IAI Accepts, published online ahead of print on 15 December 2014 Infect. Immun. doi:10.1128/IAI.02707-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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The prrF-encoded small regulatory RNAs are required for iron homeostasis and virulence of

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Pseudomonas aeruginosa

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Running Title: The prrF-encoded sRNAs are required for P. aeruginosa virulence

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Alexandria Reinhart1, Daniel A. Powell2, Angela T. Nguyen3, Maura O’Neill3, Louise Djapgne3, Angela

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Wilks3, Robert K. Ernst1,2, and Amanda G. Oglesby-Sherrouse1,3#

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University of Maryland Baltimore 1School of Medicine, Department of Microbiology and Immunology, 2

School of Dentistry, Department of Microbial Pathogenesis, 3School of Pharmacy, Department of

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Pharmaceutical Sciences, Baltimore, Maryland 21201

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To whom correspondence should be sent: [email protected].

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Keywords: iron regulation, heme regulation, small RNAs, Pseudomonas aeruginosa

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Abstract Pseudomonas aeruginosa is an opportunistic pathogen that requires iron to cause infection, but also

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must regulate the uptake of iron to avoid iron toxicity. The iron-responsive PrrF1 and PrrF2 small

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regulatory RNAs (sRNAs) are part of P. aeruginosa’s iron regulatory network and affect the expression

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of at least 50 genes encoding iron-containing proteins. The genes encoding the PrrF1 and PrrF2 sRNAs

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are encoded in tandem in P. aeruginosa, allowing for the expression of a distinct, heme-responsive

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sRNA named PrrH that appears to regulate genes involved in heme metabolism. Using a combination of

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growth, mass spectrometry, and gene expression analysis, we show here that the ∆prrF1,2 mutant,

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which lacks expression of the PrrF and PrrH sRNAs, is defective for both iron and heme homeostasis.

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We also identify phuS, encoding a heme binding protein involved in heme acquisition, and vreR,

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encoding a previously-identified regulator of P. aeruginosa virulence genes, as novel targets of prrF-

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mediated heme regulation. Finally, we show that the prrF locus encoding the PrrF and PrrH sRNAs is

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required for P. aeruginosa virulence in a murine model of acute lung infection. Moreover, we show that

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inoculation with a ∆prrF1,2 deletion mutant protects against future challenge with wild type P.

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aeruginosa. Combined, these data demonstrate that the prrF-encoded sRNAs are critical regulators of P.

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aeruginosa virulence.

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Introduction Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium and versatile opportunistic

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pathogen. Iron is required for P. aeruginosa virulence (1-6) and is obtained through several mechanisms.

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In anaerobic environments, iron in its ferrous form is freely diffusible through the outer membrane and

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transported into the cytoplasm by the Feo inner membrane transport system (7, 8). However, the

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insolubility of ferric iron in aerobic environments limits accessibility to this nutrient. Moreover, the

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sequestration of iron by host proteins creates a substantial barrier to infection (9, 10). To overcome this

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barrier, P. aeruginosa synthesizes and secretes two siderophores, pyoverdine and pyochelin, which

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scavenge ferric iron (1-4). P. aeruginosa can also acquire heme, an abundant source of iron in the human

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host (11). Once internalized, heme is sequestered by the cytosolic PhuS heme chaperone (12). PhuS

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transfers heme to the iron-regulated HemO heme oxygenase, which degrades heme to biliverdin,

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releasing carbon monoxide and iron (13, 14). Several studies have shown that iron acquisition is essential

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for P. aeruginosa virulence (1-5) and biofilm formation (15-17), demonstrating the central role of this

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element in P. aeruginosa pathogenesis.

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Despite its essentiality, iron and heme can be toxic due to their ability to catalyze the formation of

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reactive oxygen species. Thus, to maintain iron homeostasis, P. aeruginosa must be able to not only

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acquire iron, but also regulate uptake of iron and heme from the environment, as well as iron use and

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storage. Expression of genes encoding iron and heme uptake systems in P. aeruginosa is therefore

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regulated by the Fur (ferric uptake regulator) protein (18-20). In iron-replete environments, Fur binds to

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iron and becomes an active transcriptional repressor, blocking expression of genes for iron acquisition

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(21). Fur also affects the expression of genes encoding virulence traits, including toxins and extracellular

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proteases (21-23). Fur regulation in P. aeruginosa mainly occurs through the regulation of cell surface

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signaling (CSS) systems, which are comprised of a TonB-dependent outer membrane (OM) receptor, an

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extracytoplasmic function (ECF) sigma factor, and an anti-sigma factor that regulates the activity of the

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ECF sigma factor (24). As an example, Fur represses expression of pvdS, encoding an ECF sigma factor Page 3

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that directly activates expression of genes for pyoverdine biosynthesis and uptake, as well as multiple

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virulence factors (22, 25-27). Binding of ferri-pyoverdine to the FpvA OM receptor activates the PvdS

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sigma factor, which is normally sequestered at the inner membrane by its anti-sigma factor, FpvR (28,

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29). This paradigm of Fur-mediated regulation via ECF sigma factors extends to the uptake systems for

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several additional iron sources used by P. aeruginosa (30, 31).

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Fur also mediates positive regulation of gene expression via repression of the PrrF small regulatory

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RNAs (sRNAs) (32). The PrrF sRNAs negatively affect the expression of several genes, presumably by

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interaction with complementary regions of target mRNAs. Included in the PrrF regulon are genes

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encoding non-essential iron storage proteins and iron-containing metabolic enzymes (32, 33).

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Accordingly, PrrF regulation controls how P. aeruginosa uses and stores iron (iron utilization) by

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sparing this nutrient during growth in iron-limiting environments. The analogous iron-sparing system in

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Escherichia coli, which is mediated by the RyhB sRNA, was previously shown to promote growth in

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iron-depleted conditions by increasing intracellular pools of free iron (34). While most PrrF regulatory

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targets encode iron-containing proteins, it remains unknown if PrrF regulation similarly affects global

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iron usage or growth in low iron environments. Regulation by the PrrF sRNAs was previously shown to

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allow for the production of the Pseudomonas quinolone signal (PQS), a secreted signaling molecule that

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activates expression of several virulence genes (35, 36). This effect is achieved through repression of the

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antABC mRNA, encoding iron-containing enzymes that metabolize anthranilate, which is the precursor

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for PQS (33). Due to their roles in iron homeostasis and PQS production, the PrrF sRNAs are predicted

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to contribute to pathogenesis of P. aeruginosa.

