PCR Detection of Type I Polyketide Synthase Genes in Myxobacteria

The diversity of type I modular polyketide synthase (PKS) wasexplored by PCR amplification of DNA encoding ketosynthase andacyltransferase domains in myxobacteria. The sequencing of theamplicons revealed that many PKS genes were distantly relatedto the published sequences. Thus, myxobacteria may be excellentresources for novel and diverse polyketides.

INTRODUCTION

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INTRODUCTION

Myxobacteria are gram-negative, rod-shaped, gliding bacteriawith a high G+C content whose unique characteristic is the processof multicellular development that leads to fruiting body formation.Furthermore, myxobacteria are considered to be a rich sourceof antibiotics that are rarely produced by other microorganisms.Interestingly, the majority of bioactive compounds isolatedfrom myxobacteria contain polyketide structures (1, 2, 22).Therefore, myxobacterial strains seem to have many novel polyketidesynthase (PKS) genes and to produce hitherto unknown polyketides.While the presence of novel PKS genes in an organism is a goodindicator of the production of novel polyketide molecules inthis organism, reports on analyses of PKS genes in taxonomicallydiverse myxobacterial strains have been limited (17). Here,we characterized PKS genes in various myxobacterial strainsof nine different genera, which were derived not only from terrestrialbut also from marine environments.

PCR amplification of PKS genes in myxobacteria.

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PCR amplification of PKS...

Two primer sets, 5'-GCSATGGAYCCSCARCARCGSVT-3'/5'-GTSCCSGTSCCRTGSSCYTCSAC-3',reported by Schirmer et al. (23), and 5'-TTCSTSTTYMCSGGVCAGG-3'/5'-GSGGGCYSABYTCSABGAA-3',designed in this study, with conserved regions in mta (25),cta (4), mel (28), epo (27), sti (8), tub (GenBank accessionno. EAU69663), mch (GenBank accession no. AJ698723), and chi(21) were used for the amplification of DNA encoding the ketosynthase(KS) and acyltransferase (AT) domains, respectively. The reactionmixture contained a 0.2 mM concentration of each deoxynucleosidetriphosphate, 0.5% (vol/vol) dimethyl sulfoxide, a 1 µMconcentration of degenerate primers, 12.5 ng/µl of genomicDNA, and 0.025 units/µl of EX-Taq Hot Start version polymerase(TaKaRa Bio, Shiga, Japan) in 1x EX-Taq PCR buffer. Amplificationof KS sequences was performed with an initial denaturation step(94°C, 5 min), followed by 25 cycles of denaturation (94°C,30 s), annealing (66°C, 30 s), and extension (72°C,1 min). A final extension was performed at 72°C for 5 min.For the amplification of AT sequences, 35 cycles of denaturation(94°C, 30 s), annealing (64°C, 30 s), and extension(72°C, 1 min) were employed instead of the 25 cycles usedin the PCR amplification of KS. KS and AT DNAs (about 680 bpand 810 bp, respectively) were amplified from all 20 strainslisted in Table 1. The PCR products were cloned, and the sequenceswere compared to the published sequences by performing a BLASTsearch against sequences in the GenBank/EMBL/DDBJ databases.For convenience, they were classified into two categories: "knowngenes," exhibiting significant similarity (>70% identityof amino acid sequences) to published sequences, and "novelgenes," exhibiting less significant similarity (<70%) tothe published sequences. The cutoff value of 70% to separate"known" and "novel" PKS genes was chosen because the amino acidsequences of KS domains involved in the synthesis of structurallyrelated polyketide molecules were, in almost all cases, morethan 70% identical to each other in Streptomyces (our unpublishedobservations).

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TABLE 1. Tested myxobacterial strains and numbers of PKS sequences obtained in this study

Among the strains used in this study, nine have been observedto produce 10 different polyketides (Table 1). Among the 10polyketides, biosynthesis genes have been identified for 7 polyketides,namely, myxothiazol (mta), cystothiazole (cta), myxalamid (mxa/mmx)(5, 24), disorazol (dis/dsz) (2, 15), ambruticin (amb) (13),epothilone (epo), and spirangien (spi) (6). As expected, sequencessimilar to these genes were found in the corresponding producers(data not shown).

