Bacterial luciferase activity and the intracellular redox pool in Escherichia coli

Koga, Kenji; Harada, Tadashi; Shimizu, H.; Tanaka, Kan

doi:10.1007/s00438-005-0008-5

Cited by 39 publications

(30 citation statements)

References 41 publications

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“…We hypothesize that the attenuation may be directly due to the loss of luciferase, which results in the elimination of the dark luciferase reaction. The dark luciferase reaction produces toxic oxygen radicals that may serve as a direct virulence agent or a stimulant for bacterial DNA repair (15,22,23,25). Alternatively, the luciferase reaction may function as an alternative pathway to provide oxidized flavin at low oxygen tensions which may aid colonization if oxygen becomes limited (4,26).…”

Section: Discussionmentioning

confidence: 99%

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

Nelson

Tunsjø

Fidopiastis

et al. 2007

Appl Environ Microbiol

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The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host.Marine bioluminescent bacteria have been the subjects of considerable interest because of the biochemistry that drives light production and their ability to initiate specific, long-term cooperative symbioses with many species of squids and fishes (20,35,45,51). Less is known about bioluminescence in species of bacteria that have the capacity to produce light yet are found in pathogenic associations with animal hosts (32,33,38). It has always been of interest to know whether luminescence plays a role in the biology of such pathogens, either to colonize the hosts or to grow in environmental niches. However, attempts to address such questions were limited because a model system in which to study the relationship between bioluminescence and pathogenesis was not available.In the five previously characterized species of luminous bacteria (Vibrio fischeri, Vibrio harveyi, Photobacterium leiognathi, Photobacterium phosphoreum, and Photorhabdus luminescens), the six structural genes for bioluminescence are contained within a locus termed the lux operon. With the exception of a duplication of luxB (designated luxF) in one species, these genes are arranged in the order luxCDABEG (1,9,16,28). luxA and luxB, respectively, encode the alpha and beta subunits of luciferase, the enzyme responsib...

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Section: Discussionmentioning

confidence: 99%

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

Nelson

Tunsjø

Fidopiastis

et al. 2007

Appl Environ Microbiol

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“…E. coli BW25113 (W3110 lacI q rrnBT14 ⌬lacZWJ16 hsdR514 ⌬araBAD AH33 ⌬rhaBAD LD78 ) (35) and the Keio collection (a single-gene deletion mutant series derived from strain BW25113) (36) were obtained from the E. coli Stock Center (National Bio-Resource Center, Mishima, Japan). Plasmid pLXUV5, which contains the lacUV5 promoter fragment fused to the V. fischeri luxCDABE genes, was constructed previously to monitor intracellular redox pools (34).…”

Section: Methodsmentioning

confidence: 99%

“…When cell cultures were used as samples, a 0.1-ml aliquot of the culture was placed in a test tube, and the RLUs were measured immediately. Luciferase activity was calculated as RLUs/OD 600 (34).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoblot analysis was performed as described previously (38) using rabbit anti-LuxB antiserum (34). Two micrograms of total protein was analyzed during the time course.…”

Section: Methodsmentioning

confidence: 99%

“…In a previous study, we observed drastic, physiological phase-dependent changes in light emissions from E. coli cells that expressed Vibrio fischeri luciferase (34). Because the levels of luciferase proteins (LuxA and LuxB) were constant during the growth phase under the examined conditions, changes in the luciferase activity were concluded to have resulted from changes in intracellular redox pools, such as NADH and NADPH, which should have a relationship with the carbon metabolic flux that corresponds to reducing power production (34).…”

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confidence: 91%

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Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Shimada

Tanaka

2016

Appl Environ Microbiol

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Regulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employing Vibrio fischeri luciferase to probe the intracellular metabolic condition in Escherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells. IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used a Vibrio fischeri luciferase monitoring system to probe the intracellular metabolic condition in Escherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells. M olecular mechanisms of cellular metabolic regulation have been the subject of extensive studies, as understanding these mechanisms is important not only for basic biology but also for practical applications, such as improving the yield o...

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Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

et al. 2012

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Photorhabdus luminescens luxCDABE genes were integrated into E. coli K-12 using a high copy number plasmid containing modified luxABCDE genes under the control of the powerful Lac promoter. This strain emitted 10 times higher bioluminescence (BL) than P. luminescens. BL production under different growth conditions was studied. In both bacterial strains, the increase in BL signal correlated with the increase in optical density (OD) in a rich growth medium. However, at the logarithmic growth phase, the BL signal was roughly constant. By contrast, in minimal growth media, there was no substantial growth and the BL/cell was approximately five times higher than in the rich medium. The dynamic measurement range of BL was 10(2) -10(7) colony-forming units (CFU) in E. coli and 10(3) -10(7) CFU in P. luminescens. Because the decrease in the BL signal correlated with the decrease in CFU and OD, i.e. the number of bacterial cells killed, it proved to be very suitable for assessing the antibacterial effects of different antimicrobial agents. Unlike with plate counting, the kinetics of killing can be monitored on a real-time basis using BL measurements. Complement activities in different samples can be estimated using only one serum dilution. The transformed E. coli strain appeared to be superior to P. luminescens in these applications because E. coli was complement sensitive, the detection limit of E. coli was one order lower and the BL-producing system of P. luminescens appeared to be quite unstable.

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Bacterial luciferase activity and the intracellular redox pool in Escherichia coli

Cited by 39 publications

References 41 publications

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence

Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

Escherichia coli K‐12 (pEGFPluxABCDEamp): a tool for analysis of bacterial killing by antibacterial agents and human complement activities on a real‐time basis

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