However, this hydroxylation is a post-synthesis modification that requires OlsC, which is inactive in Brucella according to our genomic analysis. All these data strongly suggest that the reduction of Brucella OL PAMP is connected to the acyl chains, longer in a-Proteobacteria than in other phylogenetic groups (Figure 1 C). Indeed, these results add more weight to the hypothesis that the hydrophobic moieties of Brucella OM elements are critical in avoiding recognition of this pathogen by innate immunity [1,8,63]. Here, we also presented evidence that Brucella OLs are dispensable elements. Taking into account that Brucella OLs are quantitatively important lipids, it is surprising that they are dispensable. However, the a-Proteobacteria show a marked tendency to live in tight interactions with eukaryotic cells [64] and there is evidence that this ability is associated to a reduction in envelope molecule PAMPs. Bartonella possess a low endotoxic LPS, a reduced number of OM lipoproteins and flagellins that are not recognized by TLR5 [65–67]. Similarly, Rickettsia carry a non canonical LPS and a reduced number of lipoproteins [68], and Wolbachia do not posses genes to synthesize LPS, flagella or fimbriae, have a low number of lipoproteins and an unusual peptidoglycan [69]. Finally, the genomes of Ehrlichia and Anaplasma contain a low number of lipoprotein genes and do not have the genetic machinery to synthesize LPS, peptidoglycan or flagellin [70,71]. Accordingly, we hypothesize that free-living Brucella ancestors carried OLs that were useful OM structural elements in their environment but lost their importance once the major OM target of immunity (i.e. the LPS) became adapted to the host. This adaptation was marked by the modification in LPS PAMP connected features (i.e., acyl chains and charged groups) and had as a result that LPS no longer needed stabilizing agents in the OM. Within this perspective, the degeneracy of the olsC homologue is in keeping with the hypothesis that B. abortus OL represent dispensable ancestral structures that could be eventually eliminat- ed during the evolutionary process. Materials and Methods Ethics Statement All animals were handled and sacrificed according to the approval and guidelines established by the ‘‘Comite´ Institucional para el Cuido y Uso de los Animales’’ of the Universidad de Costa Rica, and in agreement with the corresponding law ‘‘Ley de Bienestar de los Animales No 7451’’ of Costa Rica (http://www. micit.go.cr/index.php/docman/doc_details/101-ley-no-7451-ley- de-bienestar-de-los-animales.html). BALB/c mice (Charles River, Elbeuf, France) were accommo- dated in the animal building of the CITA of Arago´n (ID registration number ES-502970012005) in cages with water and food ad libitum and under biosafety containment conditions, for 2 weeks before the start and all along the experiment. The animal handling and procedures were in accordance with the current European legislation (directive 86/609/EEC) supervised by the Animal Welfare Committee of the institution (protocol number R102/2007). Bacterial strain and growth conditions Bacteria were grown in tryptic soy broth or agar either plain or supplemented with kanamycin (Km) at 50 mg/ml, or/and nalidixic (Nal) at 25 mg/ml, or/and gentamicin (Gm) at 20 mg/ml, or/and chloramphenicol at 20 mg/ml (all from Sigma), or/and 5% sucrose. Where indicated, the defined medium of Gerhardt [72] was used. All strains were stored in skim milk at 280uC. The origin of the B. abortus and S. typhimurium strains is described in Brucella Ornithine Lipids previous works [1,73,74]. B. pertussis is a clinical isolate kindly provided by G. Mart´ınez de Tejada. Bacteriological procedures, antibiotic sensitivity and cell surface characterization The mutants were characterized according to standard Brucella typing procedures [75]: colonial morphology after 3 days of incubation at 37uC, crystal violet exclusion, catalase, oxidase, urease, acriflavine agglutination, sensitivity to Tb, Wb, Iz and R/ C phages, agglutination with anti-A and anti-M monospecific sera, CO2 and serum dependence, and susceptibility to thionine blue, fuchsine, and safranin. Moreover, the minimal inhibitory concen- trations of polymyxin B, colistin, penicillin, doxycycline, clarith- romycin, erythromycin and rifampicin were determined in M u¨ller- Hinton medium by standard procedures. Surface hydrophobicity was analyzed as described by Kupfer and Zusman [76]. Bacteria were grown in tryptic soy broth until stationary phase, incubated in 0.5% sodium azide at 37uC overnight, collected by centrifugation (70006g, 10 min, 4uC), washed twice with a solution of K2HPO4.3H20 97mM, KH2PO4 53mM, urea 21mM, MgSO4.7H2O 0.8mM and resuspended to an OD470 = 1.0. A volume of 1.5 ml of this bacterial suspension was mixed with 0.5ml of n-hexadecane and incubated during 10 min at 37uC. After shaking for 40 seconds, the tubes were settled to allow phases separation to occur. The partition was calculated as (12OD470 of water phase)/OD470 of the water phase. Western-blot analysis with monoclonal antibodies to the major Omps was carried out as described before [77]. DNA manipulations, construction of mutants and complementation Plasmid and chromosomal DNA were extracted with Qiaprep spin Miniprep (Qiagen GmbH, Hilden, Germany), and Ultraclean Microbial DNA Isolation kit (Mo Bio Laboratories) respectively. When needed, DNA was purified from agarose gels using Qiack Gel extraction kit (Qiagen). DNA sequencing analysis was performed by the Servicio de Secuenciacio´ n de DNA del Centro de Investigacio´n Me´dica Aplicada (Navarra, Spain). Primers were synthesized by Sigma-Genosys Ltd.. Searches for DNA and protein homologies were carried out using the NCBI (National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov) and the EMBL-European Bioinformatics Institute server (http://www.ebi.ac.UK/ebi_home.html). In addition, sequence data were obtained from The Institute for Genomic Research at http://www.tigr.org. Genomic sequences of B. melitensis 16M, B. abortus and B. suis were analyzed using the database of the L’Unite´ de Recherche en Biologie Mole´culaire (URBM, Namur, Belgium) (http://www.serine.urbm.fundp.ac.be/,seqbruce/GENOMES/ Brucella_melitensis). For constructing the BABDolsB mutant, oligonucleotides olsB-F1 (59 -CTTCTGTCATCGTCGCGTAG- 39) and olsB-R2 (59- GATGCGTCCCAGAATGATG-39) were used to amplify a 270-bp fragment including codons 1 to 39 of the olsB ORF, as well as 153 bp upstream of the olsB first putative start codon, and oligonucleotides olsB-F3 (59-CATCATTCTGGGACGCATCC- CAAAGGAAGCGATCAACAA-39) and olsB-R4 (59- TTAAAA- CCGGAACCGCTCTA- 39) were used to amplify a 294-bp fragment including codons 230 to 297 of the olsB ORF and 87-bp downstream of the olsB stop codon. Both fragments were ligated by overlapping PCR using oligonucleotides olsB-F1 and olsB-R4 for amplification, and the complementary regions between olsB-R2 and olsB-F3 for overlapping. The resulting fragment, containing the olsB deletion allele, was cloned into pCR2.1 (Invitrogen), to PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16030