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Journal of Bacteriology, January 2006, p. 240-248, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.240-248.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cold-Induced Putative DEAD Box RNA Helicases CshA and CshB Are Essential for Cold Adaptation and Interact with Cold Shock Protein B in Bacillus subtilis

Karen Hunger,1,{dagger} Carsten L. Beckering,1,{dagger} Frank Wiegeshoff,1 Peter L. Graumann,2 and Mohamed A. Marahiel1*

Philipps-Universität Marburg, FB Chemie, Hans-Meerwein-Str., D-35032 Marburg, Germany,1 Institut für Mikrobiologie, Albert-Ludwigs Universität Freiburg, Stefan Meier Str. 19, 79104 Freiburg, Germany2

Received 12 August 2005/ Accepted 5 October 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A sudden decrease in temperature is a serious challenge for all living microorganisms. The cellular reaction required for efficient adaptation to low temperatures is termed cold shock response. The cell has to cope with changes at different physiological levels, such as reduced fluidity of the membrane and slowdown of protein synthesis and protein folding (39). Accordingly, a general decrease of protein synthesis was observed in Escherichia coli and Bacillus subtilis cells following a cold shock (9, 17). The stress arising from a decrease in temperature can in principle be traced back to a reduction in molecular dynamics. The initiation of translation was shown to be a limiting factor for bacterial growth at low temperatures (3, 6). In addition to the delicate interaction of the complex translation machinery, the formation of secondary structures of mRNA prevents efficient initiation of translation (12). The tendency of RNA to fold and become kinetically trapped was described as a general problem and interferes with proper biological function (14). The formation of unfavorable secondary structures is even more intricate at lower temperatures. In analogy to protein folding mechanisms, RNA chaperones have been proposed to resolve and prevent misfolding of RNA molecules by either destabilizing RNA duplexes or binding to single-stranded nucleic acids (21). In the cold shock response, DEAD box RNA helicases have already been identified to destabilize RNA duplexes and the major cold shock proteins (CSPs) to bind RNA.

DEAD box RNA helicases belong to the large family of DExD/H-box proteins, which generally have an RNA-dependent ATPase activity in vitro. They are involved in various cellular processes, such as ribosome assembly, initiation of translation, and RNA turnover (31, 37). Cold-induced helicases identified so far are CsdA in E. coli (16), ChrC in the cyanobacterium Anabaena (4), DeaD in the archaeon Methanococcoides burtonii (20), and the putative helicases YdbR and YqfR in B. subtilis (here reassigned as CshA and CshB) (2). CsdA of E. coli was isolated together with the ribosomal fraction after cold shock (16) and proposed to promote translation initiation (22), to participate in assembly of the 50S ribosomal subunit at temperatures below 30°C (5), or to form the cold shock degradasome in conjunction with RNase E (29). The CSPs belong to the large family of RNA-binding proteins containing the cold shock domain that is conserved from bacteria to humans (10, 41). The fold of the cold shock domain consists of five antiparallel ß-strands forming a barrel structure (30). Two motifs, RNP1 and RNP2, containing surface-exposed basic and aromatic amino acids, are involved in binding single-stranded nucleic acids (32). In B. subtilis, three strongly cold-induced CSPs have been identified (CspB-D). They have been discussed as RNA chaperones to facilitate initiation of translation at low temperatures (8). For the homologous CspA of E. coli, RNA chaperone activity was demonstrated in vitro (15). Furthermore, in vivo complementation of B. subtilis {Delta}cspB {Delta}cspC deletion mutants by initiation factor IF1 of E. coli (38) and a transcription-dependent colocalization of B. subtilis CspB and ribosomes surrounding the nucleoid (24) supported the hypothesis that CSPs are involved in coupling transcription and translation.

Both cold-induced DEAD box RNA helicases and CSPs have been discussed in terms of RNA chaperone activity and cold shock response. Although the possibility of the two protein types working in conjunction was suggested in a review (33), no experimental evidence for a cooperative activity in the cold shock response has been reported. Therefore, we genetically characterized cshA and cshB in this work and addressed the question of a possible biological link between cold-induced DEAD box RNA helicases and CSPs. As prerequisites for a possible cooperation in vivo, we expected (i) close proximity of the two protein types within the bacterial cell, (ii) similar behavior with respect to their common cellular function, and (iii) accumulation of growth defects in strains containing combined gene deletions. The experimental results presented in this study support the hypothesis of a cooperative activity in vivo between the putative B. subtilis DEAD box RNA helicases CshA and CshB and the CSPs during the cold shock response.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and media. The bacterial strains used in this work are listed in Table 1. E. coli TOP10 (Invitrogen) was used as a host for plasmid construction and was grown in liquid LB rich medium or on LB agar plates (26) containing ampicillin (100 µg/ml) or kanamycin (50 µg/ml), as appropriate. B. subtilis strain JH642 (trpC2 pheA1) and derivatives were grown in liquid Spizizen's minimal medium (SMM) (35) containing tryptophan (50 µg/ml), phenylalanine (50 µg/ml), glucose (0.5%), and trace elements. Where necessary, glucose was replaced by fructose (0.5%) and xylose (0.5%). SMM was supplemented with chloramphenicol (5 µg/ml), lincomycin (25 µg/ml), erythromycin (1 µg/ml), or tetracycline (20 µg/ml), as required.


