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RNAIII of the Staphylococcus Aureus Agr System Activates Global Regulator Mgra by Stabilizing Mrna

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RNAIII of the Staphylococcus Aureus Agr System Activates Global Regulator Mgra by Stabilizing Mrna Correction MICROBIOLOGY Correction for “RNAIII of the Staphylococcus aureus agr system (112:14036–14041; first published October 26, 2015; 10.1073/ activates global regulator MgrA by stabilizing mRNA,” by Ravi pnas.1509251112). Kr. Gupta, Thanh T. Luong, and Chia Y. Lee, which appeared The authors note that Fig. 2 appeared incorrectly. The cor- in issue 45, November 10, 2015, of Proc Natl Acad Sci USA rected figure and its legend appear below. A B 6 6 * * * 5 ** ** * 5 * 4 4 3 3 2 2 1 1 Half-life (min) 0 Half-life (min) 0 agr agr agr (11346) Newman P1 UTR P2 UTR P1 UTR P1 UTR P2 UTR P2 UTR (1844) (1845) +RNAIII +pML100 +RNAIII 5-nt mutant(12916) (A22) +pML100 (12501) (12502) P1 UTR P2 UTR 5-nt revertant Fig. 2. Stability of mgrA mRNA. (A) Stability in various chromosomal mutants. (B) RNAIII complementation of deletion mutations in mgrA UTR. mRNA stability expressed as half-life in minutes. The numbers in parentheses represent strain number. *P < 0.05, **P < 0.01 (unpaired two-tailed Student t test between Newman and each mutant, n = 3). www.pnas.org/cgi/doi/10.1073/pnas.1523895113 E7306 | PNAS | December 29, 2015 | vol. 112 | no. 52 www.pnas.org Downloaded by guest on September 26, 2021 RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA Ravi Kr. Gupta, Thanh T. Luong, and Chia Y. Lee1 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 Edited by Richard P. Novick, New York University School of Medicine, New York, NY, and approved September 9, 2015 (received for review May 12, 2015) RNAIII, the effector of the agr quorum-sensing system, plays a key (13) to postulate that RNAIII must interact with one or more role in virulence gene regulation in Staphylococcus aureus, but how pleiotropic regulators. Here, we report that RNAIII can also affect RNAIII transcriptionally regulates its downstream genes is not com- its downstream genes through another important S. aureus global pletely understood. Here, we show that RNAIII stabilizes mgrA mRNA, transcriptional regulator, MgrA, which has been shown to affect thereby increasing the production of MgrA, a global transcriptional more than 350 genes involved in virulence, antibiotic resistance, regulator that affects the expression of many genes. The mgrA gene autolysis, and biofilm formation (21–25). Our results suggest that is transcribed from two promoters, P1 and P2, to produce two mRNA part of the MgrA regulon is affected by RNAIII. ′ transcriptswithlong5 UTR. Two adjacent regions of the mgrA mRNA Results UTR transcribed from the upstream P2 promoter, but not the P1 pro- moter, form a stable complex with two regions of RNAIII near the mgrA Gene Is Transcribed by Two Promoters. We have previously 5′ and 3′ ends. We further demonstrate that the interaction has sev- identified the mgrA gene (22). To map the mgrA promoter, we eral biological effects. We propose that MgrA can serve as an inter- performed primer extension experiments. We found three tran- mediary regulator through which agr exerts its regulatory function. scriptional start sites: at nucleotides −123, −301, and −303 up- stream from the ATG start codon (Fig. S1), which closely agree − − Staphylococcus aureus | RNAIII | MgrA | virulence regulation with the nucleotide 124 and 302 sites previously reported (21). Most likely, nucleotides −301 and −303 represent start sites from the same promoter with the former being the preferred start site. mall regulatory RNAs (sRNAs) play an important role in gene We then designated the promoters as P1 and P2, as shown in Fig. Sregulation in both eukaryotes and prokaryotes. In bacteria, 1. To further define the promoters, we constructed promoter fu- sRNAs typically act by base pairing with target mRNAs with lim- sions to the xylE reporter gene in plasmid pLL38 with successive ited or extended complementarity (antisense mechanism), but they deletions of DNA extending from 368 bp upstream of the mgrA can also modulate protein activity (1, 2). sRNAs commonly act on ATG start codon. Our results showed that deletion of P2 reduced their target mRNA near the translation initiation site by base reporter activity by about 33–43%, whereas deletion of both pro- — — pairing to block and in some cases promote translation. Some moters resulted in about 92% reduction (Fig. 1), indicating that sRNAs cause alterations in the secondary structure of the target both promoters were functional and that the predicted promoters mRNA, thereby enhancing or reducing mRNA degradation (3, 4). are most likely correct. It should be noted here that the P2 pro- Staphylococcus aureus is an important human pathogen that moter is embedded within the coding region of a divergently produces many virulence factors that