Construction of the STM6978∆T3SS and ORS285∆T3SS mutants
To be able to construct a GFP tagged T3SS mutant of ORS285 and a T3SS mutant of STM6978, two
derivative forms of the non-replicative pVO155 plasmid (Oke and Long, 1999) were constructed.
Strain STM6978 is resistant to most classical antibiotics, except for the third generation beta-lactam
cefotaxim. For this reason, a pVO155 derivative was constructed that contains the cefotaximase gene
to confer resistance to cefotaxim. To this end, the cefotaximase CTX-M-15 from Escherichia coli
strain 25104 was amplified and fused by overlapping PCR to the constitutive npt2 promoter using the
primers : Cefo-M15-f (5’- CAGGATACATGCATAGGAGGAATCCCATGGTTAAAAAATCACTGC -3’)/ CefoM15-r
(5’-
CGTTTAAGCTTTTACAAACCGTCGGTGACGATTTTAGC-3’) and
GCTAAGCTTCCACGCTGCCGCAAGCACTCAGG
-3’)/
pm-npt2-f
pm-npt2-r
(5’(5’-
ATGGGATTCCTCCTATGCATGTATCCTGTCTCTTGATCAG -3’). The resulting PCR fragment was cloned
into the HindIII site of pVO155 resulting in pVO155-npt2-Cefo. To be able to obtain a GFP tagged
T3SS mutant, a pVO155 derivative containing the gfp gene was constructed. For this purpose, the
npt2-GFP containing region from plasmid pMG103-npt2-GFP (Bonaldi et al, 2010) was isolated after
KpnI digestion and subsequently cloned into the KpnI site present in pVO155 resulting in pVO155npt2-GFP. For the construction of the T3SS mutants, a DNA region of around 400-bp inside the rhcN
gene from STM6978 and ORS285 were then amplified using the primers: RhcN-6978-f (5’GATAGCGTCGACGAGATTCGCAGCAAAGGATGTC
GAAGTCCTCTAGACACGCGAACGCCAAGCGGAAAC
CATTGCTCGAGGTGCCGTCCGCGGCCGAATCAC-3’)/
-3’)/
-3’)
RhcN-6978-r
and
RhcN-285-f
RhcN-285-r
(5’(5’(5’-
GAAATCTAGACTCAATCCCACGCCTCTTCATAG -3’). The resulting DNA fragment from STM6978 was
digested with SalI and XbaI and cloned into SalI/XbaI linearised pVO155-npt2-Cefo. The resulting
rhcN fragment from ORS285 was digested by XhoI and XbaI and cloned into plasmid pVO155-npt2GFP that had been linearized with SalI/XbaI. The resulting plasmids were then transferred into E. coli
S17-1 strain to introduce the construction in ORS285 or STM6978 strain by mating as previously
described (Giraud et al, 2010). For STM6978, mutant selection was done on YM medium
complemented with nalidixic acid (20µg/ml) and cefotaxim (20µg/ml).
References
 Bonaldi K, Gherbi H, Franche C, Bastien G, Fardoux J et al. (2010). The Nod factor-independent
symbiotic signaling pathway: development of Agrobacterium rhizogenes-mediated transformation
for the legume Aeschynomene indica. Mol Plant Microbe Interact 23: 1537-1544.
 Oke V, Long SR (1999) Bacterial genes induced within the nodule during the Rhizobium-legume
symbiosis. Mol. Microbiol. 1999, 32, 837–849.
 Giraud E, Lavergne J, Vermeglio, A. (2010). Characterization of bacteriophytochromes from
photosynthetic bacteria: Histidine kinase signaling triggered by light and redox sensing. Methods
Enzymol 471:135–159.