One of the primary goals of the Environmental Stress Pathway Project (ESPP) is to map the response of the anaerobic sulfate reducing soil bacterium, Desulfovibrio vulgaris Hildenborough to its environment. Two component systems, comprised of Histidine Kinase and Response regulator proteins, present the primary and ubiquitous mechanism in bacteria for initiating cellular response towards a wide variety of environmental conditions. In D. vulgaris Hildenborough, more than 70 such systems have been predicted, but remain mostly uncharacterized. The ability of D. vulgaris to survive in its environment is no doubt linked with the activity of genes modulated by these two component signal transduction systems. To map the two component systems to the genes they modulate, the availability of deletion mutants provides an important tool. Here we present an overview of the predicted histidine kinases in D. vulgaris and describe a strategy to create library of histidine kinase knock out mutants in D. vulgaris. We use the OmniLog(R) workflow to conduct a wide phenotypic characterization of the knock out mutants generated. To illustrate our strategy we present results from our study of the histidine kinase in the predicted kdp operon of D. vulgaris. The high-affinity potassium uptake Kdp complex is well characterized in other bacteria where it facilitates K+ uptake in low K+ or high Na+ conditions. Typically, the activity of the Kdp system is modulated by the KdpD/E two-component signal transduction system, where KdpD is the sensor histidine kinase and KdpE is the response regulator. The D. vulgaris kdp operon contains a gene with predicted response regulator function and two separate genes annotated for the sensor kinase function (kdpD and DVU3335). Interestingly, only one of these two, DVU3335, contains a conserved histidine kinase domain which is absent the D. vulgaris kdpD candidate. However, DVU3335 does not encode the well-conserved motifs associated with KdpD. We created a knock out mutant in the DVU3335 gene. The DVU3335 knock out strain showed a growth deficiency in low K+ conditions and when exposed to low K+ conditions was unable to upregulate genes in the kdp operon. Phenotypic microarrays were used to obtain a broader comparison of the mutant and wild type strains. Our results show that the major differences between the wild type and the mutant are in response to salt stress and support the role of DVU3335 in modulating K+ uptake during low K+ and high Na+ conditions.