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The PrrF sRNAs are encoded by two highly homologous genes, prrF1 and prrF2, which are located

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in tandem on the P. aeruginosa genome. This arrangement allows for the expression of a third, heme-

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responsive sRNA named PrrH (37). While the prrF1 and prrF2 genes are encoded by all pseudomonads,

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this tandem arrangement is unique to P. aeruginosa and potentially contributes to this species’ capacity

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to cause disease. PrrH is hypothesized to regulate a specific subset of genes involved in heme Page 4

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metabolism (i.e. synthesis and degradation), heme uptake (acquisition of extracellular heme), and other

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cellular processes via its unique sequence derived from the prrF1-prrF2 intergenic region. By doing so,

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the PrrH sRNA is likely to contribute to overall heme homeostasis of the cell. Accordingly, we

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previously performed in silico analysis of the prrF1-prrF2 intergenic region combined with quantitative

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real time PCR (qRT-PCR) to identify nirL as a putative target of PrrH (37). The nirSMCFDLGHJEN

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gene cluster encodes for dissimilatory nitrite reductase (NIR; cytochrome cd1) and includes genes for the

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biosynthesis of heme d1, a prosthetic group of NIR(38). Biosynthesis of heme d1 branches from the

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central heme biosynthetic pathway with NirF, NirJ, and NirE, catalyzing its production from

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uroporphyrinogen III. Thus, repression of NIR biosynthesis by PrrH may prioritize the function of the

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heme biosynthetic pathways under limiting heme concentrations, contributing to heme homeostasis (38).

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In this study, we have characterized the effects of deleting the prrF locus on multiple aspects of P.

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aeruginosa iron homeostasis and virulence. We show that the ∆prrF1,2 mutant is defective for iron and

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heme homeostasis. Additionally, we show that ∆prrF1,2 mutant biofilms are not enhanced by iron in the

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presence of aminoglycoside antibiotics, a known trait of P. aeruginosa biofilm formation (39, 40).

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Further, we demonstrate that iron and heme regulation of PrrF and PrrH is conserved in several P.

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aeruginosa clinical isolates. Moreover, we establish that the prrF locus is absolutely required for

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virulence in a murine model of acute lung infection. Combined, these results demonstrate the critical

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role of the PrrF and PrrH sRNAs in P. aeruginosa virulence.

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Methods

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Bacterial strains and growth conditions. Bacterial strains used in this work are listed in Table 1.

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Escherichia coli strains were routinely grown in L broth or on L agar plates, and P. aeruginosa strains

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were maintained in brain-heart infusion (BHI) broth or on BHI agar plates. For growth curves, qPCR,

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mass spectrometry, and northern blots, strains were diluted from L broth overnights into M9 minimal

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medium purchased from Teknova containing 2% glucose and grown for 4 hours, then subcultured into Page 5

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fresh M9 for an additional 8 hours at 37°C (41). For biofilm analysis, strains were grown in chelex-treated,

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dialyzed trypticase soy broth (DTSB) prepared as previously described (40). Ferric chloride (FeCl3) was added

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to a final concentration of 100 µM where indicated. Heme stocks were freshly prepared prior to use in

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0.1N NaOH / 20mM Tris and adjusted to pH 7.0 with HCl, and stock concentration was determined by

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pyridine hemochrome assay as previously described (42) using the following extinction coefficients: ε =

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170.0 mM-1 cm-1 at A=418 nm; ε = 17.5 mM-1 cm-1 at A=525 nm; ε = 34.5 mM-1 cm-1 at A=555 nm.

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Heme was immediately diluted into M9 medium to a concentration of 5 µM where indicated.

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Tetracycline was used at 60 µg/ml for growth of P. aeruginosa strains carrying the pVLT31 vector or its

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derivatives as indicated.

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Determination of cellular iron content. Strains were grown overnight in LB, then diluted into M9

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minimal medium purchased from Teknova containing 2% glucose and 60 µg/ml tetracycline and grown

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for 4 hours to deplete intracellular iron stores. Strains were subcultured into fresh M9 for an additional 8

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hours at 37°C, with or without iron or heme supplementation, and harvested by centrifugation.

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Harvested bacteria from the secondary cultures were dissolved in 20% nitric acid and boiled overnight at

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100°C. Inductively-coupled plasma mass spectrometry (ICP-MS) was then used to determine the metal

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content of the dissolved bacteria (Agilent 7700 ICP-MS, Agilent Technologies). Raw ICP-MS data

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(µg/L) were corrected for drift using values of internal controls (indium, scandium, and germanium) that

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were added to each sample during processing. Corrected values were then normalized to culture density

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as determined by the absorbance at 600 nm.

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Real time PCR. Primers and probes used for real time PCR (qPCR) are shown in the Supplementary

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Materials, Table S1. RNA was extracted using the Qiagen RNeasy Mini Kit according to manufacturer’s

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directions. 50 ng/µL of RNA was used to generate cDNA with the ImPromII cDNA synthesis kit

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(Promega) as previously described (37), and cDNA was analyzed using the StepOnePlus instrument

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(Applied Biosystems) and Taqman reagents (LifeTechnologies). Standard curves were produced for

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each primer probe set by analyzing cDNA generated from serial dilutions of RNA and used to determine Page 6

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relative amounts of each RNA. Relative RNA levels were then normalized to the levels of the oprF

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mRNA.

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Biofilm growth and measurement. The MBEC (minimal biofilm eradication concentration)

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Assay™ physiology and genetics biofilm device (formerly known as the Calgary Biofilm Device,

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Innovotech) was used to assay biofilm formation and the MBEC of tobramycin against PAO1 and the

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∆prrF1,2 mutant as previously described (40). Briefly, 96-well MBEC plates containing DTSB,

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supplemented with 100 µM FeCl3, were inoculated in duplicate with approximately 1 x 107 cfu ml-1

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bacteria. The plates were covered with the MBEC peg lids and incubated with gentle shaking (65 RPM)

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at 37°C for 24 hours to allow biofilm formation on the pegs. Following incubation, the peg lid was

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gently washed in PBS to remove non-adherent bacteria, then transferred to a fresh 96-well “challenge

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plate” with DTSB, with or without 100 µM FeCl3, and varying concentrations of tobramycin. Biofilms

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were incubated with tobramycin for 24 hours at 37°C, at which point the pegs were stained with 0.1%

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crystal violet solution for 10 minutes, then rinsed in water. After drying, the pegs were destained in 200

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µl 30% acetic acid, and the amount of crystal violet dye released was determined by reading the wells at

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OD595 in a Biotek plate reader.

