Construction and analysis of a coaBC-regulated E. coli strain.
In order to investigate the essentiality of coaBC
and the effects of its depletion on E. coli
, a regulated strain was constructed in which expression of coaBC
was placed under the control of the arabinose-inducible PBAD
promoter. The growth of the resulting strain, BW25113PBADcoaBC
, was dependent on the presence of arabinose, which is consistent with the reported essentiality of coaBC
). Furthermore, the lag time for detectable growth scaled with the concentration of arabinose in the growth media (), with increased lag times observable below 62 μM arabinose. In fact, the midpoint for the exponential growth phase (OD600
= 0.35) shifted from 340 min for growth in the presence of 125 μM arabinose to 380, 540, 600, 680, 820, and 960 min for growth in the presence of 63, 16, 8, 4, 2, and 1 μM arabinose, respectively ().
Fig. 2. Arabinose-dependent growth of BW25113PBADcoaBC. (A) Growth curves of BW25113PBADcoaBC grown at 37°C in LB with 30 μg/ml kanamycin and various concentrations of arabinose: 125 μM (black filled squares), 63 μM (dark gray (more ...) Nutrient supplementation.
Given that CoA biosynthesis is a primary metabolic process leading to the formation of a small-molecule cofactor, it was of interest to determine whether supplementing the media with substrates, intermediates, and/or products of the pathway could rescue deficiencies in coaBC
expression. Precedents for chemical complementation of PPCDC defects can be found in studies on Arabidopsis thaliana
. This plant possesses two genes that encode PPCDC, HAL3A and HAL3B
. While plants containing homozygous transfer DNA (T-DNA)-disrupted alleles of either hal3a-1
are viable, a hal3a-1 hal3b
double mutant is embryonically lethal, indicating a redundant and yet essential function for these genes. Interestingly, although hal3b
individuals heterozygous for HAL3A
behave similarly to wild-type plants, hal3a-1
individuals heterozygous for HAL3B
were severely impaired for seedling establishment (38
), a result that supports the notion that HAL3A
plays a dominant role with respect to HAL3B
, as suggested by differences in transcript levels (14
). This severe defect could be rescued by supplementation with pantethine, restoring normal seedling establishment.
To investigate whether a similar phenomenon could occur in E. coli, the growth phenotype of BW25113PBADcoaBC as a function of arabinose concentration was measured in the presence of 1 mM pantothenate, pantethine, dephospho-CoA, or CoA. As expected, the addition of pantothenate, the primary precursor of the CoA pathway, was unable to rescue the arabi-nose-dependent growth phenotype of BW25113PBADcoaBC (). This result is consistent with the fact that PPCS/PPCDC acts downstream of pantothenate formation in CoA biosynthesis and implies that increasing the concentration of the substrate cannot overcome a deficiency in coaBC expression. It was also noted that the lag time seen with all concentrations of arabinose increased when adding pantothenate, suggesting that E. coli is subject to additional stress imposed by the exogenous addition of this metabolite. Nonetheless, the apparent change in lag time reflects only the difference in growth conditions and does not alter the conclusion that pantothenate is unable to bypass the need for endogenous PPCS/PPCDC function. Next, addition of pantethine did deregulate arabinose-dependent growth in BW25113PBADcoaBC. As can be seen in , in the presence of 1 mM pantethine, the lag times for growth were equivalent in the presence or absence of arabinose. Finally, addition of dephospho-CoA or CoA, the penultimate and ultimate products of the pathway, did not rescue the increased lag time for growth due to underexpression of coaBC ( and E). Although dephospho-CoA and CoA formation are downstream with respect to PPCS/PPCDC function, these highly charged phosphate-containing molecules are unable to enter cells. As was the case with pantothenate, an overall increase in lag time for the bacterial cultures regardless of the arabinose concentration was observed when either dephospho-CoA or CoA was added to the medium, likely due to a change in the overall growth conditions. Additionally, there was a slight amount of growth observed with 1 mM dephospho-CoA in the absence of arabinose (), which could have been caused by the breakdown of dephospho-CoA to pantetheine over time or by the presence of a small amount of pantetheine or pantethine in the sample, as the purchased dephospho-CoA was only 90% pure. In summary, regardless of any general changes in growth that chemical supplementation may have caused, the primary phenotype of arabinose-dependent growth and increased lag time caused by regulating the expression of coaBC was rescued only in the presence of pantethine.
