Portal de Periódicos da CAPES

Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis

2001; Springer Nature; Volume: 20; Issue: 7 Linguagem: Inglês

10.1093/emboj/20.7.1681

1460-2075

Pablo S. Aguilar,

Protein Structure and Dynamics

Article2 April 2001free access Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis Pablo S. Aguilar Pablo S. Aguilar Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Ana María Hernandez-Arriaga Ana María Hernandez-Arriaga Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain Search for more papers by this author Larisa E. Cybulski Larisa E. Cybulski Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Agustín C. Erazo Agustín C. Erazo Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Diego de Mendoza Corresponding Author Diego de Mendoza Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Pablo S. Aguilar Pablo S. Aguilar Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Ana María Hernandez-Arriaga Ana María Hernandez-Arriaga Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain Search for more papers by this author Larisa E. Cybulski Larisa E. Cybulski Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Agustín C. Erazo Agustín C. Erazo Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Diego de Mendoza Corresponding Author Diego de Mendoza Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina Search for more papers by this author Author Information Pablo S. Aguilar1, Ana María Hernandez-Arriaga2, Larisa E. Cybulski1, Agustín C. Erazo1 and Diego de Mendoza 1 1Instituto de Biología Molecular y Celular de Rosario (IBR) and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina 2Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1681-1691https://doi.org/10.1093/emboj/20.7.1681 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Δ5-lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position −28 to −77 relative to the start site of the temperature-regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Δ5-desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK–DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures. Introduction Variability and adaptability are crucial characteristics of organisms possessing the ability to survive and prosper in a wide variety of environmental conditions. In order for bacteria to effectively compete and survive, they have to sense environmental conditions and respond accordingly. Temperature is one of the major stresses that all living organisms face (Phadtare et al., 2000). It has been demonstrated that bacteria respond to high growth temperatures by the induction of a group of heat shock proteins, but also to low temperatures by the induction of a group of cold shock proteins (Phadtare et al., 2000). In contrast to heat shock proteins, which include chaperones required for protein folding and peptidases (Yura et al., 2000), cold-induced proteins appear to be involved in cellular functions such as general metabolism, transcription, translation and recombination (Phadtare et al., 2000). A universally conserved adaptation response observed among bacteria and most (if not all) poikilothermic organisms is the adjustment of membrane lipid composition at low temperatures (Cronan and Rock, 1996; Vigh et al., 1998). As the growth temperature is lowered, the proportion of unsaturated fatty acids (UFAs) in the membrane lipids increases. This regulatory mechanism system, called thermal control of fatty acid synthesis, is thought to be designed to ameliorate the effects of temperature changes on the physical state of the membrane phospholipids (for a review see Cronan and Rock, 1996). There are a variety of mechanisms that can alter the membrane phospholipid composition in response to a temperature change. Bacilli cells respond to a decrease in ambient growth temperature by introducing double bonds into pre-existing fatty acids of their membrane phospholipids (Grau and de Mendoza, 1993; Aguilar et al., 1998). These double bonds are inserted by specific fatty acid desaturase enzymes (de Mendoza et al., 2001). In a previous study we reported the isolation of the des gene coding for the Δ5-desaturase of Bacillus subtilis (Aguilar et al., 1998). Studies of operon fusion as well as transcriptional analysis demonstrated that des is tightly regulated during cold shock (Aguilar et al., 1998, 1999). While the des transcript is barely detected at 37°C, the production of des mRNA is transiently induced upon a temperature downshift. Derepression of des occurs exclusively at the