The pools of constructions were transformed into E. coli strain S17-1 (> 1000 transformants/pool) and were transferred in a Brucella abortus XDB1155 strain  by mating. The XDB1155 strain produces the PdhS-CFP (cyan fluorescent protein) fusion protein from the chromosomal pdhS locus. This strain allows the quick determination of the nature of the pole marked by the
protein-YFP fusion since PdhS-CFP is known to specifically label the old pole . The diversity of the pCDSs in the pools was checked by PCR and restriction analysis on isolated clones from 5 different pools with various average pCDSs sizes, in E. coli S17-1 and B. abortus XDB1155 strains. The analysis of restriction profiles suggests that there is no main over-representation of a given clone in the examined pools. For the screening strategy, we observed the 68 pools using Selleckchem EPZ6438 CFTR activator fluorescence microscopy, and we selected pools in which a fraction of the clones exhibit a polar YFP fusion. The pooled clones were examined after cultivation on solid medium and > 1000 bacteria were observed on agarose pads. Afterwards, pools bearing polar
localization were observed clone by clone in the same way to identify clones producing polar proteins. The pCDS allowing polar localization were amplified by PCR and sequenced to allow their identification. Before analysing the 68 pools, we first screened a pool supposed to contain the pdhS coding sequence (CDS), as a positive control. The complete procedure was applied and six clones were identified as polarly localized, and all of them contained the pdhS CDS fused to YFP. This pilot study suggested that the screening procedure was working, and that PdhS was the main polar protein in this pool. The analysis of the 67 remaining pools led to the
selection of 8 pools for which a significant proportion of bacteria showed polar foci. The average size of the pCDSs contained in the 8 pools was heterogeneous, varying from 450 to 2000 bp. In one of these 8 pools, we identified a pCDS of interest (BMEII0671 and BAB2_0642 in B. melitensis 16M and B. abortus 2308 genomes, Eltanexor cost respectively), that we named aidB by homology with E. coli aidB. Brucella AidB is member of the acyl-CoA dehydrogenase Phospholipase D1 family Deduced AidB sequence is 551 amino acids long, with a predicted molecular mass of 60 kDa and without predicted transmembrane segments. The AidB sequence is similar to acyl-CoA dehydrogenases (ACADs), proteins generally involved in the fatty acid β-oxidation. In the B. melitensis 16M genome, eight pCDSs are proposed to encode enzymes similar to ACADs. B. melitensis and B. abortus AidB deduced sequences are 100% identical. Brucella AidB presents 42% identity to the Escherichia coli AidB (E value of 4 10-117 when B. abortus AidB deduced sequence is blasted against E. coli genomes), suggesting a functional conservation between these enzymes.