Genetic manipulation of the obligate chemolithoautotrophic bacterium Thiobacillus denitrificans

Chemolithoautotrophic bacteria can be of industrial and environmental importance, but they present a challenge for systems biology studies, as their central metabolism deviates from that of model organisms and there is a much less extensive experimental basis for their gene annotation than for typical organoheterotrophs. For microbes with sequenced genomes but unconventional metabolism, the ability to create knockout mutations can be a powerful tool for functional genomics and thereby render an organism more amenable to systems biology approaches. In this chapter, we describe a genetic system for Thiobacillus denitrificans, with which insertion mutations can be introduced by homologous recombination and complemented in trans. Insertion mutations are generated by in vitro transposition, the mutated genes are amplified by the PCR, and the amplicons are introduced into T. denitrificans by electroporation. Use of a complementation vector, pTL2, based on the IncP plasmid pRR10 is also addressed

Scalable and Versatile k-mer Indexing for High-Throughput Sequencing Data

International audiencePhilippe et al. (2011) proposed a data structure called Gk ar- rays for indexing and querying large collections of high-throughput sequencing data in main-memory. The data structure supports versa- tile queries for counting, locating, and analysing the coverage profile of k-mers in short-read data. The main drawback of the Gk arrays is its space-consumption, which can easily reach tens of gigabytes of main- memory even for moderate size inputs. We propose a compressed variant of Gk arrays that supports the same set of queries, but in both near-optimal time and space. In practice, the compressed Gk arrays scale up to much larger inputs with highly competitive query times compared to its non-compressed predecessor. The main applica- tions include variant calling, error correction, coverage profiling, and sequence assembly

Generalized schemes for high throughput manipulation of the Desulfovibrio vulgaris Hildenborough genome

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high- throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications including gene replacement and creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation