Genome mining reveals the genus Xanthomonas to be a promising reservoir for new bioactive non-ribosomally synthesized peptides

Abstract

Background: Various bacteria can use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in silico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by NRPS in this genus is albicidin produced by Xanthomonas albilineans. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas.

Results: We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. These two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters and share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or the secretion of peptides. In silico prediction of peptide structures according to NRPS architecture suggests eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas translucens and X. oryzae pv. oryzicola. Further genome mining revealed other novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari.

Conclusions: This study revealed the significant potential of the genus Xanthomonas to produce new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of non-ribosomally synthesized peptides in plant-bacteria interactions.

Figure 1

Physical map of the genomic region containing the XaPPTase gene in Xanthomonas oryzae strains and of the corresponding region in other sequenced species of Xanthomonas. White arrows: gene vipA (probable UDP-glucose/GDP-mannose dehydrogenase gene) and gene pgsA (probable CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase gene). Dark grey arrows: XaPPTase gene (encoding a 4′-phosphopantetheinyl transferase active on peptidyl carrier protein domains). Light grey arrows: NRPS (nonribosomal peptide synthesis) genes. Coloured arrows correspond to genes described in Figures 3 and 4. Hp: hypothetical protein gene (ortholog of accession Dd703_3065 of strain Ech703 of Dickeya dadantii). TA: transaminase gene (ortholog of accession Dd703_3064 of strain Ech703 of D. dadantii). DH: lactate dehydrogenase gene. DaT: gene involved in biosynthesis of 2,4-diaminobutyric acid (Dab). Length of arrows is not proportional to the length of genes. Salmon-coloured oval circle: Eight genes present in contig 112.1 of X. oryzae strain X11-5A that are conserved in other strains of X. oryzae and are not predicted to be involved in NRPS biosynthesis. : tRNA. Orientation of the tag indicates the orientation of the tRNA gene in the genomic regions. Amino acid specificity of each tRNA is indicated above or below each tag according to the orientation of the tRNA gene. ???? : undetermined sequence located between contigs. Two contigs separated by this tag may be located in two different genomic regions (they are not necessarily contiguous). : this tag indicates that corresponding genomic regions are not contiguous. Data on strain CFBP4834-R of X. axonopodis pv. phaseoli were obtained from an unpublished finished genome sequence (M.-A. Jacques, personal communication).

Figure 2

Representation of the NRPS loci META-A, META-B and META-C of X. albilineans strain GPE PC73. Brown arrow: staM gene predicted to be required for β−hydroxylation of an unidentified amino acid. Purple arrow: daT gene predicted to be required for biosynthesis of Dab (2,4-diamino butyric acid). Orange arrows: genes predicted to be required for biosynthesis of Dpg (3,5-dihydroxyphenylglycine). Dark blue arrows: other genes conserved in all META-B loci (ABC transporter, MbtH-like protein and transcriptional regulator AraC). Black arrows: NRPS genes. Length of arrows is not proportional to the length of genes. NRPS modules are represented by circles. Large circles indicate complete NRPS modules (containing domains C, A and PCP). The amino acid predicted to be assembled by the corresponding module is indicated within each circle. ?X: unknown amino acid specific to the NRPS signature UnknownX (Additional file 1). Brown circles: NRPS modules predicted to be specific for tyrosine. Purple circles: NRPS modules predicted to be specific to Dab. Orange circles: NRPS modules predicted to be specific to Dpg. The sequential order of amino acid incorporation from the N- to the C-terminus is illustrated by the number above each circle. Small black circles represent TE domains. The starter module circled in red exhibits a C-domain belonging to the same phylogenetic clade as starter C-domains identified by Rausch et al. [6] (Additional file 2). Starter modules circled in green indicate a C-domain belonging to the distant phylogenetic clade containing starter C-domains of loci META-A and META-C (Additional file 2). Elongation modules circled in blue indicate a C-domain belonging to the same phylogenetic clade as dual C/E domains identified by Rausch et al. [6] (Additional file 2). Amino acids predicted to be epimerized by these domains are in blue. IS: insertion sequence. Dotted box: pseudogene.

