Tested strains were initially grown in TY to late log phase (109
cells/ml; O.D600 nm 0.9-1.0). Aliquots of 1 ml of the starting cultures were centrifuged; the pelleted cells were washed with fresh TY and finally resuspended in 1 ml of the medium. Ten μl of the bacterial suspensions (~107 cells) were inoculated into 340 μl of TY broth in Bioscreen Honey comb 100-well plates which were incubated at 30°C with continuous shaking. Absorbance readings at 600 nm were selleck kinase inhibitor recorded every 2 h until the cultures reached the late Fosbretabulin price stationary phase. OD values of uninoculated media were subtracted from cultures OD readings to normalize data for background prior to plotting. Determinations were done in triplicate for each strain. Construction of the S. meliloti hfq mutant derivatives A 1,684-bp DNA region containing the 243-bp hfq ORF and flanking sequences (714-bp upstream and 727-bp downstream of hfq) was PCR amplified with Pfu polymerase using the primers pair Hfq_Fw/Hfq_Rv
SCH772984 datasheet (for all the oligonucleotides cited hereafter see the additional file 3: oligonucleotide sequences) and S. meliloti 1021 genomic DNA as template. This DNA fragment was inserted into pGEM®-T Easy vector generating plasmid pGEMhfq. For the construction of the S. meliloti 2011 hfq insertion mutant
derivatives Enzalutamide nmr two internal regions of the gene were Taq amplified from pGEMhfq with primers combinations hfqforw1/hfqrev2 and hfqforw3/hfqrev4 and subcloned into the suicide vector pK18mobsacB generating plasmids pK18_1.2 and pK18_3.4, respectively. Both plasmids were independently conjugated into the 2011 wild-type strain by triparental matings, using pRK2013 as helper, yielding mutants 2011-1.2 and 2011-3.4 which were selected as KmrSmr colonies in MM agar as a result of pK18mobsacB integration into the hfq gene by single homologous recombination events. Mutations were verified by PCR and the precise location of plasmid insertion into the hfq gene was determined by sequencing of the PCR products. For the generation of the S. meliloti 1021 hfq deletion mutant, plasmid pGEMhfq was amplified with Pfu polymerase with divergent primers (hfqi_1/hfqi_2) flanking the hfq ORF and carrying an internal HindIII restriction site. The PCR product was HindIII-digested and autoligated generating plasmid pGEMΔhfq that contains a 1,447-bp S. meliloti 1021 genomic region in which the hfq ORF was deleted and replaced by a HindIII site.
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