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  		TABLE 1. Strains and plasmids in this work

 
Plasmid and strain construction. The primers used in this work are listed in Table 2.


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  		TABLE 2. PCR primersa

 
Construction of CB30. Flanking regions of cshA were PCR amplified from the chromosomal DNA of B. subtilis JH642 using primer pairs ydbR_P1-ydbR_P2 for the 5' region and ydbR_P3-ydbR_P4 for the 3' region. A chloramphenicol cassette was cut with the restriction enzymes SpeI and PstI from the plasmid p{Delta}TE. The three overlapping fragments were fused by PCR and amplified by adding primer pair ydbR_P1-ydbR_P4. Strain B. subtilis JH642 was transformed with the resulting PCR product without further treatment, resulting in deletion strain CB30 ({Delta}cshA).

Construction of CB40. Flanking regions of cshB were PCR amplified from the chromosomal DNA of B. subtilis JH642 using primer pairs yqfR_P1-yqfR_P2 for the 5' region and yqfR_P3-yqfR_P4 for the 3' region. An erythromycin cassette was obtained from the plasmid pDG646 (11). The three overlapping fragments were fused by PCR and amplified by adding yqfR_P1 and yqfR_P4. Strain B. subtilis JH642 was transformed with the resulting PCR product without further treatment, resulting in deletion strain CB40 ({Delta}cshB).

Construction of pXkan. The pXkan plasmid is a modified pX (18) with kanamycin instead of chloramphenicol resistance. The kanamycin cassette was PCR amplified from the plasmid pDG783 (11) using primers 5'kan783(SpeI) and 3'kan783(SphI)II. The chloramphenicol cassette of pX was excised by digestion with SphI. Finally, the SphI-digested PCR fragment of the kanamycin cassette was inserted into the pX plasmid, resulting in plasmid pXkan.

Construction of CB3441. First, a copy of cshB was inserted in the amyE locus of B. subtilis JH642 under the control of a xylose-inducible promoter. Therefore, cshB was PCR amplified from B. subtilis JH642 using primer pair yqfR5'pX(SpeI)-yqfR3'pX(BamHI) and ligated into SpeI- and BamHI-digested plasmid pXkan. B. subtilis. JH642 was transformed with the resulting plasmid, giving strain CB41. Strain CB41 was successively transformed with the chromosomal DNA of CB30 and CB40 to delete the original cshA and cshB genes, resulting in strain CB3441 ({Delta}cshA {Delta}cshB {Delta}amyE::PxylcshB).

Construction of FW8. Strain B. subtilis 64D ({Delta}cspD) (8) was transformed with chromosomal DNA of strain B. subtilis CB40 ({Delta}cshB), resulting in double deletion strain FW8 ({Delta}cspD {Delta}cshB).

Construction of CB50 and CB51. 3' fragments of cshA and cshB were PCR amplified from B. subtilis JH642 using primer pairs 5'ydbRpSG51(HindIII)-3'ydbRpSG51(EcoRI) and 5'yqfRpSG51(HindIII)-3'yqfRpSG51(PstI) and ligated into the HindIII/EcoRI- or HindIII/PstI-digested green fluorescent protein (GFP) fusion plasmid pSG1151 (19). B. subtilis JH642 was transformed with the resulting plasmids, giving strains CB50 ({Delta}cshA::cshA-gfp) and CB51 ({Delta}cshB::cshB-gfp), respectively.

Construction of KH42. A 3' fragment of cshB was PCR amplified from B. subtilis JH642 using primer pair KH44-KH45 and ligated into the ApaI/EcoRI-digested yellow fluorescent protein (YFP) fusion plasmid pSG1187 (19). B. subtilis JH642 was transformed with the resulting plasmid, giving strain KH42 ({Delta}cshB::cshB-yfp).

Construction of KH44. A 3' fragment of cspB was PCR amplified from B. subtilis JH642 using primer pair KH42-KH43 and ligated into the ApaI/EcoRI-digested cyan fluorescent protein (CFP) fusion plasmid pSG1186 (19). B. subtilis JH642 was transformed with the resulting plasmid, giving strain KH44 ({Delta}cspB::cspB-cfp).

Construction of KH45. First, strain KH42 was transformed with plasmid pCm::Erm (36) to exchange the chloramphenicol cassette for an erythromycin cassette. Second, strain KH44 was transformed with plasmid pCM::Tet (36) to exchange the chloramphenicol cassette for a tetracycline cassette. Finally, the erythromycin-resistant variant of KH42 was transformed with chromosomal DNA of the tetracycline-resistant variant of KH44, resulting in strain KH45 ({Delta}cshB::cshB-yfp {Delta}cspB::cspB-cfp).