level of transcription in a promoter-dependent fashion and does not require de novo synthesis of protein (Aguilar et al., 1999). Nevertheless, the molecular mechanism by which the transcription of the B.subtilis des gene as well as the synthesis of membrane UFAs are induced transiently by low temperature remains unsolved. Previous studies suggest that membranes can sense environmental changes and, as a consequence of changes in their phase state and microdomain organization, transmit signals that activate transcription (Vigh et al., 1998; Hoppe et al., 2000; Suzuki et al., 2000). This signalling mechanism was proposed to control the expression of cold-induced desaturases from cyanobacteria and heat shock-induced genes in Saccharomyces cerevisiae and cyanobacteria (for a review see Vigh et al., 1998). For signal transduction across the cell membrane, bacteria extensively use two-component systems, which have an input-sensing domain (histidine kinase) and an output effector domain (response regulator) (Hoch, 2000). Because sensor kinases are generally integral membrane proteins that respond to environmental signals (Dutta et al., 1999), it seems likely that temperature regulation of UFAs in bacilli could be controlled by members of the family of two-component regulatory proteins. In connection with this possibility, Suzuki et al. (2000) have recently reported that two histidine kinases and a response regulator modulate the transcription of low temperature-inducible genes from cyanobacteria. Here we report the identification of the desK and desR genes from B.subtilis, which are directly responsible for the transcriptional regulation of the des gene. These genes encode products with similarity to an autophosphorylatable histidine kinase (DesK) and a DNA binding response regulator (DesR) of the two-component signal transduction system. In addition, we demonstrate that DesR interacts specifically with the regulatory region of the gene it controls and that UFAs act as negative regulators of des expression. Thus, a regulatory loop composed of the DesK–DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures. Results Inactivation of the yocF and yocG genes prevents induction by low temperature of β-galactosidase activity in a des–lacZ fusion A key issue in the regulation of UFAs synthesis in bacilli is to understand how a decrease in growth temperature induces the expression of the des gene required for oxygen-dependent desaturation of fatty acids. To gain insight into this regulatory pathway, the B.subtilis genome sequence (Kunst et al., 1997) was searched for potential two-component regulatory gene pairs involved in des environmental regulation. Among the 35 two-component signal transduction systems identified in B.subtilis, the two-gene operon formed by the yocF and yocG genes encodes a two-component system with no known function (Fabret et al., 1999). The predicted products of yocF and yocG exhibit structural similarity to the histidine kinases and response regulators of two-component regulatory systems, respectively (Fabret et al., 1999). The yocFG operon is located immediately downstream of the des gene (Kunst et al., 1997). This prompted us to investigate whether this signal transduction system could be involved in the regulation of the desaturase synthesis. To test the induction of desaturase expression upon a temperature shift we used the strain AKP3, which contains a fusion of the lacZ gene to the des promoter integrated ectopically at the non-essential amyE locus of B.subtilis JH642. When B.subtilis strain AKP3 is grown at 37°C the levels of β-galactosidase (β-gal) are very low (Figure 1A). However, when this strain is grown at 37°C and then shifted to 25°C, β-gal synthesis reaches induction levels ∼10-fold higher than the levels found at 37°C (Figure 1A). In addition, the induction of the des–lacZ fusion in strain AKP3 can be easily monitored in media containing X-gal, where the colonies turn blue at 25°C (Figure 1B, panel I). To determine whether the yocF and yocG genes are responsible for the control of des, we disrupted the operon with a kanamycin-resistance gene (KmR) cassette as described in Materials and methods. The yocFG gene disruption was introduced by homologous recombination into strain AKP3, giving strain AKP21. In contrast to strain AKP3, strain AKP21 did not form blue colonies at 25°C (Figure 1B, panel I) nor were its β-gal levels increased upon downshift from 37 to 25°C (Figure 1A). This result indicates that a mutation in