Freund oder Feind? — Die zwei Gesichter von Photorhabdus luminescens

Photorhabdus luminescens is an enteric bacterium with two faces: on the one hand these bacteria live in symbiosis with soil nematodes, on the other hand they are highly pathogenic for insects. The population is also phenotypically heterogeneous: one part lives in symbiosis with nematodes, the other part symbiotically interacts with plants. Cell-cell communication, inter-kingdom signaling, and other gene regulation processes are behind the complex decision of being friend or foe.

LitR directly upregulates autoinducer synthesis and luminescence in Aliivibrio logei

LitR is a master-regulator of transcription in the ainS/R and luxS/PQ quorum sensing (QS) systems of bacteria from Vibrio and Aliivibrio genera. Here, we for the first time directly investigated the influence of LitR on gene expression in the luxI/R QS system of psychrophilic bacteria Aliivibrio logei. Investigated promoters were fused with Photorhabdus luminescens luxCDABE reporter genes cassette in a heterological system of Escherichia coli cells, litR A. logei was introduced into the cells under control of Plac promoter. LitR has been shown to upregulate genes of autoinducer synthase (luxI), luciferase and reductase (luxCDABE), and this effect doesn’t depend on presence of luxR gene. To a much lesser degree, LitR induces luxR1, but not the luxR2 — the main luxI/R regulator. Enhanced litR expression leads to an increase in a LuxI-autoinducer synthesis and a subsequent LuxR-mediated activation of the luxI/R QS system. Effect of LitR on luxI transcription depends on lux-box sequence in luxI promoter even in absence of luxR (lux-box is binding site of LuxR). The last finding indicates a direct interaction of LitR with the promoter in the lux-box region. Investigation of the effect of LitR A. logei on luxI/R QS systems of mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio salmonicida showed direct luxR-independent upregulation of luxI and luxCDABE genes. To a lesser degree, it induces luxR A. fischeri and luxR1 A. salmonicida. Therefore, we assume that the main role of LitR in cross-interaction of these three QS systems is stimulating the expression of luxI.

Thermal stable and proteinase-K resistant insecticidal toxins produced by Photorhabdus luminescens

Photorhabdus is lives in a mutualistic association with nematodes from the family Heterorhabditis. Bacteria of the Photorhabdus can survive independently and cause toxicity in a larger variety of insects. In the present study, insecticidal activity of non-portentous heat-stable metabolites of Photorhabdus luminescens was evaluated against Galleria mellonella. For this purpose, the culture extract of P. luminescens was injected into the G. mellonella larvae, which killed almost 90% of larvae within 48 h. The extract showed 100% insecticidal activity after heat treatment of 70 C for 30 min and even 60% and 40% activity lasted at 80 C and 90 C respectively. The extract also showed a high degree of thermal stability and was 100% actives after 60 min at 70 C. In addition, insecticidal activity was preserved up to 100% after all proteinase-K treatments (0 ?g/mL to 50 ?g/mL). The results revealed that the extracts were non-portentous and showed high thermal resistance and stability. Keywords: Photorhabdus, insecticidal activity, toxins, heat stable non-proteinaceous

Two novel XRE-like transcriptional regulators control phenotypic heterogeneity in Photorhabdus luminescens cell populations

Background The insect pathogenic bacterium Photorhabdus luminescens exists in two phenotypically different forms, designated as primary (1°) and secondary (2°) cells. Upon yet unknown environmental stimuli up to 50% of the 1° cells convert to 2° cells. Among others, one important difference between the phenotypic forms is that 2° cells are unable to live in symbiosis with their partner nematodes, and therefore are not able to re-associate with them. As 100% switching of 1° to 2° cells of the population would lead to a break-down of the bacteria’s life cycle the switching process must be tightly controlled. However, the regulation mechanism of phenotypic switching is still puzzling. Results Here we describe two novel XRE family transcriptional regulators, XreR1 and XreR2, that play a major role in the phenotypic switching process of P. luminescens. Deletion of xreR1 in 1° or xreR2 in 2° cells as well as insertion of extra copies of xreR1 into 2° or xreR2 into 1° cells, respectively, induced the opposite phenotype in either 1° or 2° cells. Furthermore, both regulators specifically bind to different promoter regions putatively fulfilling a positive autoregulation. We found initial evidence that XreR1 and XreR2 constitute an epigenetic switch, whereby XreR1 represses xreR2 expression and XreR2 self-reinforces its own gene by binding to XreR1. Conclusion Regulation of gene expression by the two novel XRE-type regulators XreR1 and XreR2 as well as their interplay represents a major regulatory process in phenotypic switching of P. luminescens. A fine-tuning balance between both regulators might therefore define the fate of single cells to convert from the 1° to the 2° phenotype.

Effect of agitation speed on the density of bacteria Photorhabdus luminescens and the population dynamics of nematodes Heterorhabditis megidis in liquid culture

Liquid culture is the most scalable technology for the industrial production of entomopathogenic nematodes. Variability of the recovery after inoculation into cultures of Photorhabdus luminescens remains a persistent problem in the mass production of Heterorhabditis sp. In order to enhance infective juvenile (IJ) recovery and improve nematode population management, we analysed the correlation between the nematode Heterorhabditis megidis (strain KV – 136) development in liquid cultures, the density of bacteria of P. luminescens and the culture agitation speed. Analyses focused on the impact of different agitation speeds (160 rpm and 200 rpm) on the dynamics of population growth of H. megidis in liquid cultures at constant biotic and abiotic parameters (initial dose of nematodes introduced to the culture 2300 IJs/ml, temperature 25°C, the number of bacterial colonies 0.3 × 107/ml). The performed experiments showed that the agitation speed of 200 rpm favourably affected the density of bacteria of P. luminescens (24.14 × 107/ml). High density of bacteria at this agitation speed resulted in an earlier (on the fifth day of the culture) maximum increase in the number of hermaphroditic individuals (1239.6 H/ml) than in the culture at an agitation speed of 160 rpm.

In vivo production of entomopathogenic nematodes suppressed by asymptomatic bacterial contaminants

Summary Entomopathogenic nematodes (EPN) are excellent biological control agents possessing recycling ability as one of their major attributes. We report the presence of asymptomatic bacteria that can lead to disrupted or low progeny production in Heterorhabditis indica. In a one-to-one in vitro competitive bioassay with contaminants associated with H. indica cuticle, there was a significant suppression in the growth of Sphingomonas koreensis when stressed with the nematode symbiont Photorhabdus luminescens; however, P. luminescens was suppressed when sandwiched between Ochrobactrum anthropi. Bacillus bombysepticus associated with laboratory-reared Galleria when stressed by P. luminescens was significantly suppressed, but not so in the reverse assay. Both O. anthropi and B. bombysepticus were found to be insecticidal to Galleria larvae when fed orally. Tripartite interactive studies on the growth and multiplication of H. indica-P. luminescens symbionts in Galleria larvae, predisposed to S. koreensis, revealed no significant difference initially in the hermaphrodite formation, but subsequently there was a significant decline in the formation of amphimictic females and the final production of infective juveniles. In in vitro studies, none of the contaminants supported the growth and development of axenic H. indica. Adequate precautions should be taken to maintain proper hygiene to eliminate such contaminants while culturing the Galleria and EPN for use in the biological control of insects.