The twin-arginine protein transport (Tat) system is one of two general protein transport systems in the cytoplasmic membranes of bacteria, and is conserved in the thylakoid membranes of plants. The Tat system transports fully folded proteins of varying sizes across the membrane. This feat is achieved by the use of just three conserved integral membrane proteins, TatA, TatB and TatC. Passenger proteins are designated for transport by the system by the use of an N-terminal extension, termed a signal peptide which contains an invariant twin arginine motif. The TatC component of the transport system is responsible for recognising the signal peptide, as well as binding to TatB and the TatA components. TatC therefore emerges as the functional and organisational ‘core’ of the Tat system. There is a wealth of functional data relating to the TatC protein, but no high resolution structural information on TatC was available to interpret this data. In order to gain an understanding of the Tat system at the molecular level, a structure was required of TatC. Preliminary expression screening using green fluorescent protein had previously identified the TatC protein from the thermophilic bacterium Aquifex aeolicus as being amenable for structural work. This study purified the A. aeolicus TatC construct in a variety of detergents, used native mass spectrometry and light scattering techniques to assay the sample homogeneity and set up extensive crystallisation trials. Following optimisation of a selenomethionine minimal media growth protocol, crystals grown from selenomethionine-substituted TatC purified in lauryl maltose neopentyl glycol diffracted to 3.5Å resolution, and allowed the structure of TatC to be solved by single wavelength anomalous dispersion. Analysis of the novel structure of TatC, in combination with an isothermal titration calorimetric binding assay, gave information on how TatC binds to signal peptides. Further structural analysis combined with additional experimental data from collaborators allowed a model to be proposed for how TatC binds to the two additional integral membrane components of the system, TatB and TatA, as part of the transport mechanism. Subsequent work was undertaken in this study to attempt to gain a co-crystal structure of TatC with the signal peptide. Construct and detergent screening was carried out in order to aim towards high resolution structural characterisation of the TatBC complex.

Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. Prior to this work, mutants were generated by random transposon insertions, which yielded highly complex pools for in vivo studies. Recent advances in polymerase chain reaction (PCR)-based mutagenesis in bacteria using the lambda red recombinase, as well as whole genome sequencing, enable a more directed approach for the generation of mutants. The lambda red approach was used to construct individual mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella, but not in related genera. All the mutations were confirmed simultaneously using a novel amplification strategy to produce labeled ribonucleic acid (RNA) from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c (Bagg Albino) mice and was recovered from the spleen. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, 51 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays and eleven were confirmed in individual mixed intraperitoneal infection in mice, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to our method as Array-Based Analysis of Cistrons Under Selection (ABACUS). Among the confirmed mutants identified in the ABACUS screen was a component of the twin arginine transport (Tat) system, tatC, required for transport of folded proteins across the cellular membrane. TatC is the highly conserved component necessary for recognition of the twin arginine containing signal sequence S/T-R-R-x-FL- K. We confirmed [delta] tatC mutants are defective for colonization of the liver and spleen in competitive infections with wild type ATCC14028 after intraperitoneal infection in Salmonella- susceptible (BALB/c). We also found that [delta] tatC mutants were defective for swimming motility, but not swarming motility, which was linked to the ability to elaborate flagellins on the bacterial surface under different conditions.

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