With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their ...genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes
or
have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.
Bacterial usage of the cyclic dinucleotide c‐di‐GMP is widespread, governing the transition between motile/sessile and unicellular/multicellular behaviors. There is limited information on c‐di‐GMP ...metabolism, particularly on regulatory mechanisms governing control of EAL c‐di‐GMP phosphodiesterases. Herein, we provide high‐resolution structures for an EAL enzyme Bd1971, from the predatory bacterium Bdellovibrio bacteriovorus, which is controlled by a second signaling nucleotide, cAMP. The full‐length cAMP‐bound form reveals the sensory N‐terminus to be a domain‐swapped variant of the cNMP/CRP family, which in the cAMP‐activated state holds the C‐terminal EAL enzyme in a phosphodiesterase‐active conformation. Using a truncation mutant, we trap both a half‐occupied and inactive apo‐form of the protein, demonstrating a series of conformational changes that alter juxtaposition of the sensory domains. We show that Bd1971 interacts with several GGDEF proteins (c‐di‐GMP producers), but mutants of Bd1971 do not share the discrete phenotypes of GGDEF mutants, instead having an elevated level of c‐di‐GMP, suggesting that the role of Bd1971 is to moderate these levels, allowing “action potentials” to be generated by each GGDEF protein to effect their specific functions.
Synopsis
The cyclic‐di‐GMP hydrolase Bd1971 is a sensor‐enzyme fusion protein controlling turnover and predatory behavior of the bacterium Bdellovibrio bacteriovorus. Here, structural work combined with enzyme assays and genetics reveal regulation of Bd1971 by cAMP, with important consequences for signalling during the Bdellovibrio lifecycle.
The Bd1971 structure identifies a sensor adapted from the dimeric CRP superfamily.
Bd1971 bound to stimulus cAMP renders the protein active to hydrolyse cyclic‐di‐GMP.
Empty and part‐occupied sensor structures reveal a “swing‐out” mechanism of regulation.
Bd1971 can bind GGDEF synthases and putatively allow “action potential” spikes of cyclic‐di‐GMP to be generated.
Structural and biochemical work shows how usage of the second messenger cyclic dinucleotide c‐di‐GMP is controlled by upstream cAMP.
Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of ...the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent. This will pave the way for the development of new phage-based technologies with greater scope than lytic phage therapy.
Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of ...nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.
A continuing trend in nanotechnology is the use of materials of biological origin to develop novel nanomaterials. Accordingly, it is important to consider alteration of biomaterials to achieve ...optimal performance. One such entity that has garnered significant research attention is the filamentous M13 bacteriophage. This has been deployed in a broad range of nanotechnology applications, from the production of individually functional nanoparticles, to self-assembled materials with advanced properties reliant on their nano-scale structural characteristics. The present work had three principal aims: First to optimise the M13 bacteriophage major coat protein for labelling with exogenous compounds; second to investigate the use of dye-labelled M13 in a pathogen sensor; and third, the generation of a self-assembled nanomaterial based on dye-labelled M13. The results of this work demonstrate: 1) the generation of a mutant strain of M13 bacteriophage optimised to bind amine-directed exogenous compounds. A maximum increase in conjugation efficiency of 520 additional exogenous groups compared to the wild type was observed using absorbance spectroscopy; 2) the extension of the linear dichroism (LD) signal of M13 bacteriophage into the visible range by labelling with cyanine-based dyes; and 3) the generation of an aster-like self-assembled structure based on dye-labelled M13 bacteriophage, characterised by transmission electron microscopy, atomic force microscopy and dynamic light scattering. The results of this work will aid the further development of a pathogen sensor based on the LD signal of M13 bacteriophage. The extension of the M13 LD signal into the visible region, and the maximisation of that signal by the mutagenic optimisations made here, will enable improved portability and sensitivity of the sensor. Finally, the development of a self-assembled, dye-labelled M13-based structure will have implications in the branch of research that aims to understand how alterations to nanomaterials lead to changes in the way they self-assemble.
One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of ...biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing “adhesive” action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.
It is a challenge within the field of biomimetics to recreate the properties of light-harvesting antennae found in plants and photosynthetic bacteria. Attempts to recreate these biological structures ...typically rely on the alignment of fluorescent moieties
attachment to an inert linear scaffold,
DNA, RNA or amyloid fibrils, to enable Förster resonance energy transfer (FRET) between attached chromophores. While there has been some success in this approach, refinement of the alignment of the chromophores is often limited, which may limit the efficiency of energy transfer achieved. Here we demonstrate how linear dichroism spectroscopy may be used to ascertain the overall alignment of chromophores bound to the M13 bacteriophage, a model linear scaffold, and demonstrate how this may be used to distinguish between lack of FRET efficiency due to chromophore separation, and chromophore misalignment. This approach will allow the refinement of artificial light-harvesting antennae in a directed fashion.
It is a challenge within the field of biomimetics to recreate the properties of light-harvesting antennae found in plants and photosynthetic bacteria. Attempts to recreate these biological structures ...typically rely on the alignment of fluorescent moieties
via
attachment to an inert linear scaffold,
e.g.
DNA, RNA or amyloid fibrils, to enable Förster resonance energy transfer (FRET) between attached chromophores. While there has been some success in this approach, refinement of the alignment of the chromophores is often limited, which may limit the efficiency of energy transfer achieved. Here we demonstrate how linear dichroism spectroscopy may be used to ascertain the overall alignment of chromophores bound to the M13 bacteriophage, a model linear scaffold, and demonstrate how this may be used to distinguish between lack of FRET efficiency due to chromophore separation, and chromophore misalignment. This approach will allow the refinement of artificial light-harvesting antennae in a directed fashion.
Here we characterise four dyes and assess the complementarity of linear dichroism and FRET in biomimetic light-harvesting antennae optimisation.
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