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Originally published In Press as doi:10.1074/jbc.M703789200 on May 23, 2007

J. Biol. Chem., Vol. 282, Issue 28, 20319-20328, July 13, 2007
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In Vivo Functional Analyses of the Type II Acyl Carrier Proteins of Fatty Acid Biosynthesis*Formula

Nicholas R. De Lay{ddagger} and John E. Cronan{ddagger}§1

From the Departments of {ddagger}Microbiology and §Biochemistry University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, May 8, 2007 , and in revised form, May 22, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acyl carrier protein (ACP) is a key component of the fatty acid synthesis pathways of both type I and type II synthesis systems. A large number of structure-function studies of various type II ACPs have been reported, but all are in vitro studies that assayed function or interaction of mutant ACPs with various enzymes of fatty acid synthesis or transfer. Hence in these studies functional properties of various mutant ACPs were assayed with only a subset of the many ACP-interacting proteins, which may not give an accurate overall view of the function of these proteins in vivo. This is especially so because Escherichia coli ACP has been reported to interact with several proteins that have no known roles in lipid metabolism. We therefore tested a large number of mutant derivatives of E. coli ACP carrying single amino acid substitutions for their abilities to restore growth to an E. coli strain carrying a temperature-sensitive mutation in acpP, the gene that encodes ACP. Many of these mutant proteins had previously been tested in vitro thus providing data for comparison with our results. We found that several mutant ACPs containing substitutions of ACP residues reported previously to be required for ACP function in vitro support normal growth of the acpP mutant strain. However, several mutant proteins reported to be severely defective in vitro failed to support growth of the acpP strain in vivo (or supported only weak growth). A collection of ACPs from diverse bacteria and from three eukaryotic organelles was also tested. All of the bacterial ACPs tested restored growth to the E. coli acpP mutant strain except those from two related bacteria, Enterococcus faecalis and Lactococcus lactis. Only one of the three eukaryotic organellar ACPs allowed growth. Strikingly the ACP is that of the apicoplast of Plasmodium falciparum (the protozoan that causes malaria). The fact that an ACP from a such diverse organism can replace AcpP function in E. coli suggests that some of the protein-protein interactions detected for AcpP may be not be essential for growth of E. coli.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Both type I and type II fatty acid biosynthetic systems have a key component, the acyl carrier protein (ACP),2 that is essential for function (1–3). In both synthetic systems the ACP component carries fatty acyl intermediates among the different enzyme active sites. To perform this function ACP must be posttranslationally modified via the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of ACP. Attachment of this prosthetic group is mandatory because the thiol group of the phosphopantetheinyl moiety carries the intermediates of fatty acid biosynthesis.

In the type I systems found in the mammalian cytosol, ACP is a domain of a very large polypeptide that contains all the enzymatic domains required for fatty acid biosynthesis (4). The functional mammalian fatty-acid synthase is a dimer of two identical polypeptides in head-to-head orientation that synthesizes the fatty acids used for complex lipid biosynthesis (5–7). The finished full-length fatty acid is cleaved from the ACP domain and released into the cytosol by a thioesterase domain. In the fungal type I systems the active sites are divided between two non-identical proteins that function as hexamers with the ACP domain on the {alpha} subunit (7, 8). Although the two type I enzymes have strikingly different structures and produce different products (the fungal enzymes produce acyl-CoAs), they function by the same general mechanism (8).

In the type II systems found in bacteria, mitochondria, most plant plastids, and apicomplexan protozoans, ACPs are small (8–10 kDa), discrete, and very soluble proteins that are highly acidic (9). Escherichia coli ACP, which is encoded by the gene acpP (10), was the first such protein discovered (11, 12) and remains the paradigm of this class of proteins. The structure of the butyrylated form of E. coli ACP has been solved (13) and shows that (as originally predicted (14)) E. coli ACP consists of four {alpha} helices that form a hydrophobic pocket. The hydrophobic pocket is able to accommodate an acyl chain up to eight carbons in length (15). Near the bottom of the pocket lies Ile-54, a residue that interacts with residues of helices II, III, and IV (13, 16–19) and thereby maintains the tertiary structure of AcpP (17, 18). Serine 36 of E. coli ACP is the site of 4'-phosphopantetheine attachment and is located at the end of helix II near the opening of the hydrophobic pocket. Prosthetic group attachment is catalyzed by the AcpS phosphopantetheinyl transferase (20–22). It should be noted that, although the mammalian fatty-acid synthase ACP is a protein domain rather than a discrete protein, upon expression of the isolated domain it was found to have a structure highly similar to that of E. coli ACP and to be a substrate for two type II bacterial fatty acid synthetic enzymes (23). Therefore, studies of type II ACPs should provide data useful in understanding the functions of type I ACP domains.