Construction of AS13 and AS14. A 3' fragment of rplA was PCR amplified from B. subtilis using primer pair L1up-L1dw and cloned into ApaI/EcoRI-digested CFP fusion plasmid pSG1186 (19). B. subtilis JH642 was transformed with the resulting plasmid selecting for chloramphenicol resistance, yielding strain AS13 (rplA-cfp). Strain AS13 was transformed with chromosomal DNA from strain KH44 selecting for chloramphenicol and tetracycline resistance, generating strain AS14 ({Delta}rplA::rplA-cfp {Delta}cshB::cshB-yfP).

Cold shock experiments. Growth experiments with B. subtilis strains were carried out as follows: 200 ml of SMM was inoculated with an overnight culture to an initial optical density at 600 nm (OD600) of 0.05 and incubated in a water bath shaker at 37°C and 220 rpm. At an OD600 of 0.5, one half of the culture was rapidly transferred to a new flask in a 15°C water bath shaker, while the other half remained at 37°C. For each growth curve, at least three independent experiments were performed.

Localization and fluorescence resonance energy transfer (FRET) measurements. Fluorescence microscopy was performed on an Olympus AX70 microscope. Cells were mounted on agarose pads containing SMM on object slides. Images were acquired with a digital MicroMax charge-coupled device camera; signal intensities and cell length were measured using the METAMORPH 4.6 program. DNA was stained with 4',6'-diamidino-2-phenylindole (DAPI; 0.2 ng/ml).

FRET was performed using a CFP excitation/dichroic filter cube (or a YFP excitation/dichroic filter cube to visualize YFP fluorescence) and a MultiSpec microimager (Visitron Systems, Germany) equipped with a beam splitter and CFP and YFP emission cubes. All images were acquired using a 500-ms exposure time. FRET values were calculated by measuring fluorescence intensity in individual cells from at least 12 different fields of cells expressing no GFP fusion, CspB-CFP, L1-CFP, CshB-YFP, or CFP and YFP fusions simultaneously.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In a previous investigation, we detected two cold-induced genes, ydbR and yqfR, through transcriptional analysis of cold-shocked B. subtilis cells (2). The proteins encoded by yqfR and ydbR show highest similarity to cold-induced members of the DEAD box RNA helicase family. Therefore, we reassign YdbR and YqfR as CshA and CshB (cold shock helicase-like proteins) in this work.

Sequence analysis and 5' region of cshA and cshB. The helicase family features a well-conserved central domain as well as less-conserved N- and C-terminal domains. Therefore, the central domain is indicative for DEAD box RNA helicases. A database search with the core domain of CshA and CshB using the BLASTP program (1) revealed high similarity to the cold-induced DEAD box RNA helicases CsdA from E. coli (16), ChrC from the cyanobacterium Anabaena (4), and DeaD from the archaeon M. burtonii (20) (Table 3). Furthermore, the central domain of CshA and CshB contains all of the eight highly conserved sequence motifs that have been described for DEAD box RNA helicases. Motifs one (walker A) and five (modified walker B) are involved in ATP binding and hydrolysis (23). It has been proposed that the N- and C-terminal domains of DEAD box RNA helicases determine their specificity for different cellular functions; however, the N- and C-terminal domains of the already identified cold-induced helicases do not share significant similarity among themselves or with other known helicases (20). As this is also the case for CshA and CshB of B. subtilis, it is not possible to assign a specific cellular function starting from primary structure. CshA is 511 amino acids in length (57 kDa) with a predicted pI of 9.9, whereas CshB is 438 amino acids in length (50 kDa) with a predicted pI of 9.9. The basic pI of both helicases lies outside the pH range of an earlier proteomic analysis of cold-shocked B. subtilis cells (7) and accounts for the fact that they have not been detected in that approach.


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  		TABLE 3. Sequence identities of the central domains of DEAD box proteins

 
The 5' regions of both cshA and cshB of B. subtilis share a striking feature. Analysis of the nucleotide region upstream of the start codon of cshA and cshB revealed a sequence motif highly similar to the bacterial cold box element (Fig. 1). The cold box was proposed to be involved in the regulation of cold shock genes (28) and has been found in the 5' region of genes coding for both bacterial CSP and cold-induced DEAD box RNA helicases (20). Ten out of 11 nucleotides of the cshA cold box motif are identical to B. subtilis cspB, and 9 out of 11 nucleotides are identical to E. coli cspB. Likewise, in the cshB cold box motif 8 out of 11 nucleotides are identical to B. subtilis cspB and E. coli cspB. Despite the striking similarity of the cold box element in the 5' region of bacterial CSPs and cold-induced helicases, the exact nature of the regulatory mechanism remains to be elucidated.



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  		FIG. 1. Comparison of the cold box elements in the 5' region of genes coding for cold-induced helicases and CSPs.

