Xylella fastidiosa comparative genomic database is an information resource to explore the annotation, genomic features, and biology of different strains

Abstract

The Xylella fastidiosa comparative genomic database is a scientific resource with the aim to provide a user-friendly interface for accessing high-quality manually curated genomic annotation and comparative sequence analysis, as well as for identifying and mapping prophage-like elements, a marked feature of Xylella genomes. Here we describe a database and tools for exploring the biology of this important plant pathogen. The hallmarks of this database are the high quality genomic annotation, the functional and comparative genomic analysis and the identification and mapping of prophage-like elements. It is available from web site http://www.xylella.lncc.br.

The quality of bacterial-genome annotation varies. The lack of a direct link between annotation in public databases, and the functional information accumulated over recent years, highlights how the importance of maintaining this up-to-date is becoming a crucial task in the genomics era (Parkhill et al., 2010). Although considerable literature has accumulated on Xylella fastidiosa over the last decade, this information has not been transferred to the annotation files in public databases. Xylella is a phytopathogenic bacterium that causes economically devastating losses in the yields of such crops as grapes, citrus fruits, almonds and other plant species (Van Sluys et al., 2002). The 9a5c strain, the causal agent of citrus variegated chlorosis, was the first bacterial plant pathogen to have its genome completely sequenced (Simpson et al., 2000). Nowadays, besides the six different genomes published, additional strains are part of ongoing sequencing projects.

Genomic studies have indicated extensive lateral gene transfer (LGT) related to prophage-like regions, which in turn are related to intra-genomic deletions, insertions and rearrangements (Monteiro-Vitorello et al., 2005; da Silva et al., 2007). Moreover, the presence of phage particles has also been demonstrated by both electron microscopy (Chen and Civerolo, 2008), and plaque propagation (Summer et al., 2010), all of which implying that phages are capable of playing a major role in genomic shaping and differentiation in Xylella strains (de Mello Varani et al., 2008).

Analysis of the genomic differences between closely related strains provides, not only a starting point towards understand functional and evolutionary processes, but also clues towards defining what makes one strain more pathogenic and/or aggressive than others. This information would be useful in epidemiological studies, all of which can potentially lead to the development of novel disease management strategies by identifying potential gene targets for mitigating infection and/or disease development.

We hereby report the first comprehensive and specialized up-to-date database comprising all the sequenced genomes of the different Xylella fastidiosa strains. The web-accessible application was developed, by using the SABIA package (System for Automated Bacterial Integrated Annotation), a public-domain software for the automated identification of genome landmarks that uses a user-friendly interface for browsing and retrieving data and information (Almeida et al., 2004).

Xylella fastidiosa strains were recently grouped into subspecies (Schaad et al., 2004, 2009), although the current database version follows the original strain identification. The database includes four complete and finished public genomic sequences of strains that cause citrus variegated chlorosis (9a5c), Pierce’s disease (Temecula1), almond leaf scorch and Pierce’s diseases (M23), and almond leaf scorch disease (M12). In addition, the public draft genomes of the strains associated with oleander leaf scorch (Ann1) and almond leaf scorch (Dixon) were assembled (closed but not finished) into candidate molecules representing the main replicon and plasmids. Additional information for finishing and gap-closures can be found in the supplementary material.

Prophage-like element identification was carried out using the methodology implemented by de Mello Varani et al. (2008). Orthologous clusters were identified using the bidirectional best-hit method (Overbeek et al., 1999). This database provides access to the latest annotations that can be downloaded in raw datasets, such as flat file and GenBank file format.

The high-quality annotation process was a collaborative effort among annotator specialists. The database can be searched by gene or protein names, as well as other functional annotation terms. The search engine is capable of further refining queries using SQL rules defined by the user. Nucleotide and amino acid sequences can be searched by BLAST (Altschul et al., 1997).

A genome viewer provides a graphical overview of the position of a given selected gene on the chromosome, as well as of neighboring genes. The annotation integrates information on putative gene products, transcription regulatory sequences and ribosome binding sites. InterPro protein signatures, UniProt (Universal Protein Resource) and the NCBI non-redundant protein database were used with the BLAST program for orthology and similarity assignment. Putative protein localization is assigned by PSORT (Nakai and Horton, 1999), and possible membrane transport capacity using the TCDB database. The Enzyme Commission number (EC Number), Gene Ontology terms and COG phylogenetic classification, were used for functional categorization of the putative gene products. KEGG metabolic pathways are also available through tables and in a graphical overview interface, thereby facilitating user visualization and comparison of the complete set of pathways available in each strain.

