Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel1
Department of Genetics, University of Leicester, Leicester LE1 7RH, UK2
Author for correspondence: Eliora Z. Ron. Tel: +972 3 6409379. Fax: +972 3 6414138. e-mail: eliora{at}ccsg.tau.ac.il
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ABSTRACT |
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Keywords: Myxococcus xanthus, polyketide synthase, acyl carrier protein, ß-ketoacyl ACP synthase III, antibiotic post-modification
Abbreviations: ACP, acyl carrier protein; HMG, hydroxymethylglutaryl; KAS, ß-ketoacyl:ACP synthase; PKS, polyketide synthase
The EMBL accession number for the sequence reported in this paper is AJ132503.
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INTRODUCTION |
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Genetic studies of prokaryotic PKSs have focused on Gram-positive bacteria, particularly on actinomycetes, and not much is known about PKSs in other bacteria. Myxobacteria are Gram-negative bacteria that produce a large number of secondary metabolites, including polyketides (Reichenbach & Hofle, 1993 ). They are soil micro-organisms, feeding on proteins and peptides, that undergo a complex life cycle which includes cell-to-cell interactions, signalling, myxosporulation and fruiting body formation (Shimkets, 1990
). We have recently studied the biosynthesis of the novel macrolide antibiotic, TA (Fig. 1
), produced by Myxococcus xanthus (Rosenberg et al., 1973
), which inhibits bacterial cell wall synthesis (Zafriri et al., 1981
). It is a unique antibiotic in its ability to adhere avidly to a variety of tissues (Rosenberg et al., 1984
), a property that has been exploited in a successful preliminary clinical trial for treatment of gingivitis (Manor et al., 1989
). The production of TA and its chemical properties have been studied extensively (Rosenberg et al., 1973
, 1982
; Rosenberg & Varon, 1984
; Trowitzsch et al., 1982
). It is synthesized from the precursors acetate, methionine and glycine (Fytlovtich et al., 1982
). Genes required for the biosynthesis of TA have been identified by transposon mutagenesis, and several of them were mapped by P1-transduction to a 36 kb region of the bacterial chromosome (Varon et al., 1992
) and shown to be co-regulated (Tolchinsky et al., 1992
; Varon & Rosenberg, 1996
, Varon et al., 1997
). Several genes involved in TA post-modification and regulation, such as a cytochrome P-450 hydroxylase (Paitan et al., 1999b
) and a specific transcription anti-terminator (Paitan et al., 1999c
), were cloned and analysed. One of the cloned genes in this cluster encodes a type I PKS module, indicating that TA is produced through a type I PKS mechanism (Paitan et al., 1999a
).
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METHODS |
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Media and growth conditions.
E. coli was grown at 37 °C in Luria broth (LB), or on LB agar, with the appropriate antibiotics (Sambrook et al., 1989 ). M. xanthus was grown at 32 °C in 0·5 CTS, 1 CT or CTK medium, as required, or on media solidified with 1·5% Bacto agar (Difco) as described before (Tolchinsky et al., 1992
; Varon et al., 1992
).
General DNA procedures.
Standard genetic techniques, Southern blot analysis, colony hybridization, plasmid preparations and in vitro DNA manipulations were performed according to published protocols (Sambrook et al., 1989 ). Isolation of total DNA from M. xanthus is described elsewhere (Paitan et al., 1998
). Cosmid DNA and DNA templates for sequencing reactions and electroporation were purified by Qiagen columns. Electroporation of M. xanthus was performed as described before (Kashefi & Hartzell, 1995
) using a Gene Pulser II (Bio-Rad). Conjugative transposition of Tn1000 for sequencing is described elsewhere (Paitan et al., 1998
). Construction of plasmids for specific gene disruption was achieved by inserting a kanamycin-resistance gene to: (i) pPY11, disrupting the taF gene by replacing an 11 bp SalISalI fragment (positions 38913902), (ii) pPY10, producing a deletion of four ORFs (taCtaF) by replacing a 2·667 kb SalISalI fragment (positions 12353902), and (iii) pPY10, disrupting the taB gene by replacing a 249 bp NcoINcoI fragment (positions 429678; Fig. 2
).
