Edward A. Meighen

Physiological, Biochemical and Genetic Control of Bacterial Bioluminescence

Publisher Summary The bioluminescent bacteria comprise one of several groups of luminous organisms. Significant differences exist between the bioluminescence reactions of different organisms, including the structure and properties of the luciferases and substrates. Molecular oxygen is the only common feature of bioluminescence reactions, indicating that the luminescent systems in most organisms may have evolved independently. Luminescent bacteria are present in marine environment, freshwater, and terrestrial habitats. They can occur as free-living forms, saprophytes, commensal symbionts, parasites of animals, and specific light-organ symbionts. The luminescence produced by these bacteria, because of its inherent beauty and ease of detection, has attracted scientific attention. With the use of molecular approaches to study the luminescence systems of these bacteria, population biology, ecology, and molecular mechanisms of luminescence (lux) gene regulation can be studied. This chapter describes the current status of bioluminescent systems of luminous bacteria, emphasizing the biochemistry, lux gene organization, and the physiological and genetic regulation of lux gene expression. The effects of oxygen on luminescence illustrate the application of bacterial luminescence system as a sensor of specific molecules that affect metabolic function and gene expression. Knowledge of the basic biochemistry, molecular biology, and physiology of luminescent bacteria is thus not only of interest but of importance for future scientific endeavors.

LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization

Vibrio fischeri is the bacterial symbiont within the light‐emitting organ of the sepiolid squid Euprymna scolopes . Upon colonizing juvenile squids, bacterial symbionts grow on host‐supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities. Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization. A 606 bp open reading frame was cloned from V. fischeri that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins: Vibrio cholerae HapR, Vibrio harveyi LuxR, Vibrio parahaemolyticus OpaR and Vibrio vulnificus SmcR. When grown in culture, cells of V. fischeri strain PMF8, in which litR was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent. Protein‐binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the luxR gene. Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs. Thus, the litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacteriums competence in initiating a benign infection.

Autoregulation of luxR: the Vibrio harveyi lux-operon activator functions as a repressor

Mobility‐shift assays have been used to demonstrate that the activator of the Vibrio harveyi lux operon, LuxR, binds independently, and with similar affinity, to two sites upstream of its own open reading frame. One site was located between 52 and 107 bp upstream of, and the other site in a region 25 bp downstream of, the transcriptional start site. The luxR promoter, in a transcriptional fusion with the chloramphenicol acetyl transferase (cat) gene, could readily be expressed in Escherichia coli as well as V. harveyi in the absence of LuxR. In both species, the presence of the luxR gene product resulted in repression of luxR promotion. These results show that LuxR directly regulates its own expression by functioning as an autorepressor. A mechanism for this repression is suggested by evidence showing that LuxR has a negative effect on RNA polymerase binding to the luxR promoter. In light of the fact that LuxR is also part of a regulatory family of repressors, the mechanism by which LuxR functions as a transcriptional activator of the lux operon has been re‐examined.

Cloning and Characterization of the Bile Salt Hydrolase Genes (bsh) from Bifidobacterium bifidum Strains

ABSTRACT Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

Pheromone Biosynthesis: Enzymatic Studies in Lepidoptera

Publisher Summary This chapter presents some enzymatic studies in Lepidoptera. The pheromones of the Lepidoptera are generally a blend of long-chain unsaturated aldehydes, alcohols, and acetate esters. Specificity in the pheromone signal is achieved by variation in the chain length, the number, location, and isomeric nature of the double bonds, the nature of the functional group, and by the blending together of several compounds in a precise ratio. Consequently, investigation of the mechanisms of fatty acid biosynthesis, desaturation, and reduction and characterization of the pathways for interconversion of the aldehydes, alcohols, and acetate esters in Lepidoptera are essential for understanding the control of pheromone biosynthesis in these insects. The pheromone of the eastem spruce budworm, Choristoneura fumiferana, is a blend of (E)-11-tetradecenal and (Z)-11-tetradecenal (V) in a 96:4 ratio. The pheromone, which attracts male moths of the same species, is secreted from a specialized gland located at the end of the abdomen of the female moth.

