Characterization of the EPS clulster involved in the production of exopolysaccharides (EPS) OF Lactobacillus fermentum Lf2 and construction of the knockout mutant for the priming.

ALE, E.; BOURIN, M.; HARRIS, H.; REINHEIMER, J. A.; BINETTI, A. G.; O´TOOLE, P.

Mar del Plata

Congreso; XVI Congreso Argentino de Ciencia y Tecnología de Alimentos (CYTAL, AATA); 2017

AATA

Lactobacillus fermentum Lf2 is a strain which belongs to the INLAIN collection and it is of interest because of the high EPS amount (around 1 g/L) it produces in controlled pH and temperature conditions. Besides, this EPS was associated with immunomodulatory activities when provided in yogurt, and it was able to improve the consistency and hardness of yogurt. The genome of this strain was sequenced at the University College Cork, Ireland. Genomic DNA was purified from a single colony purified culture. The genome was sequenced on an Ion Torrent PGM platform, generating 1 million of reads (2x300 bp read lengths). De novo assembly of the trimmed, quality-filter reads were performed using Mira. The un-biased de novo assembly was then aligned to all available complete and draft L. fermentum genomes. Initial automated gene calling was performed using Glimmer 3and Genemark. Intergenic regions were examined for missed gene calls using BlastXtract. tRNAs were identified using tRNA-scan and ribosomal binding sites using RBSfinder. Protein sequences of each gene product were researched against a variety of databases with the aim of assigning a functional annotation. All predicted proteins were searched (BLASTP) against the NCBI-non-redundant protein database and, through Interproscan, against the pFAM, TigrFAM, PIR, HAMAP, PROSITE, PRINTS, PRODOM, PANTHER, SUPERFAMILY, GENE3D databases. In addition, transmembrane domains were identified with TMHMM and Signal peptides with SignalP. The automated annotation was manually curated in Artemis. Comparison to the reference complete genomes helped to identify EPS operons that may span contig boundaries in the draft L. fermentum Lf2 genome assembly. The number of contigs obtained was 272, the nucleotide coverage and the N50 were 58.1 and 32,605, respectively, and the percentage of reads used on assembly was 84.8. The isogenic mutant was obtained by insertional inactivation using a two plasmid system. Briefly, the upstream and downstream regions of the priming glycosyltransferase (PGT) were cloned into pORI19, and a transformation into L. fermentum Lf2 containing pVE6007 was done. Cells were grown at 42ºC to select the pORI19 construction only (pVE6007 replication unstable), and integration of this plasmid into the chromosome was allowed. After several passages at 37ºC, the second crossover took place and the knockout was obtained. This was confirmed by sequencing using specific primers. A bidirectional EPS gene cluster was identified (with 8 and 7 ORFs, respectively); both parts are distant and present a potential priming PGT (ORF_119 and ORF_1945, respectively), fact that could explain why the mutant with ORF_119 deleted still presents ropy phenotype. Another possible cluster was identified downstream as well, including 5 ORFs. BLAST analysis against the NCBI database was performed in order to study the presence and synteny of these genes in other Lactobacillus species, doing first a preselection of those which presented the ORF_1945 or ORF_119 with a cut-off value of 70% identity. Constructing the isogenic mutant would be useful in future assays about functionality, and it would be interesting to delete both PGTs to evaluate if the strain loses its ability to produce EPS.Keywords: exopolyssacharides, Lactobacillus fermentum, gene cluster, knockout.