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A 4·5 kb fragment was sequenced. Occasional sequence gaps were filled in by direct sequencing, using synthetic oligonucleotide primers. Analysis and assembly of the primary DNA sequence data were carried out using the MacVector 3.5 (International Biotechnologies) and Sequence Navigator (Applied Biosystems) software. Searches of databases for DNA and protein sequence homology were carried out using the BLAST (Altschul et al., 1990 ) and FASTA (Pearson, 1990
) programs.
TA production assay and developmental analysis.
The production of the antibiotic TA was determined by the disc assay described previously (Varon et al., 1992 ). Fruiting body formation and development were examined on TPM agar medium (10 mM Tris/HCl, 1 mM KH2PO4, 8 mM MgSO4, final pH 7·6, and 1·5% agar), as described by Kroos et al. (1986)
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RESULTS |
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The inserts in cosmids pPYCC64 and pPYCA111 were subcloned into pUC19 and two recombinant plasmids pPY10, carrying a HindIIISphI fragment (4·391 kb), and pPY11, carrying a XhoISphI fragment (0·948 kb) were analysed (Fig. 3d). The nucleotide sequence of the two fragments was determined, while the sequence 3' to pPY10 was obtained from the cosmid pPYCA111, used as a template, and a synthetic oligonucleotide primer complementing pPY10 sequences. The nucleotide sequence of the entire fragment consists of 4513 bp with a G+C content of 66·4 mol%, as expected for M. xanthus DNA. This fragment was found to share significant sequence homology with several genes involved in polyketide biosynthesis, such as the Bacillus subtilis pksG (Albertini et al., 1995
; Scotti et al., 1993
) and many small acyl carrier proteins (ACPs). Computer analysis identified five ORFs, all transcribed in the same direction, which were designated taB, taC, taD, taE and taF sequentially [non-standard gene names used, to conform with previous papers on this system]. The frequencies of G+C base pairs at the third position of each codon in the presumed ORFs were found to be 79·7% (taB), 91·6% (taC), 91·1% (taD), 89·2% (taE) and 90·1% (taF), values which are typical of ORFs originating from an organism with a high percentage of G+C.
Specific gene disruptions
The physiological role of the taB-taF gene products, as well as their involvment in TA biosynthesis, was examined through gene disruptions. A kanamycin-resistance gene was inserted into plasmid pPY10 (disrupting taB or producing a deletion of four ORFs, taCtaF)) and plasmid pPY11 (disrupting taF) as described in Methods. The resulting plasmids, pPY11S-Kan, pPY10S-Kan and pPY10Nc-Kan, were isolated from E. coli, linearized and electroporated into M. xanthus ER-15. As pUC19 cannot replicate in M. xanthus, kanamycin-resistant M. xanthus ER-15 colonies should result from integration of the linearized DNA into the chromosome through homologous recombination. M. xanthus transformants were plated on CTK for 5 d and the correct integration into the chromosome was verified by Southern blot analysis in several kanamycin-resistant colonies. Twenty KanR colonies from each of the transformed strains were monitored for TA production and all of them were blocked in TA biosynthesis, suggesting that both TaB and TaF are involved in the production of biologically active TA. Three mutants, one of each strain, were picked for further analysis of growth, fruiting body formation and development. The growth rates of all mutants were indistinguishable from that of ER-15, indicating that the corresponding proteins are not essential for normal growth or for fatty acid biosynthesis. Moreover, like ER-15, all strains developed to mature fruiting bodies when incubated on TPM agar plates at 30 °C, indicating that proteins encoded by these genes are not essential for the developmental process, or that their activities can be replaced by other bacterial proteins.
