Complete genome sequence of Saccharothrix espanaensis DSM 44229T and comparison to the other completely sequenced Pseudonocardiaceae
© Strobel et al.; licensee BioMed Central Ltd. 2012
Received: 27 April 2012
Accepted: 30 August 2012
Published: 9 September 2012
The genus Saccharothrix is a representative of the family Pseudonocardiaceae, known to include producer strains of a wide variety of potent antibiotics. Saccharothrix espanaensis produces both saccharomicins A and B of the promising new class of heptadecaglycoside antibiotics, active against both bacteria and yeast.
To better assess its capabilities, the complete genome sequence of S. espanaensis was established. With a size of 9,360,653 bp, coding for 8,501 genes, it stands alongside other Pseudonocardiaceae with large genomes. Besides a predicted core genome of 810 genes shared in the family, S. espanaensis has a large number of accessory genes: 2,967 singletons when compared to the family, of which 1,292 have no clear orthologs in the RefSeq database. The genome analysis revealed the presence of 26 biosynthetic gene clusters potentially encoding secondary metabolites. Among them, the cluster coding for the saccharomicins could be identified.
S. espanaensis is the first completely sequenced species of the genus Saccharothrix. The genome discloses the cluster responsible for the biosynthesis of the saccharomicins, the largest oligosaccharide antibiotic currently identified. Moreover, the genome revealed 25 additional putative secondary metabolite gene clusters further suggesting the strain’s potential for natural product synthesis.
The discovery of new antibiotics is an essential strategy to effectively combat multidrug-resistant pathogens. Two thirds of all natural products with antibiotic activity are derived from bacteria of the order Actinomycetales. However, their potential to produce new antibiotics is not exhausted . Saccharothrix is a genus of this order which harbors strains producing natural products of industrial interests . Furthermore, Saccharothrix is known for its ability to glycosylate natural products hereby increasing their biological activity .
Saccharothrix espanaensis is the producer of the saccharomicins, a new class of heptadecaglycoside antibiotics [5, 6] with activity against MRSA and VRE . In this paper we present the classification and analysis of the genome of this capable antibiotic producer.
Results and discussion
Sequencing and general features of the Saccharothrix espanaensis DSM 44229 chromosome
The genome sequence of S. espanaensis was established using a whole genome shotgun approach applying next generation sequencing techniques. The initial scaffolding was performed by 454 sequencing using a 3 k paired-end library, resulting in 352 contigs in five scaffolds. In order to obtain a single scaffold, a fosmid library of 528 clones was sequenced from the ends and the sequences were mapped onto the scaffolds. This resulted in a single scaffold and delivered templates for primer walking for 283 of the 352 remaining gaps.
General genome statistics and comparison of the completely sequenced Pseudonocardiaceae
A. mediterranei U 32
A. mirum DSM 43827
P. dioxanivorans CB 1190
S. erythraea NRRL 2338
S. espanaensis DSM 44229
S. viridis DSM 43017
T. bispora DSM 43833
G + C content [%]
For 4,297 (51.0%) of the annotated CDS, a function could be automatically inferred using a number of sequence similarity based approaches implemented in the GenDB auto-annotator METANOR .
Number of genes associated with the general eggNOG functional categories
RNA processing and modification
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane/envelope biogenesis
Intracellular trafficking, secretion, and vesicular transport
Posttranslational modification, secretion, and vesicular transport
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not hit in eggNOG
Comparison of the S. espanaensis genome with other completely sequenced Pseudonocardiaceae
This implication is backed by further analyzing the correlation between gene conservation and location relative to the oriC: the core genes are found predominantly clustered around the oriC (Figure 1, genes depicted in green in circles 3–7) while genes conserved in only some species and the 2,967 S. espanaensis singletons (Figure 1, genes depicted in red respectively light blue in circles 3–7) are found farther away from the oriC.