Pantethine is a dimeric thiol-oxidized version of the PPCS/PPCDC product that is missing the 4′-phosphate appended by PanK. The results showing that it can complement deficiencies in coaBC expression indicates that it can both enter cells and bypass the need for PPCS/PPCDC activity. To demonstrate that pantethine was not being converted to CoA by an alternative pathway, chemical complementation experiments were repeated in a strain in which coaD, which encodes the PPAT enzyme that catalyzes the step subsequent to PPCS/PPCDC formation in CoA biosynthesis, was placed under the control of the arabinose-inducible PBAD promoter. As with coaBC, coaD was confirmed to be essential, because growth of BW25113PBADcoaD was dependent on coaD expression, and the lag time for growth scaled with the concentration of arabinose in the media (see Fig. S1A in the supplemental material). As can be seen in Fig. S1B to E in the supplemental material, addition of 1 mM pantothenate, pantethine, dephospho-CoA, or CoA was unable to rescue the arabinose-regulated phenotype of BW25113PBADcoaD. Although slight differences in the overall growth could be observed when exogenous metabolites were added, especially at low concentrations of arabinose, addition of any of these nutrients did not change the fact that the lag time for growth was inversely proportional to the amount of arabinose present and that in the absence of arabinose, no growth was observed. This result confirms the idea that increasing the amount of upstream precursors such as pantothenate cannot overcome deficiencies in downstream enzyme activity and that dephospho-CoA and CoA were not entering the cells, since both were downstream of PPAT activity. More importantly, these data demonstrate that pantethine cannot bypass the expression of coaD, indicating that pantethine is ultimately converted to the PPAT substrate phosphopantetheine by a PPCS/PPCDC-independent mechanism.
Despite its inability to cross the cytoplasmic membrane in E. coli
, extracellular pantothenate can be used as a precursor to CoA biosynthesis due to its active uptake by the pantothenate permease encoded by panF
). In order to determine whether the uptake of pantethine also was dependent on panF
, knockouts were constructed in the BW25113PBADcoaBC
background. As can be seen in Fig. S2 and S3 in the supplemental material, arabinose-dependent growth is deregulated after addition of pantethine in a manner similar to that seen in comparisons of the parent BW25113PBADcoaBC
to the mutant BW25113PBADcoaBC
. This deregulation is observable as a decrease in lag time for the different concentrations of arabinose. Furthermore, the degrees of deregulation seen with increasing concentrations of pantethine were similar for the two strains (see Fig. S2B to E and S3B to E in the supplemental material). These data indicate that the ability of pantethine to enter E. coli
is not dependent on the presence of panF
. This finding is consistent with studies in which an azido-pantetheine analog was capable of entering cells and being processed to ultimately label endogenous acyl carrier proteins in a panF
-independent manner (34
Constructing coaBC knockouts.
To rule out the possibility that residual PPCS/PPCDC activity in BW25113PBADcoaBC
was responsible for cell viability due to unknown regulatory mechanisms triggered by addition of pantethine, a full deletion of coaBC
was attempted. Indeed, when pantethine-supplemented media were used, mutants could be obtained with a full knockout in the coaBC
gene. The BW25113ΔcoaBC
strain, whose identity was confirmed by sequencing, was not viable on media lacking exogenous pantethine (). Pantothenate, dephospho-CoA, and CoA could not be used as surrogates for restoring growth, and plating performed multiple times with 1012
cells did not yield any suppressors of the pantethine auxotrophy. Although these data are consistent with previous studies indicating that CoA cannot be transported across the bacterial cell envelope (23
), they alter the notion that pantothenate is the most advanced precursor to CoA that can permeate cells (24
BW25113ΔcoaBC::kan is auxotrophic for pantethine. (A) BW25113ΔcoaBC::kan grown on LB agar with 30 μg/ml kanamycin. (B) BW25113ΔcoaBC::kan grown on LB agar with 30 μg/ml kanamycin and 1 mM pantethine.
Biochemical experiments using PanK.
The use of pante-thine to chemically complement a coaBC
deficiency in a coaD
-dependent fashion suggests that E. coli
is capable of converting this molecule to the PPCDC product phosphopantetheine. This transformation could be accomplished rather directly through reduction of the disulfide to the free thiol and phosphorylation of the 4′ hydroxyl (). Although the general reducing environment of the cytoplasm could suffice for accomplishing disulfide reduction, the phosphorylation event most likely would require a specific enzyme. It has been known for some time that specific enzyme preparations purified from cellular extracts could phosphorylate both pantothenate and pantethine (1
), and ultimately this activity was attributed to the protein involved in the first step in CoA biosynthesis, PanK. There are three types of bacterial PanKs that differ with respect to structure, substrate affinity, and feedback regulation. The type I PanKs are the only type modulated through feedback inhibition by CoA and are exemplified by the prototypical member encoded by coaA
in E. coli
). Type II PanKs are related to the eukaryotic isoforms of PanK and are exemplified by the product of the coaA
gene in Staphylococcus aureus
). Finally, type III PanKs are the most divergent of the three, have high Km
values for their substrates, and are exemplified by the product of the coaX
gene in Helicobacter pylori
The coaA gene, encoding the type I E. coli PanK (EcPanK), was cloned into the C-terminal His6-tagged vector pTrcHis2B. Expression in TOP10 E. coli at 18°C with 1 mM IPTG induction yielded 30 to 40 mg/liter after purification to homogeneity using cobalt affinity and gel filtration chromatography in tandem. Initial activity titration assays performed using a pyruvate kinase/lactate dehydrogenase-coupled system demonstrated that EcPanK was active and readily phosphorylated both pantothenate and pantethine.
