Photorhabdus Luminescens Bacteria: Discovery, Life Cycle, Phenotypes, And Biosynthesis Of Secondary Metabolites

Discovery of Photorhabdus

Discuss about the Photorhabdus luminescens Bacteria.

Photorhabdus luminescens bacteria, belongs to the family Enterobactriaceae, and acts as a lethal pathogen for the insects. The bacteria reside in the gut of the nematode of family Heterorhabditidae, in a symbiotic association (Murfin et al, 2012). The life cycle of these bacteria shows a strange switch between virulent and the avirulent form, depending upon the surrounding environment. The bacteria also produce several broadspectrum antibiotics. Extensive researches are going on to understand the biosynthetic mechanism of the secondary metabolites secreted by the bacteria. The driving force behind the extensive researches is because of its application in agriculture and pest control (Ruiu, 2015). It has also found its application in the research of pharmaceuticals.

This report gives idea about the discovery of these bacteria, the different phenotypes and the different genes responsible for the different phenotypes. This report also throws light upon the different regulatory mechanisms of the operons and genes that helps the bacteria to switch between a mutualistic form and a virulent form. The latter part of the report also focuses upon the biosynthetic mechanisms of secondary metabolite produced by the bacteria, like Stilbene, Anthraquinone and antibiotic called Carbapenem. Further the report also aims at discussing the usage Nematodal formulations in the field to destroy the population of harmful insects.

Several databases have been gone through to get information about the discovery of Photorhabdus bacteria. The civil war left many soldiers wounded, dead, and impaired. The bayonets and the bullets did enough injury, but the soldiers of that era were also prone to infections. The wounds got contaminated with dirt and made a suitable environment for the growth of microbes. Some soldiers could not fight the battle of their life due to lack of medical resources. Few soldiers who waited in the mud out in the rain found their wounds are glowing in the dark. After they had been shifted to the hospital, it was found that the soldiers with glowing wound had a better survival rate than the other soldiers with unilluminated wounds. The protective effect of the strange and mysterious glow earned a nickname “Angel’s glow.”

In 2001, almost after one forty years after the battle, a seventeen year old while visiting the Shiloh battlefield with his family heard about the glowing wound. His mother was a microbiologist who had been doing research on the luminescent bacteria in the soil. They predicted that the luminescent bacteria that her mom had been studying had some connections with the Angel’s glow. It was studied that the bacteria lived in the gut of the nematodes share a strange life cycle of an avirulent symbiotic phase and a virulent phase. It was predicted that the weather and the condition of the soil was suitable for the growth of Photorhabdus bacteria. Although, they cannot survive in the normal body temperature, it can be predicted that the temperature at night would have been low enough to give a suitable environment to the bacteria.

Phenotype of Photorhabdus luminescence

Phenotype of Photorhabdus luminescence

Heterogeneity in the Phenotypes in microbial communities facilitates organisms, that are genetically identical to behave in a different way even under identical environmental conditions. Photorhabdus luminescens, a bioluminescent Gram-negative bacterium, displays a strange life cycle, which involves a mutualistic relayion with nematodes as well as a pathogenic relation with insect larvae. There are two phenotypic cell types, the primary (1°) and secondary (2°) cells. The 1° cells are pigmented, bioluminescent and  grow inside the nematodes. Individual 1° cells can undergo switching of its phenotypes after prolonged cultivation and convert to 2° cells. The LysR-type regulator hexA has been reported to be the major regulator of this switching mechanism. It has been shown that hexA controls phenotypic heterogeneity, directly as well as indirectly. Expression of hexA is increased in 2° cells, and the corresponding regulator inhibits the 1° specific traits in 2° cells. hexA does not influence bioluminescence directly, a predominant 1° specific phenotype. Since the respective lux CD ABE operon is repressed at the post-transcriptional level and transcriptional levels, chaperone gene hfq also increases its expression in 2° cells. Another phenotypic trait that is specific for 1° cells is cell clumping, mediated by quorum sensing.