E. coli ACP not only interacts with AcpS and the fatty acid biosynthetic enzymes but also with several different acyltransferases involved in complex lipid synthesis. Each acyltransferase targets an acyl group of appropriate chain length and oxidation state to a specific biosynthetic pathway. There are separate acyltransferases involved in the biosynthesis of lipid A (LpxA, LpxM, and MsbB), phospholipids (PlsB, PlsC, and PlsX), lipoic acid (LipB), acylhomoserine lactones (LuxI), and protein toxins (HlyC-type proteins).

Several recent in vitro studies have attempted to identify residues important for the interaction of E. coli and other ACPs with various enzymes of lipid synthesis. A difficulty with these studies is that only the abilities of AcpP site-directed mutants to function as substrates for only one or two enzymes were assayed, although more general conclusions have sometimes been drawn. Another function often tested is the activity of mutant ACPs in the E. coli in vitro fatty acid synthetic system. Although this system has the advantage of involving an appreciable number of enzymes, some enzymes are not required for function of the in vitro system, and because the system functions at only a few percent of the rate of fatty acid biosynthesis in vivo (17, 24, 25), the assay is rather insensitive and probably does not reflect rate-limiting reactions in vivo. Other studies have assayed the activity of several mutant ACPs as the substrate for a single fatty acid biosynthetic enzyme (25–27). Although these studies have provided useful data, they are of a piecemeal nature and hence often difficult to interpret in physiological terms. For example, some mutant ACPs function in one in vitro assay but not in a another (17, 24, 28). Therefore, our understanding of the residues critical to the overall physiological function of ACP is very limited. The above in vitro approaches were taken largely because no acpP mutants were available to assay function in vivo. The acpP gene is essential (1), and thus conditionally defective mutants were required. We recently reported the isolation and characterization of the first conditionally defective acpP mutants. These acpP(Ts) mutants encode proteins that are functional at 30 °C but that rapidly lose function and are degraded at 42 °C (1).

In this study we report the abilities of a large panel of E. coli AcpP site-directed mutants to allow growth of an acpP(Ts) mutant strain under non-permissive conditions. We also tested the abilities of ACPs from a variety of other bacteria and eukaryote organelles to allow growth of the acpP(Ts) strain.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids—The strains used in this study were all derivatives of the E. coli K-12 strain MG1655, the genome sequence of which is known (Table 1). Strains NRD29 and NRD44 were described in a previous study (1). Strain NRD44 carries a nonpolar deletion of panD and was used in the beta-[3H]alanine labeling experiments described below. Strain NRD29, which contains a temperature-sensitive allele of acpP and a chloramphenicol resistance cassette inserted into the downstream gene fabF, was used in the complementation studies.


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  		TABLE 1
Bacterial strains

 
The {Delta}araBAD strain NRD204 was constructed by {lambda} Red recombinase-mediated gene replacement (29) in strain MG1655 harboring plasmid pKD46 using the PCR product generated from the template plasmid pKD3 using primers araBADKO-For and araBADKO-Rev (see supplemental material). The araBAD deletion of strain NRD204 was transduced via phage P1vir into the {Delta}lacZY strain JT34 generating strain NRD205. The cat cassette was subsequently removed from strain NRD205 using the FLP recombinase encoded on plasmid pCP20 (29) to yield strain NRD206. The yadC::Tn10 insertion of strain CAG12025 [GenBank] was transduced using phage P1vir into the pcnB mutant strain WT286 to yield strain NRD214. The presence of the pcnB286 allele in strains NRD214 and WT286 was confirmed by sequencing of the PCR product generated using the primers pcnB-For and pcnB-Rev. The pcnB286 allele was transduced from strain NRD214 into strain NRD206 by phage P1vir. Transductants were selected for the acquisition of tetracycline resistance and then screened for the presence of the pcnB286 allele by PCR using primers pcnB-For and pcnB-Rev. The PCR products generated from the amplification of the wild type pcnB and the pcnB286 alleles using primers pcnB-For and pcnB-Rev could be distinguished by gel electrophoresis because the pcnB286 allele is an 81-bp deletion. The transductant that acquired the tetracycline resistance of the yadC::Tn10 and the pcnB286 allele was named NRD216. The acpP(Ts) mutation of strain NRD28 was transduced via phage P1vir into strain NRD216 to yield strain NRD224. Strain NRD224 harboring the lacIq-containing plasmid pMS421 was used for the complementation studies with the pMR19-derived plasmids.

