Genome mining reveals the genus Xanthomonas to be a promising reservoir for new bioactive non-ribosomally synthesized peptides
- Monique Royer1,
- Ralf Koebnik2,
- Mélanie Marguerettaz1,
- Valérie Barbe3,
- Guillaume P Robin2,
- Chrystelle Brin4,
- Sébastien Carrere5,
- Camila Gomez1,
- Manuela Hügelland6,
- Ginka H Völler6,
- Julie Noëll1,
- Isabelle Pieretti1,
- Saskia Rausch6,
- Valérie Verdier2,
- Stéphane Poussier7,
- Philippe Rott1,
- Roderich D Süssmuth6 and
- Stéphane Cociancich1Email author
© Royer et al.; licensee BioMed Central Ltd. 2013
Received: 11 January 2012
Accepted: 22 September 2013
Published: 27 September 2013
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.
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performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv.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.
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.
Various bacteria, most prominently belonging to the orders Bacillales, Pseudomonadales or Actinomycetales, use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. These molecules exhibit broad structural diversity and display biological activities that range from adaptation to unfavorable environments, communication or competition with other microorganisms in their natural habitat, or even to action as virulence factors (for review [1, 2]). Non-ribosomal peptide synthetases (NRPSs) are megaenzymes, usually with a multimodular structure, that catalyze the non-ribosomal assembly of peptides from proteinogenic and non-proteinogenic amino acids [3, 4]. A basic module consists of an adenylation domain (A-domain) responsible for amino acid activation; a peptidyl carrier protein domain (PCP-domain)—usually adjacent to the A-domain—for thioesterification of the activated amino acid; a condensation domain (C-domain), which performs transpeptidation between the upstream and downstream peptidyl and amino acyl thioesters to elongate the growing peptide chain. In addition to this basic subset of core domains, each NRPS system also has a chain-terminating thioesterase domain (TE-domain) that is responsible for detachment of the mature polypeptide. Typically, NRPS initiation modules lack a C-domain . The exceptions are initiation modules of NRPS involved in biosynthesis of cyclic lipopeptides . C-domains present in these initiation modules catalyze N-acylation of the first amino acid with a fatty acid (with a β-hydroxy-carboxylic acid). Phylogenetic analyses of C-domains in NRPSs showed that C-domains of initiation modules, termed starter C-domains, segregate in a separate phylogenetic clade distant from the C-domains of elongation NRPS modules . In addition to the essential domains mentioned above (A, PCP, C and TE), optional auxiliary domains such as epimerization (E) or heterocyclization (Cy) domains can be found within some NRPS modules. Commonly, epimerization domains are located C-terminally of PCP-domains and perform epimerization of the last amino acid of the adjacent peptidyl-thioester. Interestingly, biochemical data showed that C-domains involved in assembly of arthrofactin not only catalyze transpeptidation between the upstream peptidyl thioester and the downstream amino acyl thioester, but also catalyze epimerization of the last amino acid from the upstream peptidyl thioester from l into d configuration . These C-domains have been termed dual C/E domains . Phylogenetic analyses of C-domains in NRPSs also showed that dual C/E domains segregate in a separate phylogenetic clade . Because of their wide structural complexity and diversity, natural products synthesized by NRPSs constitute a nearly inexhaustible source of new small molecules that might yield lead compounds in drug discovery .
The A-domains are the gatekeepers of biosynthesis of the polypeptide due to the specificity of substrate binding pockets of NRPSs for their cognate substrates. Compared to the 22 proteinogenic amino acids used in ribosomal protein synthesis, the utilization of hundreds of different non-proteinogenic amino acids has been described for NRPS. Each substrate binding pocket of NRPSs is specific for its amino acid substrate, and predictive models based on domain arrangement and on the sequence of modules have been deduced. The predictive power of these models has been refined from sequence analyses of NRPS A-domains with known specificity combined with examples of crystal structures of A-domains [9–11], thus identifying amino acid residues crucial for A-domain specificity. These models postulate specificity-conferring signatures for NRPS A-domains consisting of ten critical amino acid residues putatively involved in amino acid or aryl acid substrate specificity. The number of NRPS modules and their domain organization within the enzymes determine the structure of the final peptide product. Using in silico analyses of the NRPS genes, these conserved features within the NRPS machinery allow prediction of the type, and sometimes even the structure, of the synthesized polypeptide. It is therefore possible to investigate the biosynthetic potential of a given bacterium by analysis of the architecture of its NRPS gene clusters.
The transfer of a phosphopantetheinyl group to PCP-domains is required for posttranslational activation of NRPSs. Inactive apo-NRPSs are converted to their active holo-forms by transfer of the 4′-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a serine residue in the PCP-domains. The P-pant moiety serves to tether covalently the growing polypeptide being assembled by NRPSs. Transfer of the P-pant moiety from coenzyme A to a serine residue is catalyzed by 4′-phosphopantetheinyl transferase (PPTase). PPTases are involved not only in posttranslational activation of NRPS but also in posttranslational activation of fatty acid and polyketide synthases that do not contain PCP-domains but instead have acyl carrier protein (ACP) domains. All PPTases are recognized by a common signature sequence (V/I)G(I/V)D …x40-45… (F/W)(S/C/T)xKE(S/A)xxK, but overall sequence similarities are low [12–15]. Carrier protein specificity (ACP or PCP) has been determined experimentally for some PPTases. For example, in Escherichia coli, the EntD PPTase involved in biosynthesis of the siderophore enterobactin was shown experimentally to be active only on PCP-domains and not on ACP-domains [12, 14].
The Xanthomonadaceae are a family of Gram-negative bacteria belonging to the order Xanthomonadales in the gamma subdivision of the Proteobacteria. Members of this family are typically characterized as environmental organisms, and occupy diverse ecological niches such as soil and water, as well as plant tissues. Many Xanthomonadaceae, especially species from the genera Xanthomonas and Xylella, cause plant diseases, and only one, Stenotrophomonas maltophilia, is known to be an opportunistic human pathogen. The genus Xanthomonas consists of 27 plant-associated species, many of which cause important diseases of crops or ornamental plants. Individual species comprise multiple pathovars, characterized by distinctive host specificity or mode of infection. Collectively, members of this genus cause diseases on at least 120 monocotyledonous and 260 dicotyledonous crop plants.
Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes leaf scald—a lethal disease of sugarcane (interspecific hybrids of Saccharum spp.) . Leaf scald symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. The only pathogenicity factor of X. albilineans to be extensively studied to date is albicidin—a secreted non-ribosomally synthesized peptide with phytotoxic and antibiotic properties . Albicidin is a potent DNA gyrase inhibitor with a novel mode of action . Albicidin targets chloroplastic DNA gyrase A, inhibits chloroplast DNA replication and blocks chloroplast differentiation, resulting in the white foliar stripe symptoms . Albicidin also targets bacterial DNA gyrase A and, as a consequence, exhibits a potent antibiotic activity against a wide range of Gram-positive and Gram-negative bacteria . This antibiotic activity may help X. albilineans to combat rival microorganisms during sugarcane invasion. The complete albicidin biosynthesis gene cluster, called XALB1, was cloned and sequenced from X. albilineans strain Xa23R1 . XALB1 encodes three large NRPS genes and also resistance, regulatory and tailoring genes [21–24]. A PPTase gene, annotated as xabA or albXXI, was shown to be required for albicidin biosynthesis in X. albilineans strains LS155 and Xa23R1, respectively [21, 25]. The E. coli entD gene restored the biosynthesis of albicidin in a xabA knockout mutant of X. albilineans strain LS155 , demonstrating that xabA (or albXXI) has the same PPTase activity as entD, which is active only on PCP-domains. Preliminary analyses by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) did not allow the determination of the structure of albicidin [20, 26]. However, these studies showed that albicidin contains about 38 carbon atoms and has an estimated molecular mass of 842 Da. In silico analyses of XALB1 provided further insights into the structure of this pathotoxin, and suggested that NRPSs involved in biosynthesis of albicidin incorporate as yet unknown non-proteinogenic substrates . Thus, the accumulated data obtained for albicidin and its biosynthesis gene cluster suggest that it is a potent DNA gyrase inhibitor with a novel mode of action, and therefore might constitute a lead structure for novel antibiotics.
The genome of X. albilineans strain GPE PC73 was recently sequenced . This genome contains, in addition to three NRPS genes encoding albicidin biosynthesis , seven novel NRPS genes that share similarities with NRPS genes present in the genomes of two recently sequenced American Xanthomonas oryzae pv. oryzae strains . These novel NRPS genes cannot be assigned to any structure or function since there are no orthologous genes in other bacteria. Interestingly, X. oryzae possesses an ortholog of the PPTase gene xabA (or albXXI) which is hereafter referred to as XaPPTase because of its occurence in two species of Xanthomonas.
X. oryzae pathovars are the causal agents of two important diseases of rice: bacterial leaf blight caused by X. oryzae pv. oryzae, and bacterial leaf streak caused by X. oryzae pv. oryzicola. The XaPPTase gene is found in all six genome sequences of X. oryzae published to date (the completed genome sequences of the three Asian X. oryzae pv. oryzae strains MAFF 311018, KACC10331 and PXO99A [29–31]; the non annotated draft sequences of the genomes of the two American X. oryzae pv. oryzae strains X11-5A and X8-1A ; and the completed sequence of the genome of X. oryzae pv. oryzicola strain BLS256 ). The presence of the XaPPTase gene in all six genomes indicates that all strains of X. oryzae likely possess NRPS genes and produce non-ribosomally synthesized peptides.
The objective of this study was to estimate the biosynthetic potential of X. albilineans and X. oryzae by in silico analyses of their uncharacterized NRPS gene clusters and to identify other bacterial candidates for extended genome mining by PCR screening of a collection of 94 plant pathogenic bacteria for the presence of the XaPPTase gene and other NRPS-associated genes. To complete available published genomic data, we included in our analyses unpublished draft genome sequences of the African X. oryzae pv. oryzae strain BAI3 isolated in Burkina Faso  and the Xanthomonas spp. strain XaS3 isolated in Guadeloupe (French Caribbean island). To date, no genome sequence of an African X. oryzae pv. oryzae strain is available in public databases but a draft sequence of the genome of African strain AXO1947 of X. oryzae pv. oryzae was used recently to identify candidate type III secretion system effector genes . Xanthomonas spp. strain XaS3 was isolated from the surface of a sugarcane leaf and was characterized initially as a member of the species X. albilineans. However, recent MLSA (multi locus sequence analyses) analyses showed that this strain, which does not possess the albicidin biosynthesis gene cluster, is close to X. albilineans but belongs to a separate phylogenetic clade (I. Pieretti, unpublished data).
Results and discussion
Features of the XaPPTase genes in X. albilineans and X. oryzae
Similarity between XaPPTase and other PPTases involved in NRPS and fatty acid biosynthesis in bacteria (from)
Experimentally-determined specificities (A/P)b
Spacing between domains I and II (in aa)
Overall amino acid identities / similarities with XabA
Albicidin and unknown
X. albilineans str. LS155
FS AKES LFK AAY
X. albilineans str. Xa23R1
FS AKES LFK AAY
X. albilineans str. GPE PC73
FS AKES LFK AAY
94% / 95%
Xanthomonas spp. str. XaS3
FS AKES LFK AAY
83% / 87%
All sequenced X. oryzae strains*
FS AKES LFK ASF
40% / 51%
X. sacchari str. NCPPB4393
GIG LD VERV
FS AKES FYK AAA
41% / 55%
Bradyrhizobium spp. BTAi
ALG LD IEDV
FS AKEA YYK CQY
25% / 36%
E. coli str. K12 substr. MG1655
FS AKES AFK ASE
23% / 31%
M. tuberculosis str. CSU93
FC AKEA TYK AWF
25% / 34%
Bacillus brevis str. ATCC 9999
WT IKES YIK AIG
14% / 21%
Bacillus subtilis str. RB14
WS MKES FIK QEG
17% / 25%
E. coli str. K12 substr. MG1655
GLG TD IVEI
F AVKEA AAK AFG
9% / 14%
E. coli str. K12 substr. MG1655
EVG CD IEVI
WT RKEA IVK QRG
13% / 22%
M. tuberculosis str. CSU93
W AAKEA VIK AWS
11% / 17%
X. albilineans str. GPE PC73
WC AKEA LLK AHG
25% / 31%
Xanthomonas spp. str. XaS3
WC AKEA LLK AHG
22% / 28%
All sequenced X. oryzae strains
WC AKEA LLK AYG
20% / 25%
XaPPTase proteins of strains LS155 and Xa23R1 of X. albilineans are 100% identical, but they are only 94% and 83% identical to XaPPTase proteins of strains GPE PC73 and XaS3, respectively. However, XaPPTase signatures are 100% identical in strains LS155, Xa23R1 and GPE PC73, differing only in one amino acid residue in strain XaS3 (Table 1).
