The causal agent of Huanglongbing disease, ‘Candidatus Liberibacter asiaticus’, is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from ‘Ca. L. asiaticus’ involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of ‘Ca. Liberibacter asiaticus’, using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

In our previous work, we found that feeding Lactobacillus johnsonii to BioBreeding diabetes-prone (BBDP) rats decreased the incidence of diabetes development. The aim of this study was to investigate host pathways affected by L. johnsonii, with specific focus on the rate-limiting enzyme of tryptophan catabolism, indoleamine 2,3-dioxygenase (IDO). Suspensions of L. johnsonii or an equal volume of vehicle were orally administered to BBDP rats. Tissue IDO was investigated using quantitative RT-PCR and Western blot, whereas tryptophan, kynurenine, and 5-hydroxytryptamine (5-HT) concentrations were quantified by HPLC and ELISA. IDO activity was also investigated using L. johnsonii culture cell-free supernatant (CFS) with affinity-purified IDO and HT-29 intestinal epithelial cells. L. johnsonii feeding resulted in a 17% reduction in serum kynurenine compared with that in vehicle-fed controls, correlating with a 1.4-fold elevation in 5-HT levels. H₂O₂ produced by L. johnsonii abolished IDO activity in vitro, and L. johnsonii feeding resulted in a 3.9-fold increase in ileum lumen H₂O₂. L. johnsonii CFS significantly reduced IDO activity in HT-29 intestinal epithelial cells (47% reduction) compared with that in vehicle-treated controls, an effect abolished by catalase treatment. These data support the role of H₂O₂ in commensal bacteria-host interactions and highlight the influence of commensal bacteria-derived H₂O₂ on host physiology.

CLIBASIA_01510, PrbP, is a predicted RNA polymerase binding protein in Liberibacter asiaticus. PrbP was found to regulate expression of a small subset of ribosomal genes through interactions with the β-subunit of the RNA polymerase and a short, specific sequence on the promoter region. Molecular screening assays were performed to identify small molecules that interact with PrbP in vitro. Chemical hits were analyzed for therapeutic efficacy against L. asiaticus via an infected leaf assay, where the transcriptional activity of L. asiaticus was found to decrease significantly after exposure to tolfenamic acid. Similarly, tolfenamic acid was found to inhibit L. asiaticus infection in highly symptomatic citrus seedlings. Our results indicate that PrbP is an important transcriptional regulator for survival of L. asiaticus in planta, and the chemicals identified by molecular screening assays could be used as a therapeutic treatment for huanglongbing disease.

Electron spin resonance (e.s.r.) spectra of N O adsorbed on the 4 zeolites Linde 4A, 5A, 13X, and hydrogen mordenite were measured at 77 OK. N o e.s.r. signal was observed for NO on silica gel. By use of expressions for the line shapes of polycrystalline samples, assuming axially symmetric g-tensors, and taking into account also isotropic coupling, values of g , , , g,, and line width were derived by an iterative procedure. From these, the crystal field splitting, A, of the 2pz levels, which become non-degenerate on adsorption, were calculated. By relating A to the interaction of the non-axial components of the quadrupole moment with the corresponding electric field gradients, the non-axial component of the latter is estimated for the surface fields of the zeolites.

In this study we aimed to identify small molecules with high affinity involved in the allosteric regulation of LVIS553, a MarR member from Lactobacillus brevis ATCC367. Using high throughput screening, novobiocin was found to specifically bind LVIS553 with a K D ‫؍‬ 33.8 ؎ 2.9 M consistent with a biologically relevant ligand. Structure guided site-directed mutagenesis identified Lys 9 as a key residue in novobiocin recognition. The results found in vitro were correlated in vivo. An increased tolerance to the antibiotic was observed when LVIS553 and the downstream putative transport protein LVIS552 were either expressed in a low copy plasmid in L. brevis or as a single copy chromosomal insertion in Bacillus subtilis. We provide evidence that LVIS553 is involved in the specific regulation of a new mechanism of tolerance to novobiocin.