High Metabolomic Microdiversity within Co-Occurring Isolates of the Extremely Halophilic Bacterium Salinibacter ruber

Salinibacter ruber is an extremely halophilic member of the Bacteroidetes that thrives in crystallizer ponds worldwide. Here, we have analyzed two sets of 22 and 35 co-occurring S. ruber strains, newly isolated respectively, from 100 microliters water samples from crystalizer ponds in Santa Pola and Mallorca, located in coastal and inland Mediterranean Spain and 350 km apart from each other. A set of old strains isolated from the same setting were included in the analysis. Genomic and taxonomy relatedness of the strains were analyzed by means of PFGE and MALDI-TOF, respectively, while their metabolomic potential was explored with high resolution ion cyclotron resonance Fourier transform mass spectrometry (ICR-FT/MS). Overall our results show a phylogenetically very homogeneous species expressing a very diverse metabolomic pool. The combination of MALDI-TOF and PFGE provides, for the newly isolated strains, the same scenario presented by the previous studies of intra-specific diversity of S. ruber using a more restricted number of strains: the species seems to be very homogeneous at the ribosomal level while the genomic diversity encountered was rather high since no identical genome patterns could be retrieved from each of the samples. The high analytical mass resolution of ICR-FT/MS enabled the description of thousands of putative metabolites from which to date only few can be annotated in databases. Some metabolomic differences, mainly related to lipid metabolism and antibiotic-related compounds, provided enough specificity to delineate different clusters within the co-occurring strains. In addition, metabolomic differences were found between old and new strains isolated from the same ponds that could be related to extended exposure to laboratory conditions.