Plasmids derived from pMR19 (10) that encode ACPs with cysteine substitutions were obtained from M. Lou Ernst-Fonberg (25). All other plasmids used in this study are listed in Table 2. The primers used in this study (Table 2) were purchased from Integrated DNA Technologies, Inc. Cloned species ACPs were PCR-amplified from genomic or plasmid DNA and inserted into the pCR2.1 vector by TOPO cloning (Invitrogen) and sequenced using the M13For (–21) and M13Rev (–24) primers. Site-directed mutants of the synthetic E. coli ACP gene were made by QuikChange II site-directed mutagenesis (Stratagene) and were sequenced in the pBAD322 vector using pNRD26seq-For and -Rev primers. All sequencing was performed by the Core Sequencing Unit at the Keck Center for Comparative and Functional Genomics at the University of Illinois.


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  		TABLE 2
Summary of the complementation and 4'-phosphopantetheine modification results

 
Culture Media and Growth Conditions—All strains were grown in liquid medium or agar plates of RB or minimal E salts. RB or minimal E medium was supplemented at final concentrations with the following carbon sources: arabinose (0.2%), glucose (0.2%), or glycerol (0.8%). Antibiotics were added to liquid medium or agar plates at the following concentrations: kanamycin at 50 mg/liter or a combination of ticarcillin and clavulanate (Timentin; GlaxoSmithKline) at 25 mg/liter. For beta-alanine starvation, beta-alanine was added at a final concentration of 0.5 nM. For in vivo phosphopantetheinylation assays, beta-[2,3-3H]alanine (specific activity, 60 Ci/mmol; American Radiolabeled Chemicals) was used at a final concentration of 0.5 mM.

In Vivo Phosphopantetheinylation Analysis—The in vivo phosphopantetheinylation experiments were performed by a method similar to that described by De Lay and Cronan (1, 20) using derivatives of strain NRD44 harboring plasmids that express either E. coli AcpP, a site-directed mutant of E. coli AcpP, or an ACP encoded by the genome of another organism. Alternatively the experiments were performed using strain NRD44 derivatives that carry either a plasmid expressing Sfp or an empty vector in addition to a plasmid that encoded an ACP. The strains were first starved for beta-alanine on minimal E-glycerol-clavulanate-ticarcillin agar plates containing a trace amount of beta-alanine at 42 °C overnight. Each strain was then suspended in minimal E-glycerol-clavulanate-ticarcillin liquid medium to an A600 between 0.6 and 0.7. Arabinose and beta-[3H]alanine were then added to the medium, and the cultures were incubated at 42 °C for 2 h. The cells were harvested by centrifugation, suspended in 50 mM sodium 2-(N-morpholin-o)ethanesulfonic acid (pH 6.1), and sonicated. Cell debris were removed by centrifugation, and the concentration of soluble protein was determined using a Bio-Rad protein assay kit. Equal amounts of protein from each cell extract were fractionated on a 20% native polyacrylamide gel at 120 V. Prior to fractionation, dithiothreitol was added to each sample at a final concentration of 100 mM. The polyacrylamide gel was fixed, soaked in enhancer solution (NRAMP100V, Ambion), dried, and then exposed to preflashed x-ray film (Eastman Kodak Co. BIOmax XAR film) at –80 °C.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Complementation Analyses with E. coli acpP Site-directed Mutants—We tested the importance of a large number of E. coli AcpP residues for the function of ACP residues by testing the ability of site-directed ACP mutants to complement strains carrying an acpP(Ts) mutant allele (1). In the analyses performed in strain NRD29 the ACP mutant proteins were expressed from an arabinose-inducible araBAD promotor on a plasmid of moderate copy number (30). Several of the mutants assayed were constructed and previously assayed for various in vitro functions by Worsham et al. (25). We also generated and tested a number of additional AcpP site-directed mutants. Our AcpP mutants as well as those of Worsham et al. (25) were constructed in a synthetic acpP gene previously assembled in this laboratory (10, 31) (GenBankTM accession number AF072368). Twenty-six of the codons used in the synthetic acpP gene differ from those of the natural acpP gene. This lack of homology prevents efficient homologous recombination between the plasmid-encoded and chromosome-encoded genes. It should be noted that under our standard expression conditions (0.2% arabinose) the concentration of ACP was about 12-fold greater than that normally produced. At these levels of overproduction of wild type ACP or the various mutant ACPs there was no toxicity to the growth of a wild type strain. Concentrations of arabinose lower than 0.08% failed to give complementation with the wild type gene. Each of the mutant ACPs was tested at inducer concentrations of 0.08 and 0.2% with essentially identical results. Similar results were obtained in strains proficient in and blocked in arabinose catabolism.


