In our work from April to July 2015, we moved forward to start molecular analysis of Liberibacter asiaticus rpoH effects on transcription. We also began the second major phase of work, in which we will study Liberibacter transcription regulators. We made use of new information about the effect of endogenous S. meliloti genes and the vector ribosome binding sequences (RBS) described in the March 2015 report. 1. Molecular analysis with the newly-characterized RpoH-expressing constructs: a. We have prepared RNA for S. meliloti strains containing the following plasmids: plasmid expressing Sm rpoH1; plasmid expressing optimized La rpoH; and the empty vector control, pSRK-Gm. These RNA preparations now will be processed for Affymetrix GeneChip analysis. 2. In our proposal, we aimed to study RpoH and also a set of putative transcription factors. Having completed construction of rpoH strains, we designed the optimized genes for the remaining six regulators: as before, we substituted codons of the native La rpoH gene for codons that are compatible with the GC-rich genome of S. meliloti. We ordered and received optimized DNA for these six genes. These were cloned into the pSRK-Gm vector, using the minimal background vector RBS. a. The codon-optimized La visNvisR genes were cloned as a single insert to maintain operon structure. We used the same intergenic spacer sequence between visN and visR that is present in S. meliloti. 3. We did not originally propose to create deletion strains lacking each orthologous S. meliloti regulator. However, because we found that the native rpoH genes interfered with our ability to observe the full effect of plasmid-based rpoH, we decided to construct strains with each corresponding regulator deleted. For example, we now plan to make a visNR deletion strain of S. meliloti in which to test regulation by the optimized La visNR gene; we will construct a S. meliloti ctrA deletion strain to host the La ctrA plasmid, etc. a. We are in the process of making these deletion strains using our customary sacB plasmid deletion method. In this method, a vector bearing a resistance marker and the sacB gene is used to clone DNA from regions flanking the gene to be deleted. After recombining into a wild-type S. meliloti host by a single cross-over event, the strain is plated on sucrose, which is lethal unless the sacB-containing vector has recombined a second time and been lost from the cell; among such double-crossover events, some restore the wild type gene and some have a clean deletion of the target sequence. Deletion strains are confirmed by performing PCR and sequencing. b. Results to date: We have single-crossover strains containing the sacB construct for all six chosen regulators. c. Of these six, two strains are complete and confirmed for deletion: the visNR deletion strain and ldtR deletion strain. d. We are presently screening for putative deletions of phrR1, phrR2, and lsr, and are planning a two-step construction of a ctrA deletion strain (needed since ctrA is required for basic viability).