Among the cultivable myxobacteria, Nannocystineae is the less abundant and more diverse group. Based on a recent metagenomics study, this suborder contains 11 families, 157 genera, and 309 species that only a small number of them (nine species) held in pure cultures (Liu et al. 2019).
The first study on the myxobacteria of Iran concluded that with an average of 7.4 species per sample, this region is one of the most fruitful places for the isolation of myxobacteria (Dawid 2000). This study attributed this finding to the warm summers and humid winters of Iran. Also, it reported N. exedens in 58% of the collected soil samples. The recent study on 62 collected specimens of various types from Iran could not isolate any Nannocystis species, though it used innovative culture media that seem well not suited for Nannocystis isolation (Saadatpour and Mohammadipanah 2020).
In the present study, Nannocystis species were retrieved from 22 % of sampling sites. They formed 14% of all isolated myxobacteria and included a new species (Mohr 2018). The isolation on ST21 agar was a more efficient method compared to WCX agar because this medium is more enriched and nutritious.
N. exedens strains grown mainly from rotten plants of the arid climates, as was suggested previously (Dawid 2000). In contrast, the isolation of N. pusilla strains was more successful from the topsoil layer of northern forests and Zagreus Mountains that have much more vegetation because of increased humidity. N. konarekensis again shows more affiliation to hot and dry places. The difference in habitats results in the difference in purification techniques; N. pusilla was mainly purified on media containing antibiotic solutions due to the more contaminations of samples, though purification of N. exedens was done by several subcultures. The physiology of Nannocystis species closely resembles together and needs careful examination to distinguish between strains. On the other hand, phylogenetic analysis based on partial sequences of 16S rDNA discriminates clearly between strains of Nannocystis species. Although, genetic markers such as rpoB (Ogier et al. 2019) or genotyping methods like semi-nested PCR-DGGE (Li et al. 2014), MLSA (Lalucat et al. 2020), and comparative genomics (Livingstone et al. 2018; Chambers et al. 2020) had a better discriminatory power to resolve taxonomy at the species level. These findings emphasize the importance of attention to morphological aspects of swarms and fruiting bodies. Therefore, the polyphasic approach is the most suitable for the identification and classification of Nannocystis.
Seven out of ten secondary metabolites producers have had OSMAC (one strain many compounds) potentiality (Table 4) that synthesized compounds from different structural classes. This phenomenon was previously reported from N. pusilla strains (Bader et al. 2020), but this study revealed both species had such ability. The most notable was N. exedens 215 production of the metabolites in the full spectrum of bioactivity from unknown to cytotoxic. Despite their outstanding capability to produce metabolites from different structural classes, strains of this study had a narrow range of bioactivity on Gram-positive bacteria and the E. coli TolC mutant.
Nannochelins and althiomycin were the most abundant secondary metabolites identified in both N. pusilla and N. exedens strains. Up to 70% of the isolates had produced a type of nannochelins. These compounds are citrate-hydroxamate siderophores with the activity on Gram-positive bacteria and weak inhibition of fungi by a still unknown mechanism (Kunze et al. 1992). Althiomycin is a sulfur-containing cyclic peptide, previously isolated from several myxobacteria in Cystobacterineae and Streptomyces that elicits broad antibacterial activity by inhibiting peptidyltransferase reactions [5,25]. All althiomycin producing strains except N. pussila 166 inhibited Gram-positive test microorganisms. Also, strains 34 and 51 inhibited TolC mutant E. coli in a somewhat weaker manner.
Nannopyrazinones (nannozinone) and pyrronazols are rare bioactive molecules of N-containing heterocyclic compounds with a weak antibacterial and antifungal activity. Pyrronazols have been identified in extracts of both N. pusilla and N. exedens broths (Jansen et al. 2014a). LC-MS analysis of only one strain (49) of N. pusilla showed the peak of pyrronazol consistent with its very weak inhibition of M. hiemalis. Nannopyrazinones were reported from N. pusilla (Jansen et al. 2014b) with an additional slight cytotoxicity effect on the L929 cell line, while analysis indicated these compounds in one strain of each species (3 and 215) and therefore it may contribute to the lethal effect of strain 215 on L929 cells (Herrmann et al. 2017).
In addition to nannopyrazinone, N. pusilla 3 secreted a potentially new derivative of myxoprincomide to the fermentation broth that is a linear peptide with unusual amino acids. It was discovered in M. xanthus DK 1622 broth mass spectrum by metabolome mining and statistical evaluations (Cortina et al. 2012). There is no report of biological activity or mechanism for this metabolite but, increased bioactivity of N. pusilla 3 than the reported bioactivity of nannopyrazinones may be due to the synergism between these metabolites.
Strains 7 and 212 of N. pusilla produced a single metabolite, myxopyronin, with the α-pyrone structure that inhibits bacterial RNA polymerase by binding to the “switch region” of the molecule (Mukhopadhyay et al. 2008). Myxopyronin was initially reported from M. fulvus and exhibited a broad antibacterial activity against both high and low G + C Gram-positive and some Gram-negatives (Irschik et al. 1983). Screening of bioactivities of these strains (7 and 212) support the identification based on LC-MS with inhibition of all tested Gram-positive bacteria, M. smegmatis and, TolC mutant of E. coli.
Germacrane produced by N. exedens 215 is a cyclodecane sesquiterpene previously reported from N. exedens (Reichenbach, H.; Höfle 2000). Its biological importance for producing strains is not understood, but derivatives of germacrane in medicinal plants show a broad range of antimicrobial activity and cytotoxicity (Zhang et al. 2018).
Only N. exedens 215 with the production of myxothiazol displayed toxicity to L929 fibroblasts. Myxothiazol was reported for the first time from Myxococcus fulvus strain Mx f16 and structurally belongs to the bithiazole compounds that inhibit respiratory complex III [33,34]. In addition to cytotoxicity, this antibiotic has antifungal activity as seen by slight inhibition of M. hiemalis by N. exedens 215.
The heatmap clearly shows the distinct distribution of metabolites between species of Nannocystis. Also, Nannocystis in this study shares three metabolites with Myxococcus from the Cystobacterianea suborder. Hoffmann and Krug reveal a correlation between chemical diversity and the taxonomy of myxobacteria; in this study, the profile of known and unknown metabolites of each taxon above the genus rank differs meaningfully from other taxons. Likewise, they found such a correlation between four species of Myxococcus and their metabolites, however, in a less clear state (Hoffmann et al. 2018). Another relevant finding of Hoffmann was near relatedness in secondary metabolites profiles of Myxococcus and Nannocystis genera in unknown and known metabolites (Hoffmann et al. 2018).
In conclusion, this study presents Iran as an excellent source for the isolation of Nannocystis strains with a high potential for secondary metabolites production. Especially, extensive sampling of dry and remote deserts and islands can result in the identification of new species that increase the chance of encountering new metabolites. According to the production of several natural products by most strains in our collection, novel secondary metabolites may be found by in-depth analysis of MS data and genomes for cryptic biosynthetic pathways. Our findings also emphasize the importance of well-established protocols for the isolation and cultivation of this genus.
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