Figure 1
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  		FIGURE 1.
Analyses of complementation of the acpP(Ts) mutant by and phosphopantetheinylation of several site-directed mutants of E. coli AcpP. A, derivatives of strain NRD29, an acpP(Ts) mutant, each harboring a plasmid containing a given site-directed mutant of E. coli acpP, were tested for their abilities to grow at 42 °C in the presence or absence of the inducer, arabinose. Some transformants of NRD29 with the plasmid containing the G33C AcpP grew at 42 °C in the presence of inducer, and other colonies did not. B, the {Delta}panD deletion strain NRD44 containing the same plasmids as in A were starved for beta-alanine overnight at 42 °C on minimal E-glycerol plates containing trace amounts of beta-alanine. The cells were then resuspended in minimal E-glycerol liquid medium to an A600 between 0.6 and 0.7. Arabinose andbeta-[3H]alanine were added to the medium, and the cells were incubated at 42 °C for 3 h. Cell extracts were prepared, fractionated on a native polyacrylamide gel, and exposed to film as described under "Experimental Procedures." The upper band seen in the lane containing I54C AcpP may be a disulfide-linked form produced during electrophoresis. WT, wild type.

 
An appreciable number of the site-directed mutants tested failed to restore growth of the conditional acpP mutant strain. These non-complementing mutants included the D35N, S36A, L37C, I54A, and D56C AcpPs (Fig. 1A and Table 2). Expression of other site-directed mutant proteins including E41Q, E41C, T52C, and I54C gave only partial restoration of growth. Because lack of growth upon expression of a given protein could be due to its poor modification with 4'-phosphopantetheine, we assayed modification by expression of the ACPs in NRD44, a strain auxotrophic for beta-alanine, a precursor of the 4'-phosphopantetheine component of CoA and hence of ACP. The strains were grown in a minimal medium that contained beta-[3-3H]alanine, and their degrees of modification were assessed by autofluorography of cell extracts fractionated by gel electrophoresis.

All of the site-directed mutants showed good levels of posttranslational modification except for the D35N and S36A AcpPs (Fig. 1B). The latter protein lacks the site of 4'-phosphopantetheine attachment and was included as a negative control. In the case of the D35N AcpP poor modification might have been expected because the mutated residue is adjacent to the attachment site residue, and Asp-35 forms a salt bridge between the apo-ACP and the AcpS 4'-phosphopantetheinyl transferase of Bacillus subtilis in the cocrystal structure (32). However, coexpression of B. subtilis Sfp, a much more promiscuous 4'-phosphopantetheinyl transferase (33) from plasmid pQE70-sfp (34), resulted in modification of this mutant protein and complementation of the growth of strain NRD29 (Fig. 2, A and B) indicating that the D35N protein was defective only in interaction with the AcpS 4'-phosphopantetheinyl transferase.


Figure 2
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  		FIGURE 2.
Analyses of complementation of the acpP(Ts) mutant by and phosphopantetheinylation of E. coli D35N ACP, L. lactis ACP, and E. faecalis ACP in the presence or absence of Sfp expression. A, derivatives of strain NRD29, each harboring a plasmid encoding either E. coli (pEcACP), E. coli D35N (pEcD35N), L. lactis (pLlACP), or E. faecalis ACP (pEfACP) under an araBAD promotor plus a plasmid containing Sfp (pSfp) under a lac promotor or the empty vector (pDHK29), were tested for the ability to grow at 42 °C in the presence or absence of arabinose. B, the {Delta}panD deletion strain NRD44 containing the same plasmids as in A were treated as described in Fig. 1B. Above each lane, the ACP being expressed from the plasmid is denoted as follows: E. coli ACP, EcACP; E. coli ACP D35N, EcACP D35N; L. lactis ACP, LlACP, and E. faecalis ACP, EfACP. The second plasmid present in the strain is given at the bottom of the gel. These Petri plates are divided into quadrants by plastic walls.

