CcpA, the repressor/activator mediating carbon catabolite repression and glucose activation in many Gram-positive bacteria, has been purified from Bacillus megaterium after fusing it to a His tag. CcpA-his immobilized on a Ni-NTA resin specifically interacted with HPr phosphorylated at seryl residue 46. HPr, a phospho-carrier protein of the phosphoenolpyruvate: glycose phosphotransferase system (PTS), can be phosphorylated at two different sites: (i) at His-15 in a PEP-dependent reaction catalysed by enzyme I of the PTS; and (ii) at Ser-46 in an ATP-dependent reaction catalysed by a metabolite-activated protein kinase. Neither unphosphorylated HPr nor HPr phosphorylated at His-15 nor the doubly phosphorylated HPr bound to CcpA. The interaction with seryl-phosphorylated HPr required the presence of fructose 1,6-bisphosphate. These findings suggest that carbon catabolite repression in Gram-positive bacteria is a protein kinase-triggered mechanism. Glycolytic intermediates, stimulating the corresponding protein kinase and the P-ser-HPr/CcpA complex formation, provide a link between glycolytic activity and carbon catabolite repression. The sensitivity of this complex formation to phosphorylation of HPr at His-15 also suggests a link between carbon catabolite repression and PTS transport activity.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

No abstract

Catabolite repression of a number of catabolic operons in bacilli is mediated by the catabolite control protein CcpA, the phosphocarrier protein HPr from the phosphoenolpyruvate-dependent sugar transport system (PTS), and a cis-acting DNA sequence termed the catabolite-responsive element (cre). We present evidence that CcpA interacts with HPr that is phosphorylated at Ser 46 (Ser(P) HPr) and that these proteins form a specific ternary complex with cre DNA. Titration experiments following the circular dichroism signal of the cre DNA indicate that this complex consists of two molecules of Ser(P) HPr, a CcpA dimer, and the cre sequence. Limited proteolysis experiments indicate that the domain structure of CcpA is similar to other members of the LacI/GalR family of helix-turn-helix proteins, comprised of a helix-turn-helix DNA domain and a C-terminal effector domain. NMR titration of Ser(P) HPr demonstrates that the isolated C-terminal domain of CcpA forms a specific complex with Ser(P) HPr but not with unphosphorylated HPr. Based upon perturbations to the NMR spectrum, we propose that the binding site of the C-terminal domain of CcpA on Ser(P) HPr forms a contiguous surface that encompasses both Ser(P) 46 and His 15 , the site of phosphorylation by enzyme I of the PTS. This allows CcpA to recognize the phosphorylation state of HPr, effectively linking the process of sugar import via the PTS to catabolite repression in bacilli. Catabolite repression (CR)1 in Escherichia coli has provided a general paradigm for understanding the regulation of the synthesis of various catabolic enzymes in response to the availability of rapidly metabolizable carbon sources. The mechanism for CR in bacteria such as E. coli involves the cyclic AMP-dependent action of the catabolite gene activator protein, CAP (1). In Bacillus subtilis, however, the mechanism is completely different, because no cyclic AMP or CAP homologue is present (2). CR in some bacilli and a few other Gram-positive organisms has been shown to be dependent on the catabolite control protein A (CcpA), which is a member of the LacI/GalR family of regulators (3, 4), and a cis-active operator DNA sequence, termed the catabolite-responsive element (cre), which has been identified in the promoter or in the 5Ј region of 29 B. subtilis genes (5).There is growing evidence that CR is mechanistically linked to the phosphoenolpyruvate-dependent sugar transport system (PTS), which is responsible for the import of various sugars (1). Mutation of Ser 46 to Ala in the PTS phosphocarrier protein HPr results in resistance to CR for several catabolic genes in B. subtilis (6). Ser 46 in HPr is known to be phosphorylated by an ATP-dependent kinase that is activated by glycolytic intermediates such as fructose-1,6-diphosphate (7). Confirming the link between CR and the PTS, recent DNase I protection experiments have shown that cre sequences are specifically protected by CcpA only in the presence of HPr phosphorylated at Ser 46 (Ser(P) HPr) (8). Other mechanisms for CR have been established...

