Cloning and nucleotide sequences of lux genes and characterization of luciferase of Xenorhabdus luminescens from a human wound

Abstract

Xenorhabdus luminescens HW is the only known luminous bacterium isolated from a human (wound) source. A recombinant plasmid was constructed that contained the X. luminescens HW luxA and luxB genes, encoding the luciferase a and j subunits, respectively, as well as luxC, luxD, and a portion of luxE. The nucleotide sequences of these lux genes, organized in the order luxCDABE, were determined, and overexpression of the cloned luciferase genes was achieved in Escherichia coli host cells. The cloned luciferase was indistinguishable from the wild-type enzyme in its in vitro bioluminescence kinetic properties. Contrary to an earlier report, our findings indicate'that neither the specific activity nor the size of the a (362 amino acid residues, Mr 41,389) and 1B(324 amino acid residues, M, 37,112) subunits of the X. luminescens HW luciferase was unusual among known luminous bacterial systems. Significant sequence homologies of the a and ,B subunits of the X. luminescens HW luciferase with those of other luminous bacteria were observed. However, the X. luminescens HW luciferase was unusual in the high stability of the 4a-hydroperoxyflavin intermediate and its sensitivity to aldehyde substrate inhibition.Most of the known luminous bacteria are of marine origin and fall within two genera, Vibrio and Photobacterium (references 13 and 23'and references therein). Luciferases from the marine bacteria Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi, and Photobacterium phosphoreum are all a1 heterodimers (14,19,25), with molecular weights of approximately 40,000 for a and 37,000 for P3. For the former three species, the luxA and luxB genes, encoding the luciferase a and P subunits, respectively, have been sequenced (4,9,15,17).A few nonmarine luminous bacterial species have also been identified (13, 23); among them are Xenorhabdus luminescens Hb and Hm, both of which are insect-pathogenic symbionts with nematodes of the family Heterorhabditidae (24,27,30). The luciferases from X. luminescens Hm and Hb have identical a subunits and only a single different amino acid residue in the P subunit. Moreover, they are similar in specific activity and subunit sizes to and have significant sequence homology with luciferases from marine luminous bacteria (16, 27, 29). The luxA and luxB genes from these two X. luminescens strains differ by only one and two bases, respectively (16,29). In fact, the known sequences of the lux operon, starting with the last 142 bases of luxD through the first four bases of luxE (with luxA, luxB, and noncoding regions in between) show 99% identity for X. luminescens Hm and Hb (16,29).A different luminous strain of X. luminescens has been isolated from a human wound (8), now designated strain HW. Some unusual properties have been reported for this strain and its luciferase (5) in comparison with all other known luminous bacteria, including X. luminescens Hm and Hb. X. luminescens HW bioluminescence activities both in vivo and in vitro have been found to be substantially lower in intensity but higher i...

A Unique Mechanism for Methyl Ester Formation via an Amide Intermediate Found in Myxobacteria

“…In figure 7, the protein sequence of the Mox domain is compared to those of bacterial luciferases, and the Mox domains of MelG and CtaG (CtaG represents a functionally identical homologue of MelG from a different myxobacterium [32]). Bacterial luciferases catalyze the oxidation of aldehydes to acids employing reduced FMN and releasing energy in form of bioluminescense [33]. To our knowledge, a specific FMN binding motif has not been defined and all known luciferase structures were determined in their apoforms without cofactor.…”

Mathematical model of the Lux luminescence system in the terrestrial bacterium Photorhabdus luminescens

“…5 Lux genes have been cloned and sequenced for three strains of P. luminescens, and the luciferases produced from these genes have been characterized. [6][7][8][9] The luciferase from P. luminescens has a very high thermal stability (a half life of over three hours at 45 1C), making the lux operon of this organism a very good choice as a reporter system. 7 It was for this reason that P. luminescens was chosen for study.…”

Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria

“…However, such a sequence has not been found in multiple copies in one operon or at one site. The nucleotide sequences of the luxCDAB genes, but not those of the luxE gene or flanking regions, have also been reported for the Hw strain (29). This strain is of particular interest, since it was isolated from a human wound rather than from a nematode and/or insect (4,8).…”

“…The lux genes of luminescent bacteria have been isolated from marine bacteria of the Vibrio and Photobacterium genera (3,5,6,14,15,18) and from terrestrial bacteria of the Xenorhabdus genus (9,26,29). lux genes that code for luciferase (luxAB) and the fatty acid reductase complex (luxCDE) involved in the synthesis of the fatty aldehyde substrate for the luminescence reaction have been detected (17) in all cases.…”

“…Recently, lux genes have been cloned and transferred into Eschenichia coli from three strains (Hb, Hm, and Hw) of the enteric terrestrial bacterium Xenorhabdus luminescens (9,26,29). Two of the strains, Hb and Hm, are very similar and can be isolated from the gut tracts of nematodes or from the hemocoel of insects (21).…”