Journal of Molecular Biology
Regular articleCooperative and non-cooperative DNA binding modes of catabolite control protein CcpA from Bacillus megaterium result from sensing two different signals1
Introduction
Carbon catabolite repression (CCR) is the general phenomenon to adjust expression of catabolic genes in response to the availability of rapidly metabolizable carbon sources. The mechanistic paradigm of this global regulatory pathway occurs in Escherichia coli and involves cyclic AMP and CRP (reviewed by Postma et al., 1993). The same phenomenon in Bacillus subtilis is mediated by a completely different mechanism, as no cyclic AMP or CAP homologue is available (Chambliss, 1993). Instead, CCR involves negative regulation at the level of transcription mediated by the cis-active operator sequence cre (catabolite responsive element; Weickert & Chambliss, 1990), found to be essential for CCR of numerous genes in Gram-positive bacteria (reviewed by Hueck & Hillen, 1995), and the trans-acting protein CcpA (catabolite control protein; Henkin et al., 1991), a member of the LacI GalR family of bacterial repressor proteins (Weickert & Adhya, 1992). A gene bank search for cre sequences (Hueck et al., 1994a) and an immunological screen for cross-reactivity with CcpA-antiserum (Küster et al., 1996) has suggested that both elements are widely distributed among Gram-positive bacteria. Thus, CCR mediated by cre and CcpA may represent a global regulatory mechanism shared by many bacteria.
CCR requires the sensing of various sugars according to their metabolic potential and the relay of this information to CcpA-mediated gene regulation. Several lines of evidence suggest that one important sensor is HPr, the phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In contrast to E. coli, several Gram-positive bacteria contain an HPr kinase transfering a phosphate from ATP to Ser46 of HPr at high concentrations of fructose 1,6-diphosphate (Deutscher & Saier, 1983). A non-phosphorylatable Ser46Ala mutant of HPr (Reizer et al., 1989) leads to relief of several genes from CcpA-dependent CCR (Deutscher et al., 1994).
Since expression of ccpA itself is not subject to CCR Hueck et al 1995, Miwa et al 1994, a cofactor-triggered modulation of CcpA activity seemed likely and HPr phosphorylated at Ser46 (HPr-Ser46-P) was a prime candidate. CcpA from Baccillus megaterium interacts specifically with B. subtilis HPr-Ser46-P in the absence of DNA, whereas neither the enzymatically active HPr-His15-P, the bi-phosphorylated or the unphosphorylated forms of HPr showed binding to CcpA (Deutscher et al., 1995). B. subtilis CcpA binds to the B. subtilis gnt cre in a HPr-Ser46-P dependent manner in vitro (Fujita et al., 1995). On the other hand, CcpA binds in vitro to cre of amyE in B. subtilis independent of a cofactor (Kim et al., 1995). Interaction of CcpA with cre in the xyl operon was stimulated by several small molecular mass phosphates and affected by HPr-Ser46-P (Ramseier et al., 1995). The xyl operon expression is only partially relieved from CCR in a ptsH1 background expressing only the HPr-Ser46Ala mutant (Dahl & Hillen, 1995). Thus, several sensory pathways may lead to CcpA activation.
Expression of the xylose utilization operon of B. megaterium is subject to CCR (Rygus & Hillen, 1992) mediated by CcpA (Hueck et al., 1995) and cre located within the coding region of xylA (position +130.5 with respect to the transcriptional start site; Hueck et al., 1994a). The genetic organization of the xyl operon is shown in Figure 1. We demonstrate here that purified B. megaterium CcpA must be triggered by either HPr-Ser46-P or glucose 6-phosphate (Glc-6-P) to bind specifically to cre in xylA. DNaseI footprints indicate that cre binding by CcpA is cooperative when triggered by Glc-6-P and non-cooperative when triggered by HPr-Ser46-P.
Section snippets
Overproduction and purification of CcpA
Plasmid pWH2014 (Hueck et al., 1995) contains the ccpA gene from B. megaterium transcriptionally fused to the xylA promotor (Table 1), resulting in xylose-inducible overproduction of CcpA. Addition of xylose to growing cultures of B. megaterium WH356 carrying pWH2014 leads to accumulation of a protein of approximately 38 kDa as expected for the ccpA gene product (Figure 2, lanes 1 and 2). It shows the same mobility as CcpA in Western blot analyses of crude protein extracts from wild-type B.
Discussion
CCR of many catabolic genes in the Gram-positive bacteria is mediated via CcpA and cre (reviewed by Hueck & Hillen, 1995). Direct interaction of CcpA with cre was demonstrated in vitro for amyE and gntR from B. subtilis Kim et al 1995, Fujita et al 1995. CcpA-cre interaction was achieved in the absence of cofactors in amyE (Kim et al., 1995), whereas HPr-Ser46-P was needed for binding to cre in the gnt operon (Fujita et al., 1995). The results presented here are in agreement with the latter
General methods
Restriction enzymes and bacteriophage T4 polynucleotide kinase were purchased from Biolabs (Schwalbach, FRG). Sugar phosphates and chemicals were purchased from Sigma (Munich, FRG) or Merck (Darmstadt, FRG) in the highest available purity. Recombinant DNA techniques, Southern hybridisation and DNA sequencing were as described Sambrook et al 1989, Southern 1975, Maxam and Gilbert 1980. Plasmid DNA from E. coli was prepared using the Nucleobond kit (Macherey and Nagel, Düren, FRG). B. megaterium
Acknowledgements
We thank Dr Benno Müller-Hill for kindly providing Lac repressor protein. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the EC Biotec program and the Fonds der Chemischen Industrie.
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