Control of bioluminescence in Vibrio fischeri by the LuxO signal response regulator

Bioluminescence in the marine bacterium Vibrio fischeri is controlled by the excretion of a N‐acyl homoserine lactone (HSL) autoinducer which interacts with a regulator, LuxR, and activates transcription of the lux operon at high‐cell density. This system has become the prototype for quorum sensing in many bacteria. Although light emission in Vibrio harveyi is also regulated by a N‐acyl‐HSL inducer, in sharp contrast, a completely different and more complex system is involved in quorum sensing which is mediated via LuxO, the response regulator of a phosphorelay signal transduction system. In the present work, luxO and the overlapping luxU gene, also involved in the phosphorelay system in V. harveyi, have been discovered in V. fischeri. By gene replacement technology, a V. fischeri luxO– mutant was generated whose phenotype was similar to that of V. harveyi luxO– showing that LuxO is involved in control of luminescence in V. fischeri. This mutant could be complemented with luxO from either V. fischeri or V. harveyi resulting in the restoration of the dependence of luminescence intensity on cell density. In contrast to V. harveyi luxO–, light emission of V. fischeri luxO– was stimulated by the N‐acyl‐HSL autoinducer indicating that luxO is part of a second signal transduction system controlling luminescence in this species. The presence of a luxO‐based phosphorelay regulatory system as well as the luxR‐based system in V. fischeri suggests that the former system, originally discovered in V. harveyi, may be a general regulatory mechanism in luminescent bacteria.

BIOTECHNOLOGICAL APPLICATIONS OF BACTERIAL BIOLUMINESCENCE (lux) GENES

While the phenomenon of bioluminescence has been observed in many different organisms, bioluminescent bacteria themselves comprise an extraordinarily abundant group. Although they occur as freshwater and terrestrial species, they are found predominantly in the marine environment. They exist in a variety of habitats as free-living organisms, saprophytes, gut symbionts, animal parasites and as light-organ symbionts in the teleost fishes and squid.’ Due primarily to its ease of detection, but also to its inherent beauty, luminescence from these bacteria has prompted scientific investigation for over 300

LuxO controls luxR expression in Vibrio harveyi : evidence for a common regulatory mechanism in Vibrio

Quorum‐sensing control of luminescence in Vibrio harveyi, which involves an indirect autoinducer‐mediated phosphorelay signal transduction system, contrasts with the prototypical quorum‐sensing system of Vibrio fischeri, in which the autoinducer and the transcriptional activator LuxR directly activate lux operon expression. In V. harveyi, a regulator not homologous to V. fischeri LuxR and also designated LuxR (LuxRvh), binds specifically to the lux operon promoter region and activates the expression of luminescence. A direct connection has not been identified previously between V. harveyi LuxRvh and the autoinducer‐mediated phosphorelay system. Here, we demonstrate by mobility shift assays and measurement of luxRvh mRNA levels with luxO+ and luxO– cells that the central response regulator of the V. harveyi phosphorelay system (LuxO) represses the level of LuxRvh. Expression of a luxRvh‐bearing plasmid strongly stimulated luminescence of a luxO– mutant but had no effect on luminescence of wild‐type luxO+ cells, indicating tight regulation of luxRvh by LuxO. Furthermore, luxO null mutants of V. fischeri MJ‐1 and two autoinducer mutants, MJ‐211 (luxI–) and MJ‐215 (luxI–ainS–), emitted more light and exhibited more elevated levels of litR, a newly identified V. harveyi luxRvh homologue, than their luxO+ counterparts. These results suggest that activity of the autoinducer‐mediated phosphorelay system is coupled to LuxRvh/LitR control of luminescence through LuxO in V. harveyi and V. fischeri. The presence of homologues of V. harveyi LuxRvh, LuxO and other phosphorelay system proteins in various Vibrio species and the control of LuxRvh and its homologues by LuxO identified here in V. harveyi and V. fischeri and recently in Vibrio cholerae suggest that the luxO–luxRvh couple is a central feature of this quorum‐sensing system in members of the genus Vibrio.

Proximal and distal sites bind LuxR independently and activate expression of the Vibrio harveyi lux operon

The LuxR regulatory protein of Vibrio harveyi as well as the autoinducer molecule, N‐(3‐hydroxybutanoyl) homoserine lactone, are known to be required for expression of luminescence. Although LuxR has been implicated in the activation of the promoter of the lux operon of V. harveyi, and can bind to two distinct sites upstream of the transcription initiation start site, its mode of action is unknown, in the present experiments, mobility shift assays were used to demonstrate that LuxR bound to the distal and proximal sites in an independent rather than co‐operative interaction with a much tighter binding to the distal site. Deletion and mutation analyses of DNA upstream of the lux promoter followed by transconjugation Into V. harveyi in trans using the chloramphenicol acetyl‐transferase (cat) gene as a reporter demonstrated, however, that the proximal site for LuxR was absolutely critical for promoter activation while the distal LuxR site was only necessary for maximum activation. This result was confirmed by mutation of the proximal site which blocked activation of the lux promoter and binding of LuxR to this site, but did not prevent LuxR binding to the distal site.

MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence

The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum‐sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA‐binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1000‐fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10‐fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.