Mutations in the lux Operon of Natural Dark Mutants in the Genus Vibrio

Abstract

Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete lux operons in natural isolates.Bioluminescence is a biochemically well-understood, easily identifiable physiological marker (13, 24) which, in most known species, is under quorum-sensing control (20,23). In order to be capable of bioluminescence, bacteria require a minimum of five genes arranged in an operon: luxCDABE. Most bioluminescent species have additional nonessential genes in the lux operon as well, including (among others) luxG (found in all species except Photorhabdus luminescens) and luxH (found in Vibrio harveyi) (6,11,35).For more than a century, bacterial bioluminescence has called attention to itself by its obvious visual phenotype. It is only since the advent of molecular probing techniques that we have become aware of the potentially widespread occurrence of non-visibly luminous strains (19,27,29). Palmer and Colwell (27) found a wide range of luminescence intensity levels among natural Vibrio cholerae isolates and discovered that a significant fraction of "visually non-luminescent" isolates contained a luxA gene. Lee and Ruby (19) took advantage of molecular probing to demonstrate that near-shore Hawaiian seawater contains a large number of non-visibly luminous Vibrio fischeri strains, consistent with the phenotype of the light organ symbionts of the bobtail squid Euprymna scolopes, which do not visibly glow under typical laboratory culture conditions but glow brightly in the squid. Using a V. harveyi luxA gene probe, Ramaiah et al. (29) found that an average of 1.7% of their Chesapeake Bay isolates had luxA but were not visibly bioluminescent. They suggested that either lux genes were not being expressed or that alterations or deletions had occurred in lux genes. The molecular basis of these dark phenotypes in natural populations has been, until now, unexplored.Our work shows that natural populations of vibrios in Boca Ciega Bay, Florida, also include dark strains that contain lux genes. Additionally, we have discovered the molecular basis for the dark phenotypes of our i...

Contribution of Rapid Evolution of theluxR-luxIIntergenic Region to the Diverse Bioluminescence Outputs ofVibrio fischeriStrains Isolated from Different Environments

“…Bioluminescence is a trait scattered throughout the family Vibrionaceae; however, the selective pressures driving the evolution of bioluminescence are not always clear (62). As noted here, the mystery deepens in that light output can vary by over several orders of magnitude between different environmental isolates of V. fischeri, and this is true for other Vibrio species as well (23,49). The work here provides evidence that lux has evolved relatively rapidly in V. fischeri and helps provide a foundation for understanding the mechanistic differences leading to dimmer or brighter strains.…”

“…For example, obvious visible luminescence has been frequently associated with a number of species, such as Vibrio fischeri, Vibrio harveyi, Photobacterium leiognathi, and Photobacterium phosphoreum; however, dim and even "cryptic" luminescence has also been observed upon careful examination of certain isolates, such as Vibrio salmonicida (20). Even isolates of the same species can vary in luminescence output (23,49). Such variation is dramatically evident in V. fischeri (5,32,45,62), which is a longstanding model for studying bioluminescence, its functional roles, and its regulation.…”

Polyphyly of non‐bioluminescent Vibrio fischeri sharing a lux‐locus deletion

“…The work suggests that broader, relatively unbiased environmental samplings may increase the general knowledge of the bioluminescence spectrum for any given species of Vibrionaceae . Samplings of both Vibrio cholerae and V. harveyi support this conclusion; such studies have demonstrated that non‐bioluminescent isolates of luminous species are as abundant, or more abundant, than bioluminescent isolates in marine environments (Grim et al ., 2008; O'Grady and Wimpee, 2008; Zo et al ., 2009).…”

Bacterial Quorum Sensing Stabilizes Cooperation by Optimizing Growth Strategies

“…2 and 3). These results have ecological significance, as nonluminescent luxR defectors and UC luxO mutants have all been identified in natural Vibrio populations (43)(44)(45). Importantly, this occurred in a well-mixed environment in which no biofilms or cell aggregates were observed and neither assortment of competitors nor privatization of PG would be likely.…”