Genome mining reveals three novel NRPS loci in the genome of X. albilineans strain GPE PC73
In addition to the three NRPS genes characterized previously as being involved in the biosynthesis of albicidin , the chromosome of strain GPE PC73 possesses seven large multimodular NRPS genes clustered in three loci, hereafter referred to as META-A, META-B and META-C, respectively. These non-characterized NRPS genes share no similarity with NRPS genes from the biosynthesis gene cluster of albicidin.
The META-B biosynthesis gene cluster of strain GPE PC73 contains four NRPS genes: XALc_1058 (13,686 bp), XALc_1057 (6,378 bp), XALc_1056 (32,124 bp) and XALc_1055 (1,824 bp) (Figure 2; Additional file 1). These four genes encode a NRPS system consisting of a total of 16 modules with a continuous C-A-PCP domain arrangement. Two TE-domains were identified in META-B: (i) a single TE-domain at the C-terminus of the NRPS XALc_1058, and (ii) one double TE-domain on its own encoded by a separate gene (XALc_1055) (Figure 2). Like the domain arrangement of META-A and META-C, only essential elongation domains (A, PCP, C and TE) were identified in META-B. Phylogenetic analysis showed that the C-domain of the first module of XALc_1057 belongs to the same clade as starter C-domains identified by Rausch et al. , indicating that it also corresponds to a starter C-domain (Additional file 2). Therefore, the first module of XALc_1057 was assigned as the initiation module of META-B (Figure 2). The last module of XALc_1058, which harbors a TE domain, was assigned as the termination module (Figure 2). XALc_1055 is the only gene available in GenBank to encode a ‘stand-alone’ double TE domain, indicating that this gene is not associated to any NRPS system and should be considered as a pseudogene.
Genes of strain GPE PC73
Genes of strain DSM 5908 of A. balhimycina
Overall amino acid identities / similarities with genes of strain DSM 5908
Genes of strain NRRL15009 of S. toyocaensis
Overall amino acid identities / similarities with genes of strain NRRL15009
55% / 69%
54% / 67%
Type III chalcone synthase, generates 3,5-dihydroxyphenylacetyl-CoA from four malonyl-CoA
31% / 46%
29% / 45%
Belongs to crotonase/Enoyl-CoA hydratase superfamily, enhances DpgA activity
48% / 62%
48% / 62%
Metal- and cofactor-free 3,5-dihydroxyphenylacetyl-CoA 1,2-dioxygenase, converts 3,5-dihydroxyphenylacetyl-CoA into 3,5-dihydroxyphenylglyoxylate*
43% / 58%
42% / 58%
Transaminase, generates 3,5-dihydroxyphenyl-glycine from 3,5-dihydroxyphenylglyoxylate
62% / 74%
61% / 74%
Belongs to crotonase/Enoyl-CoA hydratase superfamily, enhances DpgA activity
The identity of several amino acids recognized and activated by the NRPSs of META-A, META-B or META-C is predicted on the basis of the analysis of specificity-conferring signatures of A-domains (Figure 2; Additional file 1). Interestingly, signatures specific to Dab were identified in both loci META-A and META-C, but remarkably not in the locus of META-B, which encodes the gene required for biosynthesis of Dab (XALc_1052). This finding suggests the occurrence of cross-talk between the three NRPS loci. Four unknown substrate specificity-conferring signatures were identified in the NRPSs of META-A, META-B or META-C (Unknown1 to Unknown4). Phylogenetic analysis of C-domains also showed that C-domains of two modules of META-A, four modules of META-B and one module of META-C segregate in the same clade as dual C/E domains identified by Rausch et al.  in numerous bacteria (Additional file 2). By analogy to previously identified dual C/E domains from arthrofactin NRPS, these latter dual C/E domains are predicted to follow modules that assemble amino acids with d-configuration (Figure 2; Additional file 1).
Genome mining reveals short NRPS genes in the genome of strains GPE PC73 and XaS3
Two short NRPS genes are also present on the chromosome of strain GPE PC73 in two additional loci. These two short NRPS genes, XALc_0364 (4,047 bp) and XALc_1145 (4,011 bp), each encode only one NRPS module, with a C-A-PCP-TE domain arrangement, and overlap at their 3′ end with a glycosyltransferase gene (overlapping sequence is 4 bp and 8 bp in length for XALc_0364 and XALc_1145, respectively). XALc_0364 and XALc_1145 share 66% amino acid similarity but do not share the same substrate-specificity conferring signature (Additional file 3). XALc_0364 harbors a signature specific to Gly, and XALc_1145 harbors an unknown signature that differs from unknown signatures identified in other NRPS loci of strain GPE PC73. Their respective overlapping glycosyltransferase genes (XALc_0365 and XALc_1144) share 62% amino acid similarity (Additional file 3). The C-domains of these two short NRPS genes segregate in the same phylogenetic clade as starter C-domains identified by Rausch et al. . Genes similar to the short NRPS genes of strain GPE PC73, i.e. encoding only one NRPS module with a C-A-PCP-TE domain arrangement and overlapping a glycosyltransferase gene at their 3′-end, were found in the genome of the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis strain SK2 (Additional file 3). Interestingly, in this strain, a XaPPTase-like gene (ABO_1782) is contiguous with the glycosyltransferase gene (ABO_1783) and the short NRPS gene (ABO_1784), confirming that a PPTase specific to PCP-domains is required for the unknown function of these unusually small NRPS systems. Similar short NRPS genes were found in the genome of two other species of Xanthomonas, which nevertheless do not possess the XaPPTase gene (X. campestris pv. campestris and X. axonopodis pv. citri; Additional file 3). This indicates that these genes were present in the common ancestor of Xanthomonas but may have conserved their function only in species that conserved the XaPPTase gene. Short NRPS genes were not found in any sequenced strains of X. oryzae, including strain BAI3. However, orthologs of these genes were found in Xanthomonas spp. strain XaS3. The only short NRPS gene present in strain XaS3 shares 94% and 67% amino acid similarity with the two short NRPS genes XALc_0364 and XALc_1145 of strain GPE PC73, respectively, and exhibits the same Gly-specificity conferring signature as in XALc_0364 (Additional file 3). The overlapping glycosyltransferase gene of the short NRPS gene of strain XaS3 shares 92% and 62% amino acid similarity with the two glycosyltransferase genes XALc_0365 and XALc_1144, respectively, of strain GPE PC73 (Additional file 3).
