Journal articles: 'LuxAB'

1

Katayama, Mitsunori, Nicholas F. Tsinoremas, Takao Kondo, and Susan S. Golden. "cpmA, a Gene Involved in an Output Pathway of the Cyanobacterial Circadian System." Journal of Bacteriology 181, no. 11 (June 1, 1999): 3516–24. http://dx.doi.org