The genes of the accessory genome of S. espanaensis are either ancient and/or obtained from closely related species
Genes participating in the synthesis of saccharomicins
Our analysis of the complete genome sequence of S. espanaensis revealed the best candidate for the saccharomicin biosynthetic gene cluster (sam) (Figure 6B and Additional file 3), because this cluster harbors both the genes responsible for the production of the caffeic acid moiety of the aglycon, as well as ten glycosyltransferase genes necessary for the formation of the oligosaccharide side chain. The sam-cluster does not belong to the core genome and is located at the lower part of the circular chromosome, close to termination of replication. It comprises approximately 47,000 base pairs and is predicted to encode 38 genes. The identification of the sam-cluster allows further insights into the assembly of these new antibiotics. The product of sam7 shows similarity to acyl-CoA synthetases. It is therefore tempting to speculate that Sam7 may be involved in the synthesis of caffeoyl-CoA, which might be used to link taurine to form the entire aglycon. This reaction may be catalyzed by Sam36 which shows similarities to penicillin amidases. However, the synthesis of taurine has not been described in bacteria. In mammals, taurine originates from l-cysteine via cysteine sulfinic acid. Even though the dioxygenation of cysteine has been shown in bacteria , they are not able to produce taurine due to the lack of a cysteine sulfinic acid decarboxylase (csad). The same is true for S. espanaensis: there are potential cysteine dioxygenase genes (Ses72720, Ses71790) but no csad homologues are detectable in the genome. However, sam29 may encode the required chemistry. The protein Sam29 shows similarities to aspartate-1-decarboxylases which are responsible for the decarboxylation of l-aspartate forming β-alanine. The structures of cysteine sulfinic acid and l-aspartate are identical with the exception of one atom. While aspartate possesses a carbon as part of the carboxyl group, cysteine sulfinic acid contains sulfur in this position. As a result of this similarity we suggest that Sam29 might be able to decarboxylate cysteine sulfinic acid. If this is found to be correct, sam29 would represent the first gene responsible for the production of taurine in bacteria.
In addition to genes accountable for the formation of the aglycon, there are candidates encoding proteins involved in sugar synthesis and attachment. To link the 17 sugars to the aglycon, there are ten glycosyltransferase genes (sam11-sam20) in the cluster. Consequently, some of the glycosyltransferases may work iteratively. Because sequence analyses of those genes gave no further indication, the exact function of each glycosyltransferase will have to be experimentally investigated.
Potential for secondary metabolite production
Secondary metabolite cluster comparison of the completely sequenced Pseudonocardiaceae
A. mirum DSM 43827
A. mediterranei U32
P. dioxanivorans CB1190
S. erythraea NRRL 2338
S. espanaensis DSM 44229
S. viridis DSM 43017
T. bispora DSM 43833
Seven clusters possibly encoding terpenoid biosynthetic enzymes are distributed throughout the genome. The C5-precursors required for their biosynthesis originate from the methylerythriol phosphate pathway, for which all genes are present. Terpene derived metabolites include carotenoid pigments (crt) serving as UV protector and odorous substances providing actinomycetes with their characteristic smell [33, 34].
In the genome a total of five nonribosomal peptide synthetases (NRPS) encoding clusters could be identified (cluster 1, 2, 3, 7 and 12). Common metabolites produced by NRPS in actinomycetes are for example the antibiotic vancomycin, the cytotoxic agent bleomycin and the iron-scavenging siderophore griseobactin [35–37]. The proteins producing such polypeptides are usually composed of modules consisting of condensation, adenylation and thiolation domains. Remarkably, cluster 1 possesses a set of genes which code for only one nrps domain each. Consequently, this small cluster of about 20 kb may harbor an archetype of nrps. These genes, consisting of a condensation, an adenylation and a thiolation domain, respectively, might be ancestors of our contemporary nrps genes composed of a chain of different domains. The modules of all nrps genes and the specificity of their adenylation domains are listed in Additional file 5.
In addition to the clusters producing nonribosomal peptides, there are three clusters producing type I polyketides (cluster 4, 9 and 11). These natural products are synthesized by decarboxylative condensation of malonyl-CoA derived extender units. Polyketide synthetases (PKS) possess as well a modular assembly and are the well-known producers of the antibiotic erythromycin and the immunosuppressant tacrolimus in other actinomycetes strains [38, 39]. The modules of all polyketide synthetases identified in S. espanaensis are listed in Additional file 6. The PKS containing clusters are the largest in the genome of S. espanaensis and comprise between 50 and 86 kb.