the yocFG operon eliminates the low-temperature inducibility of the des promoter. Figure 1.Pattern of des–lacZ expression in wild-type and yocF−yocG− cells before and after temperature downshift. (A) Bacillus subtilis AKP3 cells (circles) and AKP21 cells (triangles) harbouring a des–lacZ transcriptional fusion were grown at 37°C to an optical density of 0.35 (at 525 nm) and then divided into two samples. One sample was transferred to 25°C (filled symbols), and the other was kept at 37°C (open symbols). Specific β-gal activities were determined at the indicated time intervals. (B) Bacterial strains [wild-type AKP3 (wt), yocF−yocG− (AKP21), yocF−yocG− pXyl:yocFG (AKP2147), yocF−yocG− pXyl:yocG (AKP2152), yocG− (AKP9) and yocG− pXyl:yocG (AKP952)] were streaked onto Luria–Bertani (LB) medium containing 30 μg/μl X-Gal, with (shaded quarter) or without (empty quarter) the addition of 0.8% L-xylose. The strains were incubated at 37°C for 12 h (left column) or for 5 h at 37°C, and then transferred to 25°C for 36 h (right column) before photography. Download figure Download PowerPoint The yocFG mutation can be complemented in trans To verify that the yocF and yocG genes were responsible for cold induction of des, plasmids containing the yocFG operon or the yocG gene alone under the control of the xylose-inducible Xyl promoter were integrated into the thr locus of strain AKP21 giving strains AKP2147 and AKP2152, respectively. In strain AKP2147, in which the yocF and yocG genes are provided in trans, the cold induction of the des–lacZ fusion was dependent upon the addition of xylose to the growth medium (Figure 1B, panel I). The xylose-induced expression of yocG alone, however, was unable to restablish the cold-dependent lac+ phenotype of strain AKP2152 (Figure 1B, panel II). Nevertheless, expression of this gene in strain AKP952, in which inactivation of yocG eliminated the low-temperature inducibility of the des promoter (Figure 1B, panel III), resulted in a xylose-dependent induction of the des–lacZ fusion at low growth temperatures (Figure 1B, panel III). Therefore, the yocG and yocF genes are essential for the low-temperature induction of the des gene. Inactivation of yocG inhibits the induction of the des transcript and UFA synthesis at low temperature We performed northern blot analysis to examine the expression of the des gene in the wild-type strain JH642 or in the yocG− strain AKP8, before and after a shift in temperature from 37 to 25°C (Figure 2A). As observed previously in the wild-type cells, the size of the des transcript is ∼1.1 kb, and this mRNA was only detected when cells were shifted to 25°C (Aguilar et al., 1999). However, the accumulation at 25°C of the des transcript was not observed in strain AKP8, directly demonstrating that inactivation of the yocG gene suppressed the low temperature-induced expression of the desaturase gene (Figure 2A). Figure 2.des mRNA and UFA production in wild-type and yocG− strains after a downshift temperature. (A) Northern blot analysis using formaldehyde agarose gels was carried out as described in Materials and methods. Total RNA was isolated from strains JH642 (lane 1) or strain AKP8 (yocG−, lane 2) grown until mid-exponential phase at 37°C and then shifted to 25°C by 30 min. Each lane contains 10 μg of total RNA. (B) Fatty acids synthesized by strains JH642 and AKP8 at 25°C. Cultures of strains JH642 (lane 1) and AKP8 (lane 2) were grown to mid-exponential phase at 37°C, then 2 ml of these cultures were challenged with 10 μCi of [14C]acetate and further shifted to 25°C for 12 h. The lipids were then extracted and transesterified, and the resulting methyl esters were separated into saturated (SFAs) and unsaturated (UFAs) fractions by chromatography on 20% silver nitrate-impregnated silica gel thin-layers plates. The plates were developed at −17°C and autoradiographed by 7 days. The sample in lane 1 contained 15 000 c.p.m. and 2000 c.p.m. in the SFA and UFA fractions, respectively. The sample in lane 2 contained 14 000 c.p.m. in the SFA fraction, while the UFA fraction contained only background levels of radioactivity. Download figure Download PowerPoint The fatty acid profile of the wild-type strain JH642 was compared with that of strain AKP8. The fatty acids were labelled by growth of the strains in [14C]acetate, followed by argentation chromatography of the radioactive fatty acids. While strain JH642 shifted from 37 to 20°C synthesized UFAs, strain AKP8 formed no detectable UFAs after the temperature