Fig. 1.
Phenotype characterization of the deletion mutant Xanthomonas campestris pv. campestris B100 Δcrt1 compared to the wild-type strain Xanthomonas campestris pv. campestris B100.
a) Growth behaviour of the wild-type (○) and the deletion mutant (Δ) strains cultivated in XMD minimal medium with 10 g/l glucose as sole carbon source; b) xanthan production was measured after 72 h of cultivation; xanthan was precipitated with isopropanol, dried and weighted relative to culture media; n≥ 5 biological replicates were investigated; two-tailed t-test resulted in significant p-value of 0.027 (***); viscosity of xanthan was assessed for both strains; xanthan was harvested after 72 h of cultivation, isolated and dried; all measurements were performed with 1% w/v xanthan with rheometer at room temperature; n = 4 biological replicates were investigated for each strain.
Fig. 2.
Bacterial virulence against host plant Brassica oleracea.
a)Leaf lesions in Brassica oleracea caused 6,8, and 10 dpi by infection with the X. campestris pv. campestris B100 wild-type (WT), deletion mutant strain X. campestris pv. campestris B100 Δcrt1, and a mock sample; inoculation was conducted with a sterile syringe, infection progress was documented at 6,8, and 10 days post inoculation; a representative set of leaves was chosen for the depiction of symptom propagation; b) analysis of the lesion area of the plants infected with either wild-type or deletion mutant strain, measured at 0, 6, 8, and 10 days post inoculation; depicted are calculated mean values for each condition; statistical relevance was confirmed by t-test (Supplementary Table I), for each bacterial strain n ≥11 samples were considered.
Fig. 3.
Genome-wide differential transcriptional analysis of the deletion mutant Xanthomonas campestris pv. campestris B100 Δcrt1 compared to the wild-type strain.
Ratio/intensity plot obtained from whole genome microarrays of the strain X. campestris pv. campestris B100 Δcrt1 compared to the wildtype strain X. campestris pv. campestris B100; cultivation was performed in the minimal XMD medium with 10 g/l glucose as a sole carbon source; cells were harvested at an early stationary growth stage at an oD600 nm of 1.8; shown are only targets with confidence level greater than 95%; green and red dots represent target genes with increased and decreased, respectively, transcript levels in the mutant strain X. campestris pv. campestris B100 Δcrt1; genes with transcription unaffected by the deletion of crt1 gene are represented by grey dots; annotated target genes with highest fold-change compared to the wild-type strain marked by gene name.
Fig. 4.
Differential proteome analysis of the deletion mutant Xcc B100 Δcrt1 compared to the wild-type strain.
Volcano plot obtained from proteome investigation of the strain X. campestris pv. campestris B100 Δcrt1 compared to the wild-type strain X. campestris pv. campestris B100; cultivation was performed in the minimal XMD medium with 10 g/l glucose as a sole carbon source; Cells were harvested at an early stationary growth stage at an oD600 nm of 1.8; shown are proteins with identified three unique peptides or more, and with peaks found for all replicates; green and red dots represent proteins with increased and decreased, respectively, abundance in the mutant strain Xcc B100 Δcrt1; cytosolic and membrane-bound proteins with expression unaffected by the deletion of crt1 gene are represented by grey dots; annotated proteins with highest fold-change compared to the wild-type strain marked with their respective protein name.
Table I
Effect of the crt1 deletion on X. campestris pv. campestris gene expression.
