The molecular mechanisms underlying hidden phenotypic variation among metallo-ß-lactamases

Abstract Genetic variation among orthologous genes has been largely formed through neutral genetic drift to maintain the same functional role. In some circumstances, however, this genetic variation can create critical phenotypic variation, particularly when genes are transferred to a new host by horizontal gene transfer (HGT). Unveiling “hidden phenotypic variation” through HGT is especially important for genes that confer resistance to antibiotics, which continue to disseminate to new organisms through HGT. Despite this biomedical importance, our understanding of the molecular mechanisms that underlie hidden phenotypic variation remains limited. Here we sought to determine the extent of hidden phenotypic variation in the B1 metallo-β-lactamase (MBL) family, as well as to determine its molecular basis by systematically characterizing eight MBL orthologs when they are expressed in three different organisms ( E. coli, P. aeruginosa, and K. pneumoniae ). We found that these MBLs confer diverse levels of resistance in each organism, which cannot be explained by variation in catalytic efficiency alone; rather, it is the combination of the catalytic efficiency and abundance of functional periplasmic enzyme that best predicts the observed variation in resistance. The level of functional periplasmic expression varied dramatically between MBL orthologs and between hosts. This was the result changes at multiple levels of each enzyme’s functional: 1) the quantity of mRNA; 2) the amount of MBL expressed; and 3) the efficacy of functional enzyme translocation to the periplasm. Overall, we see that it is the interaction between each gene and the host’s underlying cellular processes (transcription, translation, and translocation) that determines MBL genetic incompatibility thorough HGT. These host-specific processes may constrain the effective spread and deployment of MBLs to certain host species, and could explain the current observed distribution bias. Author Summary Orthologous genes spread among different organisms, typically maintaining the same functional role within the cell while accumulating some, presumably functionally-inert, genetic variation over time. However, these seemingly neutral gene sequence changes among orthologs can be revealed to have substantial difference in protein phenotypes, and thus, organismal fitness, when they are transferred to other host species. This so-called “hidden phenotypic variation” through horizontal gene transfer may play an important role in dissemination of antibiotic resistance genes, in particular. In this work, we systematically investigated the extent of phenotypic variation in eight orthologous antibiotic resistant genes from the metallo-β-lactamases family (MBLs), and identified the molecular causes underlying the observed phenotypic variation. We found that functional protein expression varied substantially among MBLs (causing significant variation in the level of antibiotic resistance conferred), and that this could not be explained by variation in catalytic efficiency alone. Instead, we see that functional variation is caused by multiple steps in the protein production, transcription, translation and translocation, that are necessary to provide functional enzymes in the bacterial periplasm. Thus, the successful gene transfer and dissemination of antibiotic resistance genes can be determined by complex interactions between the gene and host underlying cellular processes.