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Rovinsky, R.

Publications and source records attributed to Rovinsky, R..

2 recordsLinked to original sources
bioRxiv2024

Identification of Proteins Influencing CRISPR-Associated Transposases for Enhanced Genome Editing

CRISPR-Associated Transposases (CASTs) hold tremendous potential for microbial genome editing due to their ability to integrate large DNA cargos in a programmable and site-specific manner. However, the widespread application of CASTs has been hindered by their low efficiency in diverse, non-model bacteria. In an effort to address this shortcoming, we conducted the first genome-wide screen for host factors impacting Vibrio cholerae CAST (VchCAST) activity and used the findings to increase VchCAST editing efficiency. A genome-wide loss-of-function mutant library in E. coli was screened to identify 15 genes that impact type VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity and two were found to be inhibitory. Informed by homologous recombination involved effectors, RecD and RecA, we tested the {lambda}-Red recombineering system in our VchCAST editing vectors, which increased its insertion meditated-editing efficiency by 25.7-fold in E. coli while maintaining high target specificity and similar insertion arrangements. Furthermore, {lambda}-Red-enhanced VchCAST achieved increased editing efficiency in the industrially important bacteria Pseudomonas putida and the emerging pathogen Klebsiella michiganensis. This study improves understanding of factors impacting VchCAST activity and enhances its efficiency as a bacterial genome editor. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/612086v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@12609e5org.highwire.dtl.DTLVardef@53a128org.highwire.dtl.DTLVardef@794e63org.highwire.dtl.DTLVardef@9835f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗
bioRxiv2023

Genomic analysis of cultivated infant microbiomes identifies Bifidobacterium 2'-fucosyllactose utilization can be facilitated by co-existing species

Human milk oligosaccharides (HMOs) ensure proper infant gut microbiome establishment. Isolate studies have revealed the genetic basis for HMO metabolism, but they exclude the possibility of HMO assimilation via synergistic interactions involving multiple organisms. Here, we investigated microbiome responses to 2-fucosyllactose (2FL), a prevalent HMO and infant formula additive, by establishing individualized microbiomes using fecal samples from three different infants as the inocula. Bifidobacterium breve, a prominent member of infant microbiomes, typically cannot metabolize 2FL. Using metagenomic data, we predicted that extracellular fucosidases encoded by co-existing members such as Ruminococcus gnavus initiate 2FL breakdown, thus critical for B. breves growth. Using both targeted co-cultures and by supplementation of R. gnavus into one microbiome, we show that R. gnavus can promote extensive growth of B. breve through the release of lactose from 2FL. Overall, microbiome cultivation combined with genome-resolved metagenomics demonstrated that HMO utilization can vary with an individuals microbiome.

microbiology↗