Genome mining uncovers a META-B-like NRPS gene cluster in strains X11-5A, BAI3 and XaS3
Similarities between NRPS-associated genes present in loci META-B of Xanthomonas spp. XaS3, X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains BAI3 and X11-5A
Proteins encoded in the locus META-B of Xanthomonas spp. strain XaS3
Overall amino acid identities / similarities with proteins encoded by the locus META-B of Xanthomonas spp. XaS3
X. albilineans strain GPE PC73
X. oryzae pv. oryzae strain BAI3
X. oryzae pv. oryzae strain X11-5A
98% / 99%
90% / 95%
90% / 94%
87% / 92%
Assembly of amino acids by NRPS
95% / 96%
94% / 96%
94% / 96%
Secretion of the synthesized peptides
91% / 93%
78% / 83%
77% / 81%
Biosynthesis of Dpg
94% / 96%
86% / 90%
86% / 90%
Biosynthesis of Dpg
90% / 93%
80% / 88%
83% / 91%
Biosynthesis of Dpg
92% / 95%
80% / 87%
81% / 88%
Biosynthesis of Dpg
78% / 86%
69% / 79%
69% / 79%
Biosynthesis of Dpg
92% / 94%
85% / 89%
86% / 90%
93% / 96%
57% / 72%
57% / 71%
Biosynthesis of Dab
95% / 97%
76% / 84%
76% / 84%
74% / 82%
79% / 89%
C-domains very similar to the starter C-domain of the locus META-B of strain GPE PC73 were found in strains XaS3, X11-5A and BAI-3, confirming that these three strains possess a NRPS gene cluster similar to META-B. Additionally, a C-domain very similar to the starter C-domain of META-A was found in another contig of strain XaS3 (Figure 4). While sharing common characteristics, NRPS genes present in loci META-A, META-B or META-C in strains GPE PC73, BAI3, X11-5A or XaS3 are predicted to encode different NRPS systems, each being strain-specific. NRPS genes of each strain are described and discussed separately below.
Genomic features of NRPS genes associated with the META-B gene cluster of strain BAI3
The META-B locus of African strain BAI3 of X. oryzae pv. oryzae, which is situated next to a transcription activator-like (TAL) effector locus, contains three NRPS genes: NRPSa (14,484 bp), NRPSb (22,410 bp) and NRPSc (19,479 bp) (Figure 3). These three genes encode a NRPS system consisting of 17 modules of the characteristic domain arrangement C-A-PCP. One double TE-domain is present at the terminal module of NRPSa. Phylogenetic analysis identified the N-terminally located C-domain of the first module of NRPSb as the starter C-domain (Additional file 2). This C-starter domain shares 74% amino acid similarity with the starter C-domain of the META-B locus of strain GPE PC73 (Additional file 4). Therefore, the first module of NRPSb was assigned as the initiation module and the last module of NRPSa, which harbors the TE domain, was assigned as the termination module (Figure 3). Phylogenetic analysis of C-domains identified four modules exhibiting a dual C/E domain that are predicted to follow modules that assemble an amino acid with d-configuration in strain BAI3 (Figure 3; Additional file 1).
The META-B biosynthesis gene cluster of strain BAI3 also contains a pseudogene consisting of three degenerated NRPS modules that contain a frame-shift mutation or an insertion sequence, respectively (Figure 3). Ten out of 17 amino acids assembled by the locus META-B of strain BAI3 were predicted on the basis of analysis of specificity-conferring signatures of A-domains (Additional file 1; see Figure 3). Interestingly, one of the degenerated modules exhibits a signature specific to Dab, suggesting that a putative ancestor of strain BAI3 possessed the gene daT required for biosynthesis of Dab. Moreover, five unknown specificity-conferring signatures of A-domains were found in strain BAI3. Remarkably, the META-B locus is the only NRPS locus identified in the whole genome of strain BAI3.
Genomic features of NRPS genes associated with the META-B gene cluster of strain X11-5A
The contig 23.1 of the American strain X11-5A of X. oryzae pv. oryzae contains several NRPS-associated genes also present in the META-B gene cluster of strain GPE PC73. Two contigs (23.1 and 114.1) of strain X11-5A each contain a nucleotide sequence encoding a chain-terminating TE-domain located just upstream from a nucleotide sequence encoding a C-domain that shares more than 75% amino acid similarity with starter C-domains of the META-B loci of strains GPE PC73 and BAI3, and which segregates in the same phylogenetic clade as these starter C-domains (Additional files 2, 4). Therefore, both contigs 23.1 and 114.1 should belong to the META-B locus, suggesting that, in strain X11-5A, this locus possesses two starter C-domains and two chain-terminating TE domains, and consequently encodes two independent NRPS systems that should be involved in the biosynthesis of two different compounds. The older ancestor of META-B, considered similar to locus META-B of strain X11-5A, likely also encoded two NRPS systems. In strain BAI3, a pseudogene consisting of three degenerated NRPS modules, as well as a syrP pseudogene, may be remnants of lost ancestral genes that were required for a second NRPS system likely encoded by the older ancestor of META-B. Similarly, a pseudogene encoding a stand alone double TE-domain in strain GPE PC73 (XALc_1055) may be a remnant of a lost ancestral NRPS gene that was required for a second NRPS system. A total of 29 different nucleotide sequences encoding A-domains were found in all contigs of strain X11-5A (Figure 3; Additional file 1). The absence of nucleotide sequences encoding a TE-domain other than those encoded by contigs 23.1 and 114.1 suggests that all these 29 sequences encoding A-domains belong to the META-B gene cluster of strain X11-5A. Indeed, the total number of nucleotide sequences encoding A-domains belonging to this gene cluster may be even higher because of repeated DNA regions that are not included in the draft genome sequence and that may encode additional A-domains. Amino acids assembled by 14 of these 29 A-domains were predicted on the basis of specificity-conferring signatures of A-domains (Additional file 1; see Figure 3). Seven unknown specificity-conferring signatures of A-domains were identified in strain X11-5A (Additional file 1). Phylogenetic analysis of C-domains identified six modules exhibiting dual C/E domains in this strain (see Figure 3; Additional file 1).