All identified prophage-like elements and prophage remnants were characterized and annotated as special features in each strain. They are indicated with a special tag after the gene name, i.e. [phage-related protein, xfp3], where ‘xfp3’ represents the prophage-like element number three of the 9a5c strain. For other strains, the notation is as follows: xpd 1 to 9 for Temecula1, xap 1 to 11 for Dixon, xop 1 to 10 for Ann1, xmp 1 to 9 for M23, and xp 1 to 7 for M12 strains (for details see “Genome sequence alignment and comparative map of prophage regions of the six strains” in Supplementary Materials of the Xylella fastidiosa comparative database, http://www.xylella.lncc.br/supplementary.html).

The comparative interface consists of a pre-calculated similarity analysis of Xylella predicted genes against thirteen completely sequenced Proteobacteria genomes, by using reciprocal BLAST searches for the computation of BBH clusters (Table 1). The core and pan genome calculation was estimated, and can be accessed as tables and graphs (Figure 1). Proteins involved in a common structural complex or metabolic pathway are highlighted and this information is associated with the identification of strain-specific regions that might be related to host specificity.

Table 1.

The database includes, other than the genomes of the 6 strains of Xylella, the genomes of species considered as references for comparative analysis.

	Number of genes with products of known function			Number of conserved genes with products of unknown function			Number of hypothetical genes			Total of genes
Organism	Genome	Cluster	%	Genome	Cluster	%	Genome	Cluster	%	Genome	Cluster	%
Caulobacter crescentus	2198	2059	93%	550	424	77%	989	203	20%	3737	2686	71%
Erwinia carotovora atroseptica SCRI1043	3630	3250	89%	602	441	73%	240	7	2%	4472	3698	82%
Escherichia coli K12	2927	2854	97%	11	9	81%	1341	1162	86%	4279	4025	94%
Escherichia coli O157H7	3461	3125	90%	0	0	0%	1900	1258	66%	5361	4383	81%
Pseudomonas aeruginosa	3022	2922	96%	760	734	96%	1785	1172	65%	5567	4828	86%
Pseudomonas syringae	3917	3676	93%	944	769	81%	610	27	4%	5471	4472	81%
R. solanacearum	3601	3081	85%	670	509	75%	845	199	23%	5116	3789	74%
S. maltophilia R551-3	3129	2943	94%	510	409	80%	393	69	17%	4032	3421	84%
S. maltophilia PCC6803	2737	1370	50%	1	1	100%	429	279	65%	3167	1650	52%
Xanthomonas campestris	2691	2467	91%	1	1	100%	1489	1194	80%	4181	3662	87%
Xanthomonas campestris vesicatoria	2689	2462	91%	5	2	40%	2032	1561	76%	4726	4025	85%
Xanthomonas citri	2705	2639	97%	1276	1230	96%	331	112	33%	4312	3981	92%
Xanthomonas oryzae	3281	2561	78%	24	19	79%	1332	1001	75%	4637	3581	77%
X.f. 9a5c (CVC)	1702	1658	97%	351	330	94%	439	289	65%	2492	2277	91%
X.f. Ann1 (OLS)	1686	1587	94%	339	292	86%	432	288	66%	2457	2167	88%
X.f. Dixon (ALS)	1793	1617	90%	294	283	96%	434	311	71%	2521	2211	87%
X.f. M12 (ALS)	1496	1474	98%	275	269	97%	218	178	81%	1989	1921	96%
X.f. M23 (ALS/PD)	1535	1529	99%	263	260	98%	209	170	81%	2007	1959	97%
X.f. Temecula1 (PD)	1576	1549	98%	292	284	97%	370	309	83%	2238	2142	95%

Figure 1.

Distribution of the core and pan genome of all the strains in the database can be accessed. (A) Functional categorization of genes that are shared by at least two genomes; (B) Total number of genes vs. total number of genes in clusters for each genome within the database.

The database attempts to provide a comprehensive view of all sequence elements and their related functions in Xylella genomes, providing a valuable online resource for Xylella community researchers. Expectedly, its use will contribute to understanding the biology of Xylella, and to the study of the mechanisms involved in its pathogenicity. New sequenced Xylella genomes can be included in future versions of the database, after the complete annotation and curation process.