The activity of CobB1 protein deacetylase contributes to nucleoid compaction in Streptomyces venezuelae spores by increasing HupS affinity for DNA.

Streptomyces are soil bacteria with complex life cycle. During sporulation Streptomyces linear chromosomes become highly compacted so that the genetic material fits within limited spore volume. The key players in this process are nucleoid-associated proteins (NAPs). Among them, HU (heat unstable) proteins are the most abundant NAPs in the cell and the most conserved in bacteria. HupS, one of the two HU homologues encoded by the Streptomyces genome, is the best-studied spore-associated NAP. In contrast to other HU homologues, HupS contains a long, C-terminal domain that is extremely rich in lysine repeats (LR domain) similar to eukaryotic histone H2B and mycobacterial HupB protein. Here, we have investigated, whether lysine residues in HupS are posttranslationally modified by reversible lysine acetylation. We have confirmed that Streptomyces venezuelae HupS is acetylated in vivo. We showed that HupS binding to DNA in vitro is controlled by the acetylation. Moreover, we identified that CobB1, one of two Sir2 homologues in Streptomyces, controls HupS acetylation levels in vivo. We demonstrate that the elimination of CobB1 increases HupS mobility, reduces chromosome compaction in spores, and affects spores maturation. Thus, our studies indicate that HupS acetylation affects its function by diminishing DNA binding and disturbing chromosome organization.

Enhanced binding of an HU homologue under increased DNA supercoiling preserves chromosome organisation and sustains Streptomyces hyphal growth.

Bacterial chromosome topology is controlled by topoisomerases and nucleoid-associated proteins (NAPs). While topoisomerases regulate DNA supercoiling, NAPs introduce bends or coat DNA upon its binding, affecting DNA loop formation. Streptomyces, hyphal, multigenomic bacteria known for producing numerous clinically important compounds, use the highly processive topoisomerase I (TopA) to remove excessive negative DNA supercoils. Elongated vegetative Streptomyces cells contain multiple copies of their linear chromosome, which remain relaxed and relatively evenly distributed. Here, we explored how TopA cooperates with HupA, an HU homologue that is the most abundant Streptomyces NAP. We verified that HupA has an increased affinity for supercoiled DNA in vivo and in vitro. Analysis of mutant strains demonstrated that HupA elimination is detrimental under high DNA supercoiling conditions. The absence of HupA, combined with decreased TopA levels, disrupted chromosome distribution in hyphal cells, eventually inhibiting hyphal growth. We concluded that increased HupA binding to DNA under elevated chromosome supercoiling conditions is critical for the preservation of chromosome organisation.

A highly processive actinobacterial topoisomerase I - thoughts on Streptomyces' demand for an enzyme with a unique C-terminal domain.

Topoisomerase I (TopA) is an essential enzyme that is required to remove excess negative supercoils from chromosomal DNA. Actinobacteria encode unusual TopA homologues with a unique C-terminal domain that contains lysine repeats and confers high enzyme processivity. Interestingly, the longest stretch of lysine repeats was identified in TopA from Streptomyces, environmental bacteria that undergo complex differentiation and produce a plethora of secondary metabolites. In this review, we aim to discuss potential advantages of the lysine repeats in Streptomyces TopA. We speculate that the chromosome organization, transcriptional regulation and lifestyle of these species demand a highly processive but also fine-tuneable relaxase. We hypothesize that the unique TopA provides flexible control of chromosomal topology and globally regulates gene expression.

C-terminal lysine repeats in Streptomyces topoisomerase I stabilize the enzyme-DNA complex and confer high enzyme processivity.

Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces TopA contains the longest stretch of lysine repeats, which terminate with acidic amino acids. DNA-binding studies revealed that the lysine repeats stabilized the TopA-DNA complex, while single-molecule experiments showed that their elimination impaired enzyme processivity. Streptomyces coelicolor TopA processivity could not be restored by fusion of its N-terminal domain (NTD) with the Escherichia coli TopA CTD. The hybrid protein could not re-establish the distribution of multiple chromosomal copies in Streptomyces hyphae impaired by TopA depletion. We expected that the highest TopA processivity would be required during the growth of multigenomic sporogenic hyphae, and indeed, the elimination of lysine repeats from TopA disturbed sporulation. We speculate that the interaction of the lysine repeats with DNA allows the stabilization of the enzyme-DNA complex, which is additionally enhanced by acidic C-terminal amino acids. The complex stabilization, which may be particularly important for GC-rich chromosomes, enables high enzyme processivity. The high processivity of TopA allows rapid topological changes in multiple chromosomal copies during Streptomyces sporulation.

Global Chromosome Topology and the Two-Component Systems in Concerted Manner Regulate Transcription in Streptomyces.