| Locus | Gene name | Annotated gene product | M-value | A-value | p-Value |
|---|---|---|---|---|---|
| Up-regulated genes | |||||
| XCCB100_2325 | putative filamentous hemagglutinin-related protein | 1.22 | 11.4 | 7.42E-04 | |
| AS_XCCB100_1718 | antisense transcript gumH | 1.12 | 7.5 | 3.49E-03 | |
| XCCB100_0957 | pilP | type IV pilus assembly protein | 0.91 | 9.5 | 9.00E-04 |
| XCCB100_0907 | putative cytochrome c551 | 0.90 | 8.2 | 4.92E-03 | |
| XCCB100_0552 | groES | 10 kDa chaperonin | 0.90 | 10.2 | 1.99E-03 |
| XCCB100_0905 | putative exported protein | 0.89 | 11.6 | 2.65E-03 | |
| XCCB100_3396 | putative exported enzyme | 0.86 | 10.8 | 1.23E-09 | |
| XCCB100_1423 | murD | UDP-N-acetylmuramoylalanine-D-glutamate ligase | 0.84 | 9.0 | 3.95E-03 |
| XCCB100_1720 | gumJ | xanthan repeating unit exporter | 0.83 | 8.8 | 3.25E-02 |
| XCCB100_3538 | gfo | exported glucose-fructose oxidoreductase | 0.83 | 9.4 | 3.07E-02 |
| XCCB100_0906 | lao | L-amino-acid oxidase | 0.81 | 8.6 | 8.41E-03 |
| XCCB100_0207 | amtB | ammonium transporter | 0.80 | 12.2 | 6.28E-04 |
| XCCB100_2099 | hisH | glutamine amidotransferase | 0.77 | 8.9 | 7.83E-03 |
| XCCB100_0954 | pilM | type IV pilus assembly protein | 0.75 | 9.9 | 4.70E-03 |
| XCCB100_1209 | glk2 | glucokinase | 0.74 | 7.4 | 1.86E-02 |
| XCCB100_1615 | conserved hypothetical protein | 0.74 | 8.7 | 1.76E-02 | |
| XCCB100_2311 | hexose O-acetyltransferase | 0.73 | 8.4 | 2.52E-02 | |
| XCCB100_rna061 | putative regulatory sRNA | 0.73 | 19.7 | 1.86E-04 | |
| XCCB100_1719 | gumI | mannosyltransferase | 0.73 | 9.1 | 1.21E-02 |
| XCCB100_1672 | pilE | type IV pilus assembly protein PilE | 0.72 | 10.0 | 6.89E-03 |
| Down-regulated genes | |||||
| XCCB100_2267 | infA | translation initiation factor IF-1 | -1.50 | 11.5 | 1.11E-04 |
| XCCB100_2352 | hypothetical protein | -1.30 | 8.9 | 7.20E-04 | |
| XCCB100_1776 | hypothetical protein | -1.30 | 8.1 | 4.39E-02 | |
| XCCB100_1297 | homoserine O-acetyltransferase-like enzyme | -1.20 | 9.7 | 6.91E-06 | |
| XCCB100_2980 | conserved hypothetical protein | -1.13 | 12.6 | 7.39E-09 | |
| XCCB100_3868 | conserved hypothetical protein | -1.10 | 12.5 | 3.37E-04 | |
| XCCB100_1298 | cgl | cystathionine gamma-lyase | -1.09 | 11.5 | 1.50E-07 |
| XCCB100_4110 | hypothetical protein | -1.01 | 13.4 | 3.72E-03 | |
| XCCB100_3996 | conserved hypothetical protein | -1.00 | 10.4 | 1.31E-08 | |
| XCCB100_2290 | TonB-dependent outer membrane receptor precursor | -0.97 | 8.4 | 2.37E-03 | |
| XCCB100_rna193 | putative regulatory RNA | -0.96 | 10.2 | 2.16E-03 | |
| XCCB100_3017 | cspA2 | cold shock protein | -0.93 | 11.2 | 3.27E-06 |
| XCCB100_3810 | putative secreted protein | -0.91 | 12.4 | 8.14E-03 | |
| XCCB100_1741 | conserved hypothetical protein | -0.90 | 11.1 | 1.29E-02 | |
| XCCB100_3869 | putative manganese-containing catalase | -0.90 | 12.2 | 2.48E-03 | |
| XCCB100_3892 | conserved hypothetical protein | -0.87 | 10.8 | 8.28E-03 | |
| XCCB100_2320 | conserved hypothetical protein | -0.87 | 13.1 | 7.39E-03 | |
| XCCB100_2291 | hypothetical protein | -0.86 | 11.1 | 7.85E-03 | |
| XCCB100_1773 | hypothetical protein predicted | -0.86 | 6.6 | 1.02E-04 | |
| XCCB100_3966 | putative exported protein | -0.85 | 11.7 | 2.14E-10 | |
1 Target genes with most fold-change of transcription under deletion of crt1 gene as compared to the wild-type strain X. campestris pv. campestris B100; M-value represents the fold-change [log2] of transcript in mutant strain compared to wild-type strain; A-value denotes the intensity of the given signal; p-value has been calculated with significance test with Holm method.
