts of the purified YbeY or GFP protein in the activity buffer (10 mM Tris-HCl [pH 7.5], 50 mM KCl, 10% glycerol, and the indicated concentrations of MnCl 2 ) and incubated at 37°C for 30 min. Fifteen microliters of each sample was loaded onto a nondenaturing 8% polyacrylamide gel. Electrophoresis was performed at 100 V for 150 min in 0.5ϫ TBE buffer on ice. The RNA was visualized after staining with Gel-red (Biotium) for 10 min.
Murine acute pneumonia model. The animal infection experiments described in this study were performed following the National and Nankai University guidelines on the use of animals in research. The protocol with the permit number NK-04-2012 was approved by the animal care and use committee of the College of Life Sciences, Nankai University. The infection was performed as previously described (69). Briefly, bacteria were cultured overnight and then diluted at 1:100 in fresh LB and grown at 37°C with agitation until OD 600 reached 1. The bacterial cells were harvested by centrifugation, washed once with PBS, and resuspended in PBS at the concentration of 2 ϫ 10 8 CFU/ml. Each 6-to 8-week-old female BALB/c mouse (Vital River, Beijing, China) was anesthetized by an intraperitoneal injection of 100 l 7.5% chloral hydrate, followed by an intranasal inoculation of 20 l of the bacterial suspension, resulting in 4 ϫ 10 6 CFU per mouse. The mice were sacrificed 12 h postinfection, and the lungs were dissected and subjected to homogenization. The number of bacteria in each lung was determined by plating.
Data availability. The transcriptome (RNA sequencing) data that support the findings of this study have been deposited in the NCBI Sequence Read Archive (SRA) with the accession code PRJNA574019.

SUPPLEMENTAL MATERIAL
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