John C. Makemson

Biochemistry and Physiology of Bioluminescent Bacteria

Publisher Summary This chapter describes the biochemistry and physiology of bioluminescent bacteria. The function of bioluminescent bacteria is to emit light in biological systems. The chapter explains the variety of habitats in which these bacteria are found, the light-emitting system may play an important role in their ecology or physiology. The luciferase in bacteria, unlike that of any other luminous group (except, perhaps, the fungi), is related to the respiratory pathway, functioning as a shunt for electrons directly to oxygen at the level of reduced flavin. This luciferase is an external flavin mono-oxygenase or mixed-function oxidase, electrons for reduction of flavin mononucleotide (FMN) are provided by the reducing power derived from the electron-transport pathway. The light-emitting reaction then proceeds via the reaction of molecular oxygen with reduced flavin to form an intermediate luciferase-flavin peroxy species, whose breakdown provides energy sufficient to leave one of the products in an electronically excited singlet state, with subsequent light emission. The bacterial (luciferase-bound) peroxide chromophore, which has been isolated and characterized, provides a model in this respect for the different bioluminescent reactions.

Shewanella woodyi sp. nov., an exclusively respiratory luminous bacterium isolated from the Alboran Sea.

Thirty-four strains of nonfermentative, respiratory, luminous bacteria were isolated from samples of squid ink and seawater from depths of 200 to 300 m in the Alboran Sea. Although these strains had a few properties similar to properties of Shewanella (Alteromonas) hanedai, they did not cluster phenotypically with any previously described bacterium. The nucleotide sequence of a 740-bp segment of luxA was not homologous with other known luxA sequences but clustered with the luxA sequences of Shewanella hanedai, Vibrio logei, Vibrio fischeri, and Photobacterium species. The 16S RNA gene from two strains was sequenced and was found to be most closely related to the S. hanedai 16S RNA gene. Based on the differences observed, we describe the new isolates as members of new species, Shewanella woodyi sp. nov. Strain ATCC 51908 (= MS32) is the type strain of this new species.

Iron represses bioluminescence and affects catabolite repression of luminescence inVibrio harveyi

Bioluminescence and the synthesis of luciferase inVibrio harveyi growing in a minimal medium are repressible by iron; this is not significantly reversed by cyclic adenosine 3′,5′-monophosphate (cAMP). Cultures grown with added iron emit less light and possess less luciferase per cell than those grown under conditions of limiting iron; this may have significance in relation to the function of luciferase as an electron carrier. With iron, and with glycerol as the sole carbon and energy source, the addition of glucose causes further repression, both transient and permanent, and this is only partially reversible by cAMP. Without iron, glucose addition results in only a small and transient repression, but this is fully reversible by cAMP. The inability of cAMP to reverse iron-influenced repression may be explained by both a low rate of transport of cAMP into the bacteria and increased intracellular levels of cyclic nucleotide phosphodiesterase.

Luminous bacteria cultured from fish guts in the Gulf of Oman

The incidence of culturable luminous bacteria in Omani market fish guts was correlated to habitat type amongst 109 species of fish. Isolated representative luminous bacteria were compared to known species using the Biolog system (95 traits/isolate) and cluster analysis, which showed that the main taxa present in fish guts were clades related to Vibrio harveyi and Photobacterium species with sporadic incidence of P. phosphoreum. The luminous isolates from gut of the slip-mouth (barred pony fish), Leiognathus fasciatus, were mainly a type related to Photobacterium but phenotypically different from known species. These luminous gut bacteria were identical with the bacteria in the light organ, indicating that the light organ supplies a significant quantity of luminous bacteria to the gut. In many of the fish that lack light organs, luminous bacteria were also the dominant bacterial type in the gut, while in some others luminous bacteria were encountered sporadically and at low densities, reflecting the incidence of culturable luminous bacteria in seawater. Pelagic fish contained the highest incidence of culturable luminous bacteria and reef-associated fish the lowest. No correlation was found between the incidence of culturable luminous bacteria and the degree to which fish produce a melanin-covered gut.