Table II
Effect of the crt1 deletion on the cytosolic and membrane-bound proteome of X. campestris pv. campestris during early stationary growth stage.
| Locus | Gene name | Function | Abundance ratio | p-Value | Unique peptides |
|---|---|---|---|---|---|
| Up-regulated proteins | |||||
| XCCB100_1291 | gtrB | putative bactoprenol glucosyltransferase | 5.92 | 1.00E-17 | 3 |
| XCCB100_3258 | conserved hypothetical protein | 3.67 | 1.30E-05 | 4 | |
| XCCB100_3905 | putative carboxypeptidase | 3.50 | 4.47E-07 | 3 | |
| XCCB100_2818 | int | phage-related integrase | 3.19 | 1.05E-04 | 3 |
| XCCB100_1799 | rpfN | carbohydrate-selective porin | 3.08 | 1.00E-17 | 20 |
| XCCB100_3640 | conserved hypothetical protein | 3.08 | 1.00E-17 | 4 | |
| XCCB100_1801 | fruK | 1-phosphofructokinase | 2.89 | 1.00E-17 | 7 |
| XCCB100_1802 | fruB | PTS fructose porter | 2.71 | 1.00E-17 | 35 |
| XCCB100_2558 | ftsB | septum formation initiator protein FtsB | 2.41 | 3.35E-11 | 3 |
| XCCB100_2909 | conserved hypothetical protein | 2.19 | 6.21E-06 | 3 | |
| XCCB100_1450 | pirin-related protein | 2.14 | 5.65E-09 | 7 | |
| XCCB100_0445 | hypothetical protein | 2.08 | 1.00E-17 | 4 | |
| XCCB100_2092 | transcriptional regulator; Crp/Fnr family | 2.02 | 2.26E-07 | 3 | |
| XCCB100_1788 | phy | putative exported phytase | 1.96 | 8.66E-12 | 3 |
| XCCB100_0128 | TonB-dependent outer membrane receptor precursor | 1.95 | 4.39E-12 | 18 | |
| XCCB100_1697 | aglA1 | alpha-glucosidase | 1.95 | 1.81E-08 | 11 |
| XCCB100_1807 | gdh | glutamate dehydrogenase | 1.87 | 1.00E-17 | 124 |
| XCCB100_1800 | fruA | PTS fructose porter | 1.82 | 1.00E-17 | 12 |
| XCCB100_1894 | conserved hypothetical protein | 1.77 | 2.80E-07 | 7 | |
| XCCB100_1761 | conserved hypothetical protein | 1.74 | 6.40E-12 | 3 | |
| Down-regulated proteins | |||||
| XCCB100_0308 | agxT | aminotransferase | -2.54 | 1.66E-03 | 4 |
| XCCB100_3852 | putative aromatic ring-opening dioxygenase | -2.31 | 2.09E-04 | 3 | |
| XCCB100_0796 | putative nuclease / phosphatase | -2.31 | 3.84E-04 | 3 | |
| XCCB100_0846 | tctE | two-component system sensor histidine kinase | -2.28 | 2.85E-04 | 5 |
| XCCB100_2019 | short chain dehydrogenase | -2.22 | 5.37E-05 | 6 | |
| XCCB100_3906 | putative transcriptional regulator; LysR family | -2.21 | 1.93E-13 | 6 | |
| XCCB100_4189 | conserved hypothetical protein | -2.17 | 7.94E-12 | 3 | |
| XCCB100_1528 | ABC transporter permease and ATP-binding protein | -2.09 | 5.28E-03 | 5 | |
| XCCB100_1398 | DJ-1/PfpI family protein | -2.09 | 1.12E-07 | 8 | |
| XCCB100_2784 | conserved hypothetical protein | -2.08 | 2.49E-08 | 7 | |
| XCCB100_3277 | hsdR2 | Type I site-specific deoxyribonuclease (restriction subunit) | -1.96 | 7.02E-04 | 8 |
| XCCB100_2866 | asnB2 | asparagine synthase (glutamine-hydrolysing) | -1.95 | 1.68E-06 | 10 |
| XCCB100_0546 | conserved hypothetical protein | -1.85 | 3.77E-04 | 3 | |
| XCCB100_2526 | thiD | phosphomethylpyrimidine kinase | -1.83 | 9.26E-04 | 6 |
