Matthew James Bush- PhD - Molecular Microbiology
- PostDoc Position at John Innes Centre
Matthew James Bush
- PhD - Molecular Microbiology
- PostDoc Position at John Innes Centre
About
65
Publications
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Introduction
Current institution
Additional affiliations
October 2011 - present
Position
- Regulation of Streptomyces Development
Description
- Studying transcriptional regulation of streptomyces development
October 2007 - September 2011
Position
- PhD - interdomain repression in the Enhancer Binding Protein, NorR
Description
- PhD under the supervision of Prof. Ray Dixon (FRS): “Interdomain Repression in the Enhancer Binding Protein, NorR” NorR is an ATPase-dependent activator of transcription, required for the expression of the NO-detoxifying enzyme NorV, in E.coli.
October 2006 - June 2007
Position
- Undergraduate research project -
Description
- Undergraduate research project under the supervision of Dr. Jim G Hoggett: “Cross-linking studies of oligomeric gene activators” (Studying the bacterial Enhancer Binding Protein, PspF)
Education
October 2003 - July 2007
Publications
Publications (65)
Unlabelled:
WhiA is a highly unusual transcriptional regulator related to a family of eukaryotic homing endonucleases. WhiA is required for sporulation in the filamentous bacterium Streptomyces, but WhiA homologues of unknown function are also found throughout the Gram-positive bacteria. To better understand the role of WhiA in Streptomyces develo...
Unlabelled:
WhiB is the founding member of a family of proteins (the WhiB-like [Wbl] family) that carry a [4Fe-4S] iron-sulfur cluster and play key roles in diverse aspects of the biology of actinomycetes, including pathogenesis, antibiotic resistance, and the control of development. In Streptomyces, WhiB is essential for the process of developmen...
BldD-(c-di-GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here we report the identification and characterization of BldO, an a...
The WhiB‐like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the...
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 mutati...
In Streptomyces, the Bld (Bald) regulators control formation of the reproductive aerial hyphae. The functions of some of these regulators have been well characterized, but BldB has remained enigmatic. In addition to the bldB gene itself, Streptomyces venezuelae has 10 paralogs of bldB that sit next to paralogs of whiJ and abaA. Transcriptome sequen...
Studies of transcriptional initiation in different bacterial clades reveal diverse molecular mechanisms regulating this first step in gene expression. The WhiA and WhiB factors are both required to express cell division genes in Actinobacteria and are essential in notable pathogens such as Mycobacterium tuberculosis. The WhiA/B regulons and binding...
The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation.
DNA damage often causes an arrest of the cell cycle that provides time for genome integrity to be restored. In bacteria, the classical SOS DNA damage response leads to an inhibition of cell division resulting in temporarily filamentous growth. This raises the question as to whether such a response mechanism might similarly function in naturally fil...
DNA damage often results in a pause of cell division until damage is repaired. In bacteria, a widely conserved response to DNA damage is the SOS response which relies on two proteins: the multifunctional recombinase RecA and the transcriptional repressor LexA. Under DNA-damaging conditions, this response activates proteins involved in DNA repair an...
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-dwelli...
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...
Proteins that regulate transcription often also play an architectural role in the genome.
Thus, it has been difficult to define with precision the distinctions between transcription
factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs
as 'histone-like' implied an organizational function in a bacterial chromatin-like
c...
Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like c...
Filamentous actinobacteria like Streptomyces undergo two distinct modes of cell division, leading to the partitioning of growing hyphae into multicellular compartments via cross-walls and to the septation and release of unicellular spores. While some progress has been made towards the regulation of sporulation-specific cell division, specific deter...
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 identific...
Extracytoplasmic function (ECF) sigma factors are key transcriptional regulators that prokaryotes have evolved to respond to environmental challenges. Streptomyces tsukubaensis harbours 42 ECFs to reprogram stress-responsive gene expression. Among them, SigG1 features a minimal conserved ECF σ2–σ4 architecture and an additional C-terminal extension...
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 identifi...
Volatile compounds emitted by bacteria are often sensed by other organisms as odours, but their ecological roles are poorly understood1,2. Well-known examples are the soil-smelling terpenoids geosmin and 2-methylisoborneol (2-MIB)3,4, which humans and various animals sense at extremely low concentrations5,6. The conservation of geosmin biosynthesis...
The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae ( Sv Wh...
Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like c...
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...
The extracytoplasmic function (ECF) σ factor, σE is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprec...
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 mutati...
Streptomyces are filamentous bacteria with a complex developmental life cycle characterized by the formation of spore-forming aerial hyphae. Transcription of the chaplin and rodlin genes, which are essential for aerial hyphae production, is directed by the extracytoplasmic function (ECF) σ factor BldN, which is in turn controlled by an anti-σ facto...
Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-...
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WhiB ChIP-seq. (A) Construction of a functional FLAG-tagged version of WhiB. Shown are the phenotypes of wild-type S. venezuelae (WT), the constructed ΔwhiB::apr SV7 null mutant (ΔwhiB), SV7 carrying the empty vector pMS82 (ΔwhiB::apr attBΦBT1::pMS82), the complemented strain (ΔwhiB::apr attBΦBT1::whiB), and SV7 expressing an N-terminal, triple FLA...
