UNRAVELING Enterococcus mundtii CRL35 BEHAVIOUR IN A CHEMICALLY DEFINED MEDIUM SUPPLEMENTED WITH MEAT PROTEINS

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

San Miguel de Tucumán

Simposio; International Symposium on Lactic Acid Bacteria; 2016

CERELA_CONICET

Numerous studies have shown that enterococci of diverse origin are highly competitive inthe meat environment as a consequence of their intrinsic properties. They are able to displaya wide array of enzymatic activities such as lipolysis, glycolysis and proteolysis. In thelatter case it was previously described that meat proteases (cathepsin D-like) seems to beinvolved in the first step of protein degradation and that microbial enzymes, belonging tostrains of different genres, may act directly over the produced oligopeptides splitting themintracellularly into small peptides and free amino acids. Recent in silico studies, performedby our group, evidenced that E. mundtii CRL35 (CRL35) has no genes coding for the cell wallassociated proteinase PrtP, but clusters of genes involved in the oligopeptide and di/tripeptidetransport system, besides a large set of genes for aminopeptidases and endopeptidases,were localized. In this context, the aim of this work was to unravel which metabolic processesare triggered during CRL35 growth in the presence of meat proteins. For this purpose, CRL35was grown in a chemically defined medium (CDM) supplemented with the myofibrillar andsarcoplasmic meat protein fractions (CP), at 25 ºC for 12 h, using a non-supplemented CDMas a control. Next, the proteome changes under the influence of meat proteins, in the CRL35cell free extracts, were studied through two-dimensional polyacrylamide gel electrophoresis(2DE). Those significantly regulated proteins were first identified with the Prodigy SameSpotssoftware and then determined by MALDI TOF MS/MS analyses. As a result, 9 differentiallyexpressed proteins, when comparing the growth in CDM and CP, were successfully identified.From them, 7 were overexpressed in CP and were related to the glycolysis and gluconeogenesispathway (pyruvate kinase, type I glyceraldehyde-3-phosphate dehydrogenase and6-phosphofructokinase), the alanine, aspartate and glutamate metabolism (adenylosuccinatesynthase), the catabolite control protein A and the elongation factor G. On the otherhand, 2 proteins were down-regulated in CP, the 3-deoxy-7-phosphoheptulonate synthase ofthe phenylalanine, tyrosine and tryptophan biosynthesis pathway, and the dihydrolipoamideacetyltransferase. Parallel analysis, by one-dimensional SDS gel electrophoresis of the culturesupernatant, showed no proteolysis of the myofibrillar and sarcoplasmic proteins; andno apparent cell lysis was evidenced by measurements of the lactate dehydrogenase (LDH)activity. In conclusion, CRL35 was not able to proteolyze meat proteins, and a few glycolyticgenes and a master regulator of carbon metabolism were overexpressed when it grew in theCP medium. At present, more studies are in progress to deepen on the effect of meat environmenton CRL 35 metabolism.