| XCCB100_2607 | rpfS | DSF-sensing histidine kinase/response regulator hybrid | -1.79 | 3.06E-02 | 3 |
| XCCB100_0601 | membrane-located putative protein phosphatase | -1.76 | 4.57E-04 | 5 | |
| XCCB100_1293 | asmA | AsmA family membrane protein | -1.64 | 2.84E-03 | 7 |
| XCCB100_2313 | NDP-hexulose epimerase | -1.62 | 1.40E-02 | 3 | |
| XCCB100_4124 | birA | bifunctional biotin repressor / co-repressor biosynthesis enzyme | -1.58 | 3.02E-02 | 3 |
| XCCB100_2292 | short chain dehydrogenase | -1.56 | 1.98E-04 | 6 | |
1 Proteins with most significant abundance fold-change under deletion of crt1 gene as compared to the wild-type strain X. campestris pv. campestris B100; Abundance ratio represents the fold-change [log2] measured for replicates of deletion mutant relative to replicates of wild-type; Abundance ratio p-value has been calculated with significance background-based, nested ANOVA test, with n = 3; unique peptides describe number of peptides that occur exclusively in the given target protein were employed for its identification.
Table III
Effect of the regulator gene crt1 deletion on transcriptome and proteome of X. campestris pv. campestris.
| Locus | Gene name | Function | COG | Fold-change | Transcript/Protein abundance change | Studies in Xanthomonas |
|---|---|---|---|---|---|---|
| Cell motility | ||||||
| XCCB100_0954 | pilM | type IV pilus assembly protein | NU | 0.748 | T | Dugé de Bernonville et al, 2014 |
| XCCB100_0957 | pilP | Tfp pilus assembly protein | NU | 0.914 | T | Dugé de Bernonville et al, 2014 |
| XCCB100_1672 | pilE | Tfp pilus assembly protein | NU | 0.725 | T | Dugé de Bernonville et al, 2014 |
| Carbohydrate transport and metabolism | ||||||
| XCCB100_1697 | aglA1 | alpha-glucosidase | G | 1.950 | P | Blanvillain et al, 2007; Fatima and Kumar, 2015; Eom et al. 2015; Ruan et al. 2014; Watt et al. 2009 |
| XCCB100_1799 | rpfN | carbohydrate-selective porin | G | 3.080 | P | Moreira et al. 2004 |
| XCCB100_1800 | fruA | PTS fructose porter | G | 1.820 | P | Moreira et al. 2004 |
| XCCB100_1801 | fruK | 1-phosphofructokinase | G | 2.890 | P | Li et al. 2019 |
| XCCB100_1802 | fruB | PTS fructose porter | G | -0.507 / 2.71 | T/P | Moreira et al. 2004 |
| Signal transduction mechanisms and information processing | ||||||
| XCCB100_0846 | tctE | two-component system sensor histidine kinase | T | -2.280 | P | Timilsina et al. 2020; Wang et al. 2016; Wang et al. 2017b |
| XCCB100_2607 | rpfS | DSF-sensing histidine kinase/response regulator | T | -1.790 | P | An et al. 2014; O’Connell et al. 2013; Ryan et al. 2007 |
| Amino acid transport and metabolism | ||||||
| XCCB100_1297 | homoserine O-acetyltransferase-like enzyme | -1.204 | T | Pielken et al. 1987; Pielken et al. 1988; O’Connell et al. 2013 | ||
| XCCB100_1298 | cgl | cystathionine gamma-lyase | -1.089 | T | Pielken et al. 1987; Pielken et al. 1988; O’Connell et al. 2013 | |