 
Analysis of cshA and cshB deletion mutants. To evaluate the influence of CshA and CshB on the cold shock adaptation of B. subtilis, deletion mutants were constructed and tested for growth after temperature downshift. First, the B. subtilis single deletion strains CB30 ({Delta}cshA) and CB40 ({Delta}cshB) were constructed as described in Materials and Methods. In a control experiment at 37°C, growth of CB30 and CB40 was equal to that of the parental strain B. subtilis JH642. Also, after a temperature shift from 37°C to 15°C, the growth rates of both deletion mutants remained the same as control strain JH642 (data not shown). Thus, it appears that the deletion of either CshA or CshB alone does not result in a cold-specific growth defect. A functional complementation of the homologous CshA by CshB and vice versa in the single deletion strains CB30 and CB40 could possibly suppress a visible effect. To test this possibility, we attempted to construct a double deletion mutant of cshA and cshB. Interestingly, we were not able to create this double mutant, suggesting that the deletion of both cshA and cshB together is lethal for B. subtilis. This behavior resembles the analysis of CSPs in B. subtilis (cspB, cspC, and cspD), in which at least one member of the family must be present for viability (8, 27).

Consequently, a double deletion mutant of cshA and cshB with an inducible copy of one helicase in trans was generated. A copy of cshB was integrated into the amyE locus of B. subtilis strain JH642 under control of a xylose-inducible promoter, resulting in strain CB41. Finally, in strain CB41, but not in wild-type cells, both cshA and cshB could be deleted by successive transformation with the chromosomal DNA of the single deletion strains CB30 and CB40 in the presence of xylose, resulting in strain CB3441. These experiments show that a double csh mutant strain can be generated only with a copy in trans, showing that the presence of one csh gene is essential for viability in B. subtilis. In agreement with this, a Northern blot analysis of the in trans copy of cshB showed that even in the absence of inducer a residual transcription was present, possibly due to a leaky promoter (data not shown). However, transcription of cshB was lower in the absence of xylose than in the presence of inducer. We observed growth arrest for lower levels of CshB after cold shock in B. subtilis (Fig. 2). The absence of xylose resulted in a significant difference in growth rates after cold shock from 37°C to 15°C. Control strain JH642 still grew after cold shock, whereas the mutant CB3441 suffered a growth arrest at an OD600 of 1. In terms of cell division, this can be interpreted as a single duplication event after cold shock before the cells stop growing. CB3441 seems to grow a little more slowly prior to cold shock, possibly due to lower levels of cshB transcription compared to the wild type. These findings indicate an essential participation of the putative mRNA helicases CshA and CshB in the cold shock response of B. subtilis.



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  		FIG. 2. Growth of helicase double deletion mutant CB3441 ({Delta}cshA {Delta}cshB amyE::PxylcshB) after cold shock in the presence and the absence of 0.5% (wt/vol) inducer xylose. Strains were grown at 37°C in SMM containing 0.5% (wt/vol) fructose to an OD600 of 0.5 and then immediately shifted to 15°C. At indicated time points, the OD600 of the cells was determined. The growth experiment was repeated three times. B. subtilis JH642, open diamonds; B. subtilis JH642 with xylose, open squares; CB3441, filled diamonds; CB3441 with xylose, filled squares.

 
A combined helicase/csp deletion results in slow growth after cold shock. The cold shock phenotype of the csh double mutant strain was reminiscent of that found in csp triple mutants (8). Therefore, a genetic approach was used to establish the proposed biological link between the helicases CshA and CshB and the CSPs. As shown above, the single deletion of cshA or cshB in B. subtilis did not cause any growth defects after cold shock, as is the case with a single deletion of CSPs (8). To investigate the effect of the combined deletion of a helicase and a CSP, strain CB40 ({Delta}cshB) was transformed with the chromosomal DNA of various B. subtilis csp mutant strains. It was impossible to generate a cshB cspB double mutant strain, while in parallel, CB40 could be transformed with chromosomal DNA from strain 64D ({Delta}cspD) (8), resulting in strain FW8 ({Delta}cspD {Delta}cshB). These experiments suggest that the combined loss of CshB and the major cold shock protein CspB is detrimental for cell survival. Growth experiments of strain FW8 compared to the control strain B. subtilis JH642 showed a cold-specific phenotype after a temperature downshift from 37°C to 15°C (Fig. 3), revealing a strong synergistic effect between CshB and CspD. At 37°C, both strains showed the same growth (data not shown). These findings support the hypothesis that CSPs and helicases work in conjunction on an aspect that is vital for cold shock adaptation.



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  		FIG. 3. Growth of the cshB cspD double deletion mutant FW8 in comparison to the parental strain after cold shock. Strains were grown at 37°C in SMM containing 0.5% (wt/vol) glucose to an OD600 of 0.5 and then immediately shifted to 15°C. At indicated time points, the OD600 of the cells was determined. The growth experiment was repeated three times. B. subtilis JH642, open diamonds; FW8, filled diamonds.

