CcpA-Mediated Catabolite Activation of the Bacillus subtilis ilv-leu Operon and Its Negation by Either CodY- or TnrA-Mediated Negative Regulation

Abstract

The Bacillus subtilis ilv-leu operon functions in the biosynthesis of branched-chain amino acids. It undergoes catabolite activation involving a promoter-proximal cre which is mediated by the complex of CcpA and P-Ser-HPr. This activation of ilv-leu expression is negatively regulated through CodY binding to a high-affinity site in the promoter region under amino acid-rich growth conditions, and it is negatively regulated through TnrA binding to the TnrA box under nitrogen-limited growth conditions. The CcpA-mediated catabolite activation of ilv-leu required a helix face-dependent interaction of the complex of CcpA and P-Ser-HPr with RNA polymerase and needed a 19-nucleotide region upstream of cre for full activation. DNase I footprinting indicated that CodY binding to the high-affinity site competitively prevented the binding of the complex of CcpA and P-Ser-HPr to cre. This CodY binding not only negated catabolite activation but also likely inhibited transcription initiation from the ilv-leu promoter. The footprinting also indicated that TnrA and the complex of CcpA and P-Ser-HPr simultaneously bound to the TnrA box and the cre site, respectively, which are 112 nucleotides apart; TnrA binding to its box was likely to induce DNA bending. This implied that interaction of TnrA bound to its box with the complex of CcpA and P-Ser-HPr bound to cre might negate catabolite activation, but TnrA bound to its box did not inhibit transcription initiation from the ilv-leu promoter. Moreover, this negation of catabolite activation by TnrA required a 26-nucleotide region downstream of the TnrA box. Branched-chain amino acids (BCAAs) are the most abundant amino acids (AAs) in proteins and form the hydrophobic cores of the proteins. Moreover, these AAs are precursors for the biosynthesis of iso-and anteiso-branched fatty acids, which represent the major fatty acid species of the membrane lipids in Bacillus species (1). The initial step of isoleucine or valine synthesis is the condensation of threonine and pyruvate or two pyruvates, which leads to the formation of branched-chain keto-acids (2). Leucine is synthesized from a branched-chain keto acid, i.e., ␣-ketoisovalerate. The Bacillus subtilis ilv-leu operon comprises seven genes (ilvB, -H, and -C and leuA, -B, -C, and -D) necessary for the biosynthesis of BCAAs (3) (Fig. 1). Ludwig et al. first communicated that the ilv-leu operon was positively regulated by CcpA (4). Subsequently, it was revealed that transcription of the ilv-leu operon undergoes catabolite activation (CA) involving the complex of CcpA and P-Ser-HPr proteins (5, 6), which mediates carbon catabolite control of not only hundreds of catabolic operons and genes but also several cellular processes (7). This CcpA-mediated CA of ilv-leu, which is evoked by binding of the complex of CcpA and P-Ser-HPr to the catabolite-responsible element (cre) site (Fig. 1), links carbon metabolism to AA anabolism. Global gene expression studies on AA availability (8) and CodY regulation (9), as well as a study on metabolic linking...

Metabolic engineering of Bacillus subtilis forl‐valine overproduction

“…Accordingly, we constructed the supercompetent strain AW001‐5 that coexpressed xylose‐inducible comK and comS from the bpr locus (Figure ). Given that ilvBHC are expressed as part of a single operon and the regulatory elements controlling its expression are spatially defined, we chose to replace the 677 bp region upstream of the ilvB ribosome binding site (including a TnrA box [Tojo et al, ], all four CodY binding sites, a catabolite repression element and its upstream binding site [Fujita, Satomura, Tojo, & Hirooka, ], the tRNA Leu T‐box, and P ilv ‐ leu ) with the strong constitutive promoter P grac (Phan, Nguyen, & Schumann, ) in strain AW002‐5 (Figure ). In addition, we simultaneously mutated bcd to prevent the conversion of l ‐valine to 2‐ketoisovalerate in AW002‐5.…”

“…Given that ilvBHC are expressed as part of a single operon and the regulatory elements controlling its expression are spatially defined, we chose to replace the 677 bp region upstream of the ilvB ribosome binding site (including a TnrA box [Tojo et al, 2004], all four CodY binding sites, a catabolite repression element and its upstream binding site [Fujita, Satomura, Tojo, & Hirooka, 2014], the tRNA Leu T-box, and P ilv-leu ) with the strong constitutive promoter P grac (Phan, Nguyen, & Schumann, 2012) in strain AW002-5 ( Figure 2).…”

Deciphering the interactions between the Bacillus cereus linear plasmid, pBClin15, and its host by high-throughput comparative proteomics

“…LexA represses the SOS response to DNA damage and regulates the lytic switch in the Bacillus thuriengensis temperate phage, GIL01 [19]. CcpA plays a major role in the coordinated regulation of catabolism and anabolism to ensure optimum cell propagation [53]. It also plays a key role in toxin gene expression and virulence [54].…”

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

“…Previous studies reported that CodY often acts additively or antagonistically with another regulator named CcpA to regulate carbon and nitrogen metabolism (21)(22)(23). CcpA can mediate repression of virulence gene expression and carbon catabolism gene expression in Gram-positive bacteria, including some Clostridium spp.…”

“…CcpA is a member of the LacI/GalR family of transcriptional regulators (19,20) and often works together with CodY to regulate, directly or indirectly, the expression of hundreds of genes involved in carbon and nitrogen utilization (21)(22)(23). CcpA interacts with proteins like phosphorylated HPr, which increases its affinity for certain DNA binding sites and results in repression or activation of gene transcription (20,24).…”