downshift (Figure 2B). These experiments confirm that the yocG is essential for the synthesis of UFAs at low growth temperatures. Since both the histidine kinase YocF and its cognate regulator YocG are required for des induction, we have named the genes coding for these two-component regulatory proteins as desK and desR, respectively. h-DesR binds to the des promoter In most cases, the response partner of the two-component transduction system is a transcriptional activator for genes whose products are specifically utilized to respond to the unique nature of a given input signal (Hoch, 2000). The genetic data presented above suggested that DesR, a response regulator for low-temperature response may bind to the des promoter region. The binding of purified His-tagged DesR (h-DesR) to the des promoter was tested using the electrophoresis mobility shift assay (EMSA). The h-DesR protein was expressed by isopropyl-β-D-thio galactopyranoside (IPTG) induction with Escherichia coli M15 (pREP4, pAR18) and purified by nickel affinity chromatography (data not shown). A PCR-amplified 367 bp DNA containing the des promoter (pdesDNA) was used as target DNA. Formation of complexes between h-DesR and labelled pdesDNA was tested by EMSA in the presence of an excess of competitor poly(dI–dC) (Figure 3A). The results show that the pdesDNA exhibited changes in mobility in the presence of 21 nM h-DesR (Figure 3A, lane 1). A more dramatic shift in the mobility of this 367 bp fragment was observed with increasing concentrations of h-DesR, and at the highest concentration of protein tested (336 nM), >90% of the input DNA was complexed with h-DesR (Figure 3A, lane 5). Two bands with shifted mobilities were usually observed, indicating that two different complexes are formed. Addition of a 3-fold excess of an unlabelled DNA fragment containing the des promoter diminishes the labelled complex formation (Figure 3B, lane 2), and a 15-fold excess of unlabelled pdesDNA abolished the labelled complex formation (Figure 3B, lane 3), suggesting specific interaction. The binding specificity was confirmed by determining that h-DesR did not shift an unrelated 290 bp fragment belonging to the repAB promoter of plasmid pLS1 (del Solar et al., 1990; data not shown). Figure 3.Gel shift assay showing the binding of DesR to the des promoter region. (A) The 367 bp des promoter fragment (pdesDNA) was prepared by [α-32P]dATP PCR labelling as described in Materials and methods. The [32P]pdesDNA concentration in the binding mixtures was 1.7 nM in all cases. The concentration of h-DesR used in each binding reaction is indicated above the respective line. (B) Specific competition in binding reactions using 1.7 nM [32P]pdesDNA and 336 nM h-DesR. Lane 1 shows the retarded species in the absence of unlabelled homologous DNA. Lanes 2, 3 and 4 show the dissociation of the labelled complex in the presence of 3, 15 and 60-fold molar excess of unlabelled pdesDNA, respectively, added to the binding mixtures before the addition of h-DesR. Lane 5 shows [32P]pdesDNA without the addition of h-DesR. Download figure Download PowerPoint h-DesR protects extended DNA segments upstream from its target promoter To identify h-DesR binding sites in pdesDNA, DNase I protection analysis was performed on both strands of the des promoter. As shown in Figure 4A, binding of h-DesR resulted in the protection of DNA sequences extending from the −28 to −77 position, relative to the start of transcription. In addition to the protected regions, several hypersensitive bonds (indicative of local deformation and presumably caused by bending of the helix) were detected (Figure 4A). DNase I treatment of the h-DesR–DNA complex revealed five protected regions for both strands; these upper and lower strand protections are offset from each other towards the 3′ end of both strands. In the protected region two inverted repeats (5′-TCAT-3′) separated by nine nucleotides were found (Figure 4A). The axis of this dyad symmetry exactly matches the centre of the protected region determined by the footprinting analysis. The finding of a dyad symmetry in the centre of the protected region together with the existence of two complexes of h-DesR–DNA suggests that more than one subunit of h-DesR associates with the des promoter at one face of the DNA helix. Figure 4.DNase I footprinting assay of the des promoter region and in vivo characterization of the des promoter protected region. (A) DNase I footprinting of h-DesR protein on both strands of a 178 bp