Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

Abstract

Background: Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences.

Results: Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations.

Conclusions: This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

Figure 1

Circular representation of the chromosome and plasmids of strain 4834-R of X. fuscans subsp. fuscans. From outside to inside, circle 1 indicates the localization of the various secretion systems (T1SS to T6SS), type I pilus (T1p), type IV pilus (T4p), elements devoted to cell protection (exopolysaccharides, lipopolysaccharides), chemotaxis and motility. Circle 2 indicates the localization of type III effectectors (T3Es), and circle 3 indicates the localization of instertion sequences (ISs). The black circle shows the G + C content using a 100-base window. The green and purple circle shows the GC skew (G-C)/(G + C) using a 100-base window.

Figure 2

Pan genome of 13 Xanthomonas sp. strains. The 13 genome sequences (number of CDSs) included in the orthoMCL analysis are: X. fuscans subsp. fuscans strain 4834-R (Xff 4834-R; 4,086 CDSs), X. fuscans subsp. aurantifolii strain ICPB10535 (Xfa ICPB10535; 3,918 CDSs), X. citri subsp. citri strain 306 (Xac 306; 4,427 CDSs), X. euvesicatoria strain 85–10 (Xcv 85–10; 4,725 CDSs), X. axonopodis subsp. citrumelonis strain F1 (Xacm F1; 4,181 CDSs), X. albilineans strain GPE PC73 (Xal GPE PC73; 3,208 CDSs), X. campestris pv. campestris strain ATCC33913 (Xcc ATCC33913; 4,179 CDSs), X. campestris pv. musacearum strain NCPPB4381 (Xcm NCPPB4381; 4,209 CDSs), X. campestris pv. raphani strain 756C (Xcr 756C; 4,516 CDSs), X. gardneri strain ATCC19865 (Xg ATCC19865; 5,027 CDSs), X. oryzae pv. oryzae strain PXO99A (Xoo PXO99A; 4,988 CDSs), X. oryzae pv. oryzicola strain BLS256 (Xoc BLS256; 4,474 CDSs) and X. vesicatoria strain ATCC35937 (Xv ATCC35937; 4676 CDSs). Values represent the number of groups of orthologs, i.e. CDSs present in single copy in each genome, while values in brackets indicate the cumulated number of paralogs in the various genomes. The central disc corresponds to ubiquitous orthologs (present in the 13 genomes). The middle circle represents the variable part of the pan genome (orthologs present in 2 to 12 genomes), and the external circle represents the unique CDS of each genome.

Figure 3

Venn diagrams illustrating the comparisons of xanthomonads genomes. Venn diagrams display the number of CDSs, which are present in single copy in each genome (in bold). In addition, values in brackets indicate the cumulated number of paralogs. (A) Comparison of the genomes of Xanthomonas fuscans subsp. fuscans strain 4834-R (Xff) and two distantly related strains: Stenotrophomonas maltophilia strain R551-3 (Sm), a non-pathogenic endophyte of poplar, and Xyllella fastidiosa strain Temecula 1 (Xf), an insect-vectored pathogen of grapevine. (B) Comparison of genomes of Xff 4834-R and two strains belonging to X. oryzae: X. oryzae pv. oryzae strain PXO99A (Xoo), a vacular pathogen of rice, and X. oryzae pv. orizycola strain BLS256 (Xoc), a non-vascular pathogen of rice. (C) Comparison of genomes of Xff 4834-R with two phylogenetically close strains: X. axonopodis pv. citri strain 306 (Xac), a non-vascular pathogen of citrus, and X. fuscans subsp. aurantifolii strain ICPB10535 (Xfa), a non-vascular pathogen of citrus.

Figure 4

Comparison of T3SS clusters of eight sequenced strains of Xanthomonas. The organization of the hrp cluster encoding the T3SS and some T3-secreted proteins is compared using the R package GenoplotR for strains Xff 4834-R, Xac 306, Xcv 85–10, Xacm F1, Xcc ATCC33913, Xcr 756C, Xoo PXO99A, Xoc BLS256. Strains Xfa ICPB10535, Xcm NCPPB4381, Xg ATCC19865 and Xv ATCC35937 were not included as their hrp/hrc region is splitted on various contigs. Boxes of the same color indicate orthologous genes. Colinearity is represented by colored connectors. The hrp cluster is inserted in the vicinity of a tRNA-Arg gene, except for Xcc ATCC33913, X. campestris pv. raphani strain 756C (Xcr 756C), and X. oryzae pv. oryzae strain PXO99A (Xoo PXO99A). In strain Xoc BLS256, multiple insertions occurred between the ortholog of hpaF (aka xopAF) and the tRNA-Arg gene. These insertions in Xoc BLS256 carry virulence associated genes such as the T3E xopAD, TBDT, carbohydrate and salicylate esters degradation genes (sal operon).

Figure 5

Alignment of Xff 4834-R and Xac 306 genomes using Mauve. Colored boxes and arrows indicate synthenic fractions in the genomes. Triangles correspond to ISs suspected to be involved in the inversion of two chromosomal fragments of around 1 Mb each. Green triangles are for ISXfu2 and red triangles for ISXfu1.

Figure 6

Schematic representation of the flagellar gene clusters of Xcv 85–10 compared to the one of Xff 4834-R. Position of primers used to type flagellar cluster integrity is indicated by red arrows.

Figure 7

Non-motility of some xanthomonads strains. Illustration of non-motility as determined by soft-agar (0.2%) motility tests imaged five days after inoculation in MOKA medium containing tetrazolium chloride. The non-motile strains are a)X. albilineans CFBP 2523, b)X. arboricola pv. corylina CFBP 1159, c) X. axonopodis pv. mangiferaeindicae CFBP 1716, d)X. cucurbitae CFBP 2542, e)X. saccharii CFBP 4641, f)X. translucens pv. phlei CFBP 2062, g)X. translucens pv. translucens CFBP 2054, h)Xff 4834-R, i) CFBP 1557, j) SNES 22, k) LSV24, l) CFBP 6473, m) CFBP 6546, n) CFBP 6935, o) CFBP 6936, p)Xff 4885, q) Xap57, r) Xap59, s) Xap466, and t) Xap697. The motile Xcc ATCC33913 strain is used as positive controls in these tests.