TEXT

Top Abstract Text References

Streptomyces spp. synthesize the majority of their fatty acids from branched starters such as isobutyryl, isovaleryl, and anteisovaleryl units to give odd- and even-numbered fatty acids with a methyl branch at the -terminus (80 to 90% of total fatty acid content); the remainder are synthesized from straight starters such as acetyl and butyryl units (11, 21). The fatty acid synthase (FAS) of Streptomyces spp. is, like that found in many other bacteria (including the best-studied example, that of Escherichia coli), a type II or dissociable system (13, 18). The type II FAS consists of several discrete proteins that form loose associations to synthesize the fatty acid. The assembly of fatty acids is initiated by the condensation of an acyl coenzyme A (acyl-CoA) starter unit and a malonyl-acyl carrier protein (malonyl-ACP) extender unit; this condensation is catalyzed by -ketoacyl ACP synthase III (FabH), the product of the fabH gene. In vitro biochemical studies suggest that FabH determines the choice of starter unit to be used. E. coli FabH is specific for an acetyl-CoA starter unit, whereas Bacillus subtilis and Streptomyces glaucescens FabHs can accept a broader range of substrates, including branched- and straight-chain units (2, 8). In the case of the S. glaucescens FabH, the order of reactivity towards the different starters is isobutyryl-CoA > butyryl-CoA > acetyl-CoA. If FabH were solely responsible for the initiation of fatty acid biosynthesis in Streptomyces species, then one could hypothesize that its biochemical activity, together with the relative pool sizes of the different starter units in vivo, would account for the mix of branched and straight fatty acids (8). But when S. glaucescens was grown in the presence of high concentrations (480 µM) of the FAS inhibitor thiolactomycin, branched-chain fatty acid biosynthesis was inhibited and the proportion of straight-chain fatty acids increased (50% inhibitory concentration [IC₅₀] for purified S. glaucescens FabH 20 µM) (8). This result may be interpreted as evidence for a second; FabH-independent mechanism for fatty acid initiation in Streptomyces spp. Based on this second hypothesis, fabH should be dispensable to Streptomyces spp. In this study we provide further biochemical evidence for the role of a small cluster of presumed fab genes (which includes fabH) in Streptomyces coelicolor and a transcriptional analysis of the fab cluster, and we have attempted to disrupt fabH to determine if it is essential for the viability of the cells.