DNA fragment containing the des promoter (see Materials and methods). Sequencing reactions were performed on the same DNA fragment labelled at the coding (lanes 3 to 6) and non-coding (lanes 9 to 12) strands. Lanes 1, 2, 7 and 8 show the DNase I digestion products of pdesDNA in the presence (+) or absence (−) of h-DesR. Brackets mark the protected regions in each strand. The putative 17 bp symmetric region of the protected region is boxed, with the dyad axis of symmetry indicated by a dot. The inverted repeats are underlined with dots. DNase I footprints on both strands are shown. Arrows indicate hypersensitive bonds. (B) Promoter mutations. The sequence changes in the promoter variants are depicted along the protected region of the des promoter. The deleted region is indicated by dots, and the nucleotide changes to introduce mutations in the left inverted repeat are shown in bold characters. The inverted repeat sequences are underlined. The strains were grown at 37°C to an OD of 0.30 and then subjected to a downshift to 25°C. After 3 h of growth at 25°C, the cells were harvested and β-gal activities were determined. The average value of β-gal activity of strain AKP3 (bearing the wild-type promoter) was taken as 100% of promoter activity. The results shown are the average of three independent experiments. Download figure Download PowerPoint To determine whether this dyad symmetric element is directly involved in des transcriptional regulation, promoter variants carrying a deletion or a mutation in the DesR binding site were cloned into plasmid pJM116 to create transcriptional fusions, which were then integrated at the amyE locus of the B.subtilis chromosome. Twenty-two base pairs out of the 49 bp protected region, including both inverted repeats, were deleted, yielding strain AKL59 (Figure 4B). Under cold shock conditions this deletion almost completely abolished promoter activity (Figure 4B). This result strongly suggests that the dyad symmetric element is essential for promoter activity at low temperature. Confirming this interpretation, no promoter activity was detected when the symmetry of the dyad element was disrupted by site-directed mutagenesis of the right inverted repeat (strain AKL62, Figure 4B). DesK acts to control the activation and deactivation of DesR in response to growth temperature Previous work has shown that the overexpression of response regulators in the absence of their cognate kinases could result in constitutive expression of the gene(s) they control (Powell and Kado, 1990). This suggests that high concentrations of unphosphorylated response regulator could bind in vivo to the target promoter and cause unregulated transcription. To determine whether an excess of DesR, without the assistance of DesK, could activate transcription of des at 37°C, we constructed the desK− strains AKP2152 and AKP20, expressing the wild-type desR from the pXyl or the pKan promoters, respectively. Antibody to DesR was generated and immunoblot assays were performed with the wild-type strain JH642 or cells overexpressing desR. As shown in Figure 5A (lanes 1–3), DesR was not detected in whole-cell lysates of strain JH642 growing at either 37 or 25°C, indicating that this protein is produced at very low levels at both temperatures. However, we found that strain AKP2152, expressing desR from the pXyl promoter, showed a significant level of DesR synthesis after induction with 0.8% xylose (Figure 5A, lane 5). Although this strain overproduced DesR in the presence of the inducer, expression of des was still repressed at either 37 or 25°C (Figure 1B, panel II). Nonetheless, we found that strain AKP20, expressing desR from the constitutive pKan promoter, showed a DesR production 5-fold greater than strain AKP2152 (Figure 5B, lane 6) and was able to express the des–lacZ fusion at 37°C (Figure 5C). This experiment demonstrates that high production of DesR promotes constitutive expression of the desaturase gene without assistance from DesK. This result, therefore, agrees with the observation that unphosphorylated response regulators can activate transcription when they are overexpressed (Powell and Kado, 1990). A somewhat surprising result was that introduction of the desKR operon, under the control of pXyl promoter, into strain AKP20 (giving strain AKP2047) restored the cold-inducible expression of des (Figure 5C). This effect could not be attributed to a reduction in the synthesis of DesR, since strain AKP2047 also overproduced the DesR protein to the same