Besides pure NRPS or PKS clusters, we identified six clusters which harbor both types of these secondary metabolite synthesis genes (cluster 5, 6, 8, 10, 13 and 14). Among them, cluster 6 shows high similarity to the maduropeptin cluster from Actinomadura madurae ATCC 39144 . Consequently, cluster 6 is identified as a putative enediyne cluster.
Cluster 13 is not only a NRPS/PKS type I hybrid, but it also contains genes coding for type II PKS. The type II PKS part of the cluster is highly similar to the kinamycin gene cluster from Streptomyces murayamaensis [GenBank:AH012623.1]. All core and tailoring enzymes required for the production of kinamycin are present in cluster 13. Additionally it possesses further genes encoding tailoring enzymes like a P450 dependent monooxygenase (ses54730), an aminotransferase (ses54750) and a methyltransferase (ses54760). The type I PKS and the NRPS part of the cluster may contribute to the modification of the core structure of cluster 13, leading to a kinamycin derivative. Another hybrid, Cluster 14, shows similarities to the azinomycin B biosynthetic gene cluster from Streptomyces sahachiroi . Azinomycin B is an antitumor agent consisting of a PKS-derived naphthoate attached to a nonribosomal peptide. The peptide part of the molecule is composed of unusual building blocks like α-ketoisovaleric acid and an aziridino-[1,2a]-pyrrolidinyl amino acid. Cluster 14 harbors ses56840, whose gene product is similar to AziC1 responsible for the production of α-ketoisovaleric acid from valine. Additionally, all genes are present for the production of the aziridino-[1,2a]-pyrrolidinyl amino acid (aziC2 aziC10; ses56710, ses56680, ses56700, ses56740, ses56730, ses56750, ses56760, and ses56690) except for a homolog to aziC8. Furthermore, the cluster possesses homologues of aziD2 and aziD3 (ses56770 and ses56780) which are responsible for tailoring modifications of the molecule. However, the PKS modules of the azinomycin B cluster differ from the ones found in cluster 14. Therefore we propose that the PKS derived moiety of the compound produced by cluster 14 is not a naphthoate moiety as in azinomycin B. Another interesting fact is that cluster 14 is flanked by transposase and integrase genes. This suggests that the cluster was probably introduced into the genome of S. espanaensis by horizontal gene transfer.
In addition to the secondary metabolite gene clusters belonging to the prevalent groups of NRPS, PKS and terpene synthases, the genome of S. espanaensis also contains rare types of clusters: it harbors a putative aminocyclitol cluster, a melanin cluster and two putative lantibiotic clusters (lan1 and lan2). Lantibiotics are ribosomally produced and posttranslationally modified polypeptides which contain thioether-cross-linked amino acids .
The complete genome of S. espanaensis was sequenced and compared to the genomes of the other completely sequenced Pseudonocardiaceae. Thereby, the expected core genome of the family could be predicted to consist of about 810 genes. While the origin of the accessory genome of S. espanaensis remains unclear, some evidence provided suggests that the accessory genes are either part of the genome for quite some time and/or were obtained from bacteria with a similar polynucleotide composition.
Besides providing some insights into the genome evolution of the Pseudonocardiaceae, the genome sequence delivered a good candidate cluster for the production of the saccharomicins. The newly identified sam-cluster consists of 38 genes and comprises approximately 47,000 base pairs. It harbors a presumed operon of ten glycosyltransferase genes centered in the cluster. To our knowledge, there is no other biosynthetic gene cluster identified to date which includes this high number of glycosyltransferase genes. Nevertheless, to produce the heptadecasaccharide side chain of the saccharomicins even more than ten glycosylation steps are proposed, so some glycosyltransferases should work iteratively. We anticipate that the complete genome sequence will facilitate the production and modification of the saccharomicins, by either improving precursor supply or by engineering of genes belonging to the cluster to obtain novel variants.
Pyrosequencing of Saccharothrix espanaensis DSM 44229T
The type strain of Saccharothrix espanaensis (DSM 44229) was obtained as lyophilized culture from DSMZ (Braunschweig, Germany). Genomic DNA was isolated from 30 ml cultures grown in tryptone soy broth (TSB)  at 28°C for 24 hours. Total DNA isolation was performed according to the salting out procedure followed by RNase treatment .
