High-throughput sequencing analysis reveals genomic similarity in phenotypic heterogeneous Photorhabdus luminescens cell populations

Purpose Phenotypic heterogeneity occurs in many bacterial populations: single cells of the same species display different phenotypes, despite being genetically identical. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens is an excellent example to investigate bacterial phenotypic heterogeneity. Its dualistic life cycle includes a symbiotic stage interacting with entomopathogenic nematodes (EPNs) and a pathogenic stage killing insect larvae. P. luminescens appears in two phenotypically different cell forms: the primary (1 degrees) and the secondary (2 degrees) cell variants. While 1 degrees cells are bioluminescent, pigmented, and produce a huge set of secondary metabolites, 2 degrees cells lack all these phenotypes. The main difference between both phenotypic variants is that only 1 degrees cells can undergo symbiosis with EPNs, a phenotype that is absent from 2 degrees cells. Recent comparative transcriptome analysis revealed that genes mediating 1 degrees cell-specific traits are modulated differently in 2 degrees cells. Although it was previously suggested that heterogeneity in P. luminescens cells cultures is not genetically mediated by, e.g., larger rearrangements in the genome, the genetic similarity of both cell variants has not clearly been demonstrated yet. Methods Here, we analyzed the genomes of both 1 degrees and 2 degrees cells by genome sequencing of each six single 1 degrees and 2 degrees clones that emerged from a single 1 degrees clone after prolonged growth. Using different bioinformatics tools, the sequence data were analyzed for clustered point mutations or genetic rearrangements with respect to the respective phenotypic variant. Result We demonstrate that isolated clones of 2 degrees cells that switched from the 1 degrees cell state do not display any noticeable mutation and do not genetically differ from 1 degrees cells. Conclusion In summary, we show that the phenotypic differences in P. luminescens cell cultures are obviously not caused by mutations or genetic rearrangements in the genome but truly emerge from phenotypic heterogeneity.