Bacterial gene expression is controlled at multiple levels, with chromosome supercoiling being one of the most global regulators. Global DNA supercoiling is maintained by the orchestrated action of topoisomerases. In Streptomyces, mycelial soil bacteria with a complex life cycle, topoisomerase I depletion led to elevated chromosome supercoiling, changed expression of a significant fraction of genes, delayed growth, and blocked sporulation. To identify supercoiling-induced sporulation regulators, we searched for Streptomyces coelicolor transposon mutants that were able to restore sporulation despite high chromosome supercoiling. We established that transposon insertion in genes encoding a novel two-component system named SatKR reversed the sporulation blockage resulting from topoisomerase I depletion. Transposition in satKR abolished the transcriptional induction of the genes within the so-called supercoiling-hypersensitive cluster (SHC). Moreover, we found that activated SatR also induced the same set of SHC genes under normal supercoiling conditions. We determined that the expression of genes in this region impacted S. coelicolor growth and sporulation. Interestingly, among the associated products is another two-component system (SitKR), indicating the potential for cascading regulatory effects driven by the SatKR and SitKR two-component systems. Thus, we demonstrated the concerted activity of chromosome supercoiling and a hierarchical two-component signaling system that impacts gene activity governing Streptomyces growth and sporulation. IMPORTANCE Streptomyces microbes, soil bacteria with complex life cycle, are the producers of a broad range of biologically active compounds (e.g., antibiotics). Streptomyces bacteria respond to various environmental signals using a complex transcriptional regulation mechanism. Understanding regulation of their gene expression is crucial for Streptomyces application as industrial organisms. Here, on the basis of the results of extensive transcriptomics analyses, we describe the concerted gene regulation by global DNA supercoiling and novel two-component system. Our data indicate that regulated genes encode growth and sporulation regulators. Thus, we demonstrate that Streptomyces bacteria link the global regulatory strategies to adjust life cycle to unfavorable conditions.

Spatial rearrangement of the Streptomyces venezuelae linear chromosome during sporogenic development.

Bacteria of the genus Streptomyces have a linear chromosome, with a core region and two 'arms'. During their complex life cycle, these bacteria develop multi-genomic hyphae that differentiate into chains of exospores that carry a single copy of the genome. Sporulation-associated cell division requires chromosome segregation and compaction. Here, we show that the arms of Streptomyces venezuelae chromosomes are spatially separated at entry to sporulation, but during sporogenic cell division they are closely aligned with the core region. Arm proximity is imposed by segregation protein ParB and condensin SMC. Moreover, the chromosomal terminal regions are organized into distinct domains by the Streptomyces-specific HU-family protein HupS. Thus, as seen in eukaryotes, there is substantial chromosomal remodelling during the Streptomyces life cycle, with the chromosome undergoing rearrangements from an 'open' to a 'closed' conformation.

Compaction and control-the role of chromosome-organizing proteins in Streptomyces.

Chromosomes are dynamic entities, whose organization and structure depend on the concerted activity of DNA-binding proteins and DNA-processing enzymes. In bacteria, chromosome replication, segregation, compaction and transcription are all occurring simultaneously, and to ensure that these processes are appropriately coordinated, all bacteria employ a mix of well-conserved and species-specific proteins. Unusually, Streptomyces bacteria have large, linear chromosomes and life cycle stages that include multigenomic filamentous hyphae and unigenomic spores. Moreover, their prolific secondary metabolism yields a wealth of bioactive natural products. These different life cycle stages are associated with profound changes in nucleoid structure and chromosome compaction, and require distinct repertoires of architectural-and regulatory-proteins. To date, chromosome organization is best understood during Streptomyces sporulation, when chromosome segregation and condensation are most evident, and these processes are coordinated with synchronous rounds of cell division. Advances are, however, now being made in understanding how chromosome organization is achieved in multigenomic hyphal compartments, in defining the functional and regulatory interplay between different architectural elements, and in appreciating the transcriptional control exerted by these 'structural' proteins.

Amsacrine Derivatives Selectively Inhibit Mycobacterial Topoisomerase I (TopA), Impair M. smegmatis Growth and Disturb Chromosome Replication.

Amsacrine, which inhibits eukaryotic type II topoisomerase via DNA intercalation and stabilization of the cleavable topoisomerase-DNA complex, promotes DNA damage and eventually cell death. Amsacrine has also been shown to inhibit structurally distinct bacterial type I topoisomerases (TopAs), including mycobacterial TopA, the only and essential topoisomerase I in Mycobacterium tuberculosis. Here, we describe the modifications of an amsacrine sulfonamide moiety that presumably interacts with mycobacterial TopA, which notably increased the enzyme inhibition and drug selectivity in vivo. To analyse the effects of amsacrine and its derivatives treatment on cell cycle, we used time-lapse fluorescence microscopy (TLMM) and fusion of the ß-subunit of DNA polymerase III with enhanced green fluorescence protein (DnaN-EGFP). We determined that treatment with amsacrine and its derivatives increased the number of DnaN-EGFP complexes and/or prolonged the time of chromosome replication and cell cycle notably. The analysis of TopA depletion strain confirmed that lowering TopA level results in similar disturbances of chromosome replication. In summary, since TopA is crucial for mycobacterial cell viability, the compounds targeting the enzyme disturbed the cell cycle and thus may constitute a new class of anti-tuberculosis drugs.