CRISPR/Cas9-enhanced Targetron Insertion for Delivery of Heterologous Sequences into the Genome of Gram-Negative Bacteria.

Targetron technology, a gene-editing approach based on the use of mobile group II introns, is particularly useful for bacterial strains deficient in homologous recombination. Specifically, the Ll.LtrB intron from Lactococcus lactis can be used in a wide range of species and can be easily retargeted, that is, modified for integration into any locus of interest. Targetron technology is thus a powerful tool for generating genomic insertions in a broad range of genetic backgrounds, mainly when no other techniques can be efficiently employed. Notably, the approach can be coupled to CRISPR/Cas9 counterselection of wildtype DNA sequences to decrease the population of unmodified cells and ultimately improve Ll.LtrB insertion efficiency. Here, we describe a step-by-step protocol for delivering exogenous sequences into the genome of Gram-negative bacteria by means of targetron technology and CRISPR/Cas9 counterselection using Pseudomonas putida as a model. We describe the retargeting of the Ll.LtrB intron to the locus selected for insertion, the design of specific spacers for eliminating unmutated cells through CRISPR/Cas9 counterselection, and the cloning of exogenous sequences into Ll.LtrB. We also provide a protocol for delivering a specific cargo to the locus of choice once all necessary components of the system are ready. Lastly, we describe a general protocol for curing the engineered strain of all plasmids. CRISPR/Cas9-enhanced Ll.LtrB insertion can be an efficient alternative for overcoming low recombination-based editing efficiency and can be used in numerous bacterial species. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retargeting the Ll.LtrB intron to the target locus Support Protocol 1: Preparation of competent E. coli Basic Protocol 2: Design and cloning of CRISPR spacers to counterselect Ll.LtrB insertions Support Protocol 2: Interference assay to check efficiency of selected spacers Basic Protocol 3: Cloning cargos into Ll.LtrB Basic Protocol 4: Ll.LtrB/CRISPR/Cas9-mediated insertion Basic Protocol 5: Curing the engineered strain of plasmids.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces.

ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest.

Targetron-Assisted Delivery of Exogenous DNA Sequences into Pseudomonas putida through CRISPR-Aided Counterselection.

Genome editing methods based on group II introns (known as targetron technology) have long been used as a gene knockout strategy in a wide range of organisms, in a fashion independent of homologous recombination. Yet, their utility as delivery systems has typically been suboptimal due to the reduced efficiency of insertion when carrying exogenous sequences. We show that this limitation can be tackled and targetrons can be adapted as a general tool in Gram-negative bacteria. To this end, a set of broad-host-range standardized vectors were designed for the conditional expression of the Ll.LtrB intron. After establishing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths, to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. With these novel tools, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its ΔrecA derivative. Finally, we applied this technology to successfully tag P. putida with an orthogonal short sequence barcode that acts as a unique identifier for tracking this microorganism in biotechnological settings. These results show the value of the targetron approach for the unrestricted delivery of small DNA fragments to precise locations in the genomes of Gram-negative bacteria, which will be useful for a suite of genome editing endeavors.

Environmental Performance of Pseudomonas putida with a Uracylated Genome.

A variant of the soil bacterium Pseudomonas putida with a genome containing a ∼20 % replacement of the whole of thymine (T) by uracil (U) was made by deleting genes ung (uracil DNA glycosylase) and dut (deoxyuridine 5'-triphosphate nucleotide hydrolase). Proteomic comparisons revealed that, of 281 up-regulated and 96 down-regulated proteins in the Δung Δdut cells, as compared to the wild-type, many were involved in nucleotide metabolism. Unexpectedly, genome uracylation did not greatly change the gross environmental endurance profile of P. putida, increased spontaneous mutagenesis by only twofold and supported expression of heterologous proteins well. As U-enriched DNA is potentially degraded by the base excision repair of recipients encoding a uracil DNA glycosylase, we then tested the spread potential of genetic material originating in the Δung Δdut cells either within the same species or in a commonly used Escherichia coli strain. Transformation and conjugation experiments revealed that horizontal gene transfer of U-containing plasmids fared worse than those made of standard DNA by two orders of magnitude. Although this figure does not guarantee the certainty of containment, it suggests a general strategy for curbing the dispersal of recombinant genetic constructs.

Recombination-Independent Genome Editing through CRISPR/Cas9-Enhanced TargeTron Delivery.

Group II introns were developed some time ago as tools for the construction of knockout mutants in a wide range of organisms, ranging from Gram-positive and Gram-negative bacteria to human cells. Utilizing these introns is advantageous because they are independent of the host's DNA recombination machinery, they can carry heterologous sequences (and thus be used as vehicles for gene delivery), and they can be easily retargeted for subsequent insertions of additional genes at the user's will. Alas, the use of this platform has been limited, as insertion efficiencies greatly change depending on the target sites and cannot be predicted a priori. Moreover, the ability of introns to perform their own splicing and integration is compromised when they carry foreign sequences. To overcome these limitations, we merged the group II intron-based TargeTron system with CRISPR/Cas9 counterselection. To this end, we first engineered a new group-II intron by replacing the retrotransposition-activated selectable marker (RAM) with ura3 and retargeting it to a new site in the lacZ gene of E. coli. Then, we showed that directing CRISPR/Cas9 toward the wild-type sequences dramatically increased the chances of finding clones that integrated the retrointron into the target lacZ sequence. The CRISPR-Cas9 counterselection strategy presented herein thus overcomes a major limitation that has prevented the use of group II introns as devices for gene delivery and genome editing at large in a recombination-independent fashion.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, Central Spain).

The phylogenetic and functional structure of the microbial community residing in a Ca(2+)-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO(4) × 2 H(2)O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca(2+) and SO (4) (2-) being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical-chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.

Reactome array: forging a link between metabolome and genome.

We describe a sensitive metabolite array for genome sequence-independent functional analysis of metabolic phenotypes and networks, the reactomes, of cell populations and communities. The array includes 1676 dye-linked substrate compounds collectively representing central metabolic pathways of all forms of life. Application of cell extracts to the array leads to specific binding of enzymes to cognate substrates, transformation to products, and concomitant activation of the dye signals. Proof of principle was shown by reconstruction of the metabolic maps of model bacteria. Utility of the array for unsequenced organisms was demonstrated by reconstruction of the global metabolisms of three microbial communities derived from acidic volcanic pool, deep-sea brine lake, and hydrocarbon-polluted seawater. Enzymes of interest are captured on nanoparticles coated with cognate metabolites, sequenced, and their functions unequivocally established.