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Isotopic heme labeling studies. Heme labeling studies were performed as previously described (41,

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43). Briefly, heme was prepared from labeled [4-13C]-δ-aminolevulinic acid (ALA) according to the

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method described by Rivera and Walker (44) and the yield calculated by the pyridine hemochrome assay

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(45). P. aeruginosa strains were grown in M9 medium for 4 hours, then diluted into fresh M9 medium

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supplemented with 5 µM 13C-heme for an additional 8 hours. Bilverdin (BVIX) isomers were purified

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from the culture supernatants and separated and analyzed by liquid chromatography-tandem mass

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spectrometry (MS/MS) (Waters TQD triple quadrupole mass spectrometer with AQUITY H-Class

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UPLC) as described previously (43). Fragmentation patterns of the parent ions at 583.21 (12C-BVIX) and

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591.21 (13C-BVIX) were analyzed using multiple reaction monitoring (MRM). The fragmentation

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patterns of the respective BVIX isomers are shown in Scheme 1 (Supplementary Materials). Page 7

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Northern blots. Northern analysis of the PrrF and PrrH sRNAs was performed as previously

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described (37) with some modifications. Briefly, 1.5 µg (PrrF1 and PrrF2 probes) or 4 μg (PrrH probe)

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of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7M urea)

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gel then transferred to a BrightStar membrane (Ambion) using a semi-dry transfer apparatus.

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Biotinylated probes complementary to the PrrF1, PrrF2, or PrrH intergenic region were purchased from

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Integrated DNA Technologies (IDT) and hybridized to the blot overnight at 42°C. The membrane was

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washed using the Ambion Northern Max Low Stringency and High Stringency wash solutions according

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to the manufacturer’s instructions. Detection of the biotinylated probes was carried out using

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streptavidin alkaline phosphatase (Life Technologies) and visualized by chemiluminescence using CDP-

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Star (Sigma-Aldrich) according to the manufacturer’s instructions.

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Mouse infections. All mouse infections were performed in accordance with the Public

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Health Service Policy on the Humane Care and Use of Laboratory Animals, and with a protocol

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approved by the University of Maryland Institutional Animal Care and Use Committee.

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C57BL/6 (female, 6–8 wk old) were purchased from Jackson Laboratories. P. aeruginosa strains

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were grown overnight in L broth with 60 µg/ml tetracycline, then diluted into fresh L broth with

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60 µg/ml tetracycline and grown for an additional three hours at 37°C. Cells were harvested by

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centrifugation and resuspended in phosphate-buffered saline (PBS), and appropriate dilutions

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were prepared according to the absorbance of the cultures at 600 nm. Mice were anesthetized by

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inhalation of isoflurane, and were inoculated intranasally (i.n.) with 50 µl of inoculum (n = 5

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mice for each inoculum of each bacterial strain). Inocula were also serially diluted and plated on

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L agar to enumerate the delivered dose. Mice were observed twice daily for survival and disease

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signs, and moribund mice were euthanized by carbon dioxide inhalation followed by cervical

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dislocation. Mice that survived infection with the ∆prrF1,2/pVLT31 mutant were challenged

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with a lethal dose (~108 CFU) of wild type PAO1 at 28 days post-infection. At 28 days post final

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infection, all surviving mice were euthanized. Page 8

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Results

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The ∆prrF1,2 mutant is defective for iron utilization. It is established that the ∆prrF1,2 mutant

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displays increased expression of several genes encoding non-essential iron-containing proteins during

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iron-depleted growth (32, 33). However, it has not yet been determined how de-regulated expression of

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these genes affects overall iron homeostasis of the ∆prrF1,2 mutant. As an initial assessment of the

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ability of these strains to maintain iron homeostasis, we performed growth curves of the wild type PAO1

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strain and isogenic ∆prrF1,2 mutant under iron-depleted conditions. The wild type and ∆prrF1,2 mutant

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strains were grown for four hours in M9 minimal medium to restrict intracellular iron stores. Cells were

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subsequently sub-cultured into M9 minimal medium with or without either 100 µM iron

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supplementation, and growth was monitored for 24 hours. While the ∆prrF1,2 mutant grew similarly to

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the wild type in iron-replete conditions, growth of the ∆prrF1,2 mutant was significantly reduced as

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compared to the wild type under iron-limiting conditions (Figure 1A-B). Moreover, this growth defect

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was complemented by expression of the prrF genes in trans from the previously constructed pVLT-

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prrF1,2 vector (Figure 1A-B) (32). Thus, deletion of the prrF locus results in a growth defect under

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iron-limiting conditions.

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To confirm that the PrrF sRNAs were expressed and active under these growth conditions, we next

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analyzed expression of the PrrF sRNAs, as well as sodB, a known target of PrrF regulation (32), by

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qPCR. Our analysis showed that PrrF was expressed under these conditions, and that transcription of the

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PrrF sRNAs was strongly repressed by supplementation of the medium with 100 µM iron (Figure 2B).

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Moreover, expression of sodB was significantly de-repressed by deletion of the prrF locus, and

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complementation of ∆prrF1,2 mutant in trans restored sodB expression to wild type levels (Figure 3A).

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Surprisingly, iron exerted a very modest effect on expression of the PrrF sRNAs when expressed in

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trans (Figure 2B). Sequencing of the prrF complementation construct revealed a single nucleotide

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change four residues upstream of the prrF1 Fur box (Supplementary Materials, Figure S1), which we Page 9

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presume resulted in loss of Fur-regulated PrrF expression. Nevertheless, this construct was capable of

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restoring growth (Figure 1A) and sodB expression (Figure 3A) to wild type levels in the ∆prrF1,2

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mutant grown in low iron media. and has been used to complement previously reported phenotypes of

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the ∆prrF1,2 mutant (32, 33). Thus, our data demonstrate that the PrrF sRNAs are expressed and

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functional under low iron conditions, and that deletion of the prrF locus results in deregulated

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expression of genes in the PrrF regulon.

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Previous studies from Masse and colleagues demonstrated that the iron-regulated RyhB sRNA of E.