Once initial activities were established, a full set of Michaelis-Menten parameters for the kinase reaction were measured under optimized reaction conditions. The data in indicate that the EcPanK Km values for ATP are within a 2-fold range whether using pantothenate or pantethine as substrate and that the Km for the natural pantothenate substrate is 2-fold greater than the Km for the surrogate pantethine. These data demonstrate that EcPanK can phosphorylate pantethine and that the specific activity for this conversion is 2-fold greater than that for the natural pantothenate substrate.
The promiscuity of type I and II PanKs has been exploited in the use of pantothenamide antimetabolites that can act as substrates for PanK as well as for downstream enzymes. These molecules have been utilized as labels for carrier proteins in vivo
) and as antimicrobial agents; however, there is still debate as to whether the mechanism of action for the pantothenamide class of antimetabolites operates by direct inhibition of CoA biosynthetic enzymes (11
), by inhibition of fatty acid biosynthesis through the accumulation of nonfunctional acyl carrier proteins (29
), or by inhibition of CoA- and acetyl-CoA-utilizing enzymes (42
). Given this promiscuity, it was not surprising that E. coli
PanK was demonstrated here to efficiently utilize pantethine as a substrate in biochemical assays.
As a parallel comparison, the type III PanK enzyme was also isolated by cloning and expressing the coaX
gene from the clinically relevant pathogen Pseudomonas aeruginosa
. Possessing a rather impermeable outer membrane and a diverse range of efflux pumps (35
), P. aeruginosa
is inherently recalcitrant to antimicrobial intervention. It is of particular interest given that it has emerged in the clinic as a major cause of nosocomial infections in immunocompromised patients and is well known to be a cause of declining lung function in cystic fibrosis patients (16
Although P. aeruginosa
PanK) catalyzed robust phosphorylation of pantothenate with Michaelis-Menten parameters similar to those found for the type III PanK from H. pylori
) (), it was unable to utilize pantethine as a substrate. This result held true despite exploration of several sets of conditions in which high concentrations of enzyme and/or substrate were used and large amounts of reducing agent were supplied to form the free thiol pantetheine. The inability of Pa
PanK to utilize pantethine is also consistent with the inability of H. pylori
PanK to accept N
-pentylpantothenamide as an alternative substrate (10
Pantethine complementation dependence on PanK in vivo.
The inability of Pa
PanK to phosphorylate pantethine was further explored in vivo
in P. aeruginosa
through an attempt to create a coaBC
knockout. Initially, deletion was performed in a wild-type PAO1 background; however, single integrants never resolved to double-crossover knockout mutants. Since Pseudomonas
is known for possessing an especially impermeable outer membrane (5
), it is possible that pantethine is unable to penetrate PAO1. Therefore, the knockout experiments were repeated with the hyperpermeable Pseudomonas aeruginosa
strain Z61 (ATCC 35151) (5
). Despite the lower permeability threshold, coaBC
knockout mutants were not obtained.
The result showing that PaPanK is unable to phosphorylate pantethine and therefore is unable to form the PPAT substrate phosphopantetheine suggested that perhaps the inability to delete coaBC in P. aeruginosa and thus the inability to generate a pantethine auxotroph was not due to the lack of cellular penetration of pantethine but rather due to an inability to metabolize the molecule. If this were indeed the case, then introducing the E. coli coaA gene (EccoaA) into P. aeruginosa should allow it to utilize pantethine to bypass the PPCS/PPCDC function, rendering the coaBC deletion viable. Conversely, if the coaA gene function in E. coli were paramount for incorporating pantethine metabolism into CoA, replacing it with the coaX gene from P. aeruginosa (PacoaX) should render the pantethine bypass of coaBC ineffective.