Genomic features of NRPS genes associated with gene clusters META-A and META-B of Xanthomonas spp. strain XaS3
The nucleotide sequence of contig G129 of Xanthomonas spp. strain XaS3 encodes a TE-domain located just upstream from a sequence encoding a starter C-domain. This C-domain shares more than 71% amino acid similarity with starter C-domains of the META-B loci of strains GPE PC73 and BAI3, which segregate in the phylogenetic clade of starter C-domains (Additional files 2 and 4). Therefore, contig G129 should belong to the META-B locus. Contigs G108 and G103 are also predicted to belong to the META-B gene cluster because they contain several NRPS-associated genes specific to this locus (Figure 4). Contig G103 contains the same gene as strain GPE PC73 encoding the stand alone double TE-domain predicted to be a pseudogene (ortholog of XALc_1055). Based on these resemblances with the META-B locus of strain GPE PC73, strain XaS3 is predicted to possess a META-B gene cluster (Figure 4) encoding one NRPS system involved in the biosynthesis of one peptide. Contig G111 of strain XaS3 contains a starter C-domain located at the same position as the starter C-domain of locus META-A of strain GPE PC73, and downstream genes orthologous to XALc_1553 and XALc_1554 (Figure 4). This starter C-domain shares 82% amino acid similarity with the starter C-domain of the locus META-A of strain GPE PC73 (Additional file 4). Contig G106 of strain XaS3 contains an incomplete NRPS gene encoding six NRPS modules, with the last module containing a chain-terminating TE domain. The 5′-part of this incomplete NRPS gene is located in a region that does not encode any NRPS in strain GPE PC73, upstream from the orthologous gene of XALc_1349 (Figure 4). However, based on phylogenetic analyses of C-domains, strain XaS3 possesses only two starter C-domains, indicating that contig G106 belongs to the same META-A gene cluster as the starter C-domain of contig G111 (Figure 4). The 5′-part of META-A in strain GPE PC73 is located upstream of both the terminus of replication and the albicidin biosynthesis gene cluster XALB1, which is not present in the genome of strain XaS3. Acquisition of XALB1 by the ancestor of X. albilineans or, alternatively, loss of XALB1 by the ancestor of strain XaS3 may explain why the upstream segments of META-A from strains GPE PC73 and XaS3 are located in two different regions. A total of 33 different nucleotide sequences encoding A-domains were found in all contigs of strain XaS3 (Figure 4; Additional file 1): four of these are located in contig G129, which contains nucleotide sequences encoding the chain-terminating TE-domain and the starter C-domain of META-B; and six are located in contig G106 containing the incomplete NRPS gene of META-A. The remaining 23 nucleotide sequences encoding A-domains, which are not located in contigs encoding TE-domains, may belong either to META-A or META-B. The total number of nucleotide sequences encoding A-domains in strain XaS3 may be higher than 33 because of repeated DNA regions that are not included in the draft genome sequence and that may encode additional modules. Amino acids assembled from 21 of these 33 A-domains were predicted on the basis of specificity-conferring signatures of A-domains (Figure 4; Additional file 1). Nine unknown specificity-conferring signatures of A-domains were found in strain XaS3 (Additional file 1). Phylogenetic analysis identified nine modules exhibiting a dual C/E domain (Figure 4; Additional file 1).
Genome mining reveals a NRPS gene cluster specific to X. oryzae pv. oryzicola strain BLS256
Genome mining of Xanthomonas sacchari reveals genes required for biosynthesis of new non-ribosomal peptide(s)
A non-annotated draft sequence of the genome of X. sacchari strain NCPPB4393, isolated from an insect collected on a diseased banana plant, was published recently . The species X. sacchari, which is phylogenetically related to X. albilineans, was also isolated on sugarcane and milled rice [46, 47]. However, no disease caused by this species on any plant has been described to date. Interestingly, the genome of X. sacchari strain NCPPB4393 contains the XaPPTase gene (Table 1), which is not located at the same position as on the chromosomes of X. albilineans or X. oryzae. This genome also contains the genes daT (42% identical to XALc_1054), staM (38% identical to XALc_1066) and syrP (36% identical to XALc_1524). These three latter genes are clustered in a single locus. In addition, the genome of X. sacchari strain NCPPB4393 contains several contigs encoding NRPS-related sequences, among them adenylation domains specific to Dab, valine, glutamic acid, phenylalanine, asparagine, proline and an unknown substrate (unknown signature DAWLLGCTFK). However, the NCPPB4393 genome does not contain any sequences that are closely related to META-A, META-B, META-C, XALB1 or the short NRPS genes described above, indicating that non-ribosomal peptide(s) potentially produced by X. sacchari is/are specific to this species.
Genome mining by in silico analysis demonstrates a great biosynthetic potential for non-ribosomally synthesized peptides in the genus Xanthomonas
While sharing numerous common characteristics, the META-B gene clusters of all four investigated Xanthomonas spp. strains do not encode identical small molecules. This was concluded from the A-domain specificities of the NRPSs, and the hypothetical peptides are therefore considered strain-specific. Starter C-domains of all four loci META-B share at least 71% amino acid similarity (Additional file 4) suggesting that, although there is currently no experimental evidence, they might be expected to effect N- acylation with a structurally related β-hydroxy-carboxylic acid. Furthermore, the four META-B loci exhibit several C/E dual domains, indicating that they catalyze the assembly of residues with d-configuration. BLAST analyses identified only 188 GenBank entries that exhibit similar dual C/E domains. These 188 entries include nine genes analyzed in the present study (five NRPS genes of strain GPE PC73, two NRPS genes of strain BLS256, and two pseudogenes of strain 306 of X. axonopodis pv. citri). Draft genome sequence data of strains XaS3 and X11-5A did not indicate the number or distribution of residues in peptides assembled by their respective META-B gene cluster. However, data clearly suggest that these peptides are specific to each strain. In strain X11-5A, the META-B biosynthesis gene cluster is even predicted to control the biosynthesis of two different lipopeptides. A total of five different lipopeptides that do not resemble any compound described to date are predicted for the four Xanthomonas spp. strains due to the presence of the META-B gene cluster. These five lipopeptides could be secreted by the ABC transporter encoded by orthologs of XALc_1064, which are present in all META-B gene clusters.