Bacteria belonging to the genus Photorhabdus, are found to be in symbiotic association with entopathologic nematode of family Steinernematidae and Heterorhabditidae (Clarke, 2014). These nematodes use these bacteria that live inside the gut of the nematodes for killing their hosts. After entering the hosts these nematodes release these bacteria in the host larvae. This causes killing of the hosts within 48 hours (Nielsen-LeRoux, 2012). The virulence factors defeats the immunity of the insect and the insect die Virulence factors like toxin complexes of large tripartite ABC-type. Tcs consists of TcA, TcB, TcC proteins. TcC proteins, ADP ribosyltransferases are responsible for the cell death in the insect larvae. Transportation of the TcC componants by the TcA and TcB componants is still not specifically understood. It has been found that TcA in P. luminescense forms a transient membrane pore and the proteins are inserted into the membrane by a syringe like mechanism (Gatsogiannis et al, 2013).  This allows the nematodes to survive and thrive in the insect body. The nematodes reproduce inside the carcass of the insect. The carcass of the larva provides enough nutrients for the nematodes to grow and multiply. The nematodes undergo a number of life cycles and finally the carcass that is infected burst to give numerous progenies of the nematodes (Easom and Clarke, 2012). These new progenies then start searching for new insect hosts and start the cycle all over again. These live nematodes are sometimes used as pest control agents (Tobias et al 2012).

Life cycle of Photorhabdus luminescence

Photorhabdus bacteria have got two faces.  A mutualistic face,  while living inside the gut of the nematodes and a pathogenic face, as they are introduced in to the host larvae. In order to ensure survival the bacteria had to adapt to the different environmental changes. They can switch between a pathogenic (P form) form and a symbiotic form (M form), that does not contain the virulence factors, shows less bioluminescence and are slow growing. An inversion in the promoter helps it to switch between two forms. It has been found that when the madswitch promoter located at upstream of the genes for the mad fimbriae is switched ON, the P.luminescence is said to be in the M form, when it can colonize inside the nematode gut.  When the madswitch promoter is switched OF, the bacteria then transform into its P-form, when it can infect the insects but it loses the capacity to colonize inside the nematodes (Orozco, Hill and Stock, 2013).

A review shows that, for testing the role of the madswitch promoter an experiment had been done where the madswitch promoter had been genetically locked to either the ON or OFF orientation.  P form of the bacteria, having deleted inverted repeats and mad R, was injected into the larvae of Galleria mellonella to detect the M form colonies. Deletion of the upstream inverted repeats locked the madswitch OFF. As a result the P form cannot switch its form and initiate mutualism. Conversely, on deleting the same repeat while the madswitch is ON, switched the P form to M-form colonies without any P-form sectors (Somvanshi et al, 2012).

Photorhabdus bacteria shows underexploited novel chemical structures. Over few decades, attention has been given on the toxins and small molecules produced by Photorhabdus. A lot of research had been done in this field because of their potential applications for pest management, as well as in Pharmaceutical industries (Vizcaino, Guo and Crawford, 2014). The natural products secreted by the bacteria are important components in crop protection. Several Photorhabdus protein products and their genes have been found as an alternative to Bacillus thuringenesis for the production of transgenic crops, although their application has not been pursued (Brachmann et al., 2012).

Stilbene biosynthesis

Photorhabdus shows a secondary metabolism that is essential for the mutualism between the bacteria and the nematode. An essential secondary metabolite is a stilbene molecule, known as ST. The initial step in ST biosynthesis involves the non-oxidative deamination of phenylalanine which results in the formation of cinnamic acid. Phenylalanine-ammonium lyase, which is an enzyme, encoded by the stlA gene, catalyses the reaction. Researches have shown that the expression of stlA is regulated by nutrient limitation via a regulatory mechanism that involves by 3 regulators. It has been found that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is required for stlA expression. It had also been found that σ^S and Lrp modulates the stlA expression. These are regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria (Lango-Scholey, 2013). This report describes a regulatory pathway for secondary metabolism in Photorhabdus and, therefore, the study provides an idea about the complex regulatory network that controls secondary metabolism and mutualism, in this organism (Lango et al., 2013)

Biosynthesis of secondary metabolites

Carbapenem biosyntheses

Photorhabdus bacteria produce many broad spectrum antibiotics as their secondary metabolite (Raaijmakers and Mazzola, 2012). It has been reported that a cluster of 8 genes (cpmA to cpmH) are responsible for the formation of carbapenem like antibiotics. The cpm mRNA level gets high during the exponential phase and is regulated by Rap/Hor homolog that has been found in Photorhabdus bacteria. It has been found that marker exchanged mutagenesis in the gene leads to impaired production of carbapenem production. The luxS like signaling function also have role in the functioning of the cpm operon. It has been found the luxS gene produces an auto inducer that causes repression in the cpm gene at the end of the exponential phase of the growth cycle (Bozhüyük et al., 2017).

