For naturally competent bacteria, spatially structured growth can provide an environment for enhanced horizontal gene transfer through transformation and recombination. DNA is often present in the extracellular environment, such as in the extracellular matrix of biofilms, and the lysis of a single cell can result in high local DNA concentrations. Xylella fastidiosa is a naturally competent plant pathogen that typically lives in a surface-attached state, yet previous work characterizing the competence of this organism was conducted with planktonic cells in liquid environments. Here, we show that transformation and recombination efficiencies are two to three orders of magnitude higher for cells grown on solid compared with liquid media, with maximum recombination efficiencies of about 10(-3). Cells were highly competent throughout their exponential growth phase, with no significant change in recombination efficiencies until population growth rates began to slow. Mutations in type IV pili, competency-related, and cell-cell signalling genes significantly impacted the ability of X. fastidiosa to acquire and incorporate DNA. Because X. fastidiosa is highly competent when growing in a surface-attached state, as it does within its insect vectors and host plants, recombination of naturally transformed DNA could be a significant route by which horizontal gene transfer occurs in natural environments.

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.