| XCCB100_2099 | hisH | glutamine amidotransferase | 0.766 | T | Li et al. 2019 | |
| XCCB100_2866 | asnB2 | asparagine synthase (glutamine-hydrolysing) | E | -1.950 | P | Qian et al. 2013 |
| EPS biosynthesis | ||||||
| AS_ XCCB100_1718** | antisense transcript gumH | 1.118 | T | Chan et al. 1999; Kiraly et al. 1997; Vojnov et al. 2001; Rigano et al. 2007; Alvarez et al. 2000; Yun et al. 2006 | ||
| XCCB100_1719 | gumI | mannosyltransferase | R | 0.726 | T | Chan et al. 1999, Kiraly et al. 1997; Vojnov et al. 2001; Rigano et al. 2007; Alvarez et al. 2000; Yun et al. 2006 |
| XCCB100_1720 | gumJ | xanthan repeating unit exporter | M | 0.833 | T | Chan et al. 1999, Kiraly et al. 1997; Vojnov et al. 2001; Rigano et al. 2007; Alvarez et al. 2000; Yun et al. 2006 |
| Lipid transport and metabolism | ||||||
| XCCB100_1788 | phy | putative exported phytase | I | 1.960 | P | Raboy 2003; Blüher et al, 2017; Chatterjee et al. 2003 |
| Cell wall, membrane, envelope biogenesis | ||||||
| XCCB100_1291 | gtrB | putative bactoprenol glucosyl-transferase | 5.92 | P | Newman et al. 2001; Vorhölter et al. 2001; Li et al. 2019 | |
| Other functions | ||||||
| XCCB100_2558 | ftsB | septum formation initiator protein FtsB | D | 2.41 | P | Ferreira et al. 2017 |
1 Target genes, and cytosolic and membrane-bound proteins, involved or linked to the virulent life-style of X. campestris pv. campestris, with most significant abundance fold-change under deletion of crt1 gene as compared to the wild-type strain X. campestris pv. campestris B100; Abundance ratio represents the fold-change [log2] measured for replicates of deletion mutant relative to replicates of wild-type; Abundance ratio p-value has been calculated with significance background-based, nested ANOVA test, with n = 3; unique peptides describe how many peptides unique for a single target protein were employed for its identification.
Fig. 5.
Regulatory network of the carbohydrate related transcription factor Crt1 in Xanthomonas campestris pv. campestris.
Depicted is the schematic view of the regulatory network that is influenced by the novel regulator Crt1, as well as different virulence factors in X. campestris pv. campestris. Activating effect of Crt1 is denoted by green arrows with “+”; inhibiting effect is denoted by red arrows with “-” Virulence of X. campestris pv. campestris depends on T3S system, adhesion provided by T4 pilus, EPS, and LPS. T3S system translocates T3 effector proteins into the host plant cell, where they can modulate host gene expression. T3 effectors may also activate plant defense response cues. The adaptation of X. campestris pv. campestris lifestyle during infection includes regulation of import and utilization of plant-derived nutrients. Crt1 regulates virulence factors involved in T4P assembly, EPS and LPS biosynthesis, signaling transduction and utilization of plant-related nutrients. Furthermore, novel regulator affects enzymes implicated in methionine metabolism.