The acpP gene product stimulates long-chain fatty acid biosynthesis in vitro. The S. coelicolor FAS is still relatively poorly understood; a cluster of four fab-like genes has been identified on the S. coelicolor chromosome in the order fabD-fabH-acpP-fabF (cosmid SC4A7, S. coelicolor genome project [http://www.sanger.ac.uk/Projects/S_coelicolor/]; nucleotide sequence accession number AL133423). The deduced amino acid sequences of the fab genes are highly similar to components of the E. coli FAS, and at least some of the S. coelicolor genes are essential (13). We used a biochemical assay of fatty acid biosynthesis, dependent on the acpP gene product (ACP), to strengthen the evidence that these genes do encode the FAS of S. coelicolor. Cell extracts were prepared from S. coelicolor M145 grown for 20 h in YEME medium (12) and broken as previously described (3), with an additional clearing step by ultracentrifugation for 1 h at 100,000 × g. The supernatant was adjusted to 5 mg of protein/ml, a fresh ice-cold 10% (wt/vol) solution of streptomycin sulfate was added slowly while stirring on ice water to a final concentration of 1%; the mixture was then stirred for a further 20 min and centrifuged for 20 min at 14,000 × g. Endogenous ACP was removed from the cell extract by fractionation with a 60 to 80% ammonium sulfate cut as previously described (4), and this cut was dialyzed overnight against 1 liter of cell disruption buffer containing 2 mM dithiothreitol (DTT). Pure FAS holo-ACP was prepared as previously described (14) and reduced to the active monomeric form just prior to each assay by incubation at 30°C for 30 min in a solution containing 50 mM potassium phosphate, pH 7.2, and 10 mM DTT. Typical assay incubation conditions were as follows: 0.5 mg of S. coelicolor (60 to 80% ammonium sulfate cut) per ml, 100 mM potassium phosphate (pH 7.2), 2 mM DTT, 100 mM NADH, 100 mM NADPH, 20 µM ACP, and 100 µM [2-¹⁴C]malonyl-CoA (0.02 Bq/pmol) in a total volume of 18 µl were preincubated for 10 min at 30°C; then 2 µl of isobutyryl-CoA was added (20 µM final concentration) to initiate the reaction (alternatively, buffer alone was added as a negative control), and the incubation was continued for a total of 60 min. Assay products were analyzed in two different ways. First, conformationally sensitive polyacrylamide gel electrophoresis (CS-PAGE) was used to determine if any acyl-ACP product had been formed in the assay. The small highly acidic ACPs typically migrate faster than other proteins in CS-PAGE (9, 15), and this technique has been used previously to differentiate acyl adducts of the S. coelicolor ACP (14). After incubation in the presence of isobutyryl-CoA, ACP was depleted and a new, faster-migrating band appeared (Fig. 1a, left panel). The phosphorimage of this gel (Fig. 1a, right panel) showed that the new, faster-migrating ACP species was labeled by the extender unit, consistent with a role for this ACP in stimulating at least one round of condensation between the starter and extender units, catalyzed by the FAS components in the cell extract. Second, electrospray mass spectrometry (ESMS) was used to determine the exact mass of the acyl-ACP product identified by CS-PAGE. The assay incubation was run as described above, but it was scaled up 10-fold and cold malonyl-CoA was used. The acyl-ACP species were purified from the assay mixtures (with or without isobutyryl-CoA) using the Biocad Sprint purification system (Perkin-Elmer) with a POROS HQ/M column (4.6 by 100 mm) and eluted in a linear gradient of 0 to 800 mM NaCl in 20 mM Tris-bis-Tris propane, pH 7.2, over 15 column volumes (10 ml/min). Unmodified ACP (no isobutyryl-CoA, negative control) was eluted at 509 mM NaCl, and the acyl-ACP reaction product was eluted at 327 mM NaCl. After desalting (PD10; Pharmacia), the ACPs were analyzed by ESMS by John Crosby, School of Chemistry, University of Bristol, Bristol, England, as described previously (3). ACP purified from the assay mixture that lacked isobutyryl-CoA had a measured mass (mean ± standard deviation) of 9,126.6 ± 2 Da (expected mass, 9,128 Da), and the acyl-ACP (isobutyryl-CoA dependent) had a measured mass of 9,367.5 ± 2.9 Da, in close agreement with that expected for C₁₆ acyl-ACP (9,366 Da) (Fig. 1b). This demonstrated that the acpP gene product is able to stimulate isobutyryl-CoA-dependent long-chain fatty acid biosynthesis in cell extracts of S. coelicolor. These data provide further evidence to substantiate the argument that acpP, and by implication its surrounding genes, does encode the FAS of S. coelicolor.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Identification of the acyl-ACP products formed by in vitro FAS assay. (a) Left panel, CS-PAGE and Coomassie blue staining of material incubated in the assay. Lane 0, no acyl-CoA starter unit added; lane 1, isobutyryl-CoA added as a starter. Right panel, phosphorimage of the gel shown on the left. (b) Transformed electrospray mass spectrum of the repurified acyl-ACP after incubation with the isobutyryl-CoA starter. The major peak, with a molecular mass of 9,367.5 Da (±2.9Da) is in close agreement with the calculated mass of C16 acyl-ACP (9,366 Da).

Transcriptional analysis of the fab genes. Reverse transcriptase PCR (RT-PCR) analysis was used to detect the presence or absence of continuous mRNA spanning the junctions between each of the fab genes and between fabD and SC4A7.14 (the gene on cosmid SC4A7 upstream of and colinear with fabD, named as such in the S. coelicolor genome project) (Fig. 2a). RNA was isolated from cultures of S. coelicolor M145 grown for 20 h in YEME medium as previously described (12) and incubated with DNase (free of RNase; Roche Diagnostics) to remove traces of contaminating DNA. Cotranscription of genes was analyzed by RT-PCR of intergenic regions using the Titan One Tube RT-PCR system (Roche Diagnostics) by following the protocol recommended by the manufacturer. The temperature profile was as follows: 1 cycle at 60°C for 30 min, 30 cycles of PCR (denaturation for 1 min at 96°C, annealing for 1 min at 65°C, and extension for 4 min at 72°C), and 1 cycle at 72°C for 10 min. The total reaction volume was 50 µl, and 10 µl was analyzed on an agarose gel. Oligonucleotides were as follows (Fig. 2b): SC4A7.14 forward, 5'-AAGTCGCTGATCGGGCCGTTCG-3'; fabD reverse, 5'-CGAGATCGAGTCCGATGGCGTC-3', fabD forward, 5'-GGCGAACGTGAACGGCGCCGGT-3'; fabH forward, 5'-GGAGCGGCTCCTGGCGACCGGC-3'; acpP reverse, 5'-TGACGTCCTCGACCGGGATGCC-3'; and fabF reverse, 5'-CGATCAGCGCGAACTGCGCCGA-3'. RT-PCR products were generated across the fabD-fabH, fabH-acpP, and acpP-fabF junctions but not across the fabF-SC4A7.19c interval (SC4A7.19c is downstream of and convergent with fabH, and so this served as a negative control) or the SC4A7.14-fabD junction. In all cases, the expected PCR product was generated when genomic DNA served as the template (initial RT incubation omitted), providing a positive control for each PCR (Fig. 2c). Because the fabF-SC4A7.19c-convergent genes gave a PCR product with genomic DNA as a template, but they did not give an RT-PCR product with RNA as a template (data not shown); there was no contaminating DNA in the RNA preparation. Additional controls included the following: no DNA or RNA template (no product seen), RNA template treated with RNase (no product), and DNA template treated with DNase (no product). These results strongly suggest that one long transcript originated from a promoter upstream of fabD and continued through all four fab genes to terminate just 3' of fabF. The gene upstream of and colinear with fabD, SC4A7.14, has end-to-end similarity with genes found in every prokaryote sequenced so far, including Mycobacterium tuberculosis, in which it is also located immediately upstream of fabD, though their functions are unknown. Even though a transcript was not detected between SC4A7.14 and fabD, this does not rule out a role for the SC4A7.14 gene product in fatty acid biosynthesis; it merely indicates that it is not cotranscribed with the fab genes, at least in cells grown to mid-log phase in a rich liquid medium.

