Hyphal compartmentalization and sporulation in Streptomyces require the conserved cell division protein SepX.

Filamentous actinobacteria such as Streptomyces undergo two distinct modes of cell division, leading to partitioning of growing hyphae into multicellular compartments via cross-walls, and to septation and release of unicellular spores. Specific determinants for cross-wall formation and the importance of hyphal compartmentalization for Streptomyces development are largely unknown. Here we show that SepX, an actinobacterial-specific protein, is crucial for both cell division modes in Streptomyces venezuelae. Importantly, we find that sepX-deficient mutants grow without cross-walls and that this substantially impairs the fitness of colonies and the coordinated progression through the developmental life cycle. Protein interaction studies and live-cell imaging suggest that SepX contributes to the stabilization of the divisome, a mechanism that also requires the dynamin-like protein DynB. Thus, our work identifies an important determinant for cell division in Streptomyces that is required for cellular development and sporulation.

Bacterial chromosome segregation: New insights into non-binary replication and division.

Bdellovibrio bacteriovorus divides in a non-binary manner resulting in an even or odd number of progeny. A new study tracks the spatiotemporal dynamics of chromosome segregation in this species and shows that the process is dependent on the conserved ParA-ParB-parS system.

Evolution of a σ-(c-di-GMP)-anti-σ switch.

Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG-(c-di-GMP)2-WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2-(c-di-GMP)2-WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2-(c-di-GMP)2-WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Ecological implications of hypoxia-triggered shifts in secondary metabolism.

Members of the actinomycete genus Streptomyces are non-motile, filamentous bacteria that are well-known for the production of biomedically relevant secondary metabolites. While considered obligate aerobes, little is known about how these bacteria respond to periods of reduced oxygen availability in their natural habitats, which include soils and ocean sediments. Here, we provide evidence that the marine streptomycete strain CNQ-525 can reduce MnO2 via a diffusible mechanism. We investigated the effects of hypoxia on secondary metabolite production and observed a shift away from the antibiotic napyradiomycin towards 8-amino-flaviolin, an intermediate in the napyradiomycin biosynthetic pathway. We purified 8-amino-flaviolin and demonstrated that it is reversibly redox-active (midpoint potential -474.5 mV), indicating that it has the potential to function as an endogenous extracellular electron shuttle. This study provides evidence that environmentally triggered changes in secondary metabolite production may provide clues to the ecological functions of specific compounds, and that Gram-positive bacteria considered to be obligate aerobes may play previously unrecognized roles in biogeochemical cycling through mechanisms that include extracellular electron shuttling.

Genomic insights into the evolution of hybrid isoprenoid biosynthetic gene clusters in the MAR4 marine streptomycete clade.

BACKGROUND: Considerable advances have been made in our understanding of the molecular genetics of secondary metabolite biosynthesis. Coupled with increased access to genome sequence data, new insight can be gained into the diversity and distributions of secondary metabolite biosynthetic gene clusters and the evolutionary processes that generate them. Here we examine the distribution of gene clusters predicted to encode the biosynthesis of a structurally diverse class of molecules called hybrid isoprenoids (HIs) in the genus Streptomyces. These compounds are derived from a mixed biosynthetic origin that is characterized by the incorporation of a terpene moiety onto a variety of chemical scaffolds and include many potent antibiotic and cytotoxic agents. RESULTS: One hundred and twenty Streptomyces genomes were searched for HI biosynthetic gene clusters using ABBA prenyltransferases (PTases) as queries. These enzymes are responsible for a key step in HI biosynthesis. The strains included 12 that belong to the 'MAR4' clade, a largely marine-derived lineage linked to the production of diverse HI secondary metabolites. We found ABBA PTase homologs in all of the MAR4 genomes, which averaged five copies per strain, compared with 21 % of the non-MAR4 genomes, which averaged one copy per strain. Phylogenetic analyses suggest that MAR4 PTase diversity has arisen by a combination of horizontal gene transfer and gene duplication. Furthermore, there is evidence that HI gene cluster diversity is generated by the horizontal exchange of orthologous PTases among clusters. Many putative HI gene clusters have not been linked to their secondary metabolic products, suggesting that MAR4 strains will yield additional new compounds in this structure class. Finally, we confirm that the mevalonate pathway is not always present in genomes that contain HI gene clusters and thus is not a reliable query for identifying strains with the potential to produce HI secondary metabolites. CONCLUSIONS: We found that marine-derived MAR4 streptomycetes possess a relatively high genetic potential for HI biosynthesis. The combination of horizontal gene transfer, duplication, and rearrangement indicate that complex evolutionary processes account for the high level of HI gene cluster diversity in these bacteria, the products of which may provide a yet to be defined adaptation to the marine environment.

Phylogenetic and chemical diversity of a hybrid-isoprenoid-producing streptomycete lineage.

Streptomyces species dedicate a large portion of their genomes to secondary metabolite biosynthesis. A diverse and largely marine-derived lineage within this genus has been designated MAR4 and identified as a prolific source of hybrid isoprenoid (HI) secondary metabolites. These terpenoid-containing compounds are common in nature but rarely observed as bacterial secondary metabolites. To assess the phylogenetic diversity of the MAR4 lineage, complementary culture-based and culture-independent techniques were applied to marine sediment samples collected off the Channel Islands, CA. The results, including those from an analysis of publically available sequence data and strains isolated as part of prior studies, placed 40 new strains in the MAR4 clade, of which 32 originated from marine sources. When combined with sequences cloned from environmental DNA, 28 MAR4 operational taxonomic units (0.01% genetic distance) were identified. Of these, 82% consisted exclusively of either cloned sequences or cultured strains, supporting the complementarity of these two approaches. Chemical analyses of diverse MAR4 strains revealed the production of five different HI structure classes. All 21 MAR4 strains tested produced at least one HI class, with most strains producing from two to four classes. The two major clades within the MAR4 lineage displayed distinct patterns in the structural classes and the number and amount of HIs produced, suggesting a relationship between taxonomy and secondary metabolite production. The production of HI secondary metabolites appears to be a phenotypic trait of the MAR4 lineage, which represents an emerging model with which to study the ecology and evolution of HI biosynthesis.

Hybrid isoprenoid secondary metabolite production in terrestrial and marine actinomycetes.

Terpenoids are among the most ubiquitous and diverse secondary metabolites observed in nature. Although actinomycete bacteria are one of the primary sources of microbially derived secondary metabolites, they rarely produce compounds in this biosynthetic class. The terpenoid secondary metabolites that have been discovered from actinomycetes are often in the form of biosynthetic hybrids called hybrid isoprenoids (HIs). HIs include significant structural diversity and biological activity and thus are important targets for natural product discovery. Recent screening of marine actinomycetes has led to the discovery of a new lineage that is enriched in the production of biologically active HI secondary metabolites. These strains represent a promising resource for natural product discovery and provide unique opportunities to study the evolutionary history and ecological functions of an unusual group of secondary metabolites.