The only common characteristic shared between the loci META-A and META-C of strain GPE PC73 and the locus META-A of strain XaS3, is their unusual starter C-domain, which segregates in a distant phylogenetic clade and shares less that 42% amino acid similarity with starter C-domains of META-B loci or with starter C-domains identified in numerous bacteria by Rausch et al.  (Additional file 4). These unusual starter C-domains might catalyze linkage of the first residue with a non-amino acid substrate. A total of three different compounds, not resembling any known compound, are predicted to be produced by Xanthomonas spp. strains GPE PC73 and XaS3 by the gene clusters META-A or META-C. Short NRPS genes of strain GPE PC73 might be involved in the biosynthesis of two different small molecules consisting of an amino acid (Gly for XALc_0364 and an unknown amino acid for XALc_1145) linked to a β-hydroxy-carboxylic acid and a glycosyl residue. The only short NRPS gene of strain XaS3 might be involved in biosynthesis of a small molecule identical to that synthesized by XALc_0364 because of the high similarity of these two genes. According to in silico analyses, strain BLS256 could synthesize a cyclolipopeptide with the amino acid sequence d-Asn-d-Val-d-Leu-d-Thr-d-Leu-l- Val, which does not resemble any compound described to date (Figure 5). X. sacchari strain NCPPB4393 may also produce new non ribosomal peptide(s). Transporters involved in secretion of these unknown compounds remain to be identified as they are not encoded by the NRPS loci.
A total of 14 unknown specificity-conferring signatures of A-domains were found in the four strains GPE PC73, XaS3, X11-5A and BAI3. Only signatures “Unknown2” and “Unknown4” were found in a recently published database that includes 5,118 adenylation domains with unknown signatures . The other 12 unknown NRPS signatures are not present in this database and could be specific to the genus Xanthomonas. The signature “Unknown1” is predicted to be specific for Dpg because of its presence in all four strains and because of the absence in these strains of any signature similar to those specific to the non-proteinogenic amino acid identified by . Strains XaS3 and X11-5A possess a higher number of unknown signatures, suggesting that the three genes specific to both strains (transaminase, lactate dehydrogenase and a hypothetical protein) are involved in the biosynthesis of additional non-proteinogenic amino acids. However, these two strains share only one specific unknown signature (“Unknown9”), whereas some unknown signatures are shared by three strains (“Unknown2, -4, -6 and -7”). Eight other unknown signatures are present in only one strain (“Unknown3, -5, -8, -10, -11, -12, -13, and -14”). “Unknown5” was found only in the short gene XALc_1145 of strain GPE PC73. The presence of a high number of unknown signatures specific to the genus Xanthomonas suggests that numerous unknown non-proteinogenic amino acids are assembled by the novel NRPS genes identified in the genome of the four Xanthomonas spp. The biosynthetic capacities for those amino acids have yet to be identified. In conclusion, our results yield the expectation that numerous non-proteinogenic amino acids as well as residues with d-configuration should confer unusual structures to compounds synthesized by Xanthomonas spp. strains; identification of these compounds represents an exciting area for future study in these organisms.
PCR screening of 94 plant pathogenic bacteria for the XaPPTase gene and genes associated to NRPS in META-B
A collection of 94 strains belonging to the main genera of plant-pathogenic bacteria was screened by PCR. Primers designed to amplify DNA fragments of the XaPPTase gene, the ABC transporter gene present in META-B, the dpgB gene, the dpgC gene, the hpgT gene and the daT gene, were designed (Additional file 4). The XaPPTase gene was found in all analyzed strains of X. albilineans and X. oryzae. It was also found in strain CFBP2539 of Xanthomonas translucens pv. secalis, strain CFBP4642 of Xanthomonas cassavae, strain CFBP2431 of Pseudomonas corrugata, strain CFBP5593 of Pseudomonas brassicacearum and strain CFBP1192 of Xylophilus ampelinus (Additional file 5). The XaPPTase gene of X. cassavae is very similar to that of X. oryzae, while the XaPPTase gene of P. corrugata is very similar to that of P. brassicacearum (Additional file 5).
The ABC transporter gene was found in all four analyzed strains of X. albilineans, in all five analyzed African strains of X. oryzae pv. oryzae, in strains UPB497 and CFBP2286 of X. oryzae pv. oryzicola and in strain CFBP2539 of X. translucens pv. secalis (Additional file 5). Three additional genes of META-B (the dpgB gene, the dpgC gene and the hpgT gene) were found in all strains possessing the META-B ABC transporter, except for strain CFBP2286 of X. oryzae pv. oryzicola, which does not possess any of these genes (Additional file 5). The daT gene was found in all analyzed strains of X. albilineans, in strain UPB497 of X. oryzae pv. oryzicola and in strain CFBP2539 of X. translucens pv. secalis (Additional file 5). In summary, the META-B gene cluster seems to be present in all analyzed strains of X. albilineans, in all analyzed African strains of X. oryzae pv. oryzae, in strain UPB497 of X. oryzae pv. oryzicola and in strain CFBP2539 of X. translucens pv. secalis.
