Abstract
4'-Phosphopantetheine transferases (PPTases) transfer the 4'-phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4'-phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcB encoding AcpS from B. subtilis, which complemented an Escherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp(+) strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF260727
We have investigated the 4-phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) but only two phosphopantetheine transferases (PPTases), the acyl carrier protein synthase ACP synthase (AcpS) of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin
Construction of an E. coli acps Disruption Mutant as a Genetic Tool—Since acps is an essential gene in E. coli [20], a disruption mutant can only be generated when a complementing gene is present in trans
Summary
General Techniques—E. coli was grown on LB medium. B. subtilis was usually grown and maintained on Difco sporulation medium [11]; for preparations of chromosomal DNA, it was grown on LB medium. Construction of pQE60-ACP—The acpA gene encoding ACP was PCR-amplified with oligonucleotides 5Ј-AATTCCATGGCAGACACATTAGAGCGT-3Ј and 5Ј-TTTTGGATCCTTGCTGGTTTTGTATGTAGTTCAC-3Ј from chromosomal DNA of B. subtilis MR168 and ligated into the NcoI and BamHI sites of pQE60 to give the expression plasmid pQE60-acpA. Construction of pQE60-dltC—The dltC gene encoding the D-alanyl carrier protein (DCP) was amplified by PCR with oligonucleotides 5ЈATACCATGGATTTTAAACAAGAGG-3Ј and 5Ј-ATAAGATCTTTTCAACTCAGACAGCT-3Ј from chromosomal DNA of B. subtilis MR168 and ligated into the NcoI and BglII sites of pQE60. Reaction mixtures containing 50 mM Tris/HCl, pH 8.8, 12.5 mM MgCl2, 2 mM dithiothreitol, a 6 –100 M concentration of the respective acyl carrier protein, 2–20 M CoA, 119 – 475 nM [3H]CoA (specific activity: 40 Ci/mmol, 0.95 mCi/ml), 2.2 M to 5.6 nM AcpS of B. subtilis, or 0.8 M Sfp were incubated at 37 °C for 5–30 min. Carrier proteins were dialyzed before the assay against the respective assay buffer (50 mM Tris/HCl, pH 8.8, in the case of AcpS and 75 mM MES, pH 6.0, in the case of Sfp)
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