The Bacillus subtilis enzyme Sfp, required for production of the lipoheptapeptide antibiotic surfactin, posttranslationally phosphopantetheinylates a serine residue in each of the seven peptidyl carrier protein domains of the first three subunits (SrfABC) of surfactin synthetase to yield docking sites for amino acid loading and peptide bond formation. With recombinant Sfp and 16-17-kDa peptidyl carrier protein (PCP) domains excised from the SrfB1 and SrfB2 modules as apo substrates, kcat values of 56-104 min-1 and K(m) values of 1.3-1.8 microM were determined, indicating equivalent recognition of the adjacent PCP domains by Sfp. In contrast to other phosphopantetheinyl transferases (PPTases) previously examined, Sfp will modify the apo forms of heterologous recombinant proteins, including the PCP domain of Saccharomyces cerevisiae Lys2 (involved in lysine biosynthesis), the aryl carrier protein (ArCP) domain of Escherichia coli EntB (involved in enterobactin biosynthesis), and the E. coli acyl carrier protein (ACP) subunit, suggesting Sfp as a good candidate for heterologous coexpression with peptide and polyketide synthase genes to overproduce holo-synthase enzymes. Cosubstrate coenzyme A (CoA), the phosphopantetheinyl group donor, has a K(m) of 0.7 microM. Desulfo-CoA and homocysteamine-CoA are also substrates of Sfp, and benzoyl-CoA and phenylacetyl-CoA are also utilized by Sfp, resulting in direct transfer of acyl phosphopantetheinyl moieties into the carrier protein substrate. Mutagenesis in Sfp of five residues conserved across the PPTase family was assessed for in vivo effects on surfactin production and in vitro effects on PPTase activity.

SummaryCell-density-dependent gene expression appears to be widely spread in bacteria. This quorum-sensing phenomenon has been well established in Gramnegative bacteria, where N-acyl homoserine lactones are the diffusible communication molecules that modulate cell-density-dependent phenotypes. Similarly, a variety of processes are known to be regulated in a cell-density-or growth-phase-dependent manner in Gram-positive bacteria. Examples of such quorumsensing modes in Gram-positive bacteria are the development of genetic competence in Bacillus subtilis and Streptococcus pneumoniae, the virulence response in Staphylococcus aureus, and the production of antimicrobial peptides by several species of Gram-positive bacteria including lactic acid bacteria. Cell-densitydependent regulatory modes in these systems appear to follow a common theme, in which the signal molecule is a post-translationally processed peptide that is secreted by a dedicated ATP-binding-cassette exporter. This secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system. Moreover, genetic linkage of the common elements involved results in autoregulation of peptide-pheromone production.

Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.