Jain, Mukesh

ABSTRACT Methylglyoxal (MG) is a cytotoxic, nonenzymatic by-product of glycolysis that readily glycates proteins and DNA, resulting in carbonyl stress. Glyoxalase I and II (GloA and GloB) sequentially convert MG into d -lactic acid using glutathione (GSH) as a cofactor. The glyoxalase system is essential for the mitigation of MG-induced carbonyl stress, preventing subsequent cell death, and recycling GSH for maintenance of cellular redox poise. All pathogenic liberibacters identified to date are uncultured, including “ Candidatus Liberibacter asiaticus,” a psyllid endosymbiont and causal agent of the severely damaging citrus disease “huanglongbing.” In silico analysis revealed the absence of gloA in “ Ca . Liberibacter asiaticus” and all other pathogenic liberibacters. Both gloA and gloB are present in Liberibacter crescens , the only liberibacter that has been cultured. L. crescens GloA was functional in a heterologous host. Marker interruption of gloA in L. crescens appeared to be lethal. Key glycolytic enzymes were either missing or significantly downregulated in “ Ca . Liberibacter asiaticus” compared to (cultured) L. crescens . Marker interruption of sut , a sucrose transporter gene in L. crescens , decreased its ability to take up exogenously supplied sucrose in culture. “ Ca . Liberibacter asiaticus” lacks a homologous sugar transporter but has a functional ATP/ADP translocase, enabling it to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding MG generation via glycolysis, and (iii) directly importing ATP from the host cell. MG detoxification enzymes appear to be predictive of “ Candidatus ” status for many uncultured pathogenic and environmental bacteria. IMPORTANCE Discovered more than 100 years ago, the glyoxalase system is thought to be present across all domains of life and fundamental to cellular growth and viability. The glyoxalase system protects against carbonyl stress caused by methylglyoxal (MG), a highly reactive, mutagenic and cytotoxic compound that is nonenzymatically formed as a by-product of glycolysis. The uncultured alphaproteobacterium “ Ca . Liberibacter asiaticus” is a well-adapted endosymbiont of the Asian citrus psyllid, which transmits the severely damaging citrus disease “huanglongbing.” “ Ca . Liberibacter asiaticus” lacks a functional glyoxalase pathway. We report here that the bacterium is able to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding (significant) MG generation via glycolysis, and (iii) directly importing ATP from the host cell. We hypothesize that failure to culture “ Ca . Liberibacter asiaticus” is at least partly due to its dependence on host cells for both ATP and MG detoxification.

Host acquisition of a new microbial species can readily perturb the dynamics of preexisting microbial associations. Molecular cross talk between microbial associates may be necessary for efficient resource allocation and enhanced survival. Classic examples involve quorum sensing (QS), which detects population densities and is both used and coopted to control expression of bacterial genes, including host adaptation factors. We report that a 56-amino-acid repressor protein made by the resident psyllid endosymbiont Wolbachia can enter cells of Liberibacter crescens , a cultured proxy for the uncultured psyllid endosymbiont “ Ca . Liberibacter asiaticus” and repress “ Ca . Liberibacter asiaticus” phage lytic cycle genes. Such repression in “ Ca . Liberibacter asiaticus” may be critical to survival of both endosymbionts, since phage-mediated lysis would likely breach the immunogenic threshold of the psyllid, invoking a systemic and nonspecific innate immune reaction.

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.