 
CshA and CshB show transcription-dependent localization at subcellular sites surrounding the nucleoids. The function of proteins is related to their site of action within the cell. Accordingly, the localization of proteins can provide information on their function. To study CshA and CshB in the three-dimensional context of the cell, C-terminal GFP fusions of the proteins were generated. The GFP fusions of CshA (CB50) and CshB (CB51) are the only source of these proteins in the cell, and the transcription of the coding genes is driven by their original promoter.

Figure 4 shows exponentially growing cells of CB51 (CshB-GFP) and KH45 (CspB-CFP and CshB-YFP), monitored by fluorescence microscopy. It can be seen that the helicases are not homogeneously spread throughout the cells but localize predominantly at the cell poles (Fig. 4A). Fluorescence of CshB-GFP (Fig. 4A) was much brighter than that of CshA-GFP, suggesting that CshB is expressed at higher levels within the cell than CshA (data not shown). Strikingly, the localization of both helicases is identical to the localization observed for ribosomes and for CSPs in B. subtilis in previous studies (24, 40). This was the first hint of a biological link between the putative mRNA helicases CshA and CshB and the CSPs.



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  		FIG. 4. Cellular localization of cold-induced putative helicase CshB determined by fluorescence microscopy in B. subtilis cells. (A) Exponentially growing cells expressing CshB-GFP. (B) Cells expressing CshB-GFP after inhibition of transcription with rifampin. (C) Cells expressing CshB-GFP after entering the stationary phase. White lines indicate septa between cells. (D) Exponentially growing cells expressing CspB-CFP and CshB-YFP. White bar, 2 µm.

 
Next, the cellular localization of CshA and CshB was monitored depending on the transcriptional activity of the cell. Such a relation has been reported for CSPs and ribosomes (24, 40). By adding the transcription inhibitor rifampin to exponentially growing cells, the localization of the helicases was indeed abolished and they were dispersed throughout the cells (Fig. 4B). Furthermore, like CSPs and ribosomes, the helicases were uniformly dispersed throughout the cells in the stationary phase (Fig. 4C), where only very little transcription occurs. The transcription-dependent localization of ribosomes, CSPs, and helicases at sites surrounding the nucleoid suggests an overlapping function of CSPs and the helicases at the level between transcription and translation.

To simultaneously visualize ribosomes or CSPs together with the putative helicases, we generated C-terminal fusions of CshB to YFP and of CspB to CFP, resulting in strain KH45 (CshB-YFP and CspB-CFP). The fusions are the only source of these proteins in the cell, and the transcription of the corresponding genes is controlled by their original promoters. In strain KH45, CshB-YFP and CspB-CFP colocalized to areas surrounding the nucleoids (Fig. 4D). Likewise, CshB-GFP and a fusion of the ribosomal protein L1 and blue fluorescent protein (BFP) colocalized at the cell poles (data not shown). These results confirm the localization of the GFP fusions of CshB and CspB.

FRET interaction of CshB and CspB in vivo. The CshB-YFP and CspB-CFP fusions were used to perform FRET experiments in growing cells. If the fusion proteins are in close proximity (1 to 5 nm), the emission of CFP can excite the YFP, and an increase of transfer fluorescence can be monitored using a FRET filter. A microimager permitted the simultaneous visualization of fluorescence in both CFP and FRET channels.

Fluorescence of excited CshB-YFP (strain KH42) was detected only in the YFP channel and not in the FRET channel, where it was identical to background levels in non-YFP-labeled cells (Fig. 5A and B). Hence, the contribution of YFP in the FRET channel can be ignored. Average fluorescence in the FRET channel of cells expressing CspB-CFP (strain KH44) showed a ratio of 251/80 for signal fluorescence versus background (>200 cells imaged) (Fig. 5C). On the other hand, the cells expressing both CspB-CFP and CshB-YFP (strain KH45) showed a ratio of 351/81 for signal fluorescence versus background (250 cells analyzed) (Fig. 5D). Thus, the FRET signal of the dually labeled strain was 28% higher than that of the control strain (6 to 10% FRET values have been measured for interacting proteins in other works [25, 34]), showing that a strong FRET interaction occurs between CSPs and helicases.



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  		FIG. 5. Interaction of the putative helicase CshB with CspB determined by FRET measurements. Different filters were used to visualize either the fluorescence of CFP or YFP or the FRET signal as indicated above the figure. Fluorescence was measured in exponentially growing cells, if not stated otherwise. (A and B) CshB-YFP (KH42), (C) CspB-CFP (KH44), (D) CspB-CFP and CshB-YFP (KH45), (E) L1-CFP (AS13), (F) L1-CFP and CshB-YFP (AS14), (G) cells expressing CspB-CFP (KH44) after inhibition of transcription with rifampin, and (H) cells expressing CspB-CFP and CshB-YFP (KH45) after inhibition of transcription with rifampin. Gray bars indicate 2 µm.

