Genetic diversity of the citrate degradative pathway in Enterococcus faecium

INGRID QUINTANA; FLORENCIA EBERHARDT; CRISTIAN A. SUÁREZ; MARTÍN ESPARIZ; CHRISTIAN MAGNI

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General (SAMIGE).; 2012

Enterococci are considered one of the major opportunistic multidrug intrahospitalary nosocomial pathogens. However, these bateria as well as other lactic acid bateria (LAB) may play an important beneficial role on the production of various traditional fermented food products such us flavour development. Citrate metabolism depends on the presence of different citrate transporters which allow the specific uptake. In all cases the citrate lyase complex, which split citrate into oxaloacetate and acetate, is essential for citrate utilization by LAB. Enterococcus faecium is present in commercial and artisanal dairy products (cheeses) contributing to organoleptic features. They are poor acidifiers, and also have low proteolytic activities but they can produce bacteriocins or have probiotic properties. The capacity to ferment citrate by E. faecium was previously studied but little is known about the genetic organization of the genes involved in this pathway. We have started our studies with a comparative genomic analysis which lead us to thirty one strains of E. faecium previously sequenced. Five of these strains show a genetic organization corresponding to the CitI cluster. In this cluster there are two divergent operons; one with the transcriptional activator CitI in one direction and the other operon with the soluble oxaloacetate decarboxylase (citM), the citrate transporter (2HCT family) and genes codifying the citrate lyase complex (citDEF) in the other direction. We also found five strains with the CitO cluster which consists as well of two divergent operons but in this case with the citrate transporter (CitHM family) and the transcriptional activator CitO in one direction and the genes for the citrate lyase complex (citDEF) associated to membrane oxaloacetate decarboxylase genes (oadHABD) in the other. Finally, twenty one strains were found which belong to cit – cluster, where the genes codifying the citrate lyase complex are absent. To start our molecular characterization, seventeen E. faecium strains isolated from clinical samples and commercial and artisanal cheeses were analyzed with degenerate citrate lyase primers by PCR. Ten of these strains threw negative results, therefore we positioned them in the cit – cluster group. Among the remainig seven strains, six were positive for primer specific CitI PCR and the other was negative for both primer specific CitI and CitO PCR. Consequently, this last strain is a good candidate to be a member of the CitO cluster group, but further studies will confirm this hypothesis. The bioinformatic analysis on the gene involved in the C4 route was performed. The presence of a putative alsSD operon was identified encoding for the a-acetolactate synthetase and a-acetolactate decarboxylase gene respectively. The capacity of production of C4 compounds (acetoin and diacetyl) was analyzed and our data shown that E faecalis citI+ is able to increase the amounts of these compounds in the presence of citrate.