Characterization, identification and deduced function of the ORFs
The results, as summarized in Table 1 and displayed in Figs 4
and 5
, indicate that the proteins encoded by taC and taF are acyl enzymes possessing the activities of transacylase and condensing enzymes. This was concluded by the identification of a highly conserved acetylation site containing the conserved cysteine within the active site of condensing enzymes (C-114 in taC and C-77 in taF; Fig. 5
), and the high similarity to proteins such as the B. subtilis PksG (Fig. 4a
, b
) hydroxymethylglutaryl-CoA synthase (HMG-CoA synthetase; Fig. 5
) and the ß-ketoacyl-ACP synthase III proteins (KAS III, FabH, Fig. 5
), which are all acyl enzymes possessing these activities. In addition to the highly conserved acetylation site, a second conserved motif, identified in the N-terminal amino acid sequence of both proteins (amino acids 1624 and 3543 in TaC and TaF, respectively) is a Myc-type helixloophelix, a dimerization domain signature (PDOC00038, PS50075), which is discussed later.
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As summarized in Table 1 and displayed in Fig. 6
, the products of the other two homologous genes, taB and taE, share considerable end-to-end amino acid similarity to many small (~9 kDa) fatty acid synthase ACPs (Wang & Liu, 1991
) and PKS type I and type II ACPs (Fig. 6
). The search for conserved motifs indicates that both proteins contain a conserved active serine residue within a highly conserved amino acid motif (the 4'-phosphopantetheine binding region) found in many ACPs (Fig. 6b
). Like taC and taF, the taB and taE products also share sequence similarity (34·3% identity and 40·3% similarity; Fig. 6
), implicating them in a similar function as TA-modifying, specific ACPs in the antibiotic biosynthesis; again the presence of two similar proteins strongly suggests that each protein has a different and specific role in TA biosynthesis.
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DISCUSSION |
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The results indicate that the proteins encoded by taC and taF are homologues; they were identified as acyl enzymes, as they contain the highly conserved acetylation site, present in many condensing enzymes. The two proteins may therefore share a similar function in the post-modification process of TA. Both TaC and TaF share sequence homology with several proteins, such as members of the HMG-CoA synthetase family, KAS III proteins, including the E. coli 3-oxoacyl-[ACP] synthase III (FabH; Tsay et al., 1992 ; Fig. 4
) and proteins of the chalcone synthase family (naringenin-chalcone synthase). Such homologies imply that TaC and TaF possess, similar to KAS III proteins, the activities of both acetyl transacylase and a condensing enzyme. HMG-CoA synthetases are a defined class of enzymes, involved in ketogenesis in eukaryotes, condensing acetyl-CoA with acetoacetyl-CoA to form HMG-CoA, which is a substrate of HMG-CoA reductase in cholesterol biosynthesis (Ayte et al., 1990
; Kattar-Cooley et al., 1990
; Russ et al., 1992
). The E. coli 3-oxoacyl-[ACP] synthase III protein (FabH; Tsay et al., 1992
) is a member of the KAS III family. FabH mediates the decarboxylative condensation between acetyl-CoA and malonyl-ACP to generate acetoacetyl ACP, and unlike KAS I (FabB) and KAS II (FabF), it is the only condensing enzyme able to utilize CoA thioesters rather than acyl-ACP as the primer in the condensation reaction. FabH displays two activities: one is that of acetyl transacylase, which catalyses the transfer of a 4'-phosphopantetheine moiety from a corresponding coenzyme A to a serine residue, present in the conserved active site of ACPs. The second is that of an acetoacetyl-ACP synthase condensing enzyme (Tsay et al., 1992
). Chalcone synthase enzymes (naringenin-chalcone synthase) are ACPs which catalyse the formation of naringenin-chalcone from malonyl CoA and 4-coumaryl-CoA, which is presumably formed from polyketide intermediates (Durbin et al., 1995
). As shown in Fig. 5
, the region responsible for the acetylation/condensation activities, including the active cysteine, is highly conserved in both TaC and TaF.
The effect of disruption of taF suggests that it is probably required for the production of an active antibiotic. Moreover, the taC gene product does not complement the disrupted taF gene, located downstream of it, in the taF-disrupted mutant. However, it is possible that the block in TA production is the result of a polar effect, of the insertion in taF, on other genes downstream of taF. This possibility is difficult to exclude since there are no self-replicating plasmids in M. xanthus, which could be used for trans-complementation of the disrupted taF. This finding, and the presence of two similar homologous proteins, suggests that TaC and TaF have different and specific roles in TA biosynthesis.