 
Complementation Analyses with E. coli acpP Site-directed Mutants at an ACP Concentration Approaching Physiological Levels—The results of the in vivo complementation experiments described above are more physiologically relevant than the in vitro experiments performed by others examining the interactions of ACP with either a single fatty acid biosynthetic enzyme or a subset of enzymes under conditions ideal for the in vitro assay but far different from conditions found in an E. coli cell. Nevertheless we decided to test the ability of these site-directed mutants of ACP to complement an E. coli acpP(Ts) mutant at ACP concentrations closer to that normally found in an E. coli cell. We attempted to obtain such cellular ACP concentrations by performing the complementation analyses in strain NRD224, which carries an acpP(Ts) mutation, a pcnB mutation, and the LacIq-encoding plasmid pMS421, which is compatible with the ACP expression plasmids. This strain also was deleted for the lacY lactose permease gene, and thus transcription from the pMR19 tac promoter that drives acpP expression is proportional to the extracellular concentration of the inducer (IPTG) (35, 36). The pcnB286 mutation of strain NRD224 reduces the copy number of pBR322-derived plasmids (37). Although this pcnB allele has been characterized previously, to our knowledge the mutation of this allele had not been identified. We sequenced the PCR amplified pcnB286 allele, and the mutant gene contains an 81-bp deletion presumably resulting from a recombinational event between two 9-bp direct repeats (CCCGGAAAC). This deletion was confirmed by restriction analysis of the PCR-amplified pcnB gene from the pcnB mutant strain WT286 and the wild type strain MG1655. The amplified pcnB from strain WT286 lacked the SnaBI and FspI sites located within the deleted DNA segment, whereas the amplified pcnB from strain MG1655 contained these restriction sites. Regardless in a strain harboring the pcnB286 allele the concentration of ACP resulting from the induction of expression of ACP from the tac promoter of pMR19 by IPTG at a final concentration of 50 µM was 5-fold greater than that normally produced from the chromosomal copy of acpP. At lower IPTG concentrations no complementation of strain NRD224 by pMR19 was observed.

The complementation analyses performed in strain NRD224 harboring plasmid pMS421 were performed with all of the ACP site-directed mutants that were tested in strain NRD29 and several additional ACP site-directed mutants including the S27C, D31N, D35C, E47Q, and E53Q AcpPs. The lower ACP expression complementation data were similar to those observed for the higher level complementation experiments described above. The D35C, D35N, L37C, E53Q, and D56C AcpPs failed to complement the acpP(Ts) mutant strain NRD224 (Fig. 3A). Expression of other site-directed mutant proteins including S27C, V29C, E30C, D31N, E41C, and E47Q gave only partial restoration of growth (Fig. 3A and Table 2). Note that Gong et al. (28) recently reported in vitro data with the Vibrio harveyi ACP for those of the mutant proteins in which acidic residues were changed to their amides.

More Detailed Analysis of a Key AcpP Residue, Isoleucine 54—Isoleucine 54, which lies at the bottom of the hydrophobic pocket of AcpP, interacts with residues from helices II, III, and IV (13, 16). A previous study had shown that substitution of Ile-54 with alanine reduced the electrophoretic mobility of a recombinant V. harveyi ACP on native polyacrylamide gels and resulted in a lower content of secondary structure as assayed by circular dichroism (17). Because in E. coli ACP the I54A substitution eliminated complementation of strain NRD29 and also resulted in decreased electrophoretic mobilities similar to those seen by Flaman et al. (17) we tested a number of other residue 54 substitutions for these phenotypes. Each of the mutant proteins was efficiently phosphopantetheinylated, and each had an electrophoretic mobility that differed from that of the wild type AcpP (Fig. 4B). We tested the ability of these Ile-54 site-directed mutants to complement strain NRD29 and strain NRD224 harboring plasmid pMS421. When these site-directed mutants were tested for complementation of strain NRD29, we found that substitution of Ile-54 with valine, leucine, or methionine gave efficient complementation, whereas substitution with less conservative residues (I54A, I54C, I54S, and I54F) drastically reduced or eliminated complementation, although all of these are hydrophobic residues except serine (Fig. 4A). When the same panel of site-directed mutants that were tested at the higher ACP concentration were expressed at the lower concentration in the acpP(Ts) mutant strain NRD224 the results differed in that the I54A and I54S AcpPs failed to allow growth at the lower intracellular ACP concentration (Fig. 3B).


Figure 3
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  		FIGURE 3.
Analyses of the complementation of the acpP(Ts) mutant by ACP site-directed mutants produced at a concentration approaching physiological ACP levels. A, derivatives of strain NRD224, an acpP(Ts) and pcnB mutant harboring the LacIq-encoding plasmid pMS421 and pMR19-based plasmids, each containing a given site-directed mutant of E. coli acpP, were tested for their abilities to grow at 42 °C in the presence or absence of the inducer, IPTG. B, derivatives of strain NRD224, each harboring the LacIq-encoding plasmid pMS421 and a pMR19-based plasmid encoding a residue 54 site-directed mutant of E. coli acpP, were tested for their abilities to grow at 42 °C in the presence or absence of IPTG. Note that the only phosphopantetheinyl transferase present was the endogenous AcpS. WT, wild type.

