Thermogenetic tools to monitor temperature-dependent gene expression in bacteria

https://doi.org/10.1016/j.jbiotec.2012.01.007Get rights and content

Abstract

Free-living bacteria constantly monitor their ambient temperature. Drastic deviations elicit immediate protective responses known as cold shock or heat shock response. Many mammalian pathogens use temperature surveillance systems to recognize the successful invasion of a host by its body temperature, usually 37 °C. Translation of temperature-responsive genes can be modulated by RNA thermometers (RNATs). RNATs form complex structures primarily in the 5′-untranslated region of their transcripts. Most RNATs block the ribosome binding site at low temperatures. Translation is induced at increasing temperature by melting of the RNA structure. The analysis of such temperature-dependent RNA elements calls for adequate test systems that function in the appropriate temperature range. Here, we summarize previously established reporter gene systems based on the classical β-galactosidase LacZ, the heat-stable β-galactosidase BgaB and the green fluorescent protein GFP. We validate these systems by testing known RNATs and describe the construction and application of an optimized bgaB system. Finally, two novel RNA thermometer candidates from Escherichia coli and Salmonella will be presented.

Highlights

► Several reporter gene systems can be used to analyze RNA thermometers in vivo. ► Both inducible as well as constitutively regulated systems are applicable. ► GFP and the β-galactosidases LacZ and BgaB (thermostable) are suitable reporters. ► Besides moderate heat shock, LacZ is optimal for analyzing cold shock regulation. ► Novel RNA thermometer candidates were found upstream of the htrA gene from Salmonella and Escherichia coli.

Introduction

The expression of numerous bacterial genes is affected by temperature changes. Over a limited temperature range, there is a linear relationship between the ambient temperature and the growth rate. For Escherichia coli as a typical mesophilic model organism, this range (defined as “normal” growth temperature) reaches from 23 to 37 °C (Herendeen et al., 1979, Lim and Gross, 2011). A cold shock is defined as a temperature downshift beneath the normal growth temperature (e.g. 10–17 °C), whereas a heat shock denotes a sudden upshift to a higher, still nonlethal temperature (e.g. 42–45 °C). Deviations from the ambient temperature are recognized immediately by a number of different mechanisms involving DNA, RNA and proteins (Klinkert and Narberhaus, 2009, Schumann, 2009).

In E. coli, cold shock affects multiple cellular processes including transcription, translation and mRNA decay, leading to a growth arrest for about 3–6 h called the acclimation phase (Phadtare and Severinov, 2010). During this phase, cold shock proteins (Csps) are massively induced. Most Csps are RNA chaperones, which directly reduce cold induced secondary structures of all cellular mRNAs. Interestingly, some cold shock mRNAs exhibit unusually long 5′ untranslated regions (UTR), which control the stability of the transcript at different temperatures. Cold shock mRNAs are rapidly degraded at the “normal” growth temperature. Following a cold shock, structural rearrangements lead to a stabilization based on three different mechanisms: (i) masking of RNaseE cleavage sites at the 3′-end (Hankins et al., 2007); (ii) stalling of ribosomes, thereby occluding the major RNaseE cleavage sites; and (iii) the formation of a stabilizing 5′ stem-loop (Baker and Mackie, 2003, Bouvet and Belasco, 1992, Emory et al., 1992, Hambraeus et al., 2002, Hankins et al., 2007, Mackie, 1998, Sharp and Bechhofer, 2005).

A nonlethal temperature upshift induces the production of heat shock proteins (Hsps), most of which are chaperones or proteases that protect, refold or remove heat-damaged proteins (Lim and Gross, 2011, Meyer and Baker, 2011, Moliere and Turgay, 2009, Narberhaus, 2002, Tyedmers et al., 2010). In bacteria, transcription of heat shock genes often depends on alternative sigma factors like sigma32 (RpoH) in E. coli (Arsene et al., 2000, Nakahigashi et al., 1999, Yura, 1996).