View larger version (80K): [in this window] [in a new window]   FIG. 2.   RT-PCR analysis to detect transcriptional readthrough between fab genes. (a) Schematic representation of the fab cluster to show the organization of the genes on the chromosome (gene names are as in the S. coelicolor genome project). (b) Positions of oligonucleotides used in RT-PCR experiments and expected RT-PCR products. (c) Agarose gel of RT-PCR products showing no transcriptional readthrough between SC4A7.14 and fabD, whereas fabD and fabH, fabH and acpP, and acpP and fabF are cotranscribed. M, DNA molecular size markers; R, RNA template; D, DNA template.

View larger version (65K): [in this window] [in a new window]   FIG. 3.   High-resolution mapping of fabD promoter. A protected fragment (139 nt) of the fabD promoter probe (lane 2) comigrating with the GUG translational start point is indicated on the corresponding sequence ladder (lanes A to T). Lane 1, tRNA instead of mRNA mixed with the fabD promoter probe (negative control).

S. coelicolor fabH (encoding FabH) is essential for viability. So far, four homologues of fabH have been found in the S. coelicolor genome (with 90% of the genome complete), and each has an amino acid sequence approximately 40% identical to the fabH product (S. coelicolor genome project). The roles of two of these open reading frames are unknown, but one possibility is that they might encode alternative FabHs for fatty acid initiation. pIJ8155 (Table 1) was introduced into S. coelicolor by conjugation from E. coli (as described in reference 6); apramycin-resistant colonies were picked, and putative single-crossover recombinants were confirmed by Southern hybridization. Eleven out of 12 of the colonies showed integration of the plasmid by homologous recombination through the sequence to the left of the deletion in fabH (event 1) (Fig. 4a), and 1 (S. coelicolor WP11) out of 12 showed integration by homologous recombination through the right-hand sequence (event 2) (Fig. 4a and b, lane 2). Neither event was expected to disrupt transcription of the fab operon. WP11 was chosen as a parent from which to attempt to isolate a fabH disruptant because its low frequency of occurrence suggested that the recombination event leading to the deletion of fabH would be favored. Twenty-four apramycin-sensitive segregants were isolated among 21,553 colonies screened after three rounds of growth in the absence of apramycin. All had reverted to wild type via a reversal of the first crossover (event 2); as shown by Southern hybridization (Fig. 4b, lane 6); none had undergone the second crossover event to delete the fabH gene.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Disruption of fabH by double crossover. (a) Schematic representation of the disruption events. (b) Southern hybridization analysis of the recombinant strains at each stage of the disruption. Genomic DNA from each strain was digested with SphI and PstI. The hybridization probe was radiolabeled fabH. Lane M, -HindIII molecular size standards (sizes are indicated in kilobases); lane 1, M145 (parental strain); lane 2, WP11 (integration of pIJ8155 through event 2); lane 3, WP21 (same as WP11 but with a second copy of fabH integrated at the C31 att site on pIJ84); lane 4, WP22 (pIJ8155 excised through a reversal of the original integration event); lane 5, WP23 (pIJ8155 excised through event 1 leaving the disrupted copy of fabH in the fab cluster); lane 6, apramycin-sensitive revertant of WP11 (as M145). Note that irrelevant lanes are unidentified.