The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, lactic acid bacteria, and some actinomycetes.

SummaryThe catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram-positive bacteria. A comparison of 12 CcpA-like sequences with regulators from the LacI/ GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR-like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N-terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium. Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild-type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr-Ser-46-P in vitro, establishing a correlation between catabolite repression efficiency and HPr-Ser-46-P binding. These results support the hypothesis that the conserved region in CcpA is the HPr-Ser-46-P binding site.

Carbon catabolite repression of the gnt operon of Bacillus subtilis is mediated by the catabolite control protein CcpA and by HPr, a phosphocarrier protein of the phosphotransferase system. ATP-dependent phosphorylation of HPr at Ser-46 is required for carbon catabolite repression as ptsH1 mutants in which Ser-46 of HPr is replaced with an unphosphorylatable alanyl residue are resistant to carbon catabolite repression. We here demonstrate that mutation of His-15 of HPr, the site of phosphoenolpyruvate-dependent phosphorylation, also prevents carbon catabolite repression of the gnt operon. A strain which expressed two mutant HPrs (one in which Ser-46 is replaced by Ala [S46A HPr] and one in which His-15 is replaced by Ala [H15A HPr]) on the chromosome was barely sensitive to carbon catabolite repression, although the H15A mutant HPr can be phosphorylated at Ser-46 by the ATP-dependent HPr kinase in vitro and in vivo. The S46D mutant HPr which structurally resembles seryl-phosphorylated HPr has a repressive effect on gnt expression even in the absence of a repressing sugar. By contrast, the doubly mutated H15E,S46D HPr, which resembles the doubly phosphorylated HPr because of the negative charges introduced by the mutations at both phosphorylation sites, had no such effect. In vitro assays substantiated these findings and demonstrated that in contrast to the wild-type seryl-phosphorylated HPr and the S46D mutant HPr, seryl-phosphorylated H15A mutant HPr and H15E,S46D doubly mutated HPr did not interact with CcpA. These results suggest that His-15 of HPr is important for carbon catabolite repression and that either mutation or phosphorylation at His-15 can prevent carbon catabolite repression.The bacterial phosphotransferase system (PTS) consists of a number of phosphocarrier proteins that utilize the phosphoryl group of phosphoenolpyruvate (PEP) to phosphorylate incoming sugars. The phosphoryl transfer process is PEP3enzyme I3HPr3enzyme IIA3enzyme IIBC3sugar-P (24).HPr, the second phosphocarrier protein of the PTS, is phosphorylated on histidyl residue 15 via this phosphorylation chain, thus generating histidyl-phosphorylated HPr (P-His HPr) (17). In addition, a metabolite-activated ATP-dependent protein kinase in gram-positive bacteria is known to phosphorylate HPr on Ser-46 giving rise to seryl-phosphorylated HPr (P-Ser HPr) (6,10,12,25,26). We have recently shown that P-Ser HPr and a DNA-binding protein of the LacI-GalR family, CcpA (19, 35), interact to mediate carbon catabolite repression of the gnt operon in Bacillus subtilis (9,11,16). Further, data have demonstrated that GntR, the repressor of the B. subtilis gnt operon, is not involved in carbon catabolite repression of the gnt operon (15) and that neither inducer exclusion nor inducer expulsion is operative (11,15).The ATP-dependent HPr kinase is allosterically activated by cytoplasmic phosphorylated metabolites such as fructose-1,6-bisphosphate (FBP) (6, 26), and it specifically phosphorylates HPr at Ser-46 (10). P-Ser HPr binds to CcpA in the presence ...

Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon. Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source. Expression of XylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre. Northern analysis of the xyl mRNA reveals that a potential stem‐loop structure located in the non‐translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5′‐xylA portion is very abundant, while the 3′‐xylBT portion constitutes only a fraction of it. XylT has an apparent Michaelis constant (KM) of approx. 100 μM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.