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coli contributed to growth in iron-depleted conditions by controlling the expression of genes encoding

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iron containing proteins, a function referred to as iron sparing (34). The ∆prrF1,2 mutant is similarly

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defective for expression of genes encoding iron containing protein (32), yet to date the effects of gene

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expression changes of the ∆prrF1,2 mutant on growth and iron usage have not been examined. We

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therefore next sought to determine if the ∆prrF1,2 mutant exhibited changes in total iron content as

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compared to the wild type strain. Inductively-coupled plasma mass spectrometry (ICP-MS) was used to

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measure intracellular iron levels of the wild type and ∆prrF1,2 mutant grown under iron starved or iron-

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replete conditions. These data showed that iron content in the ∆prrF1,2 mutant under iron-depleted

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conditions was higher than that of the wild type strain (Figure 1D), indicating that the ∆prrF1,2 mutant

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induced expression of its iron uptake systems. In support of this idea, analysis of pyoverdine levels and

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iron scavenging activity in cell supernatants showed increased siderophore production by the ∆prrF1,2

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mutant as compared to wild type (Supplementary Materials, Figure S2). Combined, these data

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demonstrate that the growth defect of the ∆prrF1,2 mutant under iron-depleted conditions is not due to a

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defect in iron uptake. Instead, our data indicate that de-regulated expression of non-essential iron-

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containing proteins resulted in the ∆prrF1,2 mutant sensing iron starvation, increased production of

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siderophores, and the growth defect observed in Figure 1A.

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The ∆prrF1,2 mutant biofilm is not enhanced by sub-inhibitory levels of tobramycin. Iron

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uptake has been shown by several studies to be critical for P. aeruginosa biofilm formation (15, 16, 46),

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which is an important aspect of P. aeruginosa virulence and antibiotic resistance. Despite its defect in

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iron homeostasis as demonstrated above, the ∆prrF1,2 mutant was previously shown to form biofilms

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similar to the wild type (16). However, we wanted to determine if defective iron utilization of the

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∆prrF1,2 mutant would affect biofilms formed under different growth conditions. For example,

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aminoglycoside antibiotics have been shown to increase biofilm formation when added to P. aeruginosa

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cultures at sub-inhibitory concentrations (39). Moreover, we recently showed that induction of biofilm

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formation by exposure to tobramycin, an aminoglycoside antibiotic that is often used to treat airway

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infections in cystic fibrosis patients, is dependent upon iron (40). We therefore determined if altered iron

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homeostasis in the ∆prrF1,2 mutant would affect the capacity of aminoglycosides to enhance biofilm

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formation. Biofilms of PAO1 and the ∆prrF1,2 mutant were formed under iron-replete growth

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conditions, then exposed to varying concentrations of tobramycin in either high or low iron media. As

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expected, PAO1 biofilm mass was significantly higher when pre-formed biofilms were exposed to either

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0.5 or 1 µg/ml of tobramycin in high versus low iron conditions (Figure 4). In contrast, the ∆prrF1,2

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mutant displayed significantly reduced biofilm mass as compared to wild type when the same

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concentrations of tobramycin were added to iron-replete medium (Figure 4). Thus, our data suggest that

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the iron utilization defect of the ∆prrF1,2 mutant reduces the ability of this strain to enhance biofilm

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formation when exposed to tobramycin.

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The ∆prrF1,2 mutant is defective for heme homeostasis. The tandem organization of the prrF1

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and prrF2 genes in P. aeruginosa allows for the expression of a unique, heme-responsive sRNA named

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PrrH (37). The PrrH sRNA is predicted to contribute to heme homeostasis by regulating a distinct subset

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of genes involved in heme metabolism and uptake. As such, it is possible that the ∆prrF1,2 mutant is

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defective for growth in the presence of heme as a sole iron source. To test this idea, we analyzed growth

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of the wild type and ∆prrF1,2 mutant strains in the presence of 5 µM heme. These data showed that Page 11

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heme supplementation was not able to completely suppress the growth defect the ∆prrF1,2 mutant in

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low iron (Figure 1C). However, ICP-MS analysis demonstrated that the iron content of the ∆prrF1,2

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mutant grown in the presence of heme is increased as compared to the wild type strain (Figure 1F).

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These data suggest that the growth defect of the ∆prrF1,2 mutant under these conditions is not due to an

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inability to uptake and degrade extracellular heme as an iron source, but is instead due to other aspects

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of heme homeostasis.

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To more directly test the idea that heme homeostasis was altered in the ∆prrF1,2 mutant, we

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analyzed heme oxygenase activity of this strain using liquid chromatography-tandem mass spectrometry

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(LC-MS/MS) as previously described (43). This analysis is performed by extracting biliverdin IX

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isomers (BVIX), the breakdown products of heme by heme oxygenase, from the supernatants of P.

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aeruginosa cultures. BVIX isomers are then separated by LC, allowing spectral analysis of the BVIX

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isomers as a function of retention time. Furthermore, by providing cells with 13C-labeled heme as an

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extracellular iron source, MS/MS fragmentation can differentiate between BVIX resulting from

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degradation of extracellularly-acquired (13C-BVIX) versus intracellularly-synthesized (12C-BVIX) heme.

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Using this technique, it was previously shown that the iron-regulated HemO of P. aeruginosa degrades

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extracellular heme to BVIXδ and BVIXβ, while a second, non-iron-regulated heme oxygenase, BphO,

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degrades intracellular heme to BVIXα (see Scheme 1 in the supplemental materials) (47). Similar results

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were obtained in the current study when we analyzed the PAO1 strain grown in 5µM 13C-labeled heme:

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BphO predominantly degraded endogenous heme (blue trace) to BVIXα, while HemO degraded

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exogenous heme (red trace) to BVIXβ and BVIδ (Figure 5A). The ∆prrF1,2 mutant also turned over

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extracellular heme via the catalytic action of HemO (Figure 5B), indicating this mutant is capable of

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using heme as an iron source – a conclusion that is consistent with our ICP-MS analysis in Figure 1F.

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However, the ∆prrF1,2 mutant showed very little degradation of endogenous heme to BVIXα (Figure

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5B). qPCR analysis showed similar levels of the bphO mRNA in the ∆prrF1,2 mutant as compared to

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wild type grown in 5 µM heme (Supplementary Materials, Figure S3). Thus, decreased levels of BVIXα

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in the ∆prrF1,2 mutant were not due to PrrF- or PrrH-mediated repression of bphO.