 
Complementation by Expression of ACPs from Other Organisms—We cloned the ACP proteins from a number of diverse bacteria as well as several ACPs found in the organelles of eukaryotic organisms (Fig. 5). One rationale for these studies was that mutagenesis and structural studies performed on several other bacterial ACPs have been interpreted in terms of the E. coli AcpP paradigm. If these ACPs showed strong complementation then these interpretations would be strengthened. Each of these ACPs, those of Helicobacter pylori (38), B. subtilis (32), and Vibrio harveyi (17, 24), showed strong complementation. Other ACPs were those of organisms whose fatty acid synthetic proteins have been shown to complement mutants in E. coli fatty acid synthesis (39, 40). Surprisingly the ACPs of two of these organisms, Enterococcus faecalis and Lactococcus lactis, failed to complement, although a third (B. subtilis ACP) did. A number of other bacterial ACPs were obtained by amplification of the genes from genomic DNA samples available within our departments. All of these ACPs, those of Nitrosomonas europaea, Agrobacterium tumefaciens, Bacteroides thetaiotaomicron, Deinococcus radiodurans, and Aquifex aeolicus were expressed and efficiently modified and showed strong complementation of the growth of strain NRD29 (Fig. 6 and Table 2). Note that the N. europaea ACP we tested is that encoded within the fatty acid biosynthesis gene cluster and differs from that studied by Srisailam et al. (41).


Figure 4
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  		FIGURE 4.
Analyses of complementation of the acpP(Ts) mutant by and phosphopantetheinylation of several E. coli ACP I54 site-directed mutants. A, derivatives of strain NRD29, each harboring a plasmid encoding a residue 54 site-directed mutant of E. coli acpP, were tested for their abilities to grow at 42 °C in the presence or absence of arabinose. B, the {Delta}panD deletion strain NRD44 containing the same plasmids as in A were labeled with beta-[3H]alanine as described in Fig. 1B. The residue 54 substitution tested is given above each lane. WT, wild type.

 
The lack of complementation observed for the E. faecalis and L. lactis ACPs could have been due to lack of modification by the E. coli AcpS 4'-phosphopantetheinyl transferase (Fig. 4B). However, coexpression of these proteins with the Sfp 4'-phosphopantetheinyl transferase resulted in conversion of L. lactis and E. faecalis ACP to their modified forms (Fig. 2B). Although proper modification resulted, these proteins failed to replace AcpP function in allowing growth of the acpP(Ts) mutant strain (Fig. 2A).

Finally we tested whether or not expression of three eukaryotic ACPs, those of Bos taurus (bovine) mitochondria (42, 43), Spinacia oleracea (spinach form I) (44), and Plasmodium falciparum (the malarial parasite) (45), resulted in complementation of the acpP(Ts) mutant. The latter two proteins had been expressed and modified in E. coli, and accumulation of an acylated form of the spinach ACP was reported (44). Although all of these eukaryotic ACPs were expressed at sufficient levels and were posttranslationally modified by AcpS, only the P. falciparum ACP allowed growth of the E. coli mutant strain under non-permissive conditions (Figs. 6A and Table 2).


Figure 5
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  		FIGURE 5.
ClustalW alignment of the species ACPs analyzed in Fig. 5. The sequence identities are shaded in black, and sequence similarities are shaded in gray. Above the sequence alignment is the secondary structure of E. coli butyryl-AcpP (13).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The ability to test mutant ACPs for their in vivo functions provides a much more comprehensive and physiological assessment of the effects of sequence divergence on ACP function than was previously possible. Our finding that ACPs from a diversity of bacterial species can functionally replace E. coli AcpP in vivo justifies the historical choice of E. coli ACP as an appropriate archetype of these proteins. Moreover the fact that most of the ACPs tested were efficiently modified in E. coli strongly suggests that AcpS-like proteins are the phosphopantetheine transferases generally responsible for ACP modification.

Some of the results of complementation tests obtained with the site-directed AcpP mutants were expected from prior work. For example, Ile-54 was clearly an important residue. This residue lies in the hydrophobic cavity of AcpP where it interacts with acyl groups attached to AcpP (46) and undergoes hydrophobic interactions with the side chains of Ala-59 of helix III and Tyr-71 of helix IV. Ile-54 also interacts with the {gamma} carbon of helix II Glu-47. However, only proteins containing rather non-conservative substitutions (I54A and I54C) had been tested by in vitro assays. We found that proteins in which the more conservative residues, Val, Leu, or Met, were substituted for Ile-54 were all functional in vivo, whereas the I54F protein and those carrying small residue substitutions failed to effectively complement the acpP(Ts) mutant. When tested by gel electrophoresis under the destabilizing conditions of high pH even those proteins carrying the conservative residue substitutions had greater hydrodynamic radii than wild type AcpP as indicated by their slower electrophoretic migration rates. Stabilization as detected by gel electrophoretic mobility did not necessarily predict in vivo function because the I54F protein had a greater mobility than the Ile-54 substituted proteins that showed complementation, but the I54F protein was unable to support growth. A possible explanation is that the flat aromatic phenylalanine ring of the I54F AcpP may alter interactions between the protein and the tethered acyl chains. Two other substitutions within this segment of AcpP, T52C and D56C, also reduced or blocked in vivo function, whereas a second residue 56 mutation, D56N, allowed growth.

