Biology subjects

Bush, M. J.

Publications and source records attributed to Bush, M. J..

5 recordsLinked to original sources

bioRxiv2026

A conserved NLR activation switch governs global transcriptional control of antibiotic biosynthesis in Streptomyces

Nucleotide-binding and oligomerization domain-like receptors (NLRs) are conserved molecular switches that regulate innate immunity across diverse domains of life. While best known for their roles in immunity, bacterial NLR-related proteins also govern non-immune processes. In Streptomyces, the global transcriptional regulator AfsR employs an NLR-type architecture to control antibiotic production. Here, we identify a conserved histidine-aspartic acid motif within the nucleotide-binding and oligomerization domain of AfsR that functions as a universal NLR activation switch. Site-directed mutagenesis of this motif decouples AfsR from native upstream signals, locking it into a constitutive, autoactive "ON" state. Using integrated transcriptomics, ChIP-seq, and in vitro DNA-binding assays, we demonstrate that autoactivation expands the regulatory reach of AfsR via enhanced promoter affinity, directly controlling its canonical target afsS and uncovering wblH as an additional member of the AfsR regulon. Untargeted metabolomics further shows that autoactive AfsR drives systemic reprogramming of specialized metabolism, increasing the production of known antibiotics and awakening silent biosynthetic gene clusters in Streptomyces coelicolor and Streptomyces peucetius. Furthermore, phylogenomic analysis reveals that Actinomycetes encode multiple AfsR-like regulators with undefined roles in biosynthetic gene cluster regulation, suggesting a substantial reservoir of unexplored regulatory diversity that could be harnessed to expand natural product discovery. Together, our work provides fundamental insights into the functional diversity of NLRs and a framework for rationally engineering these regulators to unlock untapped microbial chemical diversity.

microbiology↗

bioRxiv2022

Genome-wide identification of the LexA-mediated DNA damage response in Streptomyces venezuelae

DNA damage triggers a widely conserved stress response in bacteria called the SOS response that involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and ChIP-seq analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using Surface Plasmon Resonance, we further confirm the LexA DNA binding motif (SOS box) and demonstrate that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCEThe transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced during DNA damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

microbiology↗

bioRxiv2021

Multicellular growth and sporulation in filamentous actinobacteria 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.

microbiology↗

bioRxiv2020

A conserved cell division protein directly regulates FtsZ dynamics in filamentous and unicellular actinobacteria

Bacterial cell division is driven by the polymerization of the GTPase FtsZ into a contractile structure, the so-called Z-ring. This essential process involves proteins that modulate FtsZ dynamics and hence the overall Z-ring architecture. Actinobacteria, like Streptomyces and Mycobacterium lack known key FtsZ-regulators. Here we report the identification of SepH, a conserved actinobacterial protein that directly regulates FtsZ dynamics. We show that SepH is crucially involved in cell division in Streptomyces and that it binds FtsZ via a conserved helix-turn-helix motif, stimulating the assembly of FtsZ protofilaments. Comparative in vitro studies using the SepH homolog from Mycobacterium further reveal that SepH can also bundle FtsZ protofilaments, indicating an additional Z-ring stabilizing function in vivo. We propose that SepH plays a crucial role at the onset of cytokinesis in actinobacteria by promoting the rapid assembly of FtsZ filaments into division-competent Z-rings that can go on to mediate septum synthesis.

microbiology↗

bioRxiv2017

BldC delays entry into development to produce a sustained period of vegetative growth in Streptomyces venezuelae

Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC, encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae, showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g. whiD, whiI, sigF), chromosome condensation and segregation (e.g. smeA-sffA, hupS), and sporulation-specific cell division (e.g. dynAB), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins.\n\nIMPORTANCEUnderstanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive new model system for the study of morphological differentiation in Streptomyces. This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae, we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation.

microbiology↗