The Transcriptional Regulator Crt1 is Involved in The Pathogenic Lifestyle of Xanthomonas campestris pv. campestris B100

Anna Korszańska; Petra Lutter; Vera Ortseifen; Alfred Pühler; Karsten Niehaus

doi:10.33073/pjm-2026-006

Figures & Tables

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.

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.

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.

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.

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.

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	rp^fS	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

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

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

The Transcriptional Regulator Crt1 is Involved in The Pathogenic Lifestyle of Xanthomonas campestris pv. campestris B100

Figures & Tables

Fig. 1.

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Fig. 5.

Effect of the crt1 deletion on the cytosolic and membrane-bound proteome of X_ campestris pv_ campestris during early stationary growth stage_

Effect of the crt1 deletion on X_ campestris pv_ campestris gene expression_

Effect of the regulator gene crt1 deletion on transcriptome and proteome of X_ campestris pv_ campestris_

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