The plasmid pRmM57 (nodC::lacZ fusion) [14] was used to test the expression of the nodC gene and pGD499 (npt::lacZ fusion) [15] to test the expression of the constitutive kanamycin resistance gene. The pMPTR4 plasmid is a pMP220 [24] derivative CHIR-99021 in vivo in which an EcoRI fragment of 0.6 kb harbouring

the intergenic fadD-tep1 region was cloned to create a tep1::lacZ transcriptional fusion. The pGUS3 plasmid containing an nfeD::gusA fusion was used in competition assays [25]. Triparental bacterial matings were performed using pRK2013 as helper plasmid [26]. E. coli was grown routinely at 37°C in Luria-Bertani medium (LB) [27]. S. meliloti strains were grown at 30°C in TY complex medium [28] or in defined minimal medium (MM) [29]. Growth was click here determined regularly in a spectrophotometer measuring the absorbance at 600 nm. Glucosamine and N-acetyl glucosamine were obtained from Sigma-Aldrich. Construction of a S. meliloti tep1 mutant A null-mutant in ORF SMc02161 was obtained by allelic exchange. Firstly, a 3.6 kb SacI fragment

containing this ORF was subcloned from the fadD containing cosmid pRmersf442 [2] into pUC18 [30] to give pTrans1. To disrupt the ORF SMc02161 in pTrans1, a 2 kb SmaI fragment containing the streptomycin/spectinomycin Torin 2 price resistance cassette from pHP45Ω [31] was inserted into a unique EcoRV site to give pTrans2. Next, the SacI fragment containing the disrupted ORF was treated with T4 DNA polymerase (Roche Biochemicals) to make blunt ends and then cloned into the SmaI site of the suicide vector pK18mobsac [32] to give pTrans3. This vector was then used for allelic exchange by introducing it into the S. meliloti strains GR4, and the fadD mutant QS77 via triparental mating, and selecting putative mutants by streptomycin/spectinomycin resistance and sensitivity to sucrose. The resulting SMc02161 mutant GR4T1, and double fadD, SMc02161 mutant QSTR1 were confirmed by Southern hybridization with a specific probe. Construction of a S. meliloti nodC mutant To obtain a nodC mutant in S. meliloti, a fragment was amplified from the chromosomal DNA of S. meliloti GR4 by PCR using 5′-CAGATTCAAGGTCACGAAGTGGCTAAC-3′

Digestive enzyme and 5′-ATAAGCTTGTGACAGCCAGTCGCTATTG-3′ as forward and reverse primers respectively. An EcoRI-PstI fragment of 1.5 kb derived from the PCR product and containing half of the nodB gene and most of the nodC gene was subcloned into pUC18 [30] to obtain pGRC8. To disrupt nodC, pGRC8 was digested with SalI and treated with Klenow (Roche Biochemicals) to create blunt ends. Next, the 2 kb SmaI fragment containing the streptomycin/spectinomycin resistance cassette from pHP45Ω [31] was introduced to give pNC150. The 3.5 kb EcoRI-PstI fragment from pNC150 containing the disrupted nodC gene was inserted into EcoRI-PstI digested pK18mobsac [32] to give pNC200. This suicide vector was then used to obtain the S. meliloti nodC mutant GR4C5, which was confirmed by Southern hybridization.