 
The interaction of CshB and CspB could possibly occur as a result of molecular crowding at the cell poles where both proteins and ribosomes are predominantly present. To test whether the FRET interaction of CshB and CspB is specific, a strain expressing the ribosomal protein L1-CFP and CshB-YFP was generated (AS14). The comparison of the FRET interaction in AS14 (145/81 signal versus background) and in control strain AS13 expressing L1-CFP only (142/82 signal versus background) revealed a mere 2% higher FRET signal in the dually labeled strain than the L1-CFP strain (Fig. 5E and F). These results show that there is no specific interaction between the ribosomal protein L1 and the helicases in vivo, supporting the statement that the FRET interaction between CshB and CspB is specific and not caused by molecular crowding of helicases, CSPs, and ribosomes at the cell poles.

As shown above, the localization of CshB and CspB depends on active transcription (Fig. 4B and 5G). To determine if the interaction between CshB and CspB is also dependent on active transcription, growing cells of the dually labeled strain KH45 (CshB-YFP and CspB-CFP) and of control strain KH44 (CspB-CFP) were treated with the transcriptional inhibitor rifampin. The measured FRET signal of the dually labeled strain KH45 (290/82 signal versus background) was only 5% higher than that of KH44 (276/82 signal versus background), showing that the interaction of CshB and CspB largely depends on active transcription (Fig. 5H).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cold shock response of B. subtilis has been well established over the past years and is therefore a suitable model system to study the possible interaction of the CSPs and the cold-induced DEAD box RNA helicases. Comprehensive studies have already dealt with the role of the CSPs in the cold shock response. However, not as much information is available on cold-induced helicases in B. subtilis. Consequently, we characterized the cold-induced DEAD box RNA helicases prior to experiments addressing the question of a possible interaction between CSPs and helicases.

For this purpose, we characterized the cold-induced proteins CshA and CshB of B. subtilis, which have previously been identified as putative DEAD box RNA helicases (2). In contrast to B. subtilis, only one copy of cold-induced helicases was reported for other microorganisms, namely, CsdA in E. coli, ChrC in Anabaena sp., and DeaD in M. burtonii (4, 16, 20). The sequence similarity among the cold-induced helicases (Table 3) is significantly higher than among non-cold-induced helicases. This indicates a possible similar function in the cold shock response. However, so far only CsdA of E. coli was characterized with respect to its in vivo function. Several hypotheses were proposed, ranking from translation initiation to ribosome assembly (5, 22).

With respect to the principal question of our study, it is intriguing that the cold-induced helicases CshA and CshB share several common features with the CSPs. Obviously both protein types might interact with RNA. Both are present in several copies in the chromosome. For the CSPs (CspB, CspC, and CspD), it was shown that one copy is essential for viability and that they can functionally complement each other (8). Our genetic experiments with cshA and cshB indicate the same behavior for the cold-induced putative helicases. The single deletions of cshA and cshB did not cause a growth defect, likely because the proteins complement each other. More importantly, a combination of both single deletions was possible only when an additional xylose-inducible copy was placed in trans in the chromosome of B. subtilis, showing that one copy of a csh is essential for viability. Moreover, in the absence of the inducer, this double mutant showed a growth arrest shortly after cold shock (Fig. 2). This cold-specific growth defect demonstrates the relevance of CshA and CshB for the cold shock adaptation of B. subtilis.

Furthermore, the regulation also shares a striking common feature. The bacterial cold box element is present in the 5' region of both cshA and cshB and CSPs of B. subtilis (Fig. 1). The same cold box was reported for the 5' region of CSPs and cold-induced helicases of other microorganisms (20), strengthening the general importance of this motif in the regulation of specific cold-induced genes.

To address our principal question, the interaction between putative helicases and CSPs, we first applied a genetic approach. The combination of {Delta}cshB and {Delta}cspD single mutations resulted in a strain with a cold-specific phenotype (Fig. 3). As both single mutants did not show any visible growth defects after cold shock, this observation was the first hint at a functional link of the putative helicases and CSPs in the cold shock response of B. subtilis.

Previous studies already demonstrated the colocalization of CSPs and ribosomes in areas surrounding the nucleoid in a transcription-dependent manner (24, 40). As both CSPs and ribosomes are potential interaction partners for the putative helicases, we were encouraged to initiate fluorescence microscopy experiments with CshA and CshB in B. subtilis. Our first observation of GFP fusions of CshA (data not shown) and CshB (Fig. 4A) showed that both putative helicases also localize in areas surrounding the nucleoid. In addition, the localization depended on active transcription, just as was shown for the CSPs and ribosomes before (Fig. 4B). The dependence on active transcription indicates that the localization of helicases, CSPs, and ribosomes is an active process rather than a simple exclusion from the nucleoids. These results strongly suggest a common function of the putative helicases CshA and CshB and the CSPs at a level between transcription and translation, as was already proposed for the CSPs (13, 15).