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Next, we determined if altered heme metabolism in the ∆prrF1,2 mutant was likely due to loss of the

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PrrH sRNA. For this analysis, qPCR primers and probes were designed within the prrF1-prrF2

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intergenic region to specifically detect the PrrH sRNA transcript (Figure 2A). Consistent with earlier

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studies (37), qPCR analysis demonstrated that PrrH was expressed under low iron conditions (Figure

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2C). Our analysis also showed that expression of PrrH was significantly reduced (10-fold) by the

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addition of as little as 5 µM heme (Figure 2C). However, similar to what was observed for PrrF in

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Figure 2B, expression of PrrH from the prrF complementation construct was not repressed by either iron

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or heme (Figure 2C). Thus, while PrrH expression is restored in the complemented strain grown in low

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iron, heme and iron regulation of PrrH are defective in this strain. Notably, since expression of PrrH is

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repressed by 5 µM heme in PAO1, it is unlikely that reduced BphO heme oxygenase activity in the

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∆prrF1,2 mutant when grown in the presence of heme, as shown in Figure 5B, is due to loss of PrrH.

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However, the PrrF sRNAs are still expressed under this condition (Figure 2B). Thus, it is possible that

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loss of the PrrF sRNAs is responsible for altered heme degradation by the ∆prrF1,2 mutant, potentially

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via their impact on overall iron homeostasis.

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The ∆prrF1,2 mutant shows altered expression of genes for heme acquisition and virulence.

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The ability to respond to extracellular heme via either the PrrF and/or PrrH sRNAs is likely to provide a

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competitive advantage in environments where heme is a potential iron source. We therefore sought to

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determine if the ∆prrF1,2 mutant showed altered expression of genes involved in heme uptake. Previous

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studies demonstrated that expression of phuS, which controls the flux of heme through the HemO heme

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oxygenase in PAO1, is regulated by heme (43). To determine if the sRNAs encoded by the prrF locus

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might be responsible for this regulation, we analyzed expression of phuS in PAO1 and the ∆prrF1,2

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mutant. Our analysis shows that expression of phuS is significantly increased in the ∆prrF1,2 mutant as

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compared to wild type, and is restored to wild type levels in the complemented strain (Figure 3B), Page 13

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indicating that one of the prrF-encoded sRNAs has a negative effect on phuS expression. While no

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complementarity was identified between the phuS mRNA and either of the PrrF sRNAs, analysis of the

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5’ UTR of phuS revealed a short, imperfect region of complementarity with the PrrH unique sequence

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(Figure 3B). Thus, our data are consistent with a model in which heme modulates phuS mRNA levels

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via the PrrH sRNA.

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Heme regulation via the PrrF and/or PrrH sRNAs may also provide a signaling pathway for the

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induction of virulence genes during infection. To explore this hypothesis, we used the updated

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TargetRNA2 algorithm (48) to search for potential PrrH-specific targets with links to virulence. This

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analysis identified an extensive region of complementarity between the PrrH unique sequence and the

317

translational start site and coding sequence of the vreR mRNA (Figure 3F). The vreR gene encodes an

318

anti-sigma factor of the Vre extracytoplasmic function (ECF) cell surface signaling (CSS) system, which

319

was previously shown to be required for P. aeruginosa virulence (49). To test the idea that vreR was

320

regulated by either PrrF or PrrH, we assayed vreR expression by qPCR in PAO1 and the ∆prrF1,2

321

mutant. Similarly to phuS, deletion of the prrF locus resulted in significantly increased levels of vreR (3-

322

fold, Figure 3C). While vreR expression was not completely restored to wild type levels in the

323

complemented ∆prrF1,2 mutant, expression of vreR in this strain was not statistically different from that

324

of the wild type strain (Figure 3C). Thus, our data indicate that vreR expression is negatively affected by

325

expression of either the PrrF or PrrH sRNAs.

326

Previous studies showed that the VreA ECF receptor, the VreI sigma factor, and VreR anti-sigma

327

factor are encoded by a single operon (Figure 3F) (50). The region of complementarity identified

328

between the PrrH intergenic region and vreR is located downstream of the vreA and vreI genes. We

329

therefore analyzed expression of vreA and vreI to determine if prrF1,2 deletion had a discoordinate

330

effect on this operon. Our analysis showed that deletion of the prrF locus did not result in significantly

331

increased expression of either vreA or vreI (Figure 3D-E), indicating that prrF1,2 deletion specifically

332

affected the expression of vreR. Combined, these data indicate that deletion of the prrF locus, Page 14

333

potentially through the function of the PrrH sRNA, affects the expression of genes required for virulence

334

in P. aeruginosa.

335

The ∆prrF1,2 mutant is attenuated for virulence in an acute mouse lung infection model.

336

Combined with previous studies, this report demonstrates that the prrF-encoded sRNAs contribute to

337

iron and heme homeostasis of P. aeruginosa. Our studies also indicate that the RNAs encoded by the

338

prrF locus contribute to virulence gene expression via PQS (33) and VreR (this report). We therefore

339

assayed the ∆prrF1,2 mutant for its ability to cause lung infection in an intranasal (i.n.) murine infection

340

model. C57BL/6 mice (n=5) were inoculated with either the wild type PAO1 vector control, the

341

∆prrF1,2 mutant vector control, or the complemented ∆prrF1,2 mutant. All mice inoculated with 108

342

colony forming units (CFU) of either the wild type or complemented mutant succumbed to lung

343

infection within 4 days of inoculation (Figure 6A and C). In contrast, all ∆prrF1,2 mutant-infected mice

344

survived the entire 28-day course of the experiment (Figure 6B). Thus, the prrF locus of P. aeruginosa

345

is required for virulence in this model of acute mouse lung infection.

346

We next tested whether inoculation of mice with the ∆prrF1,2 mutant generated a protective

347

immune response. Mice that received and survived the initial 108 CFU inoculum of the ∆prrF1,2 mutant

348

(Figure 6B) were subsequently inoculated i.n. with 108 CFU of wild type PAO1. All mice primarily

349

challenged with the ∆prrF1,2 mutant survived this subsequent lethal challenge (Figure 6D, black

350

squares), showing that intranasal inoculation of mice with the PAO1 ∆prrF1,2 mutant generated a

351

protective immune response against the wild type PAO1 strain. As such, the ∆prrF1,2 mutant presents a

352

potential candidate for future vaccine development.

353

Iron and heme regulation of the prrF-encoded sRNAs is conserved in clinical isolates of P.

