Functional and Comparative Genomic Analysis of Integrated Prophage-Like Sequences in " Candidatus Liberibacter asiaticus"

Abstract

Huanglongbing disease (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogen "Candidatus Liberibacter asiaticus" is an alphaproteobacterium of the Rhizobiaceae family that has been identified as the causative agent of HLB. The virulence of "Ca. Liberibacter asiaticus" has been attributed, in part, to prophage-carried genes. Prophage and prophage-like elements have been identified in 12 of the 15 available "Ca. Liberibacter asiaticus" genomes and are classified into three prophage types. Here, we reexamined all 15 "Ca. Liberibacter asiaticus" genomes using a de novo prediction approach and expanded the number of prophage-like elements from 16 to 33. Further, we found that all of the "Ca. Liberibacter asiaticus" genomes contained at least one prophage-like sequence. Comparative analysis revealed a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 "Ca. Liberibacter asiaticus" strain that had previously been reported as lacking prophages. Our findings provide both a resource for data and new insights into the evolutionary relationship between phage and "Ca. Liberibacter asiaticus" pathogenicity.IMPORTANCE Huanglongbing (HLB) disease is threatening citrus production worldwide. The causative agent is "Candidatus Liberibacter asiaticus." Prior work using mapping-based approaches identified prophage-like sequences in some "Ca. Liberibacter asiaticus" genomes but not all. Here, we utilized a de novo approach that expands the number of prophage-like elements found in "Ca. Liberibacter asiaticus" from 16 to 33 and identified at least one prophage-like sequence in all "Ca. Liberibacter asiaticus" strains. Furthermore, we identified a prophage-like sequence type that is a remnant of an integrated prophage-expanding the number of prophage types in "Ca. Liberibacter asiaticus" from 3 to 4. Overall, the findings will help researchers investigate the role of prophage in the ecology, evolution, and pathogenicity of "Ca. Liberibacter asiaticus."

Keywords: One Health; bioinformatics; environmental microbiology; microbial ecology; phage ecology; phytopathology; plant pathogens.

FIG 1

Reconstruction of type 1 and 3 prophage-like sequences using AHCA1-a and FL17-a and nearby regions. (Top panel) Sequence FL17-a is formed by a type 4 sequence followed by bacterial genes and a region resembling a type 1 prophage. Joining sequences FL17-a, FL17-b, and FL17-c reconstructs a type 1 prophage sequence with the majority of its organization similar to that predicted for the integration of SC1 (7). (Bottom panel) AHCA-1 contains a type 4 sequence followed by bacterial genes and a region highly resembling a type 3 prophage. Using nearby regions in the AHCA1 genome, a type 3 prophage can be reconstructed with an organization similar to that predicted for the integration of P-JXGC-3 (13). Brown rectangles represent assembly gaps. Alignment visualizations were done using the R package genoPlotR (v0.8.9).

FIG 2

Gene composition in prophage-like sequences. A presence-absence heatmap of predicted pangenome proteins for the prophage-like sequences and phage available in public databases is shown. A clustering approach was used to obtain gene content relationships between sequences. Colors denote sequence classification as follows: red, type 1; blue, type 2; green, type 3; yellow, type 4. Sequences from the same strain that were classified as the same phage type, e.g., FL17 type 1 and TX2351 type 2, were used as a single phage for gene content analysis. Unclassified sequences were excluded from the analysis.

FIG 3

Annotation of representative sequence Ishi-1-a of type 4. The figure represents predicted CDS of Ishi-1-a, with ORFs color coded to predicted functions.

FIG 4

Presence of prophage-like sequence type 4 in related “Candidatus Liberibacter” species. The alignment shows a comparison of type 4 prophage-like sequence Ishi-1-a to genomes of “Ca. Liberibacter africanus” ( GCA_001021085.1) and “Ca. Liberibacter americanus” ( GCA_000496595.1).