To generate the P. aeruginosa pantethine auxotroph, E. coli coaA was introduced into the wild-type PAO1 and membrane-permeable Z61 P. aeruginosa strains on either of two replicative plasmids. The pΔflp2EccoaA plasmid contains the sacB gene, which renders cells nonviable when grown on sucrose, whereas the pΔflp2ΔsacBEccoaA plasmid does not contain the sacB gene. The use of these parallel plasmids can demonstrate the essentiality of EccoaA in pante-thine complementation of coaBC deficiency. During the process of generating knockouts, single integrants of the pEX18TcΔPacoaBC::gent suicide vector are forced to resolve to double-crossover knockouts because the use of gentamicin selects for retention of the resistance cassette incorporated into the target insert and the use of sucrose selects for loss of plasmid backbone which contains the sacB gene. Concurrently, the use of sucrose in this selection also selects for loss of the pΔflp2EccoaA plasmid but would not select for loss of the pΔflp2ΔsacBEccoaA plasmid. If EcPanK were responsible for the ability to utilize pantethine, thus bypassing PPCD/PPCDC activity, only cells with pΔflp2ΔsacBEccoaA would be viable after selection on sucrose, since only they would retain a copy of EccoaA.
As evidenced in , only Z61pEX18TcΔPacoaBC::gent/pΔflp2ΔsacBEccoaA bacteria were viable when selection for knockouts on gentamicin, sucrose, and pantethine was performed. The Z61pEX18TcΔPacoaBC::gent/pΔflp2EccoaA strain failed to grow, indicating that EccoaA is required for growth on pante-thine. Colonies from this selection were isolated and sequenced to confirm that they were double-crossover knockouts of coaBC. As can be seen in and C, the resulting Z61ΔcoaBC::gent/pΔflp2ΔsacBEccoaA strain was viable only in the presence of pantethine. These results indicate that PanK phosphorylation of pantethine is necessary and sufficient for pantethine complementation of coaBC deficiency, even in P. aeruginosa, where the endogenous PanK encoded by coaX is unable to catalyze this phosphorylation. Interestingly, single integrants of the PAO1 strain containing either the pΔflp2EccoaA or pΔflp2ΔsacBEccoaA plasmid never resolved to double-crossover knockout mutants, even when pantethine was supplied at concentrations as high as 5 mM in the media. This indicates that the permeability barrier in PAO1 is too high to allow pantethine to enter.
Fig. 4. EccoaA is required for pantethine bypass of coaBC in P. aeruginosa. (A) Z61pEX18TcΔPacoaBC::gent/pΔflp2EccoaA and Z61pEX18TcΔPacoaBC::gent/pΔflp2ΔsacBEccoaA plated on PIA with 10 μg/ml gentamicin, 10 μg/ml (more ...)
To further confirm these results, the reciprocal pantethine complementation experiment was conducted using E. coli. Using the BW25113PBADcoaBC background, we replaced coaA with the coaX gene from P. aeruginosa. The growth of the resulting strain, BW25113PBADcoaBCΔcoaA::PacoaX, was assayed using various concentrations of arabinose, with or without the addition of 1 mM pantethine. As shown in , unlike the parental BW25113PBADcoaBC strain, in which addition of pantethine bypasses arabinose dependence (), in BW25113PBADcoaBCΔcoaA::PacoaX, addition of pantethine had no effect on the increased lag time observed with decreasing concentrations of arabinose. These results indicate that, when EccoaA is replaced with PacoaX, E. coli can no longer metabolize pantethine into phosphopantetheine and bypass deficiencies in coaBC. It also suggests that PanK is the only enzyme in E. coli that can phosphorylate pantethine.
Fig. 5. Arabinose-dependent growth of BW25113PBADcoaBCΔcoaA::PacoaX cannot be bypassed with pantethine. (A) Growth curves of BW25113PBADcoaBCΔcoaA::PacoaX grown at 37°C in LB with 30 μg/ml kanamycin, 25 μg/ml chloramphenicol, (more ...)
The results of this study differ somewhat from findings demonstrating pantethine rescue of pantothenate-kinase-associated neurodegeneration (PKAN) in Drosophila
studies. The neurodegenerative hereditary disease Hallervorden-Spatz syndrome, caused by mutations in human PANK2
), has been successfully recapitulated in Drosophila
through analogous mutations in dPANK2
). The mutant flies have significantly reduced levels of CoA, resulting in impaired mitochondrial function, increased oxidative damage of proteins, defective locomotor abilities, and decreased life span. All of these phenotypes could be rescued to some degree by supplementation of food with pantethine (37
), indicating that pantethine could be utilized as a substrate for generating CoA. Interestingly, these results suggest that in Drosophila
, pantethine can be utilized in a PanK-independent manner, which is in contrast to the findings of this study, where pantethine utilization was found to be absolutely dependent on PanK activity. Although an alternate kinase or pathway for pantethine metabolism in Drosophila
has yet to be found, ultimately, pantethine is most likely still converted to the intermediate 4′-phosphopantetheine, since pantethine could rescue decreased cell counts only in dPPCS-depleted cells and not in dPPAT-depleted cells (37
). This is consistent with the results of this study, which demonstrate that pantethine supplementation could bypass arabinose-regulated growth of only a coaBC
) and not a coaD