Unlocking the potential of microbiome editing: A review of conjugation-based delivery.

In recent decades, there has been a rapid increase in the prevalence of multidrug-resistant pathogens, posing a challenge to modern antibiotic-based medicine. This has highlighted the need for novel treatments that can specifically affect the target microorganism without disturbing other co-inhabiting species, thus preventing the development of dysbiosis in treated patients. Moreover, there is a pressing demand for tools to effectively manipulate complex microbial populations. One of the approaches suggested to address both issues was to use conjugation as a tool to modify the microbiome by either editing the genome of specific bacterial species and/or the removal of certain taxonomic groups. Conjugation involves the transfer of DNA from one bacterium to another, which opens up the possibility of introducing, modifying or deleting specific genes in the recipient. In response to this proposal, there has been a significant increase in the number of studies using this method for gene delivery in bacterial populations. This MicroReview aims to provide a detailed overview on the use of conjugation for microbiome engineering, and at the same time, to initiate a discussion on the potential, limitations and possible future directions of this approach.

Increasing the Scalability of Toxin-Intein Orthogonal Combinations.

Inteins are proteins embedded into host proteins from which they are excised in an autocatalytic reaction. Specifically, split inteins are separated into two independent fragments that reconstitute the host protein during the catalytic process. We recently developed a novel strategy for the specific killing of pathogenic and antibiotic resistant bacteria based on toxin-intein combinations. Bacterial type II toxin-antitoxin systems are protein modules in which the toxin can provoke cell death whereas the antitoxin inhibits toxin activity. Although our previous system was based on a split intein (iDnaE) and the CcdB toxin, we demonstrated that iDnaE is able to reconstitute four different toxins. To expand the applicability of our system by widening the repertoire of toxin-intein combinations for complex set-ups, we introduced a second intein, iDnaX, which was artificially split. We demonstrate that iDnaX is able to reconstitute the four toxins, and we manage to reduce its scar size to facilitate their use. In addition, we prove the orthogonality of both inteins (iDnaE and iDnaX) through a toxin reconstitution assay, thus opening the possibility for complex set-ups based on these toxin-intein modules. This could be used to develop specific antimicrobial and other biotechnological applications.

Fitness Cost Evolution of Natural Plasmids of Staphylococcus aureus.

Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids.IMPORTANCE Plasmids are major agents in the spread of antibiotic resistance genes among bacteria. How plasmids and their hosts coevolve to reduce the fitness cost associated with plasmid carriage when bacteria grow in an antibiotic-free environment is not well understood. Here, we investigated the cost and the genetic adaptations that occur during evolution in the absence of antibiotics when the bacterial pathogen Staphylococcus aureus acquires a new plasmid. Our results show the occurrence, at the end of evolution, of plasmid rearrangements mediated by insertion sequences that lead to the loss of antimicrobial resistance genes from the plasmid and an alleviated fitness cost. Our results thus highlight the probable benefits of reducing the use of antibiotics in management programs for the selection of S. aureus clones carrying plasmids that no longer confer resistance.

Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis.

Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels.

The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers.

Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.

Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All.

Phage-inducible chromosomal islands (PICIs) are a widespread family of mobile genetic elements, which have an important role in bacterial pathogenesis. These elements mobilize among bacterial species at extremely high frequencies, representing an attractive tool for the delivery of synthetic genes. However, tools for their genetic manipulation are limited and timing consuming. Here, we have adapted a synthetic biology approach for rapidly editing of PICIs in Saccharomyces cerevisiae based on their ability to excise and integrate into the bacterial chromosome of their cognate host species. As proof of concept, we engineered several PICIs from Staphylococcus aureus and Escherichia coli and validated this methodology for the study of the biology of these elements by generating multiple and simultaneous mutations in different PICI genes. For biotechnological purposes, we also synthetically constructed PICIs as Trojan horses to deliver different CRISPR-Cas9 systems designed to either cure plasmids or eliminate cells carrying the targeted genes. Our results demonstrate that the strategy developed here can be employed universally to study PICIs and enable new approaches for diagnosis and treatment of bacterial diseases.

A highly diverse, desert-like microbial biocenosis on solar panels in a Mediterranean city.

Microorganisms colonize a wide range of natural and artificial environments although there are hardly any data on the microbial ecology of one the most widespread man-made extreme structures: solar panels. Here we show that solar panels in a Mediterranean city (Valencia, Spain) harbor a highly diverse microbial community with more than 500 different species per panel, most of which belong to drought-, heat- and radiation-adapted bacterial genera, and sun-irradiation adapted epiphytic fungi. The taxonomic and functional profiles of this microbial community and the characterization of selected culturable bacteria reveal the existence of a diverse mesophilic microbial community on the panels' surface. This biocenosis proved to be more similar to the ones inhabiting deserts than to any human or urban microbial ecosystem. This unique microbial community shows different day/night proteomic profiles; it is dominated by reddish pigment- and sphingolipid-producers, and is adapted to withstand circadian cycles of high temperatures, desiccation and solar radiation.

Unveiling Bacterial Interactions through Multidimensional Scaling and Dynamics Modeling.

We propose a new strategy to identify and visualize bacterial consortia by conducting replicated culturing of environmental samples coupled with high-throughput sequencing and multidimensional scaling analysis, followed by identification of bacteria-bacteria correlations and interactions. We conducted a proof of concept assay with pine-tree resin-based media in ten replicates, which allowed detecting and visualizing dynamical bacterial associations in the form of statistically significant and yet biologically relevant bacterial consortia.

Standards not that standard.

There is a general assent on the key role of standards in Synthetic Biology. In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described. We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors. We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.

Engineering bacteria to form a biofilm and induce clumping in Caenorhabditis elegans.

Bacteria are needed for a vast range of biotechnological processes, which they carry out either as pure cultures or in association with other bacteria and/or fungi. The potential of bacteria as biofactories is hampered, though, by their limited mobility in solid or semisolid media such as agricultural or domestic waste. This work represents an attempt toward overcoming this limitation by associating bacterial biotechnological properties with the transport ability of the nematode Caenorhabditis elegans. We report here biofilm formation on C. elegans by engineered Escherichia coli expressing a Xhenorhabdus nematophila adhesion operon and induction of nematode social feeding behavior (clumping) through an E. coli-mediated iRNA blocking on the expression of FLP-21, a neuropeptide involved in worm solitary behavior.