Anthraquinone biosynthesis

The entomopathogenic bacterium Photorhabdus luminescens produces a red pigment and an antibiotic in insect carcasses in which it grows and in cultures. The pigment was identified as 1, 6-dihydroxy-4-methoxy-9, 10-anthraquinone, which is an Anthraquinone derivative. AQ pigment is produced by proteins encoded by the 9 gene anta-I locus. Genes present at both the end of this locus encode certain regulators of transcription are encoded by the genes of this locus. Hdfr, which is a transcription regulatory factor, acts as a repressor of ant- I expression and AQ production (Park and Crawford, 2015).

Over the years it has become necessary for humans to control the populations of harmful insects Insecticides have been used for this purpose in agricultural and horticultural sectors. Biological have replaced the synthetic insecticides, because of their harmful effects.  Recent researches have shown that species of Photorhabdus bacteria produce insecticidal toxins, which have various biotechnological, agricultural, and economic importances (Castagnola and Stock, 2014).

Conclusion

The report focuses on the recent advances in researches regarding the molecular biology of Photorhabdus luminescens, and emphasis has been given to apply the researches in the field of agriculture.  The above report signifies the Photorhabdus bacteria are the gold mine for the discovery of new type of toxins and drugs. This report also throws light upon the fact that this bacterium employs a complex regulatory mechanism of its genes and operons to control its production of secondary metabolite during its life cycle. The bacterium displays a functional heterogeneity in phase variation and interaction with different hosts. It can be concluded that the production of the secondary metabolites at the time of post exponential phase of the life cycle has a direct link with mutualistic association of the bacteria with the nematode. It has also been shown in the report that a mere switch in the promoter could regulate the bacteria’s pathogenicity towards specific host. The report gives a better understanding about the different regulatory pathways that the bacteria employ. All these characteristics of the bacteria along with its use in the field of agriculture make it a popular topic of research.

References

Bozhüyük, K.A., Zhou, Q., Engel, Y., Heinrich, A., Pérez, A. and Bode, H.B., 2017. Natural Products from Photorhabdus and Other Entomopathogenic Bacteria. The Molecular Biology of Photorhabdus Bacteria, pp.55-79.

Brachmann, A.O., Kirchner, F., Kegler, C., Kinski, S.C., Schmitt, I. and Bode, H.B., 2012. Triggering the production of the cryptic blue pigment indigoidine from Photorhabdus luminescens. Journal of biotechnology, 157(1), pp.96-99.

Castagnola, A. and Stock, S.P., 2014. Common virulence factors and tissue targets of entomopathogenic bacteria for biological control of lepidopteran pests. Insects, 5(1), pp.139-166.

Clarke, D.J., 2014. The genetic basis of the symbiosis between Photorhabdus and its invertebrate hosts. Adv Appl Microbiol, 88, pp.1-29.

Easom, C.A. and Clarke, D.J., 2012. HdfR is a regulator in Photorhabdus luminescens that modulates metabolism and symbiosis with the nematode Heterorhabditis. Environmental microbiology, 14(4), pp.953-966.

Gatsogiannis, C., Lang, A.E., Meusch, D., Pfaumann, V., Hofnagel, O., Benz, R., Aktories, K. and Raunser, S., 2013. A syringe-like injectionmechanismin Photorhabdus luminescens toxins. Nature, 495(7442), p.520.

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Orozco, R.A., Hill, T. and Stock, S.P., 2013. Characterization and Phylogenetic Relationships of Photorhabdusluminescens subsp. sonorensis (γ-Proteobacteria: Enterobacteriaceae), the Bacterial Symbiont of the Entomopathogenic Nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae). Current microbiology, 66(1), pp.30-39.

Park, H.B. and Crawford, J.M., 2015. Lumiquinone A, an α-aminomalonate-derived aminobenzoquinone from Photorhabdus luminescens. Journal of natural products, 78(6), pp.1437-1441.

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Somvanshi, V.S., Sloup, R.E., Crawford, J.M., Martin, A.R., Heidt, A.J., Kim, K.S., Clardy, J. and Ciche, T.A., 2012. A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states. Science, 337(6090), pp.88-93.

Tobias, N.J., Mishra, B., Gupta, D.K., Sharma, R., Thines, M., Stinear, T.P. and Bode, H.B., 2016. Genome comparisons provide insights into the role of secondary metabolites in the pathogenic phase of the Photorhabdus life cycle. BMC genomics, 17(1), p.537.

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