In silico predicted data on non-ribosomally synthesized peptides potentially produced by Xanthomonas strains
Non-ribosomally synthesized peptides
Lipopeptides containing non-proteinogenic amino acids Dpg, Dab and/or at least one β-hydroxy-amino acid*
X. albilineans strain GPE PC73
- Lipopeptide synthesized by META-B with the amino acid sequence Gly-Unk2-Thr-D-Asn-Tyr-Asn-D-Tyr-Ser-D-Asn-Asp-Asn-Dpg-Dpg-Ser-D-Thr-Unk2
- Glycine linked to a β-hydroxy-carboxylic acid and a glycosyl residue
- Unknown amino acid linked to a β-hydroxycarboxylic acid and a glycosyl residue
- Lipopeptide synthesized by META-A with the amino acid sequence Dab-Ser-Ser-Thr-D-Asn-Val-D-Phe-Dab-Dpg-Unk3-Unk2-Tyr
- Lipopeptide synthesized by META-C with the amino acid sequence Unk4-Phe-Dab-Asn-Ala-D-Unk3-Gln
Xanthomonas spp. strain XaS3
- Two lipopeptides synthesized by META-B and META-A, respectively.**
- Glycine linked to a β-hydroxy-carboxylic acid and a glycosyl residue
X. oryzae pv. oryzae strain BAI3
-Lipopeptide synthesized by META-B with the amino acid sequence Gly-Unk7-Unk2-Thr-Asn-D-Thr-Tyr-D-Ile-Ser-Asn-D-Asn-Ser-Dpg-Dpg-Unk6-D-Dpg-Unk4
X. oryzae pv. oryzae strain X11-5A
- Two lipopeptides synthesized by META-B.**
X. oryzae pv. oryzicola strain BLS256
- A cyclolipopeptide with the amino acid sequence D-Asn-D-Val-D-Leu-D-Thr-D-Leu-Val
X. sacchari strain NCPPB4393
No precise data on the number and nature of the non-ribosomally synthesized peptides produced by this strain because of the lack of information (only draft genome sequence including several unassembled contigs that contain NRPS sequences)
X. albilineans strain GPE PC73 isolated in Guadeloupe was sequenced recently . X. albilineans strains were grown for 48h on modified Wilbrink’s medium  or on XAS selective growth medium  at 28°C. X. oryzae pv. oryzae strain BAI3 (isolated in Burkina Faso ) was grown for 24h on PSA medium  supplemented with appropriate antibiotics at 28°C.
Design of PCR primers
Primers used to screen a collection of 94 plant pathogenic bacteria for the presence of the XaPPTase gene as well as genes associated to NRPS in META-B by PCR were designed based on genome sequence information of strains X. albilineans GPE PC73, X. oryzae pv. oryzae BAI3, and X. translucens pv. undulosa UPB513 (Claude Bragard, unpublished data). Primer sequences are listed in Additional file 4. Primers used to determine the complete sequence of the 82-kb length region containing META-B in strain BAI3 were designed based on the draft genome sequence of X. oryzae pv. oryzae strain BAI3 (Genbank accession n° JQ348075).
PCR screening for the presence of the XaPPTase gene and genes associated to NRPS in META-B
DNA templates were prepared by suspending a freshly grown colony in 100 μl sterile nuclease-free water. PCR amplifications were performed in an automated thermal cycler (GeneAmp PCR System 9700; Life Technologies, Carlsbad, CA, USA). The 20-μl PCR reaction mix consisted of 5 μl bacterial suspension, 4 μl of 5x GoTaq buffer (Promega, Madison, WI, USA), 250 μM dNTP mix, 0.2 μM of each primer, 1 unit of GoTaq Polymerase (Promega, Madison, WI, USA), and sterile nuclease-free water to final volume. The PCR program was 94°C for 4 min, 35 cycles at 94°C for 30 seconds, Tm (melting temperature) for 30 seconds, and 72°C for 30 seconds, with a final 72°C extension for 8 min. A 8-μl aliquot of each amplified product was analyzed by electrophoresis through a 1.5% agarose gel. PCR products were sequenced with primers used for their respective amplification. Screened bacteria are listed in Additional file 6.
The draft genome sequence of strain BAI3 of X. oryzae pv. oryzae was determined by a Sanger/pyrosequencing hybrid approach. A shotgun library was constructed with 10-kb sized fragments obtained after mechanical shearing of the total genomic DNA, and cloned into the vector pCNS (pSU-derived). Sequencing with vector-based primers was carried out on an ABI 3730 Applera Sequencer. A total of 5,921 reads (~1 fold coverage) were analyzed and assembled with 518,656 (~23 fold coverage) 454 GS FLX reads (Roche Applied Science; http://www.roche.com). Sequence assembly was performed using Arachne “HybridAssemble” version (Broad Institute; http://www.broad.mit.edu/), which combines the 454 contigs with Sanger reads. As the Sanger reads contribution was not quite sufficient for the scaffolding, a mate-paired 454 library with 8-kb insert size was constructed and 179,755 (~4 fold coverage) 454 GS FLX reads were added into the assembly. To further improve quality, Illumina technology was applied (36-bp reads at ~50 fold coverage), eventually resulting in an assembly of 67 scaffolds with a mean scaffold length of 80 kb.
Draft genome sequence of Xanthomonas spp. strain XaS3 (also called strain GPE39), was obtained using short read technology only. After mechanical shearing of genomic DNA, fragments with an insert size of 300–500 bp were used for Illumina library construction (http://www.illumina.com). A total of 64,891,306 GAIIx reads were produced, corresponding to a paired-end sequencing with 2 × 76 nt reading lengths. Assembly was performed using a combination of the SOAPdenovo (1.05) and Velvet (1.1.04) short read assemblers [52, 53]. In a first step, SOAPdenovo was run with kmer values ranging from 25 to 73 (step of 4) to generate contigs. In a second step, Velvet was run with parameters “-cov_cutoff 5 -min_contig_lgth 100 -max_divergence 0.05 -exp_cov auto -min_pair_count 10” to generate scaffolds, by using raw reads and SOAPdenovo contigs as input data. The range of kmer values used for Velvet was the same as for SOAPdenovo. The assembly with the maximum N50 value was selected, and remaining gaps were filled with SOAPGapCloser software. The XaS3 draft assembly spans 3,529,206 bp, and consists of 73 contigs of length ranging from 526 to 512,021 bp with a N50 of 168,272 bp. Annotated nucleotide sequences of the regions identified in these 73 contigs as containing A-domains and/or NRPS associated genes are provided in Additional file 7.