The inability of the D35N mutant AcpP to undergo 4'-phosphopantetheinylation by AcpS was not unexpected. The cocrystal structure of the B. subtilis AcpP and AcpS proteins elucidated by Parris et al. (32) showed that the AcpS primarily binds to negatively charged and hydrophobic residues along helix II of AcpP including Asp-35, Leu-37, Asp-38, Glu-41, and Asp-48. Our work has shown the importance of the negatively charged and hydrophobic residues of helix II. Substitution of Asp-35, Leu-37, or Glu-41 with a polar uncharged residue eliminated full complementation of the acpP(Ts) mutant (Fig. 1A). The lack of complementation by the D35N AcpP was shown to be due primarily to a lack of its posttranslational modification by AcpS because coexpression of another phosphopantetheinyl transferase, Sfp, resulted in both phosphopantetheine attachment and complementation of the acpP(Ts) mutation (Fig. 2, A and B). However, replacement of Asp-38 or Glu-48 with a polar uncharged amino acid gave ACPs that complemented the acpP(Ts) mutant (Fig. 1). The wild type level of complementation seen in the D38N AcpP was unexpected given that this Asp-38 residue forms a salt bridge with Arg-214 of AcpS (32) and is thought to interact with residues of two essential proteins, FabG (27) and LpxA (47). In the case of the LpxA-AcpP interaction the two proposed salt bridges involving Asp-35 and Glu-41 may serve redundant functions, and perhaps both salt bridges must be eliminated to significantly hamper lipid A biosynthesis (47).


Figure 6
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  		FIGURE 6.
Analyses of complementation of the acpP(Ts) mutant and phosphopantetheinylation of ACPs from several different species. A, derivatives of strain NRD29, each harboring a plasmid encoding an ACP from a given species under an araBAD promotor, were tested for the ability to grow at 42 °C in the presence of arabinose. B and C, the {Delta}panD deletion strain NRD44 containing the same plasmids as in A were treated as described in Fig. 1B. The ACPs tested are denoted in A–C as follows: E. coli, EcACP; A. aeolicus, AaACP; B. subtilis, BsACP; L. lactis, LlACP; B. taurus, BtaACP; D. radiodurans, DrACP; S. oleraceae, SoACP; A. tumefaciens, AtACP; B. thetaiotaomicron, BthACP; H. pylori, HpACP; N. europaea, NeACP; E. faecalis, EfACP; P. falciparum, PfACP; and V. harveyi, VhACP.

 
There also were some cases where mutant ACPs functioned as well as the wild type protein in vitro but were unable to fully complement the acpP(Ts) mutant. In vitro the T52C ACP had essentially full function in five different enzyme assays as well as in vitro fatty acid synthesis (25). However, this mutant ACP was unable to fully restore growth, although the protein was well expressed and efficiently modified (Fig. 1). It should be noted that one explanation for such discrepancies is that the defective nature of a mutant ACP would only be realized in vivo if its interaction with a rate-controlling enzyme was compromised. However, metabolic control analysis holds that the classical idea of a rate-limiting step in which one enzyme determines the rate of a pathway is incorrect. This analytical approach holds that the control of a pathway is spread among the component enzymes such that increased activity of one enzyme will not result in increased flux through the pathway (48–50). However, there are several well documented examples in which increased activity of one or two enzymes does increase the flux through a pathway in E. coli (Ref. 51 and references therein). The situation in E. coli lipid synthesis may lie between the two extreme views: that of the classical rate-limiting step and that of metabolic control analysis. It should also be noted that E. coli ACP also plays a role in several key acyltransferase reactions involved in synthesis of two essential cellular components, phospholipids and lipid A. Thus a mutant ACP could function adequately in fatty acid synthesis but block growth by failure to interact with an essential acyltransferase. The recent discovery of a new acyl-ACP-dependent soluble phospholipid acyltransferase (52) introduces another enzyme to be considered.

The complementation experiments with acpP homologs from diverse organisms produced some surprising results. All of the bacterial ACPs replaced the function of E. coli AcpP except those from L. lactis and E. faecalis, two closely related Gram-positive bacteria. These two ACPs were not modified by E. coli AcpS, but upon coexpression with Sfp, the proteins became highly modified (Fig. 2, A and B) but remained unable to replace AcpP function. These results were unexpected because expression of fatty acid synthetic proteins from both organisms in mutant E. coli strains restores fatty acid synthesis to cells that are otherwise defective in this pathway (39, 40). Moreover E. faecalis enzymes have been shown to function with E. coli ACP in vitro (53). It seems likely that the L. lactis and E. faecalis ACPs fail to function in E. coli due to their divergent helix II sequences (Fig. 5), but the fact that there is no reciprocal defect remains a puzzle.

