Syddansk Universitet

Five homologous small RNAs are involved in the response of Listeria monocytogenesto cell wall acting antibioticsNielsen, Pia Kiil; Kallipolitis, Birgitte H.

Publication date:2010

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Citation for pulished version (APA):Nielsen, P. K., & Kallipolitis, B. H. (2010). Five homologous small RNAs are involved in the response of Listeriamonocytogenes to cell wall acting antibiotics. Poster session presented at Five homologous small RNAs areinvolved in the response of Listeria monocytogenes to cell wall acting antibiotics, .

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Download date: 15. jan.. 2017

Five homologous small RNAs are involved inthe response of Listeria monocytogenes to cell wall acting antibiotics

Nielsen PK1 and Kallipolitis BH1

1Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK- 5230 Odense M, Denmark

Listeria monocytogenes is a food borne pathogen which has the ability to survive and adapt to a wide range of extremeenvironments; e.g. low pH, low and high temperatures, high osmolarity and the presence of antibiotics. Small RNAs havebeen found to play a crucial role in stress adaption. Small RNAs exist in a multitude of organisms as stable RNA transcriptswhich range from 20-500 nt in size, and are typically encodedfrom intergenic regions. Regulation by small RNAs is mainlyachieved through an antisense mechanism at the post-transcriptional level.

In L. monocytogenes more than 50 small RNAs have been identified so far, but the elucidation of their role is still inprogress (3). TheListeria Hfq-binding RNA C (LhrC) was originally found by co-immunoprecipitation with the RNAchaperone protein Hfq (1), which in both gram-positive and gram-negative bacteria, has been shown to enhance theantisense regulation of small RNAs (2,4). By computationalapproaches it was found thatL. monocytogenes contains fivehomologous copies oflhrC. LhrC1-4 are encoded from the intergenic region betweencysK andsul, whereas the fifth copy,LhrC5, is encodedfrom theintergenicregionbetweenlmo0946 andlmo0947 (fig.1a).

LhrC1-5 are fully dependent on the two component system LisRK

Fig. 1a

Fig. 1b

University of Southern Denmark

Introduction

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2% ethanol 4 µg/ml Cefuroxime 0.1 µg/ml Penicillin G

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LhrC5, is encodedfrom theintergenicregionbetweenlmo0946 andlmo0947 (fig.1a).

Alignment of all five lhrC copies shows a great variance in the promoter region, between lhrC1, lhrC2-4 and lhrC5,whereas the coding regions of lhrC1-5 are conserved (fig. 1b).

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To understand the regulatory role of LhrC1-5, transcriptional fusions of the promoter region of lhrC1, lhrC3 and lhrC5 to lacZ were constructed and introduced into L. monocytogenes EGD-e. Beta-galactosidase assays were conducted to point out which stress conditions lhrC1-5 may respond to.

The promoter activity was examined in response toa random selection of environmental stress agents.Samples were extracted at after 0, 30 and 60minutes of stress, respectively (fig. 2a-2c).

All five small RNAs are induced in response tothe presence of cell wall stress agents such asethanol and cell wall acting antibiotics.

LhrC1-5 are induced under cell wall stress

To analyze the promoter activity and transcription of LhrC1-5 in mid-exponential growing cells upon the addition ofethanol and beta-lactam antibiotics, we conducted beta-galactosidase assays (2% ethanol, 4 ug/ml Cefuroxime and 0,1ug/ml Penicillin G) and quantitative real-time PCR (4 ug/mlCefuroxime) experiments. The oligos used for qPCR weredesigned to recognize all five copies of LhrC.Results from fig. 2a-c indicate that LhrC1-5 are induced in response toconditions that activate the two-component systems LisRK and CesRK. Mutants lacking LisR and CesR were thereforeincluded in these assays.

The expression of LhrC1-5 is fully dependent on the presence of transcription factor LisR (fig. 3a and 3b).The response regulator CesR adds another layer of complexity to the regulation of LhrC1-5 expression (fig. 3a).

Perspectives

The gene regulation downstream of LhrC1-5 is currently being pursued; do they act as antisense RNAs in anadditive, redundant or hierarchical fashion, or by combination of these mechanisms? The answer may lead to anunderstanding of how L. monocytogenes endure the presence of cell wall acting agents in the environment.

References1. Christiansen, J. K., J. S. Nielsen, T. Ebersbach, P. Valentin-Hansen, L. Sogaard-Andersen, and B. H. Kallipolitis. 2006. Identification of small

Hfq-binding RNAs in Listeria monocytogenes. Rna 12:1383-96.

2. Nielsen, J.N., Lei L.K., Ebersbach T., Olsen A.S., Klitgaard J.K., Valentin-Hansen P., Kallipolitis B.H. 2010.Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes. Nucl.Ac.Res. 38:3:907-919

3. Toledo-Arana, A., O. Dussurget, G. Nikitas, N. Sesto, H. Guet-Revillet, D. Balestrino, E. Loh, J. Gripenland, T. Tiensuu, K. Vaitkevicius, M. Barthelemy, M. Vergassola, M. A. Nahori, G. Soubigou, B. Regnault, J. Y. Coppee, M. Lecuit, J. Johansson, and P. Cossart. 2009. The Listeria transcriptional landscape from saprophytism to virulence. Nature 459:950-6.

4. Valentin-Hansen P, Eriksen M, Udesen C. 2004. The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Mic.51(6):1525-33.

Fig. 2a Fig. 2b

Fig. 2c

LhrC 1-5 stability does not depend of the Hfq chaperone

LhrC 1-5 contribute to stress survival

LhrC1-5 are induced in response to cell wall stress

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5S RNA

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To summarize: • LhrC1-5 were originally identified as Hfq-binding small RNAs (2).• Alignment of all five copies oflhrC shows a great variance among the promoter regions oflhrC1, lhrC2-4 and

lhrC5, whereas the transcribed regions oflhrC1-5 are highly conserved (fig. 1b).• All five small RNAs are induced in response to the presence of cell wall stress agents (fig.2a-c).• The expression of lhrC1-5 is fully dependent on the presence of transcription factor LisR. Response regulator CesR

adds one more layer of complexity to the regulation LhrC1-5 expression (fig. 3a+b).• Hfq does not affect the stability of LhrC1-5 (fig. 4).

To examine whether the chaperone Hfq affects the stability of LhrC1-5, a stability assay was performed, including awildtype EGD-e strain and a mutant lacking Hfq. LhrC1-5 wereinduced by the addition of 0.1 µg/ml penicillin G. TheRNA turnover was followed after the addition of rifampicin at time 0 min (which equals 30 min of induction by pen G).Samples were extracted at time -2, +2, +4, +8, +16 and +24 min relative to addition of rifampicin.

By nothern blotting using a probe recognizing all five small RNAs, it is observed that Hfq does not affect thestability of LhrC1-5 (fig. 4).

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