Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae.

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.

An educational guide for nanopore sequencing in the classroom.

The last decade has witnessed a remarkable increase in our ability to measure genetic information. Advancements of sequencing technologies are challenging the existing methods of data storage and analysis. While methods to cope with the data deluge are progressing, many biologists have lagged behind due to the fast pace of computational advancements and tools available to address their scientific questions. Future generations of biologists must be more computationally aware and capable. This means they should be trained to give them the computational skills to keep pace with technological developments. Here, we propose a model that bridges experimental and bioinformatics concepts using the Oxford Nanopore Technologies (ONT) sequencing platform. We provide both a guide to begin to empower the new generation of educators, scientists, and students in performing long-read assembly of bacterial and bacteriophage genomes and a standalone virtual machine containing all the required software and learning materials for the course.

Discovering novel hydrolases from hot environments.

Novel hydrolases from hot and other extreme environments showing appropriate performance and/or novel functionalities and new approaches for their systematic screening are of great interest for developing new processes, for improving safety, health and environment issues. Existing processes could benefit as well from their properties. The workflow, based on the HotZyme project, describes a multitude of technologies and their integration from discovery to application, providing new tools for discovering, identifying and characterizing more novel thermostable hydrolases with desired functions from hot terrestrial and marine environments. To this end, hot springs worldwide were mined, resulting in hundreds of environmental samples and thousands of enrichment cultures growing on polymeric substrates of industrial interest. Using high-throughput sequencing and bioinformatics, 15 hot spring metagenomes, as well as several sequenced isolate genomes and transcriptomes were obtained. To facilitate the discovery of novel hydrolases, the annotation platform Anastasia and a whole-cell bioreporter-based functional screening method were developed. Sequence-based screening and functional screening together resulted in about 100 potentially new hydrolases of which more than a dozen have been characterized comprehensively from a biochemical and structural perspective. The characterized hydrolases include thermostable carboxylesterases, enol lactonases, quorum sensing lactonases, gluconolactonases, epoxide hydrolases, and cellulases. Apart from these novel thermostable hydrolases, the project generated an enormous amount of samples and data, thereby allowing the future discovery of even more novel enzymes.

A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts.

The use of bioreporters in high-throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l-arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l-arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts.

OsdR of Streptomyces coelicolor and the Dormancy Regulator DevR of Mycobacterium tuberculosis Control Overlapping Regulons.

Two-component regulatory systems allow bacteria to respond adequately to changes in their environment. In response to a given stimulus, a sensory kinase activates its cognate response regulator via reversible phosphorylation. The response regulator DevR activates a state of dormancy under hypoxia in Mycobacterium tuberculosis, allowing this pathogen to escape the host defense system. Here, we show that OsdR (SCO0204) of the soil bacterium Streptomyces coelicolor is a functional orthologue of DevR. OsdR, when activated by the sensory kinase OsdK (SCO0203), binds upstream of the DevR-controlled dormancy genes devR, hspX, and Rv3134c of M. tuberculosis. In silico analysis of the S. coelicolor genome combined with in vitro DNA binding studies identified many binding sites in the genomic region around osdR itself and upstream of stress-related genes. This binding correlated well with transcriptomic responses, with deregulation of developmental genes and genes related to stress and hypoxia in the osdR mutant. A peak in osdR transcription in the wild-type strain at the onset of aerial growth correlated with major changes in global gene expression. Taken together, our data reveal the existence of a dormancy-related regulon in streptomycetes which plays an important role in the transcriptional control of stress- and development-related genes. IMPORTANCE Dormancy is a state of growth cessation that allows bacteria to escape the host defense system and antibiotic challenge. Understanding the mechanisms that control dormancy is of key importance for the treatment of latent infections, such as those from Mycobacterium tuberculosis. In mycobacteria, dormancy is controlled by the response regulator DevR, which responds to conditions of hypoxia. Here, we show that OsdR of Streptomyces coelicolor recognizes the same regulatory element and controls a regulon that consists of genes involved in the control of stress and development. Only the core regulon in the direct vicinity of dosR and osdR is conserved between M. tuberculosis and S. coelicolor, respectively. Thus, we show how the system has diverged from allowing escape from the host defense system by mycobacteria to the control of sporulation by complex multicellular streptomycetes. This provides novel insights into how bacterial growth and development are coordinated with the environmental conditions.

Reporter-based screening and selection of enzymes.

The biotech industry is continuously seeking for new or improved biocatalysts. The success of these efforts is often hampered by the lack of an efficient screening assay. Thus, to be able to extend the number of enzymes available for industrial applications, high-throughput screening and selection methods are required. In the last few years an impressive range of screening and selection strategies has been developed. In this review, we will mainly focus on in vivo reporter systems in which the activity of a reporter is controlled by the activity of an enzyme of interest. Different mechanisms can be distinguished: (a) binding of the product of the enzymatic reaction to a transcriptional regulator and thereby turning on transcription of the reporter; (b) direct modification of a transcriptional regulator by the enzyme resulting in expression of the reporter; (c) binding of the product to a regulatory riboswitch or ribozyme, resulting in translation of the reporter; and (d) direct modification of the reporter by the enzyme, altering the reporter's activity. The choice for either a selection or a screening strategy depends on the type of reporter, e.g. providing antibiotic resistance (selection) or transmitting a fluorescent signal (screening). Although developing the specificity of each of these reporter-based selection or screening systems towards a certain enzymatic reaction is not yet straightforward, their adjustable modular design appears to be a promise for general applicability in the near future.