Interestingly, translation of the rpoH transcript and some other heat shock mRNAs is controlled by the action of thermosensitive RNA elements (RNA thermometers, RNAT) (Gaubig et al., 2011, Morita et al., 1999, Waldminghaus et al., 2009). Typical RNA thermometers are located in the 5′-UTR of the temperature-controlled mRNAs, but can also employ additional parts of the coding region as it is the case for the rpoH thermometer. At low temperatures, they form secondary structures consisting of one or several hairpins. Usually, the Shine-Dalgarno (SD) sequence and/or the AUG start codon are sequestered by base pairing to prevent translation. The structure melts in response to a temperature upshift by which the SD sequence is liberated and free to interact with the 30S ribosomal subunit (Narberhaus, 2010, Narberhaus et al., 2006).

Virulence-related RNA thermometers are used by pathogenic bacteria to recognize successful invasion of their warm-blooded host, i.e. a rise to 37 °C for mammals (Hoe and Goguen, 1993, Johansson et al., 2002). They enable translation of virulence factors, which often act on top of a virulence cascade (Gripenland et al., 2010, Papenfort and Vogel, 2010). The first experimentally described virulence-related transcriptional regulator controlled by an RNA thermometer is Listeria PrfA (Johansson et al., 2002). Another virulence factor, LcrF from Yersinia pestis, was proposed to be controlled translationally by temperature-dependent accessibility of the SD (Hoe and Goguen, 1993). The lcrF 5′-UTR contains four consecutive uridines. This fourU motif is able to pair with the SD sequence and could mediate its accessibility by a temperature-dependent zipper-like melting as shown for the agsA gene from Salmonella enterica (Rinnenthal et al., 2010, Rinnenthal et al., 2011, Waldminghaus et al., 2007b).

Apparently, translational control by structured RNA regions plays an important role in the regulation of cold shock, heat shock and virulence genes. Several reporter gene constructs have been developed to examine this type of control in vivo, usually in E. coli (Neupert et al., 2008, Nocker et al., 2001, Waldminghaus et al., 2005, Waldminghaus et al., 2007a). Here, we briefly survey previously described systems, report on the optimization of a thermo-tolerant reporter system and present two novel RNA thermometer candidates, regulating translation of the htrA gene in both E. coli and Salmonella.

Section snippets

Bacterial strains and growth conditions

The bacterial strains, plasmids and primers used in this study are summarized in Table 1.

E. coli cells were grown in Luria-Bertani (LB) medium supplemented with ampicillin (Ap, 150 mg/ml) if appropriate at the indicated temperatures. For induction of the PBAD promoter in strains carrying translational lacZ or bgaB fusions, l-arabinose was added to a final concentration of 0.01% (w/v).

Construction of pBAD2-bgaB

All recombinant DNA work was performed according to standard protocols (Sambrook and Russel, 2001). Automated

pBAD-lacZ – new applications for an old vector

The pBAD-lacZ system has previously been used to study the translational regulation by ROSE (repression of heat shock gene expression)-like RNA thermometers from various alpha- and gamma-proteobacteria (Waldminghaus et al., 2005). In this vector system, the 5′-UTR of interest is cloned downstream of the arabinose-inducible PBAD promoter (Guzman et al., 1995) and in frame with the lacZ reporter gene. To ascertain that transcription strictly depends on the addition of arabinose the plasmid

Acknowledgments

We would like to thank Ralph Bock for the generous gift of plasmid pBS-U0, and Jörg Vogel for providing pXG-10 and E. coli DH5α-Z1. We thank Björn Schulte for initial experiments on the construction of the pBAD2 system. Stefanie Krajewski and Jens Kortmann are acknowledged for critical reading of the manuscript. This work was supported by the German Research Foundation (DFG priority program SPP 1258: Sensory and regulatory RNAs in prokaryotes) to FN and a Ruhr University Research School grant

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