Catabolite Regulation of the pta Gene as Part of Carbon Flow Pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase

Design of artificial cell–cell communication using gene and metabolic networks

Artificial transcriptional networks have been used to achieve novel, nonnative behavior in bacteria. Typically, these artificial circuits are isolated from cellular metabolism and are designed to function without intercellular communication. To attain concerted biological behavior in a population, synchronization through intercellular communication is highly desirable. Here we demonstrate the design and construction of a gene-metabolic circuit that uses a common metabolite to achieve tunable artificial cell–cell communication. This circuit uses a threshold concentration of acetate to induce gene expression by acetate kinase and part of the nitrogen-regulation two-component system. As one application of the cell–cell communication circuit we created an artificial quorum sensor. Engineering of carbon metabolism in Escherichia coli made acetate secretion proportional to cell density and independent of oxygen availability. In these cells the circuit induced gene expression in response to a threshold cell density. This threshold can be tuned effectively by controlling ΔpH over the cell membrane, which determines the partition of acetate between medium and cells. Mutagenesis of the enhancer sequence of the glnAp(2) promoter produced variants of the circuit with changed sensitivity demonstrating tunability of the circuit by engineering of its components. The behavior of the circuit shows remarkable predictability based on a mathematical design model

Transcriptional Activation of the Bacillus subtilis ackA Promoter Requires Sequences Upstream of the CcpA Binding Site

Carbon catabolite protein A (CcpA) is a global regulator of carbon metabolism in gram-positive bacteria, repressing transcription of genes for the utilization of secondary carbon sources in the presence of a readily metabolized carbon source and activating transcription of genes, such as ackA and pta, that are required for carbon excretion. The promoter region of the Bacillus subtilis ackA gene contains two catabolite responsive elements (cre sites), of which only the site closest to the promoter (cre2) binds CcpA to activate transcription. A region immediately upstream of the cre2 site is also important for transcriptional activation. The required elements in this region were further defined by mutagenesis. CcpA binds to the ackA promoter region in gel shift assays even in the presence of mutations in the upstream element that block transcriptional activation, indicating that this region has a function other than promoting binding of CcpA

Catabolite Repression of the Bacillus subtilis FadR Regulon, Which Is Involved in Fatty Acid Catabolism ▿

The Bacillus subtilis fadR regulon involved in fatty acid degradation comprises five operons, lcfA-fadR-fadB-etfB-etfA, lcfB, fadN-fadA-fadE, fadH-fadG, and fadF-acdA-rpoE. Since the lcfA-fadRB-etfBA, lcfB, and fadNAE operons, whose gene products directly participate in the β-oxidation cycle, had been found to be probably catabolite repressed upon genome-wide transcript analysis, we performed Northern blotting, which indicated that they are clearly under CcpA-dependent catabolite repression. So, we searched for catabolite-responsive elements (cre's) to which the complex of CcpA and P-Ser-HPr binds to exert catabolite repression by means of a web-based cis-element search in the B. subtilis genome using known cre sequences, which revealed the respective candidate cre sequences in the lcfA, lcfB, and fadN genes. DNA footprinting indicated that the complex actually interacted with these cre's in vitro. Deletion analysis of each cre using the lacZ fusions with the respective promoter regions of the three operons with and without it, indicated that these cre's are involved in the CcpA-dependent catabolite repression of the operons in vivo