Substituted lactam and cyclic azahemiacetals modulate Pseudomonas aeruginosa quorum sensing

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure γ-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The γ-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.

Differentiation of marine luminous bacteria using commercial identification plates

Differentiation of marine luminous bacteria using Biology GN plate combined with API 20e or the BBL Crystal ID plate inoculated with cell suspensions in artificial seawater was accomplished by comparison to type species using cluster analysis. Inoculum density affected the results from Biolog GN plates, but had less of an effect on the reactions obtained from API 20e strip or BBL Crystal ID plate. In a few cases, combination of the Biolog GN traits along with either the API 20e or Crystal ID traits was necessary to differentiate some marine luminous bacteria.

Stabilization of luciferase intermediates by fatty amines, amides, and nitriles.

Long-chain aliphatic amides, mono- and diamines, mono- and dialcohols, and nitriles were found to inhibit the bacterial luciferase reaction by binding with an enzyme intermediate (II, the luciferase-bound 4 alpha-flavin hydroperoxide). Inhibition was determined by measuring the decay rates of the inhibitor-intermediate II complex at different inhibitor concentrations. The data fit a model which was used to estimate the KI. At high concentrations, a plot of the decay rate (k) vs 1/[I] produced a straight line; extrapolation of this to 1/[I] = 0 yields an estimate of the decay rate at infinite inhibitor concentration which we defined as the inhibitor-enzyme-substrate stabilization constant, kESI.

The cytochromes of luminous bacteria and their coupling to bioluminescence

Vibrio fischeri andV. harveyi possess cytochromes a, b, and c, whereasPhotobacterium leiognathi andP. phosphoreum also contain cytochrome d. In all, cytochrome a as well as some of c binds carbon monoxide. Carbon monoxide does not inhibit bioluminescence (in vivo or in vitro), but carbonyl cyanidem-chlorophenylhydrazone inhibits only in vivo bioluminescence. This inhibition is due to dissipation of the proton motive force which indirectly inhibits bioluminescence by interruption of aldehyde recycling. Bioluminescence is thereby indirectly coupled to the proton motive force.

Transient and dynamic DNA supercoiling potently stimulates the leu-500 promoter in Escherichia coli

The inactive prokaryotic leu-500 promoter (Pleu-500) contains a single A-to-G point mutation in the −10 region of the leucine operon promoter, which causes leucine auxotrophy. This promoter can be activated by (−) DNA supercoiling in Escherichia coli topA strains. However, whether this activation arises from global, permanent, or transient, dynamic supercoiling is still not fully understood. In this article, using a newly established in vivo system carrying a pair of divergently coupled promoters, i.e. an IPTG-inducible promoter and Pleu-500 that control the expression of lacZ and luc (the firefly luciferase gene), respectively, we demonstrate that transient, dynamic (−) DNA supercoiling provided by divergent transcription in both wild-type and topA strains can potently activate Pleu-500. We found that this activation depended on the promoter strength and the length of RNA transcripts, which are functional characteristics of transcription-coupled DNA supercoiling (TCDS) precisely predicted by the twin-supercoiled domain model of transcription in which a (+) supercoiled domain is produced ahead of the RNA polymerase and a (−) supercoiled domain behind it. We also demonstrate that TCDS can be generated on topologically open DNA molecules, i.e. linear DNA molecules, in Escherichia coli, suggesting that topological boundaries or barriers are not required for the production of TCDS in vivo. This work demonstrates that transient, dynamic TCDS by RNA polymerases is a major chromosome remodeling force in E. coli and greatly influences the nearby, coupled promoters/transcription.

A simple assay of cyclic-AMP phosphodiesterase in the presence of interfering enzymes in Vibrio harveyi

Abstract PEI-cellulose thin layer chromatography is used to separate the reaction products formed from cyclic-AMP initiated by phosphodiesterase in crude extracts of bacteria.