extent as AKP20 (Figure 5B, lanes 4 and 5). It should be noted that the pXyl promoter is ∼5-fold weaker than the pKan promoter (as estimated from the levels of production of DesR from desR fusions to these promoters; Figure 5A and B). It is more likely, therefore, that the cellular levels of DesK are much lower than the levels of DesR in strain AKP2047. The fact that in strain AKP2047 the xylose-induced expression of desK results in deactivation of DesR at 37°C suggests that DesK acts as a phosphatase that dephosphorylates DesR in response to an increase in growth temperature. A downshift in temperature, however, would suppress the phosphatase activity of DesK, favouring the phosphorylation of DesR by DesK, resulting in cold-induced transcriptional activation of the desaturase gene. The constitutive expression of desR in strain AKP20 could be explained by the existence of a second kinase or another phosphodonor, such as acetylphosphate (Lukat et al., 1992), capable of phosphorylating this response regulator irrespective of the growth temperature. This putative phosphate donor would have low affinity for DesR and would require a high concentration of this response regulator to phosphorylate it. Figure 5.Overexpression of DesR leads to constitutive expression of des. (A) JH642 cells (wild type) were cultured at 37°C until mid-exponential phase (lane 1) and then shifted to 25°C for 30 min (lane 2) or 3 h (lane 3). Samples were taken and cell extracts were analysed for the presence of DesR with DesR antiserum. AKP2152 (2152) cells were cultured at 37°C in the absence (lane 4) or presence of 0.8% L-xylose (lane 5) to mid-exponential phase and then shifted to 25°C by 2 h. Samples were taken and analysed for the presence of DesR as described above. (B) Strains JH642 (wt, lane 1), AKP2152 (2152) (lanes 2 and 3), AKP2047 (2047) (lanes 4 and 5) and AKP20 (20) (lane 6) were cultured at 37°C until mid-exponential phase and then shifted to 25°C by 2 h. AKP2152 and AKP2047 cultures were supplemented (+) or not (−) with 0.8% xylose. Samples were taken and proteins from the same quantity of cells were analysed for the presence of DesR. (C) Bacterial strains AKP3 (wt), AKP20 (desK−,pkan-desR) and AKP2047 (AKP20 pXyl-desKdesR::thr) were streaked onto LB medium containing 30 μg/μl X-Gal, with (shaded quarter) or without (empty quarter) the addition of 0.8% L-xylose. The strains were incubated at 37°C for 12 h (left) or for 5 h at 37°C, and then transferred to 25°C for 36 h (right) before photography. Download figure Download PowerPoint UFAs regulate the expression of the des gene Previous work has shown that a downshift of B.subtilis cells to low temperature produces a transient increase in des mRNA due to shutoff of transcription rather than to the instability of des mRNA (Aguilar et al., 1999). Since DesK and DesR are necessary for induction of des mRNA, it is possible that des expression is downregulated by shutoff of desKR transcription at low temperatures. To test this hypothesis, total cellular RNA was isolated from cultures of strain JH642 grown at 37°C and then shifted to 25°C for various times. Northern blot analysis indicated that the 1.9 kb desKR mRNA is constitutively expressed both at 37 and 25°C, although its level is slightly increased at 25°C, probably due to increased stability of the transcript at the low temperature (Figure 6). This result shows that downregulation of des transcription cannot be attributed to shutoff of desKR transcription at low temperature. Therefore, the transient induction of des should be due to inhibition of the pathway that senses or transduces low-temperature signals. Since UFAs are sophisticated signalling molecules that can mediate a myriad of cellular processes (Dowhan, 1997), including gene expression (Choi et al., 1996; Hoppe et al., 2000), we reasoned that the UFAs formed at low temperatures could act as negative regulators of the low-temperature signalling transduction system that induces the synthesis of the Δ5-desaturase. To test this hypothesis we inserted a KmR cassette into the des gene of strain AKP3, giving strain AKP4. This strain allows monitoring of the low-temperature inducibility of a des–lacZ fusion in the absence of UFAs synthesis. We assayed the β-gal activity of strains AKP3 and AKP4 upon a temperature downshift. While the β-gal levels of the des+ strain AKP3 began to decrease after 6 h at 25°C, the β-gal activity of the des–lacZ fusion contained into the des− strain AKP4 continued increasing duri