354

aeruginosa. Numerous studies have demonstrated the plasticity of the P. aeruginosa genome, resulting

355

in highly variable expression of virulence factors in different strains (51-53). We therefore determined

356

the ability of several P. aeruginosa laboratory strains and clinical isolates to express and regulate the

357

PrrF and PrrH sRNAs in response to iron and heme. Included in our analysis were PA14 and WR5, Page 15

358

which are both wound isolates (54-56), and FRD1, a mucoid strain isolated from the lung of a cystic

359

fibrosis patient (57). Northern analysis demonstrated that the PrrF1, PrrF2, and PrrH sRNAs are

360

expressed by each of these strains when grown in M9 minimal media, although PrrH expression was

361

barely apparent in strain WR5 (Figure 7A). We also assayed iron- and heme-regulated expression of the

362

PrrF and PrrH sRNAs in each of these isolates by qPCR. Supplementation of the PAO1, PA14, and

363

FRD1 strains with either 100 µM FeCl3 or 5 µM heme resulted in significant repression of PrrH

364

expression (Figure 7B-C). In contrast, heme supplementation of WR5 cultures had no effect on PrrH

365

expression, and iron repression of PrrH in strain WR5 was less substantial than that of other strains

366

analyzed in this study (Figure 7B-C). Consistent with our northern blot analysis, PrrH also appeared to

367

be expressed at lower levels in the WR5 strain grown in low iron than in PAO1 and other isolates that

368

we tested, although this difference was not statistically significant (Figure 7C). Overall, our results

369

indicate that expression of the PrrF and PrrH sRNAs is conserved across P. aeruginosa, although the

370

mechanisms that guide iron and heme regulation of these sRNAs are somewhat variable.

371 372

Discussion

373

Several studies have demonstrated the essentiality of iron for pathogenesis of P. aeruginosa (1-5).

374

While iron acquisition plays a critical role in maintaining iron homeostasis of P. aeruginosa, appropriate

375

utilization of this element is also essential for bacterial survival, as evidenced by the broad conservation

376

of iron regulatory factors. Iron-responsive regulators in bacteria have largely been shown to contribute

377

to iron homeostasis by one of two mechanisms: repressing the expression of iron uptake systems in iron-

378

replete conditions, and reducing the production of iron-containing proteins in iron-limiting

379

environments. The latter of these functions is mediated in many bacterial species by iron-responsive

380

sRNAs (32, 34, 58-65), and, in some pathogenic species, these iron-regulated sRNAs affect the

381

expression of virulence traits (33, 63, 64, 66, 67). Here, we show that deletion of the locus encoding the

382

iron-responsive PrrF sRNAs of P. aeruginosa causes defects of iron and heme homeostasis, alters Page 16

383

biofilm formation, affects virulence gene expression, and most strikingly, attenuates virulence.

384

Combined with a previous study demonstrating the role of the PrrF sRNAs in production of the PQS

385

quorum sensing molecule (33), these results demonstrate the central role of the prrF locus in the

386

physiology and pathogenesis of P. aeruginosa.

387

A recent report demonstrated that a mutant for the iron-regulated RyhB sRNA of uropathogenic E.

388

coli (UPEC) was defective for bladder colonization in a murine model of urinary tract infection (68).

389

Notably, the ryhB mutant of E. coli was also shown in this study to be defective for siderophore

390

production, a known contributor to UPEC virulence (69). As previously shown in non-pathogenic E. coli

391

(70), the UPEC ∆ryhB mutant displayed decreased expression of shiA, encoding a permease for

392

shikimate, a required substrate of enterobactin biosynthesis. Moreover, this report also showed that

393

genes encoding biosynthetic enzymes for the hydroxamate siderophore aerobactin were down-regulated

394

in the ∆ryhB mutant (68). Thus, the RyhB sRNA of UPEC contributes to siderophore production by

395

multiple mechanisms, likely promoting colonization and survival during urinary tract infection. These

396

findings are in stark contrast to the P. aeruginosa ∆prrF1,2 mutant, which is not defective for

397

siderophore production (Figure S2). Moreover, we show that the ∆prrF1,2 mutant is capable of utilizing

398

heme as an iron source (Figure 5B). Thus, our data indicate that defects in iron uptake, via either

399

siderophore or heme acquisition pathways, are not the source of virulence attenuation in the P.

400

aeruginosa ∆prrF1,2 mutant.

401

Iron is a known requirement for P. aeruginosa biofilm formation (16, 40, 46). Results of a previous

402

study indicated that either active uptake of chelated iron (through siderophores or ferric citrate) or

403

intracellular iron levels were responsible for this dependency (16). This same study established that the

404

∆prrF1,2 mutant formed biofilms similar to the wild type strain. Thus, our finding that the ∆prrF1,2

405

mutant shows reduced biofilm formation as compared to wild type in the presence of iron and sub-

406

inhibitory concentrations of tobramycin is intriguing. While sub-inhibitory concentrations of tobramycin

407

and other aminoglycosides are know to induce biofilm formation (39), the underlying mechanism for this Page 17

408

phenomenon remains unclear. We previously showed that iron supplementation is required for this

409

phenomenon (40). Our data here suggest that appropriate iron utilization, mediated by the PrrF sRNAs,

410

may also play a role in tobramycin-induced biofilm formation. The PrrF sRNAs are also know to

411

mediate production of the PQS quorum sensing molecule, which itself contributes to multicellular

412

behavior (33). Thus, the inability of the ∆prrF1,2 mutant biofilm to respond to iron may be the result of

413

pleiotropic effects on iron utilization, PQS biosynthesis, and potentially other unknown factors. More

414

studies into the formation and structure of the PAO1 and ∆prrF1,2 mutant biofilms in the presence of

415

sub-inhibitory concentrations of aminoglycosides will be required to fully understand the basis for this

416

phenotype.

417

A notable characteristic of P. aeruginosa is the heterogeneity of genome sequences from different

418

clinical and environmental isolates. In many cases, this genetic variation results in altered expression of

419

virulence factors, or loss altogether of genes encoding certain virulence determinants (51-53). To date, all

420

sequenced isolates of P. aeruginosa possess the tandem arrangement of the prrF genes (71), indicating

421

that the region encoding both the PrrF and PrrH sRNAs is part of the core genome of this species. In this

422

study, we present additional evidence that iron and heme regulation of the PrrF and PrrH sRNAs is

423

conserved across several clinical isolates of P. aeruginosa. Moreover, we recently showed that the PrrF

424

and PrrH sRNAs are maintained and expressed by a variety of isolates of P. aeruginosa during CF lung

425

infection (41). Combined, these data support the hypothesis that the unique arrangement of the P.

426

aeruginosa prrF genes is a critical asset during infection of the human host.

