Biology subjects

Dalia, T. N.

Publications and source records attributed to Dalia, T. N..

4 recordsLinked to original sources

bioRxiv2017

ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.

microbiology↗

bioRxiv2017

Systematic Genetic Dissection Of Chitin Degradation And Uptake In Vibrio cholerae

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2, and (GlcN)2, play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.\n\nORIGINALITY-SIGNIFICANCE STATEMENTVibrio cholerae, the causative agent of the diarrheal disease cholera, interacts with the chitinous shells of crustacean zooplankton in the aquatic environment, which serves as an environmental reservoir for this pathogen. It degrades and utilizes chitin-derived products as a source of carbon and nitrogen. Also, chitin serves as an inducing cue for natural transformation - an important mechanism of horizontal gene transfer in this species. Here, we systematically dissect the genes required for chitin degradation and uptake, and characterize the role of these genes for growth on chitin as a nutrient and during chitininduced natural transformation. Thus, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

microbiology↗

bioRxiv2017

Enhancing Multiplex Genome Editing by Natural Transformation (MuGENT) via inactivation of ssDNA exonucleases

Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ~50% without selection in cis. Consequently, editing constructs are generated in a single PCR reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.

microbiology↗

bioRxiv2017

Multiplex genome editing for synthetic biology in Vibrio natriegens

Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of large genome edits at high-efficiency on unprecedented timescales, which will extend the utility of this species for diverse applications.\n\nV. natriegens is the fastest growing organism known, with a doubling time of <10 min1,2. With broad metabolic capabilities, lack of pathogenicity, and its rapid growth rate, it is an attractive alternative to E. coli for diverse molecular biology and biotechnology applications3. Methods for classical genetic techniques have been developed for V. natriegens, but these are rel ...

synthetic biology↗