Identification of the Genes Required for the Culture of Liberibacter crescens, the Closest Cultured Relative of the Liberibacter Plant Pathogens

Abstract

Here Tn5 random transposon mutagenesis was used to identify the essential elements for culturing Liberibacter crescens BT-1 that can serve as antimicrobial targets for the closely related pathogens of citrus, Candidatus Liberibacter asiaticus (Las) and tomato and potato, Candidatus Liberibacter solanacearum (Lso). In order to gain insight on the virulence, metabolism, and culturability of the pathogens within the genus Liberibacter, a mini-Tn5 transposon derivative system consisting of a gene specifying resistance to kanamycin, flanked by a 19-base-pair terminal repeat sequence of Tn5, was used for the genome-wide mutagenesis of L. crescens BT-1 and created an insertion mutant library. By analyzing the location of insertions using Sanger and Illumina Mi-Seq sequencing, 314 genes are proposed as essential for the culture of L. crescens BT-1 on BM-7 medium. Of those genes, 76 are not present in the uncultured Liberibacter pathogens and, as a result, suggest molecules necessary for the culturing these pathogens. Those molecules include the aromatic amino acids, several vitamins, histidine, cysteine, lipopolysaccharides, and fatty acids. In addition, the 238 essential genes of L. crescens in common with L. asiaticus are potential targets for the development of therapeutics against the disease.

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

“…The genes that could not foster a Tn5 insertion were considered to be essential for growth in culture, which accounted for 314 protein-coding genes [Lai et al, 2016]. The results of the mutagenesis are consistent with those of Fagen et al [2014], and the authors suggest targeting ABC transporters and lipopolysaccharide biosynthesis genes for the development of antimicrobial treatments [Lai et al, 2016].…”

Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen “Candidatus Liberibacter asiaticus” an Energy Scavenger

“…It has been transformed with plasmids expressing "Ca. Liberibacter asiaticus" genes and reporter constructs (8,9; the present study) and used for Tn5 mutagenesis (29). In the present study, site-directed mutagenesis of the L. crescens sugar transporter sut (B488_RS00965) via marker interruption (Fig.…”

Crystal structures of a putative periplasmic cystine‐binding protein from Candidatus Liberibacter asiaticus: insights into an adapted mechanism of ligand binding

“…Curiously, the expression of one of them specific for cationic amino acids mainly occurs in citrus psyllid host while the second one having a maximum sequence homology to cystine-binding receptors mainly expresses in the plant [5]. Furthermore, the expression of the transmembrane permease suggested to be specific for the cystine-binding receptor is quite high in case of host plant [20]. These reports clearly suggest the importance of the putative cystine-binding receptor in survival of the CLas in phloem of host plant.…”