10 μg was used to construct both a 3 k PE and a WGS library for the pyrosequencing on a Genome Sequencer FLX (Roche Applied Science). Assembly of the shotgun reads was performed with the GS Assembler software (version 2.0.00.22). A total of 1,536,941 reads (404,849,780 bp) was assembled into 352 contigs in five scaffolds.
Completion of the draft sequence
For gap closure and assembly validation, the genomic contigs were bridged by (i) a fosmid library of 528 clones spanning all but 69 gaps and (ii) 69 PCR products addressing the remaining gaps. For finishing of the genome sequence, the CONSED software package  was used.
Sequencing of fosmid ends was carried out by IIT GmbH (Bielefeld, Germany). Gaps between contigs of the whole genome shotgun assembly were closed by sequencing on PCR products and fosmid clones carried out by IIT GmbH on ABI 377 sequencing machines. To obtain a high quality genome sequence, all bases of the consensus sequence were polished to at least phred40 quality by primer walking. Collectively, 854 sequencing reads were added to the shotgun assembly for finishing and polishing of the genomic sequence.
Genome analysis and annotation
In a first step, gene finding was done by using GISMO  and an automatic annotation was performed using the genome annotation system GenDB 2.0 . In a second annotation step, all predicted ORFs were manually re-inspected to correct start codon and function assignments. Intergenic regions were checked for ORFs missed by the automatic annotation using the BLAST programs .
For comparative analyses, the annotated genome sequences of the following bacteria were imported into EDGAR : Actinosynnema mirum DSM 43827 [GenBank:NC_013093], Amycolatopsis mediterranei U32 [GenBank:NC_014318], Pseudonocardia dioxanivorans CB1190 [GenBank:NC_015312, GenBank:NC_015313, GenBank:NC_015314], Saccharopolyspora erythraea NRRL 2338 [GenBank:NC_009142], Saccharomonospora viridis DSM 43017 [GenBank:NC_013159], and Thermobispora bispora DSM 43833 [GenBank:NC_014165]. The project is available as an open EDGAR project (http://edgar.cebitec.uni-bielefeld.de/cgi-bin/edgar_login.cgi?cookie_test=1&open=1) called EDGAR_BMC_Pseudonocardiaceae.
The gene content comparisons were done via a BLASTP analysis against the bactNOG subset of the eggNOG database . Two genes were considered to be orthologs if the reciprocal BLASTP hit had a sequence identity of at least 40% and a coverage of at least 75%. For the phylogenetic analysis, all predicted CDS were compared against the RefSeq protein database  (from August 2011) using BLASTP. The species for each best hit (e-value cutoff 1e-10, hit must cover at least 75% of query and subject) was retrieved and counted.
All other genome comparisons were done using EDGAR, the PCA analyses were done using R (http://cran.r-project.org/).
Analysis of secondary metabolite clusters
For the identification of secondary metabolite clusters the genome of S. espanaensis was scanned for homologues to known secondary metabolite synthases via BLAST search. These manual investigations were supported by antiSMASH . A set of genes was considered to be a cluster, when there was at least one gene encoding a secondary metabolite synthase. Consequently, a locus possessing a gene with only a single domain, for example an A domain, was not considered to be a cluster. The boundaries of the clusters were defined by the last gene upstream and downstream of a secondary metabolite synthase with homology to a gene encoding a regulator, transporter or tailoring enzyme. In cases where this gene was part of a putative operon, the whole operon was included into the cluster. The modular organization of the type I polyketide and nonribosomal peptide megasynthases were determined using web tools [48, 49]. The A domain specificities were investigated using NRPSpredictor2 [50, 51].
Nucleotide sequence accession numbers
The sequences reported here have been deposited in the EMBL database (accession no.: [EMBL:HE804045]). The locus tag prefix "BN6_" assigned by the ENA is replaced by "ses" throughout.
CR and RS acknowledge funding by a research grant awarded by the MIWFT within the BIO.NRW initiative (grant 280371902).The project was funded by a grant from the Federal Ministry of Education and Research (BMBF, grant 0313805A).
We gratefully acknowledge John Quimby at MIT for his very helpful improvements in choice of language.
We acknowledge support of the publication fee by Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University.
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