Figure 3

Representation of NRPS sequences of strains BAI3 and X11-5A of X. oryzae pv. oryzae. A. Representation of the NRPS locus META-B of X. oryzae pv. oryzae strain BAI3. B. Representation of contigs of X. oryzae pv. oryzae strain X11-5A that contain a nucleotide sequence encoding at least one A domain and/or a nucleotide sequence encoding non-NRPS genes similar to genes found in the locus META-B of strain X. albilineans GPE PC73. ???? : undetermined sequence located between contigs. Two contigs separated by this tag may be located in two different genomic regions (they are not necessarily contiguous). Brown arrows: staM and staM’ genes predicted to be required for β-hydroxylation of two unidentified amino acids, respectively. Purple arrow: daT gene predicted to be required for biosynthesis of Dab (2,4-diamino butyric acid). Orange arrows: genes predicted to be required for biosynthesis of Dpg (3,5-dihydroxyphenyl-glycine). Dark blue arrows: other genes conserved in all META-B loci (ABC transporter, MbtH-like protein and transcriptional regulator AraC). Green arrows and yellow arrow: genes conserved in strains X11-5A and XaS3 predicted to be required for biosynthesis of an unknown non-proteinogenic amino acid (Hp: hypothetical protein gene, TA: transaminase gene, DH: lactate dehydrogenase gene). Light blue arrow: syrP gene predicted to be required for β-hydroxylation of an unknown amino acid. Black arrows: NRPS genes. Length of arrows is not proportional to the length of genes. All information regarding characteristics and color-code for NRPS modules are as detailed in Figure 2. IS: insertion sequence. FS: frameshift mutation. Dotted box or dotted arrow: pseudogene. Dotted circle: non-functional module.

Figure 4

Representation of NRPS sequences of Xanthomonas spp. strain XaS3. A. Representation of contigs of Xanthomonas spp. strain XaS3 that contain a nucleotide sequence encoding at least one A domain and/or a nucleotide sequence encoding non-NRPS genes similar to genes found in the locus META-B of strain X. albilineans GPE PC73. B. Comparison of the locus META-A of X. albilineans strain GPE PC73 with the two contigs of Xanthomonas spp. strain XaS3 predicted to belong to a locus similar to this locus META-A. Brown arrows: staM and staM’ genes predicted to be required for β-hydroxylation of two unidentified amino acids, respectively. Purple arrow: daT gene predicted to be required for biosynthesis of Dab (2,4-diamino butyric acid). Orange arrows: genes predicted to be required for biosynthesis of Dpg (3,5-dihydroxyphenyl-glycine). Dark blue arrows: other genes conserved in all META-B loci (ABC transporter, MbtH-like protein and transcriptional regulator AraC). Green arrows and yellow arrow: genes conserved in strains X11-5A and XaS3 predicted to be required for biosynthesis of an unknown non-proteinogenic amino acid (Hp: hypothetical protein gene; TA: transaminase gene; DH: lactate dehydrogenase gene). Black arrows: NRPS genes. Length of arrows is not proportional to the length of genes. All information regarding characteristics and color-code for NRPS modules are as detailed in Figure 2. IS: insertion sequence. XALB1: albicidin biosynthesis gene cluster. Incomplete circles: incomplete nucleotide sequence located at the end or at the beginning of a contig encoding only one part of a module.

Figure 5

Representation of the NRPS locus present in the same region as XaPPTase in X. oryzae pv. oryzicola strain BLS256. Comparison with the similar genomic region of X. axonopodis pv. citri strain 306. NRPS modules are represented by circles. Large circles indicate complete NRPS modules (containing domains C, A and PCP). The amino acid predicted to be assembled by the corresponding module is indicated within each circle. Small black circles represent TE domains. Black arrows: NRPS genes. The starter module circled in red exhibits a C-domain belonging to the same phylogenetic clade as starter C-domains identified by Rausch et al. [6] (Additional file 2). Elongation modules circled in blue exhibit a C-domain belonging to the same phylogenetic clade as dual C/E domains identified by Rausch et al. [6] (Additional file 2). Amino acids predicted to be epimerized by these domains are in blue. : tRNA. Orientation of the tag indicates the orientation of the tRNA gene in the genomic regions. Amino acid specificity of each tRNA is indicated above or below each tag according to the orientation of the tRNA gene. IS: insertion sequence. Dotted arrows: pseudogenes. Dotted and incomplete circles: incomplete and non-functional modules.