The abilities of the three eukaryotic ACPs tested, B. taurus mitochondrial ACP, S. oleracea ACP, and P. falciparum ACP, to complement the acpP(Ts) mutant and become posttranslationally modified in E. coli was also examined. All three ACPs studied were phosphopantetheinylated in E. coli, but only the ACP of the malarial parasite, P. falciparum, complemented the acpP(Ts) strain suggesting that the interactions occurring between P. falciparum ACP and its fatty acid biosynthetic proteins are very similar to those of the corresponding E. coli proteins. Such differences in protein-protein interactions between these two microbes and B. taurus may allow the development of compounds that can inhibit type II fatty acid biosynthesis in bacteria and P. falciparum without disrupting the synthesis of lipoic acid precursors in the mitochondria of their mammalian host cells (43).

The P. falciparum data should be considered in light of recent results on the protein interaction network of E. coli (54–56). These studies report that AcpP is found in readily isolated complexes with a large number of proteins. Indeed ACP is the most interactive of any discrete protein of E. coli and is one the four major interaction nodes of the organism; the others are the multiprotein complexes RNA polymerase, DNA polymerase, and the ribosome (54). An unexpected result of the interaction work is that AcpP was reported to interact with several proteins that are involved in physiological processes other than lipid metabolism in E. coli (47, 48). In light of these data the fact that expression of the P. falciparum ACP allowed growth of an acpP(Ts) strain was very surprising because this ACP is located in an organelle called the apicoplast (45). Apicoplasts are considered vestigial chloroplasts acquired via secondary endosymbiosis from an alga (57). Although apicoplasts are essential sites of fatty acid synthesis in their host cells, they are thought to contain only about 500 proteins (57) and lack recognizable homologs of the non-lipid synthetic proteins reported to interact with E. coli ACP. Apicoplasts are thought to have arisen at least 400 million years ago (57) so apicoplastic proteins have long been isolated from virtually all of bacterial metabolism. These considerations argue that either the interactions of AcpP with those proteins that are not involved in lipid synthesis are either non-essential or that the sites of interaction involve sequences that have been conserved between the E. coli and P. falciparum ACPs despite their many millions of years of segregation from one another.

Finally we must address the puzzling finding that complementation of the acpP(Ts) mutation required levels of ACP about 5-fold greater than the level present in wild type cells. Lower levels of acpP expression invariably failed to give complementation. One possibility is that the mutant A68T/N73D ACP encoded from chromosome may be an inhibitor of various lipid biosynthetic enzymes, and ACP overproduction is required to overcome the inhibition by mass action. This seems unlikely because the mutant ACP is unstable at the nonpermissive temperature (1), although it remains possible that some of the mutant ACP molecules could be protected from proteolysis by being bound to lipid synthetic proteins and thereby remain inhibitory. However, we found that complementation of a strain carrying a deletion of the entire acpP gene (1) requires the same high IPTG concentrations required to complement the point mutant strains (data not shown). A second possibility is raised by the recent realization that proteins are not expressed at a constant rate in individual E. coli cells but rather are produced in bursts that may be spaced out over an appreciable fraction of the cell cycle (58–62). This results in situations where the ratio of two proteins may differ greatly among the cells of a putatively homogeneous population (60, 61). Thus, the ratios of ACP to its various cognate proteins may show significant fluctuations within wild type cells. Although a consistent picture emerges from extant reports of protein production in single E. coli cells, the reports are few due to the formidable technology required. For example, we do not yet have data comparing plasmid-borne and chromosomal expression of a given gene. Hence the greater level of plasmid-encoded ACP required to replace function of the chromosomally encoded protein could be due to a lack of coordinate expression of the plasmid-encoded ACP with some of its cognate proteins. Despite this complication we believe that our in vivo data provide the most comprehensive picture of the residues important in ACP function. Some of our data directly conflict with those obtained in vitro, whereas others agree well. In this regard it should be noted that in none of the in vitro reports were the concentrations of ACP and its substrate enzymes matched to those found in vivo. Indeed data on the in vivo concentrations of most of these proteins have not been available.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant AI15650. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed: Dept. of Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Tel.: 217-333-7919; Fax: 217-244-6697; E-mail: j-cronan{at}life.uiuc.edu.

2 The abbreviations used are: ACP, acyl carrier protein; IPTG, isopropyl 1-thio-beta-D-galactopyranoside. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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