Characterization of the gen locus involved in β-1,6-oligosaccharide utilization by Enterococcus faecalis.

Abstract

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-β-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β-1,6- rather than β-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.