WhiA is detected after WhiB ChIP. Data represent results of WhiA Western blot analysis (anti-WhiA polyclonal antibody; 1:2,500) following anti-FLAG ChIP (A) or anti-WhiB ChIP (B) in the strains indicated. WhiA is detected after anti-FLAG immunoprecipitation in the 3×FLAG-[Gly4Ser]3-WhiA strain (WhiA-FLAG, positive control) and in the 3×FLAG-[Gly4Se...
Strains, plasmids, and oligonucleotide primers used in this study.
Western blot analysis. (A). Stabilities of FLAG-tagged versions of WhiB—with or without the [4Fe-4S] cluster and in the presence or absence of WhiA. Data represent results of Western blot analysis of WhiB (ΔwhiB::apr attBΦBT1::3×FLAG-[Gly4Ser]3-whiB) (1), WhiB in the absence of whiA (ΔwhiA ΔwhiB::apr attBΦBT1::3×FLAG-[Gly4Ser]3-whiB) (2), the WhiB...
(A) Complete ChIP-seq data set for S. venezuelae WT (anti-WhiB) and the ΔwhiB::apr attBΦBT1::3×FLAG-[Gly4Ser]3-whiB (anti-FLAG) strain. Each row represents a ChIP “peak” based on the analysis of 25-bp segments of the S. venezuelae genome. Only those peaks with significance at a P value of <E-04 for at least one of the ChIP samples are included in t...
Supplemental Materials and Methods. Download
Complementation of the whiA whiB double mutant. Shown are the phenotypes of wild-type S. venezuelae (WT), the constructed ΔwhiA ΔwhiB null mutant SV51 (ΔwhiA ΔwhiB::apr), SV51 carrying the empty vector pMS82 (ΔwhiA ΔwhiB::apr attBΦBT1::pMS82), and the complemented strains SV51-pIJ6760 (ΔwhiA ΔwhiB::apr attBΦBT1::whiA), SV51-pIJ6761 (ΔwhiA ΔwhiB::ap...
WhiA and WhiB cannot promote sporulation independently of one another. (A) Phenotypes of the constructed ΔwhiA SV11 null mutant (ΔwhiA::apr), the constructed ΔwhiB SV7 null mutant (ΔwhiB::apr), and the ΔwhiA ΔwhiB double mutant SV51 (ΔwhiA ΔwhiB::apr). The phenotype of each carrying the empty vector pIJ10257, whiA under ermE* control (pIJ10605), or...
Comparison between anti-FLAG and anti-WhiB ChIP-seq data. ChIP traces are shown for 12 selected WhiB target genes: sigN, infA, sven6396/wblH, cslA, pyrR/bldD, sven5479/nrdR, cvnA4/sven0992, sven1586, cvnA1/sven5239, sven5277/5278, sven5692, and sven2270. Color coding of the ChIP samples is as follows: 3×FLAG-[Gly4Ser]3-WhiB anti-FLAG strain (WhiB a...
Cysteine variants of WhiB are unable to promote sporulation. (A) Comparison of the phenotypes of wild-type S. venezuelae (WT), the ΔwhiB SV7 null mutant (ΔwhiB::apr), SV7 carrying the empty vector pMS82 (ΔwhiB::apr attBΦBT1::pMS82), the complemented strain (ΔwhiB::apr attBΦBT1::whiB), the WhiB Cys–Ala variant complemented strain (ΔwhiB::apr attBΦBT...
The complex life cycle of streptomycetes involves two distinct filamentous cell forms: the growing (or vegetative) hyphae and the reproductive (or aerial) hyphae, which differentiate into long chains of spores. Until recently, little was known about the signalling pathways that regulate the developmental transitions leading to sporulation. In this...
σ54-dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to σ70, the σ54- RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerizes into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activ...
The orphan, atypical response regulators BldM and WhiI each play critical roles in Streptomyces differentiation. BldM is required for the formation of aerial hyphae, and WhiI is required for the differentiation of these reproductive structures into mature spores. To gain insight into BldM function, we defined the genome-wide BldM regulon using ChIP...
Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these s...
A flavorubredoxin and its associated oxidoreductase (encoded by norV and norW respectively) detoxify NO (nitric oxide) to form N2O (nitrous oxide) under anaerobic conditions in Escherichia coli. Transcription of the norVW genes is activated in response to NO by the σ54-dependent regulator and dedicated NO sensor, NorR, a member of the bacterial enh...
Bacterial enhancer binding proteins (bEBPs) are specialized transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ(54). Transcriptional activation by the NorR bEBP is controlled by a regulatory GAF domain tha...
The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to
the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding sit...
Questions
Question (1)
I am trying to optimise overexpression of a particular protein and it contains a number of codons not commonly used in E.coli. I have tried to use Rosetta stains but without much success. Would you suggest codon optimisation? Are there any advantages of engineering a codon-optimised construct rather than expressing a non-optimised construct in Rosetta?