Interestingly, while examining the end-to-end similarities between the deduced proteins encoded by taC, taF and pksG, we observed that the two former proteins are longer than the pksG product, displaying an extension at their N-termini (75 and 38 amino acids, respectively). In both TaC and TaF, a Myc-type helixloophelix, a dimerization domain signature, has been identified within the N-terminal amino acid sequence. This motif is absent from the putative PksG, HMG-CoA synthetase, and KAS III proteins. The Myc-type helixloophelix domain is thought to be a dimerization domain of several eukaryotic transcriptional regulators and DNA binding proteins. In the case of the TaC and TaF, this motif could have a physiological importance, either as a dimerization site, or perhaps as a recognition site for an interacting protein or ligand.
The identification of TaB and TaE as TA-modifying specific ACPs is based on their end-to-end homology to many small (~9 kDa) ACPs. Furthermore, in both proteins, a presumed active serine residue is present within a highly conserved amino acid motif found in other polyketide-ACPs (the 4'-phosphopantetheine binding motif; Fig. 6). A ~9 kDa ACP is a well-characterized component of all fatty acid synthase systems and of several PKS systems. The small ACPs found in the TA cluster could act as specific post-modifying enzymes in TA biosynthesis. The exact role(s) of TaB and TaE is as yet unknown, although gene disruption of taB suggests that it may be essential for TA production; but in-frame deletion of taB needs to be tested for the production of biologically active TA. In addition, the presence of the two ACPs, the effect of gene disruption of taB and the finding that TaE does not complement the disrupted taB suggest that each ACP performs a specific step in TA biosynthesis and probably recognizes a different substrate. This suggestion is supported by a recent study (Ritsema et al., 1998
) describing the structurefunction relationships of the chimeric protein AcpPNodF. That study suggested that the N-terminal domain of ACP proteins determines the activity, while the C-terminal part carries a specialized domain for protein recognition. Again, it is possible in the taB taE case that the lack of complementation is the result of a polar effect (of the insertion in taB) on other genes 3' to taB.
Considering the structure of TA, the chemical pattern of type I PKS condensation steps and the pattern of precursor incorporation to TA, as studied by NMR (Rosenberg & Varon, 1984 ; Rosenberg et al., 1982
; Trowitzsch et al., 1982
), several post-modification steps, including addition of 3 carbon atoms (at C-13, C-17 and C-32) originating from acetate carbon atoms, specific hydroxylation (at C-20), O-methylation (at C-34) and three C-methylations (at C-2, C-4 and C-28) should exist. The identification of taB, taC, taE and taF as two sets of genes (taBtaC and taEtaF) sharing sequence similarity to ACPs and KAS III proteins suggests that they could be involved in the addition of two of the carbon atoms which, as indicated by NMR, originate from acetate (C-32, C-33 and C-34). Their organization further implies a possible coupling of activities, as each ACP synthase (TaC and TaF) probably processes the corresponding ACP (TaB and TaE, respectively). However, further work is required to examine this possibility, as there are no other data from other polyketide systems which display a similar genomic structure. Future analysis of a non-polar disruption of these genes followed by NMR analysis of TA intermediates should resolve the exact functions and the specificities of these proteins.
The most meaningful similarities found between TaD and proteins in the databases were to membrane-associated cell-surface proteins. The inability to assign a function to taD from the sequence analysis may reflect a unique feature of TA as a polyketide. TaD could be involved in TA regulation, or in an as yet unknown physiological role of TA. The partial similarities to several membrane/cell surface-associated proteins, involved in signal transduction, suggest that TaD is likely to be a membrane/transmembrane protein involved in extracellular signalling/sensing related to TA biosynthesis.
In summary, the present study represents the first molecular analysis of genes involved in post-modification of a type I PKS, complex polyketide antibiotic in Gram-negative bacteria. This information should make it possible to compare the Gram-negative systems to the better-characterized polyketide systems of the Gram-positive actinomycetes. In addition, it may reveal novel genetic and biochemical features, which could provide the basis for developing new biologically active polyketides using combinatorial genetics.
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ACKNOWLEDGEMENTS |
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Received 16 February 1999;
revised 10 May 1999;
accepted 9 July 1999.