UNRAVELING Enterococcus mundtiiCRL35 BEHAVIOUR IN A CHEMICALLY DEFINED MEDIUM SUPPLEMENTED WITH MEAT PROTEINS

ORIHUEL, A; BONACINA, J.; FADDA S; SAAVEDRA L

San Miguel de Tucuman

Simposio; V Simposio Internacional de Bacterias Lácticas-SIBAL CERELA- CONICET; 2016

CERELA CONICET

Numerous studies have shown that enterococci of diverse origin are highly competitive in the meat environment as a consequence of their intrinsic properties. There they are able to display a wide array of enzymatic activities such as lipolysis, glycolysis and proteolysis. In the latter case it was previously described that meat proteases (cathepsin D-like) seems to be involved in the first step of protein degradation step and that microbial enzymes, belonging to strains of different genres, may act directly over the produced oligopeptides splitting them intracellularly into small peptides and free amino acids. Recent in silico studies, performed by our group,evidenced that E.mundtii CRL35 (CRL35) has no genes coding for the cell wall associated proteinase PrtP, but clusters of genes involved in the oligopeptides and di/tripeptides transportation, besides a large set of genes for aminopeptidases and endopeptidases were registered.In this context, the aim of this work was to unravel which metabolic processes are triggered during CRL35 growth in the presence of meat proteins.For this purpose, E. mundtii CRL35 was grown in a chemically defined medium (CDM) supplemented with the myofibrillar and sarcoplasmic meat protein fractions (CP), at 25ºC for 12 h (late log phase), using a non-supplemented CDM as a control. Next, the proteome changes under the influence of meat proteins, in the CRL35 cell free extracts, were studied through two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) according to Fadda et al.Those significantly regulated proteins were first localized with the Prodigy SameSpots software and then identified by MALDI TOF/ TOF MS/MS analyses. As a result, 12 differentially expressed proteins, when comparing the growth in CDM and CP, were found. Of From them, 9 9 were successfully identified. Nine were overexpressed in CP and were assigned to different metabolic pathway such as glycolysis and gluconeogenesis (and included: a pyruvate kinase, a type I glyceraldehyde-3-phosphate dehydrogenase type I and a 6-phosphofructokinase, all assigned to the glycolysis and gluconeogenesis pathway, and and alanine, aspartate and glutamate metabolism (a catabolite control protein A, an elongation factor G, and an adenylosuccinate synthase) that could be related to alanine, aspartate and glutamate metabolism. The 2 remaining spots could not be determined. On the other hand, only 3 proteins were under regulated in CP, the dihydrolipoamide acetyltransferase, the 3-deoxy-7-phosphoheptulonate synthase of the pPhenylalanine, tyrosine and tryptophan biosynthesis pathway, and another one that could not be identified. A pParallel analysis by one-dimensional SDS gel electrophoresis of the culture supernatant showed no proteolysis of the myofibrillar and sarcoplasmic proteins; or apparent cell lysis was evidenced by measurements of the lactate dehydrogenase (LDH) activity. In conclusion, and according to our previous results, CRL35 was not able to proteolyze meat proteins, and a few glycolytic genes and regulators are differentially expressed when growing in their presence.