LazyAF, a pipeline for accessible medium-scale in silico prediction of protein-protein interactions.

Artificial intelligence has revolutionized the field of protein structure prediction. However, with more powerful and complex software being developed, it is accessibility and ease of use rather than capability that is quickly becoming a limiting factor to end users. LazyAF is a Google Colaboratory-based pipeline which integrates the existing ColabFold BATCH software to streamline the process of medium-scale protein-protein interaction prediction. LazyAF was used to predict the interactome of the 76 proteins encoded on the broad-host-range multi-drug resistance plasmid RK2, demonstrating the ease and accessibility the pipeline provides.

Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145.

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor. However, despite this early work, the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 300-fold, explaining the increase in actinorhodin production. However, while ChIP-seq identified 85 CutR binding sites in S. coelicolor none of these are in the actinorhodin biosynthetic gene cluster, meaning the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved HtrA-family foldases: HtrA3 and HtrB, and a putative VKOR enzyme, which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. Thus, we tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins, its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.

Advances in actinomycete research: an ActinoBase review of 2019.

The actinomycetes are Gram-positive bacteria belonging to the order Actinomycetales within the phylum Actinobacteria. They include members with significant economic and medical importance, for example filamentous actinomycetes such as Streptomyces species, which have a propensity to produce a plethora of bioactive secondary metabolites and form symbioses with higher organisms, such as plants and insects. Studying these bacteria is challenging, but also fascinating and very rewarding. As a Microbiology Society initiative, members of the actinomycete research community have been developing a Wikipedia-style resource, called ActinoBase, the purpose of which is to aid in the study of these filamentous bacteria. This review will highlight 10 publications from 2019 that have been of special interest to the ActinoBase community, covering 4 major components of actinomycete research: (i) development and regulation; (ii) specialized metabolites; (iii) ecology and host interactions; and (iv) technology and methodology.

Sensing and responding to diverse extracellular signals: an updated analysis of the sensor kinases and response regulators of Streptomyces species.

Streptomyces venezuelae is a Gram-positive, filamentous actinomycete with a complex developmental life cycle. Genomic analysis revealed that S. venezuelae encodes a large number of two-component systems (TCSs): these consist of a membrane-bound sensor kinase (SK) and a cognate response regulator (RR). These proteins act together to detect and respond to diverse extracellular signals. Some of these systems have been shown to regulate antimicrobial biosynthesis in Streptomyces species, making them very attractive to researchers. The ability of S. venezuelae to sporulate in both liquid and solid cultures has made it an increasingly popular model organism in which to study these industrially and medically important bacteria. Bioinformatic analysis identified 58 TCS operons in S. venezuelae with an additional 27 orphan SK and 18 orphan RR genes. A broader approach identified 15 of the 58 encoded TCSs to be highly conserved in 93 Streptomyces species for which high-quality and complete genome sequences are available. This review attempts to unify the current work on TCS in the streptomycetes, with an emphasis on S. venezuelae.

Genetic economy in picornaviruses: Foot-and-mouth disease virus replication exploits alternative precursor cleavage pathways.

The RNA genomes of picornaviruses are translated into single polyproteins which are subsequently cleaved into structural and non-structural protein products. For genetic economy, proteins and processing intermediates have evolved to perform distinct functions. The picornavirus precursor protein, P3, is cleaved to produce membrane-associated 3A, primer peptide 3B, protease 3Cpro and polymerase 3Dpol. Uniquely, foot-and-mouth disease virus (FMDV) encodes three similar copies of 3B (3B1-3), thus providing a convenient natural system to explore the role(s) of 3B in the processing cascade. Using a replicon system, we confirmed by genetic deletion or functional inactivation that each copy of 3B appears to function independently to prime FMDV RNA replication. However, we also show that deletion of 3B3 prevents replication and that this could be reversed by introducing mutations at the C-terminus of 3B2 that restored the natural sequence at the 3B3-3C cleavage site. In vitro translation studies showed that precursors with 3B3 deleted were rapidly cleaved to produce 3CD but that no polymerase, 3Dpol, was detected. Complementation assays, using distinguishable replicons bearing different inactivating mutations, showed that replicons with mutations within 3Dpol could be recovered by 3Dpol derived from "helper" replicons (incorporating inactivation mutations in all three copies of 3B). However, complementation was not observed when the natural 3B-3C cleavage site was altered in the "helper" replicon, again suggesting that a processing abnormality at this position prevented the production of 3Dpol. When mutations affecting polyprotein processing were introduced into an infectious clone, viable viruses were recovered but these had acquired compensatory mutations in the 3B-3C cleavage site. These mutations were shown to restore the wild-type processing characteristics when analysed in an in vitro processing assay. Overall, this study demonstrates a dual functional role of the small primer peptide 3B3, further highlighting how picornaviruses increase genetic economy.

