The complete genome sequence of 'Candidatus Liberibacter solanacearum', the bacterium associated with potato zebra chip disease

Abstract

Zebra Chip (ZC) is an emerging plant disease that causes aboveground decline of potato shoots and generally results in unusable tubers. This disease has led to multi-million dollar losses for growers in the central and western United States over the past decade and impacts the livelihood of potato farmers in Mexico and New Zealand. ZC is associated with 'Candidatus Liberibacter solanacearum', a fastidious alpha-proteobacterium that is transmitted by a phloem-feeding psyllid vector, Bactericera cockerelli Sulc. Research on this disease has been hampered by a lack of robust culture methods and paucity of genome sequence information for 'Ca. L. solanacearum'. Here we present the sequence of the 1.26 Mbp metagenome of 'Ca. L. solanacearum', based on DNA isolated from potato psyllids. The coding inventory of the 'Ca. L. solanacearum' genome was analyzed and compared to related Rhizobiaceae to better understand 'Ca. L. solanacearum' physiology and identify potential targets to develop improved treatment strategies. This analysis revealed a number of unique transporters and pathways, all potentially contributing to ZC pathogenesis. Some of these factors may have been acquired through horizontal gene transfer. Taxonomically, 'Ca. L. solanacearum' is related to 'Ca. L. asiaticus', a suspected causative agent of citrus huanglongbing, yet many genome rearrangements and several gene gains/losses are evident when comparing these two Liberibacter. species. Relative to 'Ca. L. asiaticus', 'Ca. L. solanacearum' probably has reduced capacity for nucleic acid modification, increased amino acid and vitamin biosynthesis functionalities, and gained a high-affinity iron transport system characteristic of several pathogenic microbes.

Figure 1. Schematic representation of the ‘Candidatus Liberibacter solanacearum’ genome.

Circular representation of the 1.26 Mbp genome. The tracks from the outmost circles represent (1) Forward CDS (green) and (2) Reverse CDS (green) with pseudogenes in red; (3) tRNA (blue); (4) bacteriophage derived regions and probable phage remnants (red); (5) three copies of rRNA operon (16S, 23S and 5S) (purple); (6) % G+C content and (7) GC skew [(GC/(G+C))].

Figure 2. Comparison of the ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter asiaticus’ predicted proteomes.

‘Ca. L. solanacearum’-encoded sequences are delimited by the red line and ‘Ca. L. asiaticus’-encoded sequences are delimited by the blue line. 884 bidirectional best hits (BBH) were identified in both genomes using an e-value cutoff of 10⁻¹⁵. The remaining sequences consist of both unidirectional hits and unique sequences. Using the above cutoff values, 236 BBH ‘Ca. L. solanacearum’ sequences are considered unique to ‘Ca. L. solanacearum’ and 186 BBH ‘Ca. L. asiaticus’ sequences are considered unique to ‘Ca. L. asiaticus’. Most of the species-specific predicted translations are annotated as hypothetical proteins.

Figure 3. Comparison of the ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter asiaticus’ chromosomal features.

Locally collinear blocks (LCBs) identified genomes of ‘Ca. L. solanacearum’ and ‘Ca. L. asiaticus’. Each contiguously colored region is a LCB, a region without rearrangement of homologous backbone sequence. LCBs below a genome's center line are in the reverse complement orientation relative to the reference genome, ‘Ca. L. solanacearum’. Lines between genomes trace each orthologous LCB through every genome. The ‘Ca. L. solanacearum’ and ‘Ca. L. asiaticus’ genomes have undergone considerable genome rearrangements. Two rectangles represent bacteriophage-derived regions in ‘Ca. L. solanacearum’ which was matched with ‘Ca. L. asiaticus’.

Figure 4. Predicted ‘Candidatus Liberibacter solanacearum’ metabolic pathways and general features.

Schematic representation of an ‘Ca. L. solanacearum’ cell bounded by inner and outer membranes. Cofactors (aqua) and amino acids (violet) that are predicted to be synthesized by ‘Ca. L. solanacearum’ are indicated by dark bold text; exogenously-supplied vitamins and amino acids are indicated in faint text. Ions, amino acids, and nucleotides are indicated by common abbreviations. Glucose-6-phosphate (G6P); fructose-6-phosphate (F6P); fructose bisphosphate (FBP); glyceraldehyde-3-phosphate (G3P); 1,3-bisphosphoglycerate (3PGP); 3-phosphoglycerate (3PG); 2-phosphoglycerate (2PG); phosphenolpyruvate (PEP); pyruvate (PYR); orotate (ORO); 5-phosphoribosyl diphosphate (PRPP); ornithine (Orn); citrulline (Ctn); arginosuccinate (ARS); acetyl-CoA (AcCoA); dihydroxyacetone phosphate (DHAP); oxaloacetate (OAA); citrate (CIT); 2-oxoglutarate (2OG); succinate (SUCC); fumarate (FUM); malate (MAL); erythrose-4-phosphate (E4P); S-adenosylmethionine (SAM); gamma-glutamylcysteine (GGC); glutathione (GSH); inorganic phosphate (Pi); flavin adenine dinucleotide (FAD); p-aminobenzoic acid (PABA); dihydroneopterin triphosphate (DHNTP); dihydronepterin (DHN); dihydrofolate (DHF); tetrahydrofolate (THF); polyglutamylated tetrahydrofolate (THF-Glun); dicarboxylate compounds (C4); dicarboxylate transporter (DctA); three-component ABC transporter (ABC);ATP/ADP nucleotide transporter (NttA); glucose permease (Per); phosphotransferase system (PTS); secretion system (SS); cation diffusion facilitator (CDF); high-afinity iron transporter (FTR1); Tol-import pathway (Tol); signal recognition particle secretion pathway (Sec-SRP); complex I (CI); complex II (CII); complex IV (CIV); complex V (CV); small-conductance mechanosensitive ion channel (MscS); large-conductance mechanosensitive ion channel (MscL); oxidative phosphorylation (OxPhos); and pentose phosphate pathway (PPP).

Figure 5. Analysis of the ‘Candidatus Liberibacter solanacearum’ arginine biosynthesis pathway.

The typical prokaryotic arginine biosynthesis pathway. The NAGK family of enzymes (COG0548) catalyze the second step in arginine biosynthesis and are known as ArgB in many bacteria , , , . In general, NAGKs come in two forms: hexameric arginine-sensitive enzymes and dimeric arginine-insensitive enzymes. The arginine-sensitive varieties of these enzymes typically function as a critical point of feedback inhibition for arginine biosynthesis , , .

Figure 6. Analysis of the ‘Candidatus Liberibacter solanacearum’ folate biosynthetic pathway.

A typical prokaryotic folate biosynthetic pathway. Enzymes in red are those encoded by ‘Ca. L. solanacearum’ that are not encoded by ‘Ca. L. asiaticus’.

Figure 7. The iron transport and assimilation (ITA) gene cluster.

Structure of the ITA gene cluster from several pathogenic microbes.