Genome based analysis of type-I polyketide synthase and nonribosomal peptide synthetase gene clusters in seven strains of five representative Nocardia species

Abstract

Background: Actinobacteria of the genus Nocardia usually live in soil or water and play saprophytic roles, but they also opportunistically infect the respiratory system, skin, and other organs of humans and animals. Primarily because of the clinical importance of the strains, some Nocardia genomes have been sequenced, and genome sequences have accumulated. Genome sizes of Nocardia strains are similar to those of Streptomyces strains, the producers of most antibiotics. In the present work, we compared secondary metabolite biosynthesis gene clusters of type-I polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS) among genomes of representative Nocardia species/strains based on domain organization and amino acid sequence homology.

Results: Draft genome sequences of Nocardia asteroides NBRC 15531(T), Nocardia otitidiscaviarum IFM 11049, Nocardia brasiliensis NBRC 14402(T), and N. brasiliensis IFM 10847 were read and compared with published complete genome sequences of Nocardia farcinica IFM 10152, Nocardia cyriacigeorgica GUH-2, and N. brasiliensis HUJEG-1. Genome sizes are as follows: N. farcinica, 6.0 Mb; N. cyriacigeorgica, 6.2 Mb; N. asteroides, 7.0 Mb; N. otitidiscaviarum, 7.8 Mb; and N. brasiliensis, 8.9 - 9.4 Mb. Predicted numbers of PKS-I, NRPS, and PKS-I/NRPS hybrid clusters ranged between 4-11, 7-13, and 1-6, respectively, depending on strains, and tended to increase with increasing genome size. Domain and module structures of representative or unique clusters are discussed in the text.

Conclusion: We conclude the following: 1) genomes of Nocardia strains carry as many PKS-I and NRPS gene clusters as those of Streptomyces strains, 2) the number of PKS-I and NRPS gene clusters in Nocardia strains varies substantially depending on species, and N. brasiliensis strains carry the largest numbers of clusters among the species studied, 3) the seven Nocardia strains studied in the present work have seven common PKS-I and/or NRPS clusters, some of whose products are yet to be studied, and 4) different N. brasiliensis strains have some different gene clusters of PKS-I/NRPS, although the rest of the clusters are common within the N. brasiliensis strains. Genome sequencing suggested that Nocardia strains are highly promising resources in the search of novel secondary metabolites.

Figure 1

Phylogenetic positions of Nocardia strains studied in the present work. The phylogenetic tree was constructed using 16S rRNA gene sequences of type strains (http://www.bacterio.cict.fr/, http://www.ncbi.nlm.nih.gov/nuccore). MEGA5 software [34] was used to draw non-rooted neighbor-noining phylogenic tree. Positional information of Mycobacterium tuberculosis (Genbank accession # X58890) and Streptomyces coelicolor (#AB184196) was added to the tree. Bootstrap values of 1000 re-samplings are shown only for the main branches. The five species studied in the present work were marked with red arrows.

Figure 2

PKS-I, NRPS, and PKS-I/NRPS hybrid gene clusters identified in genome sequences of Nocardia strains.

Figure 3

Comparison of nbt -like, siderophore-synthesizing gene clusters (Figure 2 #20) among Nocardia species. N. farcinica (A), N. otitidiscaviarum (B), N. asteroides (C), N. brasiliensis NBRC 14402^T (D), and N. brasiliensis IFM 10847 (E). An nbtF-like gene was located distantly from other nbt-like genes in N. otitiduscaviarum and N. asteroides genomes. Homologous NRPSs are marked with the same colors (green, purple, and yellow), while PKS-Is are colored orange. In N. otitidiscaviarum, nbtA, nbtB, nbtC, nbtE-like genes were not found. F. Domains and module structures of a putative siderophore synthetic gene cluster (line #40 in Figure 2) found only in N. otitidiscaviarum IFM 11049. Putative gene functions were inferred by BLASTP search [26] and MOTIF search [21] and are indicated in the figure.

Figure 4

A representative example of PKS-I gene cluster (#46 in Figure  2 ) (A), and its putative product (B). The cluster is specific to N. asteroides NBRC 15531^T. A. Domain organization. LM, loading module; m1 – m11, modules; KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; DH, dehydratase; KR, ketoreductase; ER, enoyl-reductase domains. Gray ’DH” domains are probably inactive. B. Chemical structure of intermediate polyketide chain predicted from the gene cluster #46 based on assembly line rule [46, 49].

Figure 5

Large PKS-I/NRPS hybrid gene clusters found in N. otitidiscaviarum , N. brasiliensis HUJEG-1 and IFM 10847 (Figure 2 #20). A; The hybrid gene cluster found in N. otitidiscaviarum. B and C; N. brasiliensis HUJEG-1 and IFM 10847 have only the left half of the hybrid cluster shown in A. A similar cluster in N. brasiliensis NBRC 14402^T is truncated at an edge of a contig, and not shown here. D and E; Clusters similar to the one in A are found in Rhodococcus opacus B4 (D) [53], and Rhodococcus jostii RHA1 (E) [54]. Loci of the left clusters and the right two NRPS clusters are separated in Rhodococcus genomes.