Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

Abstract

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.

FIG. 1

Plasmid construction. Various B. subtilis chromosomal DNA fragments (horizontal lines) containing the pta gene (bold arrow or bar) were inserted into the vectors listed at the right. Designations for the resulting plasmids are given at the left. The B. subtilis chromosomal DNA fragments are numbered relative to the identified transcriptional start site of the pta gene. The promoter region of the pta gene (P) and the CRE (open box) are indicated. Some restriction sites are also shown.

FIG. 2

pta promoter region. The nucleotide sequence of a 291-bp-long DNA fragment containing the pta promoter and the beginning of the pta gene is presented. The vertical arrow indicates the position of the transcription start site, +1. The primer used for the primer extension experiment is indicated by the long horizontal arrow. The −10 and −35 regions, corresponding to the RNA polymerase binding site, are indicated. The potential ribosome-binding site (Shine-Dalgarno [SD]) and the CRE sequence are boxed. The bent arrows at positions −109, −49, and −22 indicate the start of the CRE, ΔCRE, and ΔP fragments used for the construction of the pta-lacZ fusions in pDIA5377, pDIA5378, and pDIA5381 (Fig. 1).

FIG. 3

Mapping of the transcription start site of the pta gene by primer extension. Total RNAs was extracted from B. subtilis 168 grown in CSK medium in the presence (lane 1) or absence (lane 2) of 0.4% glucose. In lane 3, the labelled oligonucleotide was loaded as a control. The labelled primer used for reverse transcription is indicated in Fig. 2. Sequencing reactions were performed with the same oligonucleotide as a primer and pDIA5373 as the template.

FIG. 4

Importance of the CRE and −35 promoter region for pta expression. We monitored the expression of pta-lacZ expression over time for strains BSIP1114 (□), BSIP1115 (○), and BSIP1116 (▵). These strains contain the CREpta, ΔCREpta, and ΔPpta fragments (Fig. 2) fused to the lacZ gene inserted at the amyE locus. The strains were grown in CSK medium in the absence (open symbols) or presence (closed symbols) of 0.4% glucose.

FIG. 5

DNase I footprinting experiments with the pta promoter region and trans-acting CCR proteins. (A) Footprinting with the 248-bp EcoRI-HindIII DNA fragment containing the CREpta (−109 to +139) (Fig. 2); (B) footprinting with the 188-bp EcoRI-HindIII DNA fragment ΔCREpta (−49 to +139) (Fig. 2). The CREpta and ΔCREpta fragments were obtained from plasmids pDIA5379 and pDIA5380, respectively, and labelled at the 3′ end as described in Materials and Methods. A+G standards for Maxam and Gilbert reactions were made for each fragment. The DNA sequence of the promoter region of the two fragments (from positions −84 to −33 [A] and −14 to +34 [B]) as well as the protected areas are indicated. DNase I digestions were performed as indicated in Materials and Methods with both fragments. In each case, the DNA was digested in the absence of proteins (lane 1) or in the presence of 2 μM CcpA (lane 2), 2 μM CcpA and 10 μM HPr (lane 3), 2 μM CcpA and 10 μM HPrSer-P (lane 4), 2 μM CcpA, 10 μM HPrSer-P, and 20 mM FBP (lane 5), 2 μM CcpA and 10 μM Crh (lane 6), 2 μM CcpA and 10 μM CrhSer-P (lane 7), or 2 μM CcpA, 10 μM CrhSer-P, and 20 mM FBP (lane 8).

FIG. 6

pta promoter regions protected in DNase I footprinting experiments. The sequence of the pta promoter region from positions −70 to +29 is presented. The CRE sequence is boxed, and the CRE consensus sequence is indicated. The bases protected against digestion with DNase I are indicated by asterisks.