Determination of the complete sequence of the 82-kb length region containing META-B in X. oryzae pv. oryzae strain BAI3
On the basis of the draft genome sequence of strain BAI3, three scaffold DNA sequences were identified as belonging to META-B. The sizes of these DNA regions are 35,406 bp, 16,409 bp and 22,029 bp, respectively. Seventeen clones from a 10-kb shotgun library of strain BAI3 were identified by BLAST analyses to contain fragments of the META-B NRPS cluster. These clones are ABP0AAB3YI02, ABP0AAB3YP05, ABP0AAB8YK02, ABP0AAB8YE14, ABP0AAB5YJ09, ABP0AAB4YK08, ABP0AAB3YL17, ABP0AAB6YE03, ABP0AAB6YC14, ABP0AAB5YG12, ABP0AAB7YB12, ABP0AAB5YK06, ABP0AAB5YJ04, ABP0AAB7YD04, ABP0AAB3YG10, ABP0AAB5YE07 and ABP0AAB3YM11. Sequences of the insert ends were mapped onto the three scaffold DNA sequences. Four clones (ABP0AAB4YK08, ABP0AAB3YL17, ABP0AAB7YB12 and ABP0AAB5YK06) were identified as bridging two of the three scaffold DNA sequences. All other clones were mapped to within one of the three scaffold DNA sequences. Clones ABP0AAB4YK08, ABP0AAB3YL17, ABP0AAB7YB12 and ABP0AAB5YK06 were digested with restriction enzyme Xho I and partial digestion with restriction enzyme Sal I followed by re-ligation. The restriction site Xho I is present only in the polylinker of pCNS vector and several Sal I restriction sites are present in the inserts. Clone ABP0AAB7YB12 was also digested with restriction enzyme Bam HI followed by re-ligation. The borders of the resulting clones were sequenced using universal primers (M13R for Bam HI borders and M13F for Xho I borders). One clone resulting from an internal Sal I-mediated deletion of clone ABP0AAB5YK06, and harboring an insert of 3.6 kb, was sequenced with primers MRK16, MRK17, MRK18, MRK19 and MRK20 (Additional file 8). A clone resulting from an internal Sal I-mediated deletion of clone ABP0AAB7YB12 and harboring an insert of 1.9 kb was sequenced with primers MRK19 and MRK20 (Additional file 8). Clone ABP0AAB5YK06 was sequenced with primers MRK7, MRK8, MRK18, MRK22R and MRK23R. The new sequence information allowed the three scaffold DNA sequences to be merged into a contiguous scaffold sequence of 82 kb. Contigs of strain BAI3 were mapped to this sequence and three sets of primers were designed in order to sequence (i) gaps between contigs, (ii) regions between NRPS and a tal gene, and (iii) regions with frameshifts, respectively (Additional file 8). These primers were used to sequence a clone resulting from an internal Sal I-mediated deletion of clone ABP0AAB4YK08 and harboring an insert of 5.0 kb and to sequence clones ABP0AAB3YG10, ABP0AAB3YM11, ABP0AAB5YE07, ABP0AAB5YJ04, ABP0AAB6YC14, ABP0AAB6YE03, ABP0AAB7YB12 and ABP0AAB7YD04 (Additional file 8). DNA sequencing was performed by Beckman Coulter Genomics (Takely, UK). Sequence reads were used to assemble the complete 81,740-bp META-B region of strain BAI3 (Genbank accession n° JQ348075).
In silico analyses of NRPS
Specificity of adenylation domains in NRPS and signatures were predicted using software available at http://nrps.informatik.uni-tuebingen.de/Controller?cmd=SubmitJob. In silico analyses were performed on NRPS genes present in the finished annotated genome sequence of X. albilineans strain GPE PC73 (GenBank accession n°: NC_013722.1), in the non-annotated draft genome sequence of X. oryzae pv. oryzae strain X11-5A (GenBank accession n°: AFHK00000000.1; sequences analyzed in the current study are annotated in Additional file 9), in the unpublished annotated sequence of the 82-kb length region containing META-B in X. oryzae pv. oryzae strain BAI-3 (Genbank accession n° JQ348075), in the unpublished sequence of the contigs of strain Xanthomonas spp. XaS3 (sequences analyzed in the current study were annotated in Additional file 7), in the finished annotated genome sequence of X. oryzae pv. oryzicola strain BLS256 (nucleotide GenBank accession n°: CP003057.1), in the finished annotated genome sequence of X. axonopodis pv. citri strain 306 (nucleotide GenBank accession n°: AE008923.1), and in the non-annotated draft genome sequence of X. sacchari strain NCPPB4393 (nucleotide GenBank accession n°: AGDB00000000.1).
The phylogenetic tree presented in Additional file 2 was reconstructed using the maximum likelihood method implemented in the PhyML program. The LG substitution model was selected, assuming an estimated proportion of invariant sites (of 0.01) and four gamma-distributed rate categories to account for rate heterogeneity across sites. The locus analyzed was the C-domain. Multiple alignments of the amino acid sequences of the C-domain and for all taxa were performed using ClustalW. The phylogenetic tree was calculated with PhyML; http://atgc.lirmm.fr/phyml/, version 2.4.4. Five hundred bootstrap replicates were performed with PhyML program. Data are available from the Dryad Digital Repository: http://doi.org/10.5061/dryad.gh7h8.
Acyl carrier protein domain
2,4-diamino butyric acid
- C/E domain:
Nuclear magnetic resonance
Non-ribosomal peptide synthesis
Non-ribosomal peptide synthetases
Multi locus sequence analyses
Peptidyl carrier protein domain
We thank Claude Bragard for access to sequence data of X. translucens. We thank Helen Rothnie for English editing. CG was supported by a post-doctoral fellowship awarded by the Agence Nationale de la Recherche, France (ANR-09-BLAN-0413-01). MM was supported by a PhD Fellowship awarded by the Région Languedoc-Roussillon and by the Centre de Coopération Internationale en Recherche Agronomique pour le Développement, France. GPR was supported by a PhD fellowship awarded by the Région Languedoc-Roussillon and by the Centre National de la Recherche Scientifique, France (grant 07-845). PR was supported by a Marie Curie Outgoing International Fellowship from the European Commission. The project was supported by a joint research grant of the Agence Nationale de la Recherche to MR (ANR-09-BLAN-0413-01) and the Deutsche Forschungsgemeinschaft to RDS (DFG SU239/11-1).
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