427

One of the most fascinating aspects of the PrrF sRNAs in P. aeruginosa is the tandem arrangement

428

of the prrF1 and prrF2 genes, allowing for expression of the heme-responsive PrrH sRNA (37). Heme is

429

an abundant source of iron in the human host, and strains that are capable of using and responding to this

430

nutrient are likely to exhibit a competitive advantage during infection. It is therefore predicted that heme

431

regulated expression of PrrH and PrrF contributes to heme homeostasis and, potentially, the pathogenic

432

capacity of this organism. In support of this hypothesis, the present study demonstrates that the ∆prrF1,2 Page 18

433

mutant is altered for heme metabolism; specifically, our data indicate that the BphO heme oxygenase,

434

which degrades endogenously-synthesized heme, is expressed yet relatively inactive in the ∆prrF1,2

435

mutant (Figures S3 and 5B). However, the mechanism underlying this phenotype is not yet known. One

436

possibility is that the iron starvation phenotype of the ∆prrF1,2 mutant, resulting from increased

437

production of iron-containing proteins in iron-depleted conditions, precludes endogenous heme

438

biosynthesis. Alterantively, altered heme metabolism in the ∆prrF1,2 mutant may be due to changes in

439

the production of distinct factors required for endogenous heme biosynthesis and/or degradation. Future

440

studies into how the ∆prrF1,2 mutant responds to changes in heme availability on a genomic scale will

441

be key to distinguishing between these possibilities.

442

We previously showed that the prrF locus allows for heme-regulated expression of the nirL gene,

443

potentially due to a region of complementarity identified between the unique region of the PrrH sRNA

444

and the 5’ end of the nirL mRNA (37). In silico analysis in the current study identified additional

445

mRNAs that shared complementarity with the PrrH unique sequence: vreR, encoding a regulator of

446

virulence gene expression (49), and phuS, encoding a heme-responsive regulator of heme acquisition and

447

iron homeostasis (43). Our data further showed that the ∆prrF1,2 mutant exhibits increased levels of the

448

vreR and phuS mRNAs, suggesting the PrrH sRNA is capable of interacting with and modulating the

449

expression of these genes. Notably, deletion of the prrF locus had no significant effect on the expression

450

levels of vreA and vreI, which are co-transcribed with vreR (50). Combined with the finding that the

451

unique region of PrrH shares complementarity with a region of the vreAIR operon immediately upstream

452

of vreR, these data support the model that PrrH is responsible for prrF-mediated regulation of vreR.

453

However, more studies are clearly required to determine the mechanism by which the sRNAs encoded

454

by the prrF locus affect expression of each of these genes, as well as whether this regulation occurs by a

455

direct or indirect mechanism.

456

One complicating factor of our studies was the finding that iron- and heme-regulation of the PrrF

457

and PrrH sRNAs expressed from the pVLT-prrF1,2 complementation construct was diminished (Figure Page 19

458

2B-C). Sequencing analysis of the complementation construct identified a single nucleotide substitution

459

immediately upstream of the prrF1 Fur binding site, within the -35 region of the prrF1 promoter (Figure

460

S1, Supplementary Materials), which is presumably the cause of de-regulated PrrF and PrrH expression.

461

While this construct complements many the previously described phenotypes of the ∆prrF1,2 mutant

462

(32, 33), as well as those described in the current study, it is clear that more refined genetic studies will

463

be necessary for teasing apart the individual regulatory roles of the PrrF and PrrH sRNAs during

464

pathogenesis. Unfortunately, subsequent attempts at cloning the prrF locus have resulted in similar

465

promoter mutations, and clones lacking the native prrF promoters carry mutations within the prrF

466

genes. We are therefore pursuing alternative strategies to assess the individual functions of the PrrF and

467

PrrH sRNAs.

468

In spite of these issues, the current prrF expression construct was able to complement the virulence

469

defect of the ∆prrF1,2 mutant, demonstrating the requirement of at least one of the prrF-encoded

470

sRNAs in virulence. Even more striking, our studies show that intranasal inoculation of mice with the

471

∆prrF1,2 mutant generated a protective immune response, presenting this strain as a potential candidate

472

for future vaccine development in vulnerable populations. To date, our studies indicate that virulence

473

attenuation of the ∆prrF1,2 mutant may be due to any one or a combination of the following

474

phenotypes: reduced PQS production, loss of iron homeostasis, loss of heme homeostasis via de-

475

regulated expression of nirL and/or phuS, and/or de-regulated expression of the Vre regulon. Because of

476

the potential multifactorial role of the PrrF and PrrH sRNAs in regulating virulence, developing

477

resistance to a PrrF and/or PrrH inhibitor would likely require mutations in both the PrrF and PrrH

478

sRNAs and several of the target mRNAs they regulate. Thus, this strategy may raise the bar for infecting

479

strains to develop resistance, potentially reducing future burdens of antimicrobial resistant P.

480

aeruginosa. Delivery of antisense oligonucleotides (AS-ODNs) via anionic liposomes has been

481

successful in several bacterial species (72-74), and may provide a novel means for targeting sRNAs, such

482

as PrrF and PrrH, to block virulence. Future work determining how each of the ∆prrF1,2 mutant Page 20

483

phenotypes contributes to P. aeruginosa virulence attenuation will be critical for the development of

484

AS-ODN molecules that effectively block pathogenesis.

485 486

Page 21

487 488

Acknowledgements We are grateful to Michael Schurr, Ph.D. for sharing the FRD1 mucoid CF isolate, and to Mike

489

Vasil, Ph.D. for the WR5 acute wound isolate. We also thank Lauren Hittle, Ph.D. for helpful

490

discussions about heme regulation and metabolism in P. aeruginosa. This work was supported by NIH-

491

NIAID grant AI089776 and start-up funding from the University of Maryland School of Pharmacy (to

492

AOS) and NIH grant AI102883 (to AW).

