Identification, Characterization, and Regulation of a Cluster of Genes Involved in Carbapenem Biosynthesis in Photorhabdus luminescens

Abstract

The luminescent entomopathogenic bacterium Photorhabdus luminescens produces several yet-uncharacterized broad-spectrum antibiotics. We report the identification and characterization of a cluster of eight genes (named cpmA to cpmH) responsible for the production of a carbapenem-like antibiotic in strain TT01 of P. luminescens. The cpm cluster differs in several crucial aspects from other car operons. The level of cpm mRNA peaks during exponential phase and is regulated by a Rap/Hor homolog identified in the P. luminescens genome. Marker-exchange mutagenesis of this gene in the entomopathogen decreased antibiotic production. The luxS-like signaling mechanism of quorum sensing also plays a role in the regulation of the cpm operon. Indeed, luxS, which is involved in the production of a newly identified autoinducer, is responsible for repression of cpm gene expression at the end of the exponential growth phase. The importance of this carbapenem production in the ecology of P. luminescens is discussed.Photorhabdus luminescens, a gram-negative luminescent gamma-proteobacterium, forms an entomopathogenic symbiosis with a soil nematode of the genus Heterorhabditis. This bacterium undergoes a complex life cycle that involves a symbiotic stage, in which bacteria colonize the intestine of the nematodes, and a pathogenic stage, in which susceptible insects are killed by the combined action of the nematode and the bacteria. After entering the insect host, the nematodes expel their bacterial symbionts into the insect hemocoel. Once released, the bacteria proliferate, kill the insect within 24 h to 48 h, and enhance conditions for nematode reproduction by providing nutrients and other growth factors. Remarkably, the insect carcass does not putrefy over a period of several weeks, because P. luminescens produces several broad-spectrum antibiotics that prevent contamination of the insect cadaver by other microorganisms. These products are believed to be secreted into the insect hemolymph when the bacteria enter stationary-phase conditions. Two P. luminescens antibiotic molecules have now been identified. They consist of a red pigment with antimicrobial activity, identified as an anthraquinone derivative, and a hydroxystilbene derivative, 1,3-dihydroxy-4-isopropylstilbene, which showed strong fungicidal activity in previous studies (20,30).Carbapenems are a class of ␤-lactam antibiotics synthesized by a biochemical route distinct from that found in the biosynthetic pathways of the sulfur-ring ␤-lactams, namely, penicillins, cephamycins, and cephalosporins (5, 43). As naturally produced metabolites, they were originally isolated in the late 1970s from Streptomyces species. The first carbapenem antibiotic discovered was thienamycin, produced in small quantities

luxS mutants of Serratia defective in autoinducer-2-dependent ‘quorum sensing’ show strain-dependent impacts on virulence and production of carbapenem and prodigiosin

“…This is in stark contrast to the situation in Photorhabdus luminescens, where carbapenem production is increased in the luxS mutant (Derzelle et al, 2002). However, the regulation of carbapenem production is clearly different in the two genera.…”

“…However, the regulation of carbapenem production is clearly different in the two genera. In Serratia 39006, in addition to this luxS modulation, carbapenem production is under strict aHSL-mediated QS control (Slater et al, 2003), whereas in Photorhabdus luminescens, no aHSL system was detected (Derzelle et al, 2002).…”

Carbon source regulation of antibiotic production

“…In addition, in contrast to E. carotovora, P. luminescens carbapenemlike synthesis is not induced by the quorum-sensing molecule N-acyl-homoserine lactone. 30 An additional example of carbon regulation has been reported in the ethanol-utilizing strain, Serratia marcesens. In this microorganism (strain S389), Cang et al 31 observed that a low glucose concentration (27.7 mM) added to the growth medium, almost completely repressed formation of the red tripirrole antibiotic, prodigiosin.…”

“…strain American Type Culture Collection 39006 and Photorhabdus luminescens strain TT01. 30 Interestingly, in P. luminescens, the carR gene is not present, suggesting a different mechanism for the regulation of antibiotic formation. In addition, in contrast to E. carotovora, P. luminescens carbapenemlike synthesis is not induced by the quorum-sensing molecule N-acyl-homoserine lactone.…”

In Vivo Cell Tracking with Bioluminescence Imaging

“…Lux is isolated from Photorhabdus luminescens; its luminescence reaction involves the oxidation of a long-chain aldehyde (RCHO) and reduced flavin mononucleotide, resulting in the production of oxidized flavin mononucleotide and a long-chain fatty acid (RCOOH), along with the emission of blue-green light at 490 nm [22,23] which has been used for BLI study of bacterial infections [46]. It is a unique bioreporter system with no addition of exogenous substrate due to generation of its own substrate [47].…”