Functional studies of acyl carrier protein and the phosphopantetheinyl transferase AcpT in Escherichia coli K-12

Acyl carrier protein (ACP) is a small, acidic protein that shuttles intermediates between fatty acid biosynthetic enzymes. Escherichia coli ACP is post-translationally modified by the enzyme AcpS which transfers a 4'-phosphopantetheine group from CoA to a conserved serine residue on the carrier protein. The thiol group provided by the phosphopantetheinyl moiety is the attachment site for biosynthetic intermediates being shuttled by the carrier protein between fatty acid biosynthetic enzymes.; The lack of a conditional mutant of acpP, the gene encoding E. coli ACP, was an obstacle preventing thorough in vivo studies of the functions of ACP and the importance of particular residues of ACP in performing these functions. I have isolated and characterized three temperature-sensitive acpP mutants of E. coli. I have tested several site directed mutants of E. coli ACP and ACPs from several diverse bacterial and eukaryotic species for the ability to complement one of these acpP(Ts) mutants. I found that several mutant ACPs containing substitutions of ACP residues previously reported to be required for ACP function in vitro support normal growth of the acpP mutant. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth.; A phosphopantetheinyl transferase called AcpT, which is encoded in the genome of E. coli strains, was previously reported to suppress an acpS mutant upon overexpression, but the purified AcpT failed to modify E. coli ACP in vitro. I have found that the growth of E. coli in the absence of AcpS activity is dependent on having a functional copy of acpT. I have also identified two carrier protein substrates for AcpT that are encoded in the genome of E. coli O157:H7.; Overexpression of AcpT was reported to suppress an E. coli K-12 mutant that has a temperature-sensitive phenotype and lipid A biosynthetic defect. I have mapped the mutation responsible for the lipid A biosynthetic defect and temperature-sensitive phenotype of this strain and have further characterized the suppression of the temperature-sensitive phenotype of strain LH530 by overexpression of AcpT.