The localization experiments gave us some general clues about the possible interaction and cooperative work. However, the results obtained by FRET experiments led us to a more detailed picture of the interplay between the putative helicases CshA and CshB, CSPs, and ribosomes. We detected a strong FRET interaction between CshB and CspB, indicating a close proximity of these two proteins in the cell (Fig. 5D). The observed FRET signal could have arisen from molecular crowding at the cell poles, because helicases, CSPs, and ribosomes all localize in the same area. However, we ruled out this possibility by showing that there is no significant FRET interaction between the putative helicase CshB and the ribosomal protein L1 (Fig. 5F). Therefore, we conclude that there is a specific interaction between the helicase and CSPs in B. subtilis. Furthermore, the interaction of CshB and CspB was strongly reduced after inhibition of transcription (Fig. 5H). This means that the interaction is an active process occurring at or on mRNA and not an unspecific attraction of the two proteins.

In general, CSPs bind to single-stranded nucleic acids, and DEAD box RNA helicases target double-stranded RNA. The close proximity observed by FRET interaction and the transcription-dependent behavior suggest that the common physiological target of both proteins is the mRNA. Based on these observations, we propose the following model (Fig. 6). After cold shock, mRNA forms secondary structures that prevent initiation of translation at the ribosome. To rescue the mRNA, the cold-induced putative DEAD box RNA helicases CshA and CshB destabilize the unfavorable secondary structures. Freed of the secondary structures, the single-stranded mRNA can successively be bound by CSPs to prevent refolding until translation is initiated at the ribosome. Thus, we conclude that the cold-induced putative helicases CshA and CshB work in conjunction with the CSPs to ensure proper initiation of translation at low and optimal temperatures in B. subtilis.



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  		FIG. 6. Model of the cooperation of RNA helicases with the CSPs and FRET interaction of CshB-YFP with CspB-CFP.

 


   ACKNOWLEDGMENTS
 
We thank Astrid Steindorf and Melanie Wittmann for technical assistance and the generation of strains and Jenny Rood for critical reading of the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft (M.A.M.), Heisenberg Programm (P.L.G.), and Fonds der Chemischen Industrie.


   FOOTNOTES
 
* Corresponding author. Mailing address: Philipps-Universität Marburg, FB Chemie, Hans-Meerwein-Str., D-35032 Marburg, Germany. Phone: 49 6421-282-5722. Fax: 49 6421-282-2191. E-mail: marahiel{at}chemie.uni-marburg.de. Back

