Bacterium Photorhabdus Luminescens Research Articles

Survival of insect pathogenic and human clinical isolates of Photorhabdus luminescens in previously sterile soil.

Most characterized strains of the bacterium Photorhabdus luminescens are symbionts of entomopathogenic nematodes, whereas other strains have been isolated from human clinical specimens. The ability of P. luminescens strains to survive and grow in soil has received limited attention, with some studies indicating these bacteria have little or no ability to persist in soil. Survival and (or) growth of P. luminescens strains in previously sterilized soil, and examination of different soil amendments on their numbers in soil, have not been previously reported. Entomopathogenic P. luminescens (ATCC 29999) and a human clinical isolate (ATCC 43949) were introduced into a soil that had been sterilized by autoclaving, with or without different soil amendments, and bacterial numbers were estimated over time by viable plate count. In the previously sterilized soil receiving no exogenous amendments, numbers fell drastically over a week's time, followed by an increase in numbers by day 30. Treatments involving the addition of calcium carbonate and gelatin or casamino acids to soil usually resulted in higher bacterial numbers. For some sampling dates and soil treatments, there were statistically significant differences between the numbers of the two bacterial strains recovered from soil. The two strains of P. luminescens used in this study were able to survive and grow after being inoculated into previously sterilized soil.

Purification and characterization of a high-molecular-weight insecticidal protein complex produced by the entomopathogenic bacterium photorhabdus luminescens

Photorhabdus luminescens is a gram-negative enteric bacterium that is found in association with entomopathogenic nematodes of the family Heterorhabditidae. The nematodes infect a variety of soil-dwelling insects. Upon entering an insect host, the nematode releases P. luminescens cells from its intestinal tract, and the bacteria quickly establish a lethal septicemia. When grown in peptone broth, in the absence of the nematodes, the bacteria produce a protein toxin complex that is lethal when fed to, or injected into the hemolymph of, Manduca sexta larvae and several other insect species. The toxin purified as a protein complex which has an estimated molecular weight of 1,000,000 and contains no protease, phospholipase, or hemolytic activity and only a trace of lipase activity. The purified toxin possesses insecticidal activity whether injected or given orally. Analyses of the denatured complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed it to be composed of several protein subunits ranging in size from 30 to 200 kDa. The complex was further separated by native gel electrophoresis into three components, two of which retained insecticidal activity. The purified native toxin complex was found to be active in nanogram concentrations against insects representing four orders of the class Insecta.

The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.

Insecticidal toxins from the bacterium Photorhabdus luminescens.

Transgenic plants expressing Bacillus thuringiensis (Bt) toxins are currently being deployed for insect control. In response to concerns about Bt resistance, we investigated a toxin secreted by a different bacterium Photorhabdus luminescens, which lives in the gut of entomophagous nematodes. In insects infected by the nematode, the bacteria are released into the insect hemocoel; the insect dies and the nematodes and bacteria replicate in the cadaver. The toxin consists of a series of four native complexes encoded by toxin complex loci tca, tcb, tcc, and tcd. Both tca and tcd encode complexes with high oral toxicity to Manduca sexta and therefore they represent potential alternatives to Bt for transgenic deployment.

Insertional inactivation of genes encoding the crystalline inclusion proteins of Photorhabdus luminescens results in mutants with pleiotropic phenotypes.

The entomopathogenic bacterium Photorhabdus luminescens exhibits phase variation when cultured in vitro. The variant forms of P. luminescens are pleiotropic and are designated phase I and phase II variants. One of the characteristic phenotypes of phase I cells is the production of two types of intracellular protein inclusions. The genes encoding the protein monomers that form these inclusions, designated cipA and cipB, were cloned and characterized. cipA and cipB encode hydrophobic proteins of 11,648 and 11,308 Da, respectively. The deduced amino acid sequences of CipA and CipB have no significant amino acid sequence similarity to any other known protein but have 25% identity and 49% similarity to each other. Insertional inactivation of cipA or cipB in phase I cells of P. luminescens produced mutants that differ from phase I cells in bioluminescence, the pattern and activities of extracellular products, biochemical traits, adsorption of dyes, and ability to support nematode growth and reproduction. In general, the cip mutants were phenotypically more similar to each other than to either phase I or phase II variants.

Anaerobic metabolism of primary and secondary forms of Photorhabdus luminescens

An oxygen electrode inserted into a dead Manduca sexta larva infected with Heterorhabditis nematodes carrying the bacterium Photorhabdus luminescens showed barely detectable levels of oxygen in a 1 to 2 mm zone below the cuticle, and virtual anaerobiosis deeper in the carcass. This observation indicates that the bacteria in this habitat, where they are actively growing, are probably carrying out a fermentative metabolism. Therefore, the anaerobic metabolism of the primary and secondary form variants of P. luminescens Hm and NC1 was compared. Amino acids were not fermented by either strain, either singly or in mixtures. Glucose was fermented by both forms of both organisms, forming products typical of mixed acid fermentation by Enterobacteriaceae. The fermentation patterns were the same in the primary and secondary forms. Growth rates of the secondary form cells were higher in defined medium with glucose as energy and carbon source. Growth yields of the primary and secondary forms of strain Hm were nearly identical, whereas the growth yield of secondary form cells of strain NC1 was slightly higher than that of the primary form. The results of this study indicate that the observed predominance of primary form cells in infected insect larvae cannot be explained by an advantage over the secondary form cells related to the efficiency of anaerobic growth or fermentative metabolism.

Anaerobic metabolism of primary and secondary forms of Photorhabdus luminescens

An oxygen electrode inserted into a dead Manduca sexta larva infected with Heterorhabditis nematodes carrying the bacterium Photorhabdus luminescens showed barely detectable levels of oxygen in a 1 to 2 mm zone below the cuticle, and virtual anaerobiosis deeper in the carcass. This observation indicates that the bacteria in this habitat, where they are actively growing, are probably carrying out a fermentative metabolism. Therefore, the anaerobic metabolism of the primary and secondary form variants of P. luminescens Hm and NC1 was compared. Amino acids were not fermented by either strain, either singly or in mixtures. Glucose was fermented by both forms of both organisms, forming products typical of mixed acid fermentation by Enterobacteriaceae. The fermentation patterns were the same in the primary and secondary forms. Growth rates of the secondary form cells were higher in defined medium with glucose as energy and carbon source. Growth yields of the primary and secondary forms of strain Hm were nearly identical, whereas the growth yield of secondary form cells of strain NC1 was slightly higher than that of the primary form. The results of this study indicate that the observed predominance of primary form cells in infected insect larvae cannot be explained by an advantage over the secondary form cells related to the efficiency of anaerobic growth or fermentative metabolism.

Identification of two pigments and a hydroxystilbene antibiotic from Photorhabdus luminescens

Two yellow pigments were isolated for the first time from the entomopathogenic bacterium Photorhabdus luminescens in liquid culture and were identified as the anthraquinone derivatives 3,8-dimethoxy-1-hydroxy-9,10-anthraquinone (minor) and 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone (major). A known antibiotic, 3,5-dihydroxy-4-isopropylstilbene, was also detected and for the first time showed strong fungicidal activity against several fungi of medical and agricultural importance.