Halobacillus trueperi as major active microbial component with high biotechnological potential in extreme saline system of Southern Tunisia

Several bacterial and archeal species have evolved and adapted to extreme hypersaline environments. In particular spore-forming bacteria, although generally regarded as deposit spores and not active cells, have been reported to be the most abundant habitants of these ecological niches. In this work, we assessed the phylogenetic diversity and biotechnological potential of a collection of 336 spore-forming isolates recovered from six salt saturated brines (Chott and Sebkhas) located in the hyper-arid regions of Southern Tunisia. Requirement and abilities for growth at a wide range of salinities from 0 to 30% (w/v) of NaCl, showed that 45% of the isolates were extremely halotolerant (15% NaCl), among them, 8 strains were able to grow at 20% NaCl. Only 27% of the isolates were strict halophiles and were not able to grow in the absence of NaCl. Phylogenetic diversity was analyzed basing on the amplification of the internal transcribed spacers between the 16S and 23S rRNA genes (ITS) and by 16S rRNA sequencing. A total of 65 distinct ITS haplotypes were detected on agarose matrix. Partial 16S rRNA gene sequencing performed on 78 isolates showed high level of identity with known sequences of spore-formers except for 4 isolates that had a relatively low sequence identity (95-97%) and that potentially represent new species. Isolates were clustered into seven genera: Bacillus, Gracilibacillus, Halobacillus, Oceanobacillus, Paenibacillus, Pontibacillus, and Virgibacillus. Bacillus genus was the most abundant, representing 62% of the isolates. The second group was represented by Halobacillus genus (19.3%), mainly Halobacillus licisalis and the Halobacillus trueperi/H. litoralis group (99% of identity in 16S rRNA genes). Halobacillus trueperi was the most encountered species in all the sampled sites, particularly 42 isolates showing haplotype ITS-25. The intraspecific diversity of this sub-collection was further assessed by BOX-PCR fingerprinting and generated 14 genotypes which indicate a high level of microdiversity. These results suggest that H. trueperi constitute one of the major active microbial components adapted to the harsh desert environment and saline systems. Besides quenching quorum sensing, the H. trueperi strains isolated in this study were also shown to possess several hydrolytic activities (amylase, cellulase, DNase and lipase) together with high antagonistic potential against phytopathogenic fungi. These biotechnological traits make H. trueperi species a good candidate for application in plant growth promotion (PGPR) in salty soils, one of the major agriculture problems in southern Mediterranean countries