Deletion Mutants, Archived Transposon Library, and Tagged Protein Constructs of the Model Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

ABSTRACT The dissimilatory sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris Hildenborough (ATCC 29579) was chosen by the research collaboration ENIGMA to explore tools and protocols for bringing this anaerobe to model status. Here, we describe a collection of genetic constructs generated by ENIGMA that are available to the research community.

Probing the Structure of [NiFeSe] Hydrogenase with QM/MM Computations

The geometry and vibrational behavior of selenocysteine [NiFeSe] hydrogenase isolated from Desulfovibrio vulgaris Hildenborough have been investigated using a hybrid quantum mechanical (QM)/ molecular mechanical (MM) approach. Structural models have been built based on the three conformers identified in the recent crystal structure resolved at 1.3 Å from X-ray crystallography. In the models, a diamagnetic Ni2+ atom was modeled in combination with both Fe2+ and Fe3+ to investigate the effect of iron oxidation on geometry and vibrational frequency of the nonproteic ligands, CO and CN-, coordinated to the Fe atom. Overall, the QM/MM optimized geometries are in good agreement with the experimentally resolved geometries. Analysis of computed vibrational frequencies, in comparison with experimental Fourier-transform infrared (FTIR) frequencies, suggests that a mixture of conformers as well as Fe2+ and Fe3+ oxidation states may be responsible for the acquired vibrational spectra.

Development of genome-wide genetic assays in Desulfovibrio vulgaris Hildenborough

Illumina sequencing and other massively-parallel, short read sequencing technologies have become prevalent methods for querying the genetic systems of organisms. This trend was initially driven by the demand for general de novo and resequencing applications in eukaryotes, but uses for these instruments have recently grown in scope to also include more specific assays and methods intended for use in prokaryotic systems. This thesis seeks to develop two such assays for use in the sulfate reducing bacteria type species Desulfovibrio vulgaris Hildenborough. These novel assays continue advances made in other bacteria, and the use of this environmentally relevant obligate anaerobe will ensure their extension to other bacteria outside the easily-manipulated groups studied previously. The first assay is a variation on existing transposon sequencing (Tn-seq) assays, which seek to determine gene fitness profiles and essential genes by simultaneous analyses of whether the absence of each gene product alters the growth kinetics of the bacterium. Here we modify the standard Tn-seq procedure by including delivery of the transposon through conjugation and liquid culture enrichment of the mutant pool, creating transposon liquid enrichment sequencing (TnLE-seq). This simplifies and shortens the process, and also reduces barriers to application of the technique in microbes lacking a facile genetic system. The second assay provides a means of mapping consensus 3' end sites of RNA transcripts across a reference genome. This method is known as 3' RNA-seq and compliments an established technique for determining 5' start sites of transcripts. Together these facilitate the application of current sequencing technology to a wider array of microbes and a new type of biologically relevant genetic data.