Publications

3807

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.

‘Candidatus Liberibacter solanacearum’ is a bacterium associated with several vegetative disorders on solanaceous and apiaceous crops. Following the recent detection of the bacterium in carrots in Europe, and particularly carrot plants used for seed production in France, two independent laboratories conducted experiments on the transmission of this pathogen by seed and had discordant results: one study showed no bacterial transmission to plants, and the other showed transmission to carrot seedlings starting from the fourth month of culture. To test the hypothesis that growing conditions affect seed transmission efficiencies, trials were renewed in 2015 on four lots of 500 carrot seeds naturally contaminated with ‘Ca. L. solanacearum’ and two lots of 100 healthy seeds. The plants were grown for 6 months in an insect-proof NS2 greenhouse. Sets of 108 plants from the contaminated lots and 24 plants from the healthy lots were individually analyzed each month using real-time PCR to detect the bacterium. The detection tests on seeds and plants from healthy lots were always negative. During the 6 months of the trial, no plants from the contaminated seed lots tested positive for the bacterium or showed any infection symptoms. These results indicate that transmission of ‘Ca. L. solanacearum’ by carrot seed is rare and difficult to reproduce.