Novel PKS genes observed in myxobacteria.

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Novel PKS genes observed...

In addition to the known PKS genes described so far, many PKSgene sequences whose sequence identities to known genes areless than 70% were also found in this analysis. All PKS genesdetected in the Myxococcus xanthus type strain were almost identical(>98% identity) to those in M. xanthus DK 1622, whose genomeproject has been completed (9), and therefore no novel PKS genewas found. On the other hand, all 19 of the other strains possessednovel PKS genes (Table 1).

The percentages of novel genes in terrestrial Cystobacterineaeand terrestrial Sorangiineae strains were 24% and 40%, respectively,while that in marine Sorangiineae was 100% (Fig. 1). Furthermore,the sequence similarities of the PKS genes found in the marinestrains were quite low compared to those found in terrestrialstrains: almost half of the marine PKS genes exhibited lessthan 55% identity to the reported sequences.

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FIG. 1. Percentages of novel PKS sequences in myxobacteria of different categories.

Phylogenetic diversity of PKS sequences.

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Phylogenetic diversity of PKS...

The phylogenetic relationship of myxobacterial PKS sequenceswas reconstructed by the NJ method (Fig. 2; see Fig. S1 in thesupplemental material). Most of the sequences detected in Sorangiumcellulosum EW-4 were similar to those in S. cellulosum YA-2.Often, but not always, PKS in one strain of Cystobacterineaewas similar to that in another strain of Cystobacterineae; however,most other sequences were nonredundant, indicating that differentstrains have different PKS genes.

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FIG. 2. Phylogenetic trees of of AT domains in PKSs from myxobacteria. Bootstrap values of >500 calculated from 1,000 bootstrap trees (neighborhood-joining algorithm) are indicated at the nodes. Bars indicate 5% amino acid sequence divergence. Identity to the best-matched PKS genes (or their genomes) found in the BLAST search are indicated in brackets. DK, PKS genes in the genome of M. xanthus DK 1622 (9). Sequences with <70% identity are shaded in gray or black. Symbols: open star, terrestrial Cystobacterineae; filled star, terrestrial Sorangiineae; open circle (and black background), marine Sorangiineae.

PKS sequences were organized into many clades. In addition tothe clades of reported PKS genes, many novel clades were constructed.These clades were often composed of only Cystobacterineae sequencesor only Sorangiineae sequences, suggesting the relationshipbetween the taxonomy and the distribution of PKS genes, althoughsome PKS clades included sequences derived from different suborderstrains. In general, the correlation between the phylogeneticpositions based on 16S rRNA gene sequences and those based onPKS genes is low, and the distribution of PKS genes in differentbacterial strains is often explained by horizontal gene transfer(12, 18). However, in myxobacteria, horizontal gene transferbetween different suborders might not be so frequent, and differenttaxa of myxobacteria seem to possess different PKS genes.

Interestingly, some marine Sorangiineae sequences formed marine-specificclades which were well separated from the sequences of terrestrialstrains (Fig. 2). Such phylogenetically novel clades found inthis study would be especially interesting for exploring PKSgenes involved in the synthesis of novel metabolites. The remarkablenovelty and diversity of PKS genes in marine strains indicatethe importance of marine myxobacteria as sources for exploringnovel polyketide compounds.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

DNA sequences obtained in this study were deposited in the DDBJunder accession numbers AB376371 to AB376541.

ACKNOWLEDGMENTS

This work was supported by a grant from the New Energy and IndustrialTechnology Development Organization, Japan (P02038).

FOOTNOTES

* Corresponding author. Mailing address: NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu, Chiba 292-0818, Japan. Phone: 81-438-20-5764. Fax: 81-438-52-5766. E-mail: hskomaki{at}nbrc.nite.go.jp

Published ahead of print on 7 July 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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