Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface

Abstract

Background Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace’s Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens. Results The evaluation of the growth parameters—pH, aeration, temperature, and buffering capacity—reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium. Conclusion L. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments—the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis—the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3−), light and other photosynthates might lead to develop better strategies for disease management.

The Genome of “Candidatus Liberibacter asiaticus” Is Highly Transcribed When Infecting the Gut of Diaphorina citri

“…A relation that can be seen in reduced genomes with adaptive pressure and evolutive process is the loss of redundant and non-functional gene sequences ( Mira et al, 2001 ; Sällsträm and Andersson, 2005 ), a factor that certainly renders these Liberibacters highly host-dependent for growth. L. crescens is the only Liberibacter available in axenic culture, which makes it suitable to functional studies and growth assays ( Leonard et al, 2012 ; Jain et al, 2015 ; Jain et al, 2017b ; Cruz-Munoz et al, 2019 ; Zuñiga et al, 2020 ). In Las strains and other Liberibacters that have their genomes sequenced, type II secretion system, avr and hrp genes, plant cell wall degrading enzymes, and several known virulence determinants also found in other plant pathogenic bacteria are absent ( Duan et al, 2009 ; Lin et al, 2011 ; Wulff et al, 2014 ; Lin et al, 2015 ) but potential Las effectors ( Thapa et al, 2020 ) were expressed in the psyllid gut in our analysis, except for CLIBASIA_04530.…”

Proteomic and bioinformatic analyses of proteins in the outer membrane and extracellular compartments and outer membrane vesicles of Candidatus Liberibacter species

“…Liberibacter crescens BT-1was cultured in Basal Medium 7 (BM7) medium consisting of 2 g alpha-ketoglutarate, 10 g N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer, 3.75 g KOH, 150 ml of fetal bovine serum (Gibco) and 300 ml of TMN-FH insect medium (Sigma) per litter, adjusted to pH 6.5 ( Cruz-Munoz et al, 2019 ). Bacterial culture was grown at 250 rpm and 28°C.…”