Comparative Genomics of Cultured and Uncultured Strains Suggests Genes Essential for Free-Living Growth of Liberibacter

Abstract

The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host.

Liberibacter crescens gen. nov., sp. nov., the first cultured member of the genus Liberibacter

“…BT-1 T was grown for 4 days in BM7 broth (Fagen et al, 2014). Cultures were maintained at 28 u C in a shaking incubator at 125 r.p.m.…”

“Candidatus Liberibacter asiaticus” Prophage Late Genes May Limit Host Range and Culturability

“…L. crescens growth conditions and transformation. L. crescens strain BT-1 was maintained in liquid BM7 medium containing 2 g of alphaketoglutarate, 10 g of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer, and 3.75 g of KOH in 550 ml water, pH 6.9, followed by the addition of 300 ml of filter-sterilized fetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 300 ml of modified Grace's insect culture medium (TNM-FH; HyClone Laboratories) and gentle shaking at 150 rpm at 28°C (14). Transformation of BT-1 cells was done by electroporation.…”

Understanding the Physiology of <b><i>Liberibacter asiaticus</i></b>: An Overview of the Demonstrated Molecular Mechanisms

“…The genome of L. asiaticus was compared with that of L. solanacearum and L. crescens BT-1, the closest cultured relative, to acquire knowledge regarding the current inability to culture most Liberibacter species [Fagen et al, 2014]. Compared to L. crescens, L. asiaticus was found to lack the following: pathways for synthesis of 6 amino ac-DOI: 10.1159/000492386 ids, the shikimic acid pathway, the (R)-pantothenate biosynthetic pathway or transporter, a functional folate production pathway, polyamine synthesis pathway, glucose-6-phosphate isomerase necessary in Embden-Meyerhof glycolytic pathway, a cytochrome d type ubiquinol oxidase for aerobic respiration, 5 ABC transporters (niaP, ftsEX, fhuBCD, oppABCD, and raxB), the twin arginine transport system, 4 two-component signal transduction systems, multi-copper oxidase, zinc uptake regulation (ZUR) protein, the stringent response, LpxL protein involved in lipid A biosynthesis, several peptidoglycan recycling proteins, indole-3-acetamide production pathway.…”