are regulated by an equally transcribed upstream gene encoding a putative ABC transporter. impressive number of regulators (5, 6). A key regulatory system involved in virulence regulation in S. aureus is the agr quorum- P2 5′ UTR Affects the Stability of mgrA mRNA. The 123-nt and 301/ sensing system. A 514-nt RNA, RNAIII, is the effector through 303-nt UTRs transcribed from the P1 and P2 promoters, re- which most genes in the agr regulon are regulated (7, 8). RNAIII is spectively, are unusually long for bacterial mRNAs. For conve- one of the largest known sRNAs that regulates gene expression via nience, herein we refer to the region between the P2 and P1 antisense mechanism by base pairing with target mRNAs (5, 9). transcriptional start sites as the P2 UTR and the region between RNAIII is capable of forming a stable structure that is characterized by 14 stem-loop motifs and three long-distance interacting helices (9). Several examples of regulation by direct interaction between Significance RNAIII and its target mRNAs have been reported (10–14). A common denominator in these regulatory interactions is the tar- In Staphylococcus aureus,theagr quorum-sensing system is geting of translation initiation sequences, through base pairing of critical for regulation of many virulence genes, primarily through the CU-rich loop domains of RNAIII with regions containing the RNAIII, a small RNA. RNAIII functions by interacting with trans- Shine and Dalgarno (SD) sequence or AUG start codon of the lational elements of its downstream genes typically by inhibiting target mRNA, resulting in negative control of translation (15, 16). translation initiation. Only a few direct targets of RNAIII have A notable exception is the activation of Hla by RNAIII, in which been identified, one of which is global regulator Rot. However, the interaction unmasks the SD sequence to facilitate translation of Rot can only partially account for RNAIII regulation. We show hla mRNA (10). Although a recent report shows that RNAIII here that RNAIII can affect gene expression through another activates map expression, the mechanism of activation is un- global regulator MgrA by enhancing MgrA production via mRNA known (17). More recently, RNAIII has also been shown to neg- atively control sbi translation through direct interaction with the stabilization, a novel mechanism of RNAIII regulation. Our find- translation initiation sequences of the sbi mRNA. However, in this ings provide evidence linking two major master regulators, case, three distant RNAIII domains that do not include the CU- thereby advancing our understanding of virulence regulation in rich loop domains have been shown to directly interact with three this important human and animal pathogen. distinct sites in the sbi mRNA to repress sbi translation (18). Rot is a pleiotropic regulator that affects many toxins and sur- Author contributions: R.K.G. and C.Y.L. designed research; R.K.G. and T.T.L. performed face proteins (19). The finding that RNAIII represses rot mRNA research; R.K.G., T.T.L., and C.Y.L. analyzed data; and R.K.G. and C.Y.L. wrote the paper. translation has advanced the understanding of agr regulation The authors declare no conflict of interest. (12, 13). However, RNAIII and Rot transcriptomes only partially This article is a PNAS Direct Submission. overlap (19, 20). Thus, RNAIII repression of Rot cannot com- 1To whom correspondence should be addressed. Email: [email protected]. pletely account for how RNAIII regulates the majority of its This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. downstream genes. This led Geisinger et al. (12) and Boisset et al. 1073/pnas.1509251112/-/DCSupplemental. 14036–14041 | PNAS | November 10, 2015 | vol. 112 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1509251112 -368 P2 P1 100bp +82 Promoter mRNA level of the genes involved (27). Thus, it is possible that Plasmid activity (%) -35 -10 -35 -10 mgrA pTL3234 100±16.3 RNAIII could affect NWMN_0656 expression, thereby affecting Δ59bp pTL3219 57.8±2.6 mgrA mRNA levels. However, we showed that RNAIII had no effect Δ103bp pTL3235 65.8±5.0 Δ152bp pTL3236 67.7±4.9 on NWMN_0656 transcription (Fig. S4), indicating that RNAIII Δ216bp pTL3221 8.3±2.6 does not regulate mgrA mRNA levels through NWMN_0656. Δ95bp pTL3245 66.8±7.8 -492 pTL3289 100±22.3 Δ138bp pTL3271 53.4±10.2 mgrA UTR Interacts with RNAIII at Two Separate Domains. To determine whether the complementary regions identified above are involved in Fig. 1. Analysis of mgrA promoter region. DNA fragments with various de- the interaction between RNAIII and mgrA P2 UTR, we used RNA- letions, as indicated, were fused to the xylE reporter in pLL38. All deletions RNA EMSA. RNA corresponding to the mgrA P2 UTR was syn- were derived from pTL3234 with a 450-bp insert containing both promoters and 82 bp of the mgrA coding sequence, except pTL3271, which was derived thesized in vitro and labeled with biotin.
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