Simultaneous Analysis of Bacterioferritin Gene Expression and Intracellular Iron Status in Pseudomonas putida KT2440 by Using a Rapid Dual Luciferase Reporter Assay

A dual luciferase reporter (DLR) system utilizing firefly andRenilla luciferases was developed and tested in a model rhizobacterium,Pseudomonas putida KT2440. The DLR was applied to simultaneouslyanalyze expression of three putative bacterioferritin genes(bfr ${alpha}$ , bfrβ, and bfr) and assess the cellular iron statusof strain KT2440 by monitoring expression of the Fur-regulatedfepA-fes promoter. The DLR proved to be reproducible and sensitive.Expression of bfr ${alpha}$ (PP0482) and bfrβ (PP1082) was consistentwith expectations for bacterioferritin and varied directly withthe iron level. However, expression of bfr (PP4856) was inverselyrelated to the iron concentration and it was thus more likelyto encode a Dps-like protein rather than a bacterioferritin.

INTRODUCTION

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INTRODUCTION

Iron is an essential element, but excessive levels can alsoinduce oxidative stress (7). Ferritins are in a class of iron-bindingproteins that reversibly sequester excess iron and can therebyserve to mitigate iron-related oxidative stress (1). Ferritinshave been well studied in mammalian systems, but comparativelylittle is known regarding the roles that these proteins havein bacterial survival in soil (15).

Bacteria classified as Pseudomonas putida are ubiquitous insoil and in plant rhizospheres (6, 7), and the rhizobacteriumP. putida KT2440 has been widely used as a model of this group(16). The following three types of ferritins have been identifiedin bacteria: bacterial ferritin, bacterioferritin (Bfr), anddodecameric ferritin (also called Dps; DNA-binding protein fromstarved cells) (4). The genome of strain KT2440 lacks a ferritingene but contains one predicted Dps gene, and three are annotatedas encoding Bfr (bfr ${alpha}$ , PP0482; bfrβ, PP1082; and bfr, PP4856)(13). The Bfr pattern is unusual, as bacteria that produce Bfrtypically possess one copy of bfr (homopolymeric Bfr) (2) ortwo nonidentical copies, bfr ${alpha}$ and bfrβ (heteropolymericBfr) (9). Thus, the goal of this study was to determine if bfr ${alpha}$ ,bfrβ, and bfr were all expressed and, if so, how expressionvaried as a function of iron levels in the environment and inthe cell.

A dual luciferase reporter (DLR) system was applied to strainKT2440 to assay its intracellular iron status (as indicatedby Fur binding to the fepA-fes dual promoter) (10) and Bfr geneexpression. Bacterial dual reporter systems have been describedpreviously (10, 12). However, these have combined genes forwhich different technologies were required to assay expression(e.g., phoA and lacZ). A potential drawback of such systemsis that results may be complicated by data obtained from separatesamples. In contrast, dual reporters utilizing luciferases fromfireflies (Fluc) and sea pansies (Renilla reniformis; Rluc)can be assayed in a single sample by sequential activation ofluminescence from Fluc, quenching of Fluc, and then activationof Rluc (14). The DLR system has been used extensively in mammaliancells to assay transient gene expression but, to the best ofour knowledge, has not been reported for bacteria. In this report,the DLR reporters constructed in strain KT2440 were designatedas follws: SCH119 (fes-fepA-fluc; bfr::rluc), SCH115 (fes-fepA-fluc;bfr ${alpha}$ ::rluc), and SCH121 (fes-fepA-fluc; bfrβ::rluc). Detailsregarding the construction of these reporters are provided inthe supplemental material (see Tables S1 and S2 and Fig. S1,S2, and S3 in the supplemental material).

fepA-fes promoter from Escherichia coli was regulated by Fur in P. putida KT2440 in vivo.

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fepA-fes promoter from...

Firefly luciferase activity was readily detected in strain SCH35and increased in response to increasing levels of the iron-sequesteringagent 2,2'-bipyridyl (Fig. 1A). The fes-fepA-fluc promoter wasconfirmed to be Fur-regulated in P. putida KT2440 as indicatedby reporter activity in fur and the fur mutant background (Fig.1B). With the wild type, Fluc intensity varied inversely withthe iron level, as would be expected for Fur-regulated expressionof fluc (Fig. 1B). In contrast, in fur deletion mutants, Flucactivity increased in response to progressively greater ironlevels (Fig. 1B).

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FIG. 1. Effect of iron levels and fur deletion on the expression of fes-fepA-fluc. (A) Regulation of fes-fepA-fluc by iron. The medium contained 15 µM of iron (final concentration); the iron availability concentration was adjusted by adding different concentrations of 2,2'-dipyridyl as indicated. (B) Comparison of fes-fepA-fluc expression in the wild type (strain SCH35) and fur deletion mutant (strain SCH113). The iron level was adjusted by adding 2,2'-dipyridyl (200 µM) or iron at various concentrations as indicated. The solid bar represents the wild type (strain SCH35), and the striped bar represents the fur deletion mutant (strain SCH113). Values are means of data from triplicate cultures (with standard deviation).

Sensitivity and stability of the DLR system in P. putida KT2440.

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Sensitivity and stability of...

