Phenotypic heterogeneity in microbial communities enables genetically identical organisms to behave differently even under the same environmental conditions. Photorhabdus luminescens, a bioluminescent Gram-negative bacterium, contains a complex life cycle, which involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic cell types, designated as the primary (1°) and secondary (2°) cells. The 1° cells are bioluminescent, pigmented and can support nematode growth and development. Individual 1° cells undergo phenotypic switching after prolonged cultivation and convert to 2° cells, which lack the 1° specific phenotypes. The LysR-type regulator HexA has been described as major regulator of this switching process. Here we show that HexA controls phenotypic heterogeneity in a versatile way, directly and indirectly. Expression of hexA is enhanced in 2° cells, and the corresponding regulator inhibits 1° specific traits in 2° cells. HexA does not directly affect bioluminescence, a predominant 1° specific phenotype. Since the respective luxCDABE operon is repressed at the post-transcriptional level and transcriptional levels of the RNA chaperone gene hfq are also enhanced in 2° cells, small regulatory RNAs are presumably involved that are under control of HexA. Another phenotypic trait that is specific for 1° cells is quorum sensing mediated cell clumping. The corresponding pcfABCDEF operon could be identified as the first direct target of HexA, since the regulator binds to the pcfA promoter region and thereby blocks expression of the target operon. In summary, our data show that HexA fulfills the task as repressor of 1° specific features in 2° cells in a versatile way and gives first insights into the complexity of regulating phenotypic heterogeneity in Photorhabdus bacteria.

BackgroundPhotorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR.ResultsUnexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes.ConclusionsAccording to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5121-z) contains supplementary material, which is available to authorized users.

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°) and the secondary (2°) cell variants. While 1° cells are bioluminescent, pigmented, and produce a huge set of secondary metabolites, 2° cells lack all these phenotypes. The main difference between both phenotypic variants is that only 1° cells can undergo symbiosis with EPNs, a phenotype that is absent from 2° cells. Recent comparative transcriptome analysis revealed that genes mediating 1° cell-specific traits are modulated differently in 2° 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° and 2° cells by genome sequencing of each six single 1° and 2° clones that emerged from a single 1° 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° cells that switched from the 1° cell state do not display any noticeable mutation and do not genetically differ from 1° 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.