HexA is a versatile regulator involved in the control of phenotypic heterogeneity of Photorhabdus luminescens.

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.

One-shot NMR analysis of microbial secretions identifies highly potent proteasome inhibitor.

Natural products represent valuable lead structures for drug discovery. However, for most bioactive compounds no cellular target is yet identified and many substances predicted from genome analysis are inaccessible due to their life stage-dependent biosynthesis, which is not reflected in common isolation procedures. In response to these issues, an NMR-based and target-directed protease assay for inhibitor detection of the proteasome was developed. The methodology is suitable for one-shot identification of inhibitors in conglomerates and crude culture broths. The technique was applied for analysis of the different life stages of the bacterium Photorhabdus luminescens, which resulted in the isolation and characterization of cepafungin I (CepI), the strongest proteasome inhibitor described to date. Its biosynthesis is strictly regulated and solely induced by the specific environmental conditions determined by our methodology. The transferability of the developed technique to other drug targets may disclose an abundance of novel compounds applicable for drug development.

A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states.

Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

Stability of entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus luminescens, during in vitro culture.

The entomopathogenic nematode-bacteria complexes Heterorhabditis bacteriophora/Photorhabdus luminescens and Steinernema carpocapsae/Xenorhabdus nematophila are mass produced for use as biological insecticides. Stability of the bacterial partner in culture is essential for maintaining traits important for both biological control and production. Two geographically distinct strains of each bacterial species were isolated from their nematode partners and serially subcultured on in vitro media to assess trait stability. Subculturing resulted in a shift to secondary cell production in one P. luminescens strain and both X. nematophila strains within ten in vitro culture cycles. However, when cell phenotypic variation was controlled in X. nematophila strains by regular selection for primary variants, no trait change was detected in the primary variant after prolonged subculture. When P. luminescens cell phenotypic variation was controlled by selection for primary variants, changes in the primary variant of both strains were noted including reductions in cell and inclusion body size and inclusion body prevalence. Bacterial ability to cause lethal infections following injection into the hemocoel of Tenebrio molitor larvae declined by more than half in primary variants of one P. luminescens strain. Conversely, yield was enhanced, with the subcultured P. luminescens strains showing 53.5 and 75.8% increases in primary cell density. Field adapted traits of primary variant P. luminescens strains tend to deteriorate during in vitro culture as tradeoffs for gains in yield. In vitro producers of the P. luminescens/H. bacteriophora complex must weigh the need for superior bacterial yield against the need to preserve traits important for biological control.