In the quarter ending September 15, we focused on study of 6 Liberibacter regulators: RpoH, VisNR (a dimeric regulator encoded by two genes), LdtR, LsrB, PhrR, and CtrA. We chose the following target promoters, based on our previous global transcription studies: Regulator: target promoter(s): – RpoH: ibpA, groESL5 – LdtR: tacA, SMc04059, ldtP – VisNR: rem – CtrA: minCDE – LsrB: SMc01834, lrp3-lpsCDE – PhrR: SMc00404, ldtR To assess strength of expression, we constructed plasmids in which the target promoter fusion was on the same plasmid as the transcription factor gene. The fusions used enhanced GFP (F64L, S65T), with codon usage optimized for S. meliloti. On this modular plasmid, one can exchange regulatory gene, reporter gene, and terminators with straightforward cloning. For example, we made a plasmid in which CLas rpoH (controlled by lac promoter) was on the same plasmid as the ibpA-eGFP fusion. Therefore activity of CLas RpoH protein would be detected as fluorescence; inhibition of CLas RpoH would be evident by a decrease in fluorescence. Using plates with induction of the lac promoter, we examined the level of fluorescence for each regulator-promoter pair. The data from these tests shows us that of our six original transcription regulators, 3 gave robust fluorescence, and therefore would be suitable to look for inhibitory compounds. We chose the best-responding promoter target for each. The final constructs are: RpoH (target promoter ibpA); VisNR (target promoter rem); and LdtR (target promoter SMc04059). The test plates used were 96 well Costar black plates with clear bottom. In initial trials, cells were diluted to various concentrations, and we tried several culture media including two rich media (TY and LB supplemented with Mg and Ca ions) as well as minimal (defined) medium. To assay fluorescence, we used the following conditions: Excitation wavelength = 489 nm Emission wavelength = 509 nm. In addition to fluorescence we measured optical absorbance at 600 nm in order to detect inhibition of bacterial growth itself. This is important because we want to know whether the compounds inhibit the regulator (desired) or just kill the cell (undesired). As of September, we had begun the first test run for control strains.