{dagger} These authors contributed equally to this work. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.[CrossRef][Medline]
  2. Beckering, C. L., L. Steil, M. H. Weber, U. Voelker, and M. A. Marahiel. 2002. Genomewide transcriptional analysis of the cold shock response in Bacillus subtilis. J. Bacteriol. 184:6395-6402.[Abstract/Free Full Text]
  3. Broeze, R. J., C. J. Solomon, and D. H. Pope. 1978. Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens. J. Bacteriol. 134:861-874.[Abstract/Free Full Text]
  4. Chamot, D., W. C. Magee, E. Yu, and G. W. Owttrim. 1999. A cold shock-induced cyanobacterial RNA helicase. J. Bacteriol. 181:1728-1732.[Abstract/Free Full Text]
  5. Charollais, J., M. Dreyfus, and I. Iost. 2004. CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit. Nucleic Acids Res. 32:2751-2759.[Abstract/Free Full Text]
  6. Friedman, H., P. Lu, and A. Rich. 1971. Temperature control of initiation of protein synthesis in Escherichia coli. J. Mol. Biol. 61:105-121.[CrossRef][Medline]
  7. Graumann, P., K. Schroeder, R. Schmid, and M. A. Marahiel. 1996. Cold shock stress-induced proteins in Bacillus subtilis. J. Bacteriol. 178:4611-4619.[Abstract/Free Full Text]
  8. Graumann, P., T. M. Wendrich, M. H. Weber, K. Schroeder, and M. A. Marahiel. 1997. A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures. Mol. Microbiol. 25:741-756.[CrossRef][Medline]
  9. Graumann, P. L., and M. A. Marahiel. 1999. Cold shock response in Bacillus subtilis. J. Mol. Microbiol. Biotechnol. 1:203-209.[Medline]
  10. Graumann, P. L., and M. A. Marahiel. 1998. A superfamily of proteins that contain the cold-shock domain. Trends Biochem. Sci. 23:286-290.[CrossRef][Medline]
  11. Guerout-Fleury, A. M., K. Shazand, N. Frandsen, and P. Stragier. 1995. Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167:335-336.[CrossRef][Medline]
  12. Hall, M. N., J. Gabay, M. Debarbouille, and M. Schwarz. 1982. A role for messenger-RNA secondary structure in the control of translation initiation. Nature 295:616-618.[CrossRef][Medline]
  13. Hanna, M. M., and K. Liu. 1998. Nascent RNA in transcription complexes interacts with CspE, a small protein in E. coli implicated in chromatin condensation. J. Mol. Biol. 282:227-239.[CrossRef][Medline]
  14. Herschlag, D. 1995. RNA chaperones and the RNA folding problem. J. Biol. Chem. 270:20871-20874.[Free Full Text]
  15. Jiang, W., Y. Hou, and M. Inouye. 1997. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J. Biol. Chem. 272:196-202.[Abstract/Free Full Text]
  16. Jones, P. G., M. Mitta, Y. Kim, W. Jiang, and M. Inouye. 1996. Cold shock induces a major ribosomal-associated protein that unwinds double-stranded RNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 93:76-80.[Abstract/Free Full Text]
  17. Jones, P. G., R. A. VanBogelen, and F. C. Neidhardt. 1987. Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169:2092-2095.[Abstract/Free Full Text]
  18. Kim, L., A. Mogk, and W. Schumann. 1996. A xylose-inducible Bacillus subtilis integration vector and its application. Gene 181:71-76.[CrossRef][Medline]
  19. Lewis, P. J., and A. L. Marston. 1999. GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227:101-110.[CrossRef][Medline]
  20. Lim, J., T. Thomas, and R. Cavicchioli. 2000. Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii. J. Mol. Biol. 297:553-567.[CrossRef][Medline]
  21. Lorsch, J. R. 2002. RNA chaperones exist and DEAD box proteins get a life. Cell 109:797-800.[CrossRef][Medline]
  22. Lu, J., H. Aoki, and M. C. Ganoza. 1999. Molecular characterization of a prokaryotic translation factor homologous to the eukaryotic initiation factor eIF4A. Int. J. Biochem. Cell. Biol. 31:215-229.[CrossRef][Medline]
  23. Luking, A., U. Stahl, and U. Schmidt. 1998. The protein family of RNA helicases. Crit. Rev. Biochem. Mol. Biol. 33:259-296.[CrossRef][Medline]
  24. Mascarenhas, J., M. H. Weber, and P. L. Graumann. 2001. Specific polar localization of ribosomes in Bacillus subtilis depends on active transcription. EMBO Rep. 2:685-689.[CrossRef][Medline]
  25. Matroule, J. Y., H. Lam, D. T. Burnette, and C. Jacobs-Wagner. 2004. Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter. Cell 118:579-590.[CrossRef][Medline]
  26. Miller, J. 1972. A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  27. Phadtare, S., J. Alsina, and M. Inouye. 1999. Cold-shock response and cold-shock proteins. Curr. Opin. Microbiol. 2:175-180.[CrossRef][Medline]
  28. Phadtare, S., and M. Inouye. 1999. Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli. Mol. Microbiol. 33:1004-1014.[CrossRef][Medline]
  29. Prud'homme-Genereux, A., R. K. Beran, I. Iost, C. S. Ramey, G. A. Mackie, and R. W. Simons. 2004. Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a ‘cold shock degradosome.’ Mol. Microbiol. 54:1409-1421.[CrossRef][Medline]
  30. Schindelin, H., M. A. Marahiel, and U. Heinemann. 1993. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature 364:164-168.[CrossRef][Medline]
  31. Schmid, S. R., and P. Linder. 1992. DEAD protein family of putative RNA helicases. Mol. Microbiol. 6:238-292.
  32. Schroeder, K., P. Graumann, A. Schnuchel, T. A. Holak, and M. A. Marahiel. 1995. Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif. Mol. Microbiol. 16:699-708.[Medline]
  33. Sommerville, J. 1999. Activities of cold-shock domain proteins in translation control. Bioessays 21:319-325.[CrossRef][Medline]
  34. Sourjik, V., and H. C. Berg. 2002. Binding of the Escherichia coli response regulator CheY to its target measured in vivo by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA. 99:12669-12674.[Abstract/Free Full Text]
  35. Spizizen, J. 1958. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. USA 44:407-408.
  36. Steinmetz, M., and R. Richter. 1994. Plasmids designed to alter the antibiotic resistance expressed by insertion mutations in Bacillus subtilis, through in vivo recombination. Gene 142:79-83.[CrossRef][Medline]
  37. Tanner, N. K., and P. Linder. 2001. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell 8:251-262.[CrossRef][Medline]
  38. Weber, M. H., C. L. Beckering, and M. A. Marahiel. 2001. Complementation of cold shock proteins by translation initiation factor IF1 in vivo. J. Bacteriol. 183:7381-7386.[Abstract/Free Full Text]
  39. Weber, M. H., and M. A. Marahiel. 2003. Bacterial cold shock responses. Sci. Prog. 86:9-75.[Medline]
  40. Weber, M. H., A. V. Volkov, I. Fricke, M. A. Marahiel, and P. L. Graumann. 2001. Localization of cold shock proteins to cytosolic spaces surrounding nucleoids in Bacillus subtilis depends on active transcription. J. Bacteriol. 183:6435-6443.[Abstract/Free Full Text]
  41. Wolffe, A. P., S. Tafuri, M. Ranjan, and M. Familari. 1992. The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man. New Biol. 4:290-298.[Medline]

Journal of Bacteriology, January 2006, p. 240-248, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.240-248.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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