Strain SCH115 was grown to exponential phase in defined mineralsalt medium (10 mM benzoate, 15 µM iron), and variousamounts of cells were taken for analysis by the DLR assay. Aliquotswere also plated on LB agar for viable cell counting. Activitiesof both luciferases were readily measurable, with as few as500 cells being linear over at least 2 orders of magnitude (seeFig. S4 in the supplemental material). Collectively, as appliedto P. putida KT2440, the DLR assay was a stable, reproducible,and sensitive means of studying gene expression (see Fig. S5in the supplemental material).

Transient activity of Fluc and Rluc firefly in vivo.

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Transient activity of Fluc...

Temporal activity of Fluc and Rluc was tested by exposing P.putida KT2440 DLR strains to shifts in environmental iron levels.In a shift from conditions with low levels of iron to thosewith high levels of iron, responses of both reporters were detectedwithin 30 min (Fig. 2). Consistent with expectations, Fluc activitydropped in this transition (Fig. 2), demonstrating that cellsquickly responded to an alteration in iron levels as indicatedby changes in expression of Fur-regulated genes. An increasein Fluc production was recorded after 90 to 150 min; the mechanismfor this response is not yet known. In contrast to Fluc, Rlucdisplayed immediate and consistent increases throughout the150-min incubation; luminescence increased by 100% after 60min and by nearly 500% after incubation for 150 min, indicatingthat the transition from low levels of iron to high levels ofiron resulted in rapid and sustained expression of bfr ${alpha}$ .

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FIG. 2. Transient activity of Fluc and Rluc in P. putida KT2440 in response to various iron levels. DLR strain SCH115 (fepA-fes-rluc bfr ${alpha}$ ::rluc) was cultured in LB medium overnight and then transferred to mineral salts medium supplemented with benzoic acid and incubated for 12 h. Cells were harvested by centrifugation, resuspended in mineral salts medium supplemented with benzoic acid supplemented with 15 µM iron, and subjected to dual luciferase activity analysis after incubation at room temperature for the indicated amounts of time. The solid and open bars represent firefly and Renilla luciferase activity, respectively. Values are means of data from triplicate cultures (with standard deviation).

Application of the DLR system to simultaneously monitor cellular iron status and expression of Bfr-encoding genes.

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Application of the DLR...

Cultures were grown to early stationary phase under conditionswith low and high levels of iron, and luciferase activitieswere measured immediately. Two response patterns were displayedby the three genes annotated in P. putida KT2440 as encodingBfr. For bfr ${alpha}$ and bfrβ, expression levels decreased $~$ 50%under conditions with low levels of iron compared with thosewith high levels of iron (Fig. 3). In contrast, expression ofbfr was enhanced 300% under conditions with low levels of iron(Fig. 3). These contrasting expression patterns reflected divergentregulation and indicated that the function of bfr (PP4856) likelydiffers from that of bfr ${alpha}$ and bfrβ. Expression patternsof the latter two were consistent with an iron storage functionexpected for bacterioferritin. The concordant expression ofbfr ${alpha}$ and bfrβ would also be consistent with a heteropolymericstructure for bacterioferritin in P. putida, as has been determinedfor bacterioferritin in P. aeruginosa (11). However, the expressionpattern of PP4856 was similar to that expected for a Dps-likeprotein (8) and not bacterioferritin. Furthermore, sequencesof both Bfr ${alpha}$ and Bfrβ contained ferroxidase and heme-bindingmotifs that are associated with Bfr (3); the latter also containeda conserved Met-52 residue that is typically present in Bfrβbut absent in Bfr ${alpha}$ (5). In contrast, PP4856 possessed none ofthe aforementioned bacterioferritin signatures but did havemotifs associated with Dps. Thus, the protein predicted forPP4856 is more likely to serve in the capacity of a Dps-likeprotein and not bacterioferritin.

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FIG. 3. Effect of iron levels on expression of bfr, bfr ${alpha}$ , and bfrβ. DLR strains SCH115 (fes-fepA-fluc; bfr ${alpha}$ ::rluc), SCH119 (fes-fepA-fluc; bfr::rluc), and SCH121 (fes-fepA-fluc; bfrβ::rluc) were cultured in iron-replete (15 µM of iron) and iron-depleted medium (200 µM of 2,2'-dipyridyl) for 48 h and then analyzed by the dual luciferase assay. Values are expressed as the level of luciferase activity measured under treatments with low levels of iron divided by that from cells cultured in the presence of high levels of iron. The solid and open bars represent firefly and Renilla luciferase activity, respectively. Values are means of data from triplicate cultures (with standard deviation).

ACKNOWLEDGMENTS

These studies were funded by a grant from the USDA NRI-CGP (awardnumber 2006-02624; to W.F.B. and W.J.H.).

We thank J. M. Tiedje and M. Bagdasarian for constructive adviceand for kindly providing the plasmid pJK100 and the P. putidastrain.

FOOTNOTES

* Corresponding author. Mailing address: Department of Soil Science, University of Wisconsin, 1525 Observatory Dr., Madison, WI 53706-1299. Phone: (608) 262-9018. Fax: (608) 265-2595. E-mail: wjhickey{at}wisc.edu

Published ahead of print on 1 December 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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