Starr, J. R. | Search bioRxiv

bioRxiv2018

SyngenicDNA: stealth-based evasion of restriction-modification barriers during bacterial genetic engineering

Restriction-modification (RM) systems hinder the use of genetic approaches in the vast majority of bacteria. Here, we describe a systematic approach to adapt genetic tools for use in bacteria that are genetically intractable or poorly tractable owing to active RM defenses. In this process, we determine the genome and methylome of a bacterial strain and use this information to define the bacteriums RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent SyngenicDNA tool and propagate the tool as novel minicircle plasmids, termed SyMPL tools, before transformation. Using SyngenicDNA and SyMPL tools, we achieved a profound, >100,000- fold, improvement in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus demonstrating the efficacy of these approaches for evading RM systems. The SyngenicDNA and SyMPL approaches are effective, flexible, and should be broadly applicable in microbial genetics. We expect these will facilitate a new era of microbial genetics free of the restraints of restriction-modification barriers.

microbiology↗

bioRxiv2017

Resolving Rapid Radiations Within Families Using Anchored Phylogenomics

Despite the promise that molecular data would provide a seemingly unlimited source of independent characters, many plant phylogenetic studies are based on only two regions, the plastid genome and nuclear ribosomal DNA (nrDNA). Their popularity can be explained by high copy numbers and universal PCR primers that make their sequences easily amplified and converted into parallel datasets. Unfortunately, their utility is limited by linked loci and limited characters resulting in low confidence in the accuracy of phylogenetic estimates, especially when rapid radiations occur. In another contribution on anchored phylogenomics in angiosperms, we presented flowering plant-specific anchored enrichment probes for hundreds of conserved nuclear genes and demonstrated their use at the level of all angiosperms. In this contribution, we focus on a common problem in phylogenetic reconstructions below the family level: weak or unresolved backbone due to rapid radiations ([≤] 10 million years) followed by long divergence, using the Cariceae-Dulichieae-Scirpeae clade (CDS, Cyperaceae) as a test case. By comparing our nuclear matrix of 461 genes to a typical Sanger-sequence dataset consisting of a few plastid genes (matK, ndhF) and an nrDNA marker (ETS), we demonstrate that our nuclear data is fully compatible with the Sanger dataset and resolves short backbone internodes with high support in both concatenated and coalescence-based analyses. In addition, we show that nuclear gene tree incongruence is inversely proportional to phylogenetic information content, indicating that incongruence is mostly due to gene tree estimation error. This suggests that large numbers of conserved nuclear loci could produce more accurate trees than sampling rapidly evolving regions prone to saturation and long-branch attraction. The robust phylogenetic estimates obtained here, and high congruence with previous morphological and molecular analyses, are strong evidence for a complete tribal revision of CDS. The anchored hybrid enrichment probes used in this study should be similarly effective in other flowering plant groups. [Carex, coalescent based species tree, flowering plants, low-copy nuclear genes, low-level phylogenetics, universal hybrid enrichment probes]

plant biology↗