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost.

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

CTP switches in ParABS-mediated bacterial chromosome segregation and beyond.

Segregation of genetic material is a fundamental process in biology. In many bacterial species, segregation of chromosomes and low-copy plasmids is facilitated by the tripartite ParA-ParB-parS system. This system consists of a centromeric parS DNA site and interacting proteins ParA and ParB that are capable of hydrolyzing adenosine triphosphate and cytidine triphosphate (CTP), respectively. ParB first binds to parS before associating with adjacent DNA regions to spread outward from parS. These ParB-DNA complexes bind to ParA and, through repetitive cycles of ParA-ParB binding and unbinding, move the DNA cargo to each daughter cell. The recent discovery that ParB binds and hydrolyzes CTP as it cycles on and off the bacterial chromosome has dramatically changed our understanding of the molecular mechanism used by the ParABS system. Beyond bacterial chromosome segregation, CTP-dependent molecular switches are likely to be more widespread in biology than previously appreciated and represent an opportunity for new and unexpected avenues for future research and application.

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Dissolution of the Disparate: Co-ordinate Regulation in Antibiotic Biosynthesis.

Discovering new antibiotics is vital to combat the growing threat of antimicrobial resistance. Most currently used antibiotics originate from the natural products of actinomycete bacteria, particularly Streptomyces species, that were discovered over 60 years ago. However, genome sequencing has revealed that most antibiotic-producing microorganisms encode many more natural products than previously thought. Biosynthesis of these natural products is tightly regulated by global and cluster situated regulators (CSRs), most of which respond to unknown environmental stimuli, and this likely explains why many biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions. One approach towards novel natural product discovery is to awaken these cryptic BGCs by re-wiring the regulatory control mechanism(s). Most CSRs bind intergenic regions of DNA in their own BGC to control compound biosynthesis, but some CSRs can control the biosynthesis of multiple natural products by binding to several different BGCs. These cross-cluster regulators present an opportunity for natural product discovery, as the expression of multiple BGCs can be affected through the manipulation of a single regulator. This review describes examples of these different mechanisms, including specific examples of cross-cluster regulation, and assesses the impact that this knowledge may have on the discovery of novel natural products.

Coordinate Regulation of Antimycin and Candicidin Biosynthesis.

Streptomyces species produce an incredible array of high-value specialty chemicals and medicinal therapeutics. A single species typically harbors ~30 biosynthetic pathways, but only a few them are expressed in the laboratory; thus, poor understanding of how natural-product biosynthesis is regulated is a major bottleneck in drug discovery. Antimycins are a large family of anticancer compounds widely produced by Streptomyces species, and their regulation is atypical compared to that of most other natural products. Here we demonstrate that antimycin production by Streptomyces albus S4 is regulated by FscRI, a PAS-LuxR family cluster-situated regulator of the polyene antifungal agent candicidin. We report that heterologous production of antimycins by Streptomyces coelicolor is dependent on FscRI and show that FscRI activates the transcription of key biosynthetic genes. We also demonstrate through chromatin immunoprecipitation sequencing that FscRI regulation is direct, and we provide evidence that this regulation strategy is conserved and unique to short-form antimycin gene clusters. Our study provides direct in vivo evidence of the cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products and expands the paradigmatic understanding of the regulation of secondary metabolism. IMPORTANCE Natural products produced by members of the phylum Actinobacteria underpin many industrially and medically important compounds; however, the majority of the ~30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. Understanding the diversity of regulatory strategies controlling the expression of these pathways is therefore critical if their biosynthetic potential is to be explored for new drug leads. Our findings reveal that the candicidin cluster-situated regulator FscRI coordinately controls the biosynthesis of both candicidin and antimycin, which is the first observation of cross-regulation of disparate biosynthetic gene clusters specifying unrelated natural products. We anticipate that this will emerge as a major strategy by which members of the phylum Actinobacteria coordinately produce natural products, which will advance our understanding of how the expression of secondary metabolism is controlled and will aid the pursuit of "silent" biosynthetic pathway activation.