Specific identification and detection of Pantoea stewartii subsp. stewartii using a membrane-based multi-gene oligonucleotide array

Figures & data

Table 1. Putative housekeeping genes, location on the array, code and oligonucleotide sequences evaluated in this study.

		Location
Gene	Gene product/comments	on array	Code	Sequence (5ʹ → 3ʹ)
16S rRNA	Structural and translational roles	a1	Uni506_16S	GTGCCAGCAGCCGCGGTAATAC
		b1	si447_16S	GTGGTGAGGTTAATAACCTCATCA
		c1	s994_16S	GCGAACTTGGCAGAGATGCCTT
		d1	442rRNA_ana	GGAAGGCGATGTGGTTAATA
leuS	leucyl-tRNA synthetase-translational role	a2	uni_leu128	GGAAGAGTGGCCTGAGCAGGTC
		b2	si305leu	GGCACAGGCGGCGGCCAGCAAC
		c2	i452leu	GAACGCCTGCCTGTCTGGGTC
		d2	442leu_ana	CAGGTGAGGCTGTTCCTGTCTG
		d6	218leu _agglo	CGAAGAGAAGGTCACTGTCTACA
cpsD	Production of the exopolysaccharide stewartan – role in pathogenicity and virulence of Pantoea stewartii	a3	Uni_cps470	CCATCGTAATTCCGGCTTATA
		b3	cps_308	TGGTTGACTCGCTAATCAAC
		c3	cps_339	TCCCGTGAAAGGTGTCACGCAG
		d3	cps_ana320	ACGGACCCGGCGGCGCGCCC
rpoB	Subunit beta of DNA-directed RNA polymerase	a4	Uni_rpoB1940	CCGCGATCAGGTTGACTACATG
		b4	i1967rpoB	CGACCCAACAGGTCGTTTCCG
		c4	s1854rpoB	GGCGAATACCAACCTGTCAGAA
		d4	1967rpoB_ana	CCACCCAGCAGGTGGTTTCTG
		b6	s1967rpoB	CGACCCAACAGGTTGTTTCCG
		c6	s1985rpoB	CCGTCGGTGCATCACTGATCC
gyrB	Gyrase subunit B-DNA replication, supercoiling of chromosomal DNA	b6	848gyb	GATGACGCGCGTGAGGGCCT
		c6	557gyb	AGGTGGGATTAAAGCGTTTG
		d6	uni_gyb678	TGCAGTGGAACGACGGCTTCC
Other	Negative control	a6	ns*	N/A^#
		c5	ns*	N/A^#

* not spotted, used as negative control; ^# Not applicable.

Fig. 1 Layout of multilocus oligonucleotides spotted on array. Uni506_16S was a positive control used to normalize hybridization intensity; NS, not spotted, negative control. 1, 16S rRNA; 2, leuS, 3, cpsD; 4, rpoB; 5, gyrB; 6, rpoB (b6 & c6) and leuS (d6). The corresponding sequences of all oligonucleotides are listed in Table 1.

Fig. 2 Maximum likelihood inferred phylogenetic tree of Pantoea species from gyrB-leuS-rpoB concatenated sequences. Pantoea stewartii subsp. stewartii strains seem to be homogeneous while those of P. stewartii subsp. indologenes are heterogeneous. General time reversible substitution model was used with 1000 bootstrap replicates. Tatumella spp. used as outgroup.

Fig. 3 Summary of hybridization patterns of multiplex digoxigenin-labelled PCR amplicons of Pantoea stewartii and other bacterial strains to an array of subspecies-, species- and group-specific oligonucleotides on nylon membranes. Chemiluminograms were scanned at 600 dpi using HP ScanJet 5590 scanner, and grey scale values of each dark spot, computed with ImageJ software and normalized using oligonucleotide Uni506_16S. Normalized signals are indicated by the following symbols: □, <0.1 (not detected); ▪, 0.15–0.39; ◘, 0.40–0.79; ■, >0.80. The locations of the oligonucleotides given at the top correspond to their locations on the membranes in the array. Oligonucleotides are presented in Table 1. The type strains were obtained from BCCM/LMG bacterial collection, Belgium or the Collection of Plant associated Bacteria (CFBP), France. Strains with prefixes SW or DC were kindly provided by Dr David Coplin.

Fig. 3 (Continued.)

Fig. 4 Hybridization profiles of 18 Pantoea species and subspecies, Pectobacterium and Dickeya (former Erwinia members) and Tatumella punctata. Note specificity of detection of Pantoea stewartii subsp. stewartii LMG 2715^T and differentiation from Pantoea stewartii subsp. indologenes based on oligonucleotide s1854rpoB (location c4).

Fig. 5 Chemiluminograms showing specific detection of Pantoea stewartii subsp. stewartii DOAB 021 on corn plants infected or not under growth chamber conditions. a, non-infected plant, and b, infected plant. Leaf lesions (40 mg) were processed as described in Tambong et al. (2008). No DNA extraction was performed. Four plants per treatments were analysed with consistent hybridization pattern obtained. Row numbers represent the different genes used: 1, 16S rRNA; 2, leuS; 3, cps; 4, rpoB; 5, gyrB; 6, mix: rpoB (b6 & c6) and leuS (d6) spotted on the membrane-based array.

Fig. 6 Alignment of rpoB-derived oligonucleotide (s1854rpoB) sequences showing nucleotide mismatches and specificity to P. stewartii subsp. stewartii compared with other strains of Pantoea and Tatumella. Note nucleotide mismatches of oligonucleotide s1854rpoB are almost at the centre for effective differentiation.

Tambong JT, Mwange KN, Bergeron M, Ding T, Mandy F, Reid LM, Zhu X. 2008. Rapid detection and identification of the bacterium Pantoea stewartii in corn by Taqman real-time PCR assay targeting the cpsD gene. J Appl Microbiol. 104:1525–1537.

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