It has been reported that Pseudomonas fluorescens grown under conditions that increase OL content becomes more resistant to the polycationic lipopeptide polymyxin B indicating a connection between these amino lipids and the resistance to bactericidal peptides [12,13]. Moreover, OLs of Bordetella pertussis, Flavobacterium meningosepticum and Achromobacter xylosoxidans display antagonistic effects on LPS endotoxicity as well as proinflammatory and inflammatory activity [14–18]. Such an activity in Brucella OL would be in apparent contradiction with the furtive behavior of these bacteria with respect to innate immunity. Therefore, it was of interest to know whether Brucella OLs play a role in the OM stability and resistance to polycations and whether they display a biological activity different from that of other OLs, including the evasion of pathogen recognition receptors. Results OLs are OM components of B. abortus To determine the cellular localization of OLs, we first examined the free-lipids in virulent S B. abortus 2308 NalR grown in tryptic soy broth to the stationary phase, in the OM fragments released spontaneously during growth [19] and in non-delipidated B. abortus LPS [20]. Thin-layer chromatography of the corresponding chloroform:methanol:water extracts [21] confirmed the presence of OLs in B. abortus and showed their enrichment in the OM fragments (Figure 1 A), thus demonstrating that they are OM components. Although in amounts lower than PE, OLs were also detected in non-delipidated B. abortus LPS suggesting an association in the OM (Figure 1 A). The levels of OLs did not change when the bacteria were cultured in tryptic soy broth or in Brucella Gerhardt’s minimal medium (lactate-glutamate-glycerol, mineral salts, vitamins) [22] (Figure 1 A). B. abortus OLs are synthesized through a two step pathway We searched the B. abortus 2308 genome for orthologs of the genes involved in OL synthesis in other a-2 Proteobacteria. In Sinorhizobium meliloti, OlsB acylates the ornithine a amino group with C18:0(3-OH) and OlsA generates the acyloxyacyl group by esterification with C18:0 (Figure 1 B) [23,24]. In Rhizobium tropici, an additional gene (olsC) codes for an oxygenase that generates a 2- hydroxy substitution on the ester-linked acyl group [25] (Figure 1 B). We found that ORF BAB1_0147 (annotated as encoding a hypothetical protein) codes for a protein with 55% identity and 66% similarity with OlsB, and that the product of ORF BAB1_2153 (annotated as a phospholipid-glycerol acyltransferase) has 45% identity and 61% similarity with OlsA. Moreover, we located similar ORFs in the genomes of B. melitensis 16M and B. suis 1330 (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). ORF BAB1_0147 (henceforth BABolsB) maps as an isolated gene. BAB1_2153 (henceforth BABolsA) is in a possible operon formed by at least BAB1_2151 (putative glycoprotease), BAB1_2152 (putative acetyltransferase) and probably by BAB1_2154 (hypo- thetical protein; the stop codon of BAB1_2152 and the start codon of BAB1_2153 overlap). Both BABOlsA and BABOlsB contain a sequence [H73 (X)4 D78 and H76 (X)4 D81, respectively] that could correspond to the consensus motif [H(X)4D] of glycerolipid acyltransferases [26]. Moreover, BABOlsA contains two regions, NHTS (amino acids 72–75) and AEGTT (amino acids 143–147), closely resembling the NHQS and PEGTR motifs highly conserved in lysophosphatidic acid acyltransferases [27]. We also found a DNA sequence with homology to olsC. However, the possible BABolsC carried a frame shift in the same position in all accessible B. abortus, B. melitensis and B. suis genomes so that it Brucella Ornithine Lipids corresponds to two ORF (annotated as BMEI0464 and BMEI0465 in B. melitensis 16M). Amino acids 1 to 155 of BMEI0464 have a 87% identity (92% homology) with amino acids 31 to 185 of S. meliloti OlsC, and amino acids 1 to 94 of BMEI0465 are 87% identical (91% homology) to the 187 to 280 stretch of OlsC [28]. Most of the consensus of the OslC-LpxO family of proteins is in BMEI0464 but at the very end of the protein and truncated in the last four amino acids, including the last isoleucine conserved in all OlsC homologues [29]. All these characteristics strongly suggest that the structure of Brucella OLs is similar to those described previously for Rhodospseudomonas sphaeroides (Figure 1C). Using the above-described evidence, we constructed internal in- frame deletion mutants of the virulent B. abortus 2308 NalR strain (henceforth BAB-parental) devoid of the consensus motifs following a PCR overlap strategy [30] (Table S1). For BABolsB, we removed amino acids 40 to 229, which resulted in a truncated protein of 107 amino acids (mutant BABDolsB). The deletion in BABolsA (mutant BABDolsA) eliminated amino acids 48 to 245 and resulted in a truncated protein of 69 amino acids. Figure 1 A shows that BABDolsA lacked OLs but synthesized a new ninhydrin-positive component corresponding to the lyso-ornithine lipid (lyso-OL) precursor [31]. In contrast, the only ninhydrin-positive lipid generated by mutant BABDolsB was PE. When we complemented mutant BABDolsB with plasmid pLPI-6 (carrying BABolsB; Table S1), OL synthesis was restored (Figure 1 A). Similarly, BABDolsA complemented with plasmid pYLI-1 (carrying BABolsA) was able to produce OLs (not shown). These results are consistent with a two step pathway in which BABOlsB and BABOlsA act consecutively and where deletion of the former abrogates OL and lyso-OL synthesis (Figure 1 B). Characterization of mutants BABDolsA and BABDolsB showed no change in colonial morphology, or in catalase, oxidase, and urease activities. They were S according to lysis by B. abortus S- specific phages, agglutination with anti-A and anti-M monospe- cific sera, crystal violet exclusion and acriflavine agglutination test. OLs are not required for Brucella OM resistance to bactericidal peptides and complement Due to the OL abundance in Brucella and their zwitterionic nature, it has been proposed that they play a relevant role in the stabilization of negative charges of LPS and, therefore, in the stability of the OM [32]. To test this, we first examined the sensitivity to bactericidal peptides. We found no significant differences in the minimal inhibitory concentrations of polymyxin B and colistin on BAB-parental, BABDolsA and BABDolsB strains. Since bactericidal peptides are also OM permeabilizing agents, we probed the mutants with polymyxin B plus lysozyme under hypotonic conditions in comparison with Escherichia coli. This treatment was effective in killing E. coli but had no action on mutants BABDolsB or BABDolsA or on BAB-parental (Figure 2). Moreover, EDTA alone or combined with polymyxin B did not promote lysozyme uptake, proving that divalent cations were not taking over the hypothetical role of OLs in OM stabilization (Figure 2). Finally, sensitivity to a set of antibiotics (penicillin, doxycycline, clarithromycin, erythromycin and rifampicin) that penetrate the OM by hydrophilic or hydrophobic pathways, or of dyes like thionine blue, fuchsine and safranin remained unchanged (not shown). All these results indicate that OLs are neither necessary to stabilize the OM of B. abortus against bactericidal peptides nor influence its permeability. B. abortus mutants altered in PC synthesis, with a truncated LPS or an upset OM protein profile are sensitive to killing by non- immune serum [33–35]. However, OL deficiency did not have a similar effect because we observed only a small and not significant (p.0.05) increase in serum sensitivity in mutant BABDolsB PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16030