Subject(s)
Genetic Engineering/methods , Genetic Therapy/methods , Genetics/education , Genomics/education , Information Dissemination/methods , Interdisciplinary Communication , Research/organization & administration , Delivery of Health Care/methods , Delivery of Health Care/organization & administration , Genetic Therapy/trends , Genomics/methods , Genomics/trends , United KingdomABSTRACT
Enterococcus flavescens CCM 439 is phenotypically similar to Enterococcus casseliflavus; it possesses intrinsic low-level resistance to vancomycin and has the VanC phenotype. The complete vanC-3 vancomycin resistance gene cluster was cloned and sequenced, and found to contain five open reading frames. These encoded five proteins that displayed a high degree of amino acid identity to the proteins of the vanC-2 cluster of E. casseliflavus. The serine racemases displayed the lowest degree of identity (97%), whereas the response regulators VanR(C-2) and VanR(C-3) were 100% identical. Long-PCR-RFLP analysis of the vanC-3 and vanC-2 gene clusters distinguished E. flavescens CCM 439 from E. casseliflavus ATCC 25788 due to the absence of a single EcoRV restriction endonuclease site from the E. flavescens gene cluster. However, the lack of nucleotide divergence between the sequences of the vanC-2 and vanC-3 clusters casts doubt on the validity of E. flavescens and E. casseliflavus being classed as distinct species.
Subject(s)
Bacterial Proteins/genetics , Enterococcus/genetics , Multigene Family/genetics , Vancomycin Resistance/genetics , Bacterial Proteins/isolation & purification , Enterococcus/isolation & purification , Humans , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Polymorphism, Restriction Fragment LengthABSTRACT
The vanC-2 cluster of Enterococcus casseliflavus ATCC 25788 consisted of five genes (vanC-2, vanXY(C-2), vanT(C-2), vanR(C-2), and vanS(C-2)) and shared the same organization as the vanC cluster of E. gallinarum BM4174. The proteins encoded by these genes displayed a high degree of amino acid identity to the proteins encoded within the vanC gene cluster. The putative D,D-dipeptidase-D,D-carboxypeptidase, VanXY(C-2), exhibited 81% amino acid identity to VanXY(C), and VanT(C-2) displayed 65% amino acid identity to the serine racemase, VanT. VanR(C-2) and VanS(C-2) displayed high degrees of identity to VanR(C) and VanS(C), respectively, and contained the conserved residues identified as important to their function as a response regulator and histidine kinase, respectively. Resistance to vancomycin was expressed inducibly in E. casseliflavus ATCC 25788 and required an extended period of induction. Analysis of peptidoglycan precursors revealed that UDP-N-acetylmuramyl-L-Ala-delta-D-Glu-L-Lys-D-Ala-D-Ser could not be detected until several hours after the addition of vancomycin, and its appearance coincided with the resumption of growth. The introduction of additional copies of the vanT(C-2) gene, encoding a putative serine racemase, and the presence of supplementary D-serine in the growth medium both significantly reduced the period before growth resumed after addition of vancomycin. This suggested that the availability of D-serine plays an important role in the induction process.
