Generalized Schemes for High-Throughput Manipulation of the Desulfovibrio vulgaris Genome

Abstract

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 the 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 vulgarisHildenborough, 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.

A Post-Genomic View of the Ecophysiology, Catabolism and Biotechnological Relevance of Sulphate-Reducing Prokaryotes

“…The marker-exchange deletion procedure also formed the foundation for the generation of 1404 D. vulgaris protein-encoding genes (41% of those predicted in the genome) fused with sequences encoding the STF tandem tag (Strep tag ® (IBA, G€ ottingen)-TEV (Tobacco Etch Virus)-FLAG) (Chhabra, Butland, et al, 2011). PCR amplicons that fused the tag sequences with the encoding genes producing proteins with tag fusions at the carboxy-termini were captured in suicide plasmids by application of the sequence ligation-independent cloning (SLIC) procedure (Li & Elledge, 2007).…”

“…Further studies along this experimental line included investigations on the energetic consequences of nitrite stress and periplasmic hydrogenases (Caffrey et al, 2007). Proteomic analysis of D. vulgaris Hildenborough was concerned, e.g., with adaptation to different energy sources and growth phases (Pereira, He, Valente, et al, 2008;Zhang, Gritsenko, Moore, et al, 2006), posttranslational modification of sulphate reduction enzymes (Gaucher et al, 2008) and protein-protein interactions (Chhabra, Butland, et al, 2011;Chhabra, Joachimiak, et al, 2011). During the last 10 or so years, D. vulgaris served as a model system for a large group of scientists to explore the global response to a variety of stress conditions (Zhou et al, 2011).…”

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported

“…vulgaris Hildenborough wild-type ATCC29579 was genetically engineered to encode locus-specific affinity purification (AP)-tagged fusion proteins using electroporation of nonreplicating "suicide constructs" (28). Of the 3525 predicted D. vulgaris protein-coding genes, we attempted to create tagged strains for a priority list of 2086 genes.…”

“…A non-redundant total of 1401 unique genes are represented as AP-tagged alleles in the 1498 strains constructed. All affinity purifications were performed as described previously (28). In all cases, Strep-TEV-FLAGHis 6 strains were treated exactly as Strep-TEV-FLAG strains for the purposes of affinity purification of protein complexes.…”

Rapid Transposon Liquid Enrichment Sequencing (TnLE-seq) for Gene Fitness Evaluation in Underdeveloped Bacterial Systems

“…These include high-resolution microarrays (11,12), transcript start site information (13), affinity-tagged proteins (14), markerless deletion mutants (15), and an isolated library of over 1.2 ϫ 10 4 individually maintained transposon mutants with known unique insertion sites (http://desulfovibriomaps.biochem.missouri.edu/mutants/). The availability of these tools for confirmation of gene fitness information, combined with the impracticality of employing preexisting Tn-seq methods (2-5) for analysis of DvH, makes it a challenging proof-of-principle organism for the establishment of this technique.…”