RESUMO

Non-ribosomal peptide synthetases (NRPSs) and NRPS-like enzymes are abundant in microbes as they are involved in the production of primary and secondary metabolites. In contrast to the well-studied NRPSs, known to produce non-ribosomal peptides, NRPS-like enzymes exhibit more diverse activities and their evolutionary relationships are unclear. Here, we present the first in-depth phylogenetic analysis of fungal NRPS-like A domains from functionally characterized pathways, and their relationships to characterized A domains found in fungal NRPSs. This study clearly differentiated amino acid reductases, including NRPSs, from CoA/AMP ligases, which could be divided into 10 distinct phylogenetic clades that reflect their conserved domain organization, substrate specificity and enzymatic activity. In particular, evolutionary relationships of adenylate forming reductases could be refined and explained the substrate specificity difference. Consistent with their phylogeny, the deduced amino acid code of A domains differentiated amino acid reductases from other enzymes. However, a diagnostic code was found for α-keto acid reductases and clade 7 CoA/AMP ligases only. Comparative genomics of loci containing these enzymes revealed that they can be independently recruited as tailoring genes in diverse secondary metabolite pathways. Based on these results, we propose a refined and clear phylogeny-based classification of A domain-containing enzymes, which will provide a robust framework for future functional analyses and engineering of these enzymes to produce new bioactive molecules.

Assuntos

Assuntos

RESUMO

Actinobacteria are prokaryotes with a large biotechnological interest due to their ability to produce secondary metabolites, produced by two main biosynthetic gene clusters (BGCs): polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS). Most studies on bioactive products have been carried out on actinobacteria isolated from soil, freshwater or marine habitats, while very few have been focused on halophilic actinobacteria isolated from extreme environments. In this study we have carried out a comparative genomic analysis of the actinobacterial genus Saccharomonospora, which includes species isolated from soils, lake sediments, marine or hypersaline habitats. A total of 19 genome sequences of members of Saccharomonospora were retrieved and analyzed. We compared the 16S rRNA gene-based phylogeny of this genus with evolutionary relationships inferred using a phylogenomic approach obtaining almost identical topologies between both strategies. This method allowed us to unequivocally assign strains into species and to identify some taxonomic relationships that need to be revised. Our study supports a recent speciation event occurring between Saccharomonospora halophila and Saccharomonospora iraqiensis. Concerning the identification of BGCs, a total of 18 different types of BGCs were detected in the analyzed genomes of Saccharomonospora, including PKS, NRPS and hybrid clusters which might be able to synthetize 40 different putative products. In comparison to other genera of the Actinobacteria, members of the genus Saccharomonospora showed a high degree of novelty and diversity of BGCs.

RESUMO

Rhodococcus erythropolis S43 is an arsenic-tolerant actinobacterium isolated from an arsenic contaminated soil. It has been shown to produce siderophores when exposed to iron-depleting conditions. In this work, strain S43 was shown to have the putative heterobactin production cluster htbABCDEFGHIJ(K). To induce siderophore production, the strain was cultured in iron-depleted medium in presence and absence of sodium arsenite. The metabolites produced by S43 in the colorimetric CAS and As-mCAS assays, respectively, showed iron- and arsenic-binding properties reaching a chelating activity equivalent to 1.6 mM of desferroxamine B in the supernatant of the culture without arsenite. By solid-phase extraction and two subsequent HPLC separations from both cultures, several fractions were obtained, which contained CAS and As-mCAS activity and which were submitted to LC-MS analyses including fragmentation of the major peaks. The mixed-type siderophore heterobactin B occurred in all analyzed fractions, and the mass of the "Carrano heterobactin A" was detected as well. In addition, generation of a molecular network based on fragment spectra revealed the occurrence of several other compounds with heterobactin-like structures, among them a heterobactin B variant with an additional CH2O moiety. 1H NMR analyses obtained for preparations from the first HPLC step showed signals of heterobactin B and of "Carrano heterobactin A" with different relative amounts in all three samples. In summary, our results reveal that in R. erythropolis S43, a pool of heterobactin variants is responsible for the iron- and arsenic-binding activities. KEY POINTS: • Several heterobactin variants are the arsenic-binding compounds in Rhodococcus erythropolis S43. • Heterobactin B and the compound designated heterobactin A by Carrano are of importance. • In addition, other heterobactins with ornithine in the backbone exist, e.g., the new heterobactin C.

Assuntos

RESUMO

Members of the genus Cylindrospermopsis represent an important environmental and health concern. Strains CS-508 and MVCC14 of C. raciborskii were isolated from freshwater reservoirs located in Australia and Uruguay, respectively. While CS-508 has been reported as non-toxic, MVCC14 is a saxitoxin (STX) producer. We annotated the draft genomes of these C. raciborskii strains using the assembly of reads obtained from Illumina MiSeq sequencing. The final assemblies resulted in genome sizes close to 3.6 Mbp for both strains and included 3202 ORFs for CS-508 (in 163 contigs) and 3560 ORFs for MVCC14 (in 99 contigs). Finally, both the average nucleotide identity (ANI) and the similarity of gene content indicate that these two genomes should be considered as strains of the C. raciborskii species.