Yasuhiko Miwa

Specific recognition of the Bacillus subtilis gnt cis‐acting catabolite‐responsive element by a protein complex formed between CcpA and seryl‐phosphorylated HPr

Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis‐acting catabolite‐responsive element (ORE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR‐Lacl family of bacterial transcriptional repressors/activators, and the seryl‐phosphorylated form of HPr, a phosphocarrier protein of the phosphoenol‐pyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P‐ser‐HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to Tat position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P‐ser‐HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P‐ser‐HPr.

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite‐responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis‐acting catabolite‐responsive element (cre) by a complex of CcpA, which is a member of the GalR‐LacI family of bacterial regulatory proteins, and the seryl‐phos‐phorylated form of HPr (P‐ser‐HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere located in the promoter region (creup). A translational gntR‐lacZ fusion expressed under the control of various combinations of wild‐type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of p‐galac‐tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P‐ser‐HPr, and catabolite repression exerted by credown was partially independent of P‐ser‐HPr. DNase I footprinting experiments indicated that a complex of CcpA and P‐ser‐HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose‐6‐phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.

Possible function and some properties of the CcpA protein of Bacillus subtilis

The ccpA mutations alsA1 (alsA1 is allelic to ccpA) and ccpA::Tn917 completely abolished catabolite repression of gluconate kinase and sorbitol dehydrogenase synthesis in Bacillus subtilis, whereas they only partially affected the catabolite repression of inositol dehydrogenase, histidase and xylose isomerase synthesis. The alsA1 mutation also partially affected catabolite repression of sporulation. Analysis of revertants from the alsA1 mutant by direct sequencing indicated that this mutation comprises a base substitution of guanine at nucleotide -14 to adenine within the Shine-Dalgarno sequence of the ccpA gene (ccpA translation starts at nucleotide +1). A 1.37 kb EcoRI fragment carrying the ccpA gene was cloned into Escherichia coli plasmid pUC19 and B. subtilis plasmid pUB110, resulting in plasmids pCCPA19 and pCCPA110, respectively. The ccpA gene carried in pCCPA110 complemented the alsA1 mutation. Western blotting revealed that the level of the CcpA protein in B. subtilis cells, which seemed to be constitutively synthesized, was approximately 10 times lower for the alsA1 mutant than for the wild-type. The CcpA protein synthesized by either E. coli cells bearing pCCPA19 or B. subtilis cells bearing pCCPA110 was purified to over 90% homogeneity; the latter cells were grown in the presence of glucose. The molecular mass of the protein purified from E. coli was 74 kDa, suggesting that this protein exists as a dimer because its subunit molecular mass was 38 kDa as determined by SDS-PAGE. Gel retardation analysis indicated that the purified CcpA protein in both cases did not bind to the cis sequence for catabolite repression of the gnt operon, but it bound non-specifically to DNA.

Sequencing of a 65 kb region of the Bacillus subtilis genome containing the lic and cel loci, and creation of a 177 kb contig covering the gnt-sacXY region

Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, this paper communicates the sequencing of a chromosome region containing the lic and cel loci (65 kb), which creates a 177 kb contig covering the region from gnt to sacXY. This 65 kb region contains 64 ORFs (62 complete and two partial genes). The 14th, 15th and 17th genes correspond to licT, licS and katE, encoding the antiterminator for licS transcription, beta-glucanase (lichenase) and catalase 2, respectively. The 11th, 30th, 36th, 39th, 41st, 45th-48th, 51st and 58th genes are designated deaD, pepT, galE, aldY, msmX, cydABCD, sigY and katX because their products probably encode ATP-dependent RNA helicase, tripeptidase, UDP-glucose 4-epimerase, aldehyde dehydrogenase, multiple sugar-binding transport ATP-binding protein, the respective components of cytochrome d ubiquinol oxidase and ATP-binding cassette transporter, sigma-factor of RNA polymerase and catalase, respectively. The 60th-64th genes are celRABCD, which are probably involved in cellobiose utilization. Gene organization and gene features in the gnt-sacXY region are discussed.

Cloning and nucleotide sequencing of a 15 kb region of the Bacillus subtilis genome containing the iol operon

Within the framework of an international project on the sequencing of the whole Bacillus subtilis genome, a 15 kb chromosome segment, which contains the iol operon involved in inositol utilization, has been cloned and sequenced. This region (14,974 bp) contains 12 complete open reading frames (ORFs; genes) and two partial ones; the seventh gene (E83G) is the idh gene encoding inositol dehydrogenase. All the genes identified are transcribed in the same direction as that of the movement of the replication fork. A homology search for their products deduced from the 12 complete ORFs revealed that eight of them exhibit significant homology to known proteins such as fructokinase, acetolactate synthase, fructose-1,6-bisphosphate aldolase (B. subtilis), and PhoB and FtsE proteins (Escherichia coli). It also implied that two genes (B65D and B65E) might encode a set of two-component regulatory proteins and that the B65F gene might encode a protein belonging to the ATP-binding cassette (ABC) family. Based on the features of the nucleotide sequence determined and the results of the homology search, the primary structure of the iol operon is predicted.

Analysis of an insertional operator mutation (gntOi) that affects the expression level of theBacillus subtilis gnt operon, and characterization ofgntOi suppressor mutations

TheBacillus subtilis gnt operon is negatively regulated via interaction of thegnt repressor (GntR) with an operator upstream ofgntR, which is antagonized by gluconate. An 8 bp insertional operator mutation (gntOi) of thegnt operon was constructed which affected the expression level of this operon. Two suppressors of thisgntOi mutation, exhibiting normal expression, were also isolated; one involved a threonine substitution for the Ala-48 residue (gntR48T) within the helix-turn-helix DNA-binding motif of GntR, and the other an adenine substitution for the guanine at nucleotide — 4 within thegntOi operator (gntOiM4A) (+1 is the transcription initiation site). ThegntR48T mutation by itself rendered thegnt operon partially constitutive. When thegntR43L mutation, which renders thegnt operon fully constitutive, was introduced into thegntOi orgntOiM4A mutant, the operator mutations were found not to affect the promoter activity of thegnt operon. These in vivo results indicate that thegntOi mutation affects the operator interaction with GntR, causing a low expression level even in the presence of gluconate. In vitro gel retardation and DNase I footprint analyses demonstrated that even when gluconate was present, GntR still bound to thegntOi operator region.