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709

Tables Table 1. PrrF/PrrH phenotype of Pseudomonas aeruginosa strains Strain

Description

PAO1

Wild type strain used for mutational analysis in this and previous studies; originally isolated from a human wound in 1955 in Australia

(75)

ΔprrF1-2

Deletion mutant of entire prrF locus in PAO1

(32)

PAO1/pVLT31

Source/ Reference

R

Wild type PAO1 strain carrying the pVLT31 broad host range vector (tc ) R

(33)

∆prrF1,2/pVLT31

The ∆prrF1,2 mutant carrying the pVLT31 broad host range vector (tc )

(33)

∆prrF1,2/pVLTprrF1,2

The ∆prrF1,2 mutant carrying the prrF1,2 complementation construct (tcR)

(32)

PA14

Clinical isolate from 1995 at the Massachusetts General Hospital, Boston; virulent in a variety of plant and animal models of infection

(56)

FRD1

Mucoid clinical isolate in a sputum sample from a cystic fibrosis patient

(57)

WR5

Clinical isolate from a burn patient at Walter Reed Army Hospital, Washington, DC, in 1976; natural toxA mutant isolate; virulent in experimental mouse models

710 711 712

Page 28

(54, 55)

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Figures Legends Figure 1. The ∆prrF1,2 mutant is defective for growth in low iron medium and with heme as a sole iron source. A-C. The indicated strains were grown for 4 hours in M9 minimal medium with 60 µg/ml tetracycline to deplete intracellular iron stores and diluted into fresh M9 medium with or without FeCl3 (100 µM) or heme (5µM) supplementation as indicated. Growth was monitored for 24 hours in a Bioscreen C instrument. Significant differences in culture density of the wild type and ∆prrF1,2 mutant, as determined by two-tailed Student’s t test, are indicated by a vertical bar flanking the relevant time points. D-F. The indicated strains were grown for 4 hours in M9 minimal medium to deplete intracellular iron stores, then diluted into fresh M9 medium with or without FeCl3 (100 µM) or heme (5µM) supplementation and grown for additional 8 hours in M9 minimal media as indicated, and subsequently analyzed for iron content by ICP-MS analysis. Error bars indicate the standard deviation of three independent experiments. The indicated p values were obtained by two-tailed Student’s t test. Figure 2. Iron and heme regulation of the PrrF and PrrH sRNAs in M9 media. A. Map of the prrF locus, showing approximate locations of the PrrF.for and PrrF.rev primers and PrrF TaqMan probe, which will detect the PrrF and PrrH sRNAs (panel B), and the PrrH.for and PrrH.rev primers and PrrH TaqMan Probe, which will only detect PrrH (panel C). B-C. The indicated strains were grown for 4 hours in M9 medium with 60 µg/ml tetracycline to deplete intracellular iron stores, then sub-cultured into fresh M9 medium without iron supplementation (black bars), with 100 µM FeCl3 supplementation (dark gray bars), or 5 µM heme supplementation (light gray bars). Relative expression of PrrF and PrrH combined (B) and PrrH (C), detected using the primers shown in panel A, was determined by standard curves as described in the materials and methods, and expression of each sample was normalized to the PAO1/vector low iron sample. Error bars indicate the standard deviation from three independent experiments. Asterisks indicate the following p value as determined by a two-tailed Student’s t test when comparing values upon heme or iron supplementation to that of the low iron sample, or as otherwise indicated: * p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001. Figure 3. The ∆prrF1,2 mutant shows altered expression of genes involved in heme homeostasis and virulence. A-E. The indicated strains were grown for 4 hours in M9 medium with 60 µg/ml tetracycline to deplete intracellular iron stores, then sub-cultured into fresh M9 medium without iron supplementation. Relative expression of sodB (A), phuS (B), vreR (C), vreA (D), and vreI (E) was determined by standard curves as described in the materials and methods, and expression of each sample was normalized to the PAO1/vector sample. Primers and probes used are shown in the supplementary materials. Error bars indicate the standard deviation from three independent experiments. Complementarity between PrrF1 and the sodB mRNA as previously reported (32) and the PrrH unique region (“PrrH IG”) and the phuS mRNA are shown below the graphs in panels A and B, respectively. The translational start site of phuS is underlined. Asterisks indicate the following p value as determined by a two-tailed Student’s t test when comparing values upon heme or iron supplementation to that of the low iron sample, or as otherwise indicated: ^ p < 0.1, * p < 0.05, ** p < 0.001, *** p < 0.0001. F. Map of the vreAIR operon, indicating the approximate location of complementarity with the PrrH unique region (PrrH IG). Figure 4. The ∆prrF1,2 mutant is defective for antibiotic-induced biofilm formation. PAO1 and ∆prrF1,2 mutant biofilms were allowed to form on MBEC device pegs for 24 hours in DTSB supplemented with 100 µM FeCl3. The biofilms were then washed in saline, then challenged for 24 hours with varying concentrations of tobramycin in the presence or absence of 100 µM FeCl3 as indicated. Biofilm formation was then determined as described in the materials and methods. Error bars Page 29

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show the standard deviation of three independent experiments. Asterisks indicate the following p values as determined by a two-tailed Students t-test: ** p < 0.01. Figure 5. Heme metabolism is altered in the ∆prrF1,2 mutant. MS/MS fragmentation of BVIX isomers extracted from the culture supernatants of PAO1 (A) and the ∆prrF1,2 mutant (B) grown for 8 hours in M9 minimal medium supplemented with 5 µM 13C-heme. LC-MS/MS was performed as described in the methods with multiple reaction monitoring. Figure 6. The prrF locus is required for acute murine lung infection. A-C. Eight-week old C57Bl/6 mice were inoculated intranasally with 106, 107, or 108 CFU of wild type PAO1 with vector (A), the ∆prrF1,2 mutant with vector (B) or the complemented ∆prrF1,2 mutant (C). Five mice received each inoculum of each of the indicated P. aeruginosa strains. Mice were monitored for survival as described in the materials and methods. D. C57Bl/6 mice that survived infection with 108 CFU of the ∆prrF1,2 mutant (n = 5) were challenged with 108 CFU of the wild type PAO1 strain (black squares) and monitored for survival as described in the materials and methods. White squares indicate historical data obtained with naive C57Bl/6 mice inoculated with 108 CFU of PAO1. Figure 7. The PrrF and PrrH sRNAs are expressed by multiple clinical isolates of P. aeruginosa. A. The indicated strains were grown for 4 hours in M9 medium to deplete intracellular iron stores, then sub-cultured into fresh M9 medium without iron supplementation and grown for an additional 8 hours. RNA was analyzed by northern blot as described in the materials and methods to detect the PrrF1, PrrF2, and PrrH sRNAs. B-C. The indicated strains were grown for 4 hours in M9 medium to deplete intracellular iron stores, then sub-cultured into fresh M9 medium without iron supplementation (black bars), with supplementation of 100 µM FeCl3 (dark gray bars), or 5 µM heme (light gray bars). Relative expression of PrrF and PrrH combined (B) and PrrH (C), detected using the primers shown in Figure 2A, was determined by standard curves as described in the materials and methods, and expression of each sample was normalized to the PAO1 low iron sample. Error bars indicate the standard deviation from three independent experiments. Asterisks indicate the following p value as determined by a two-tailed Student’s t test when comparing values upon heme or iron supplementation to that of the low iron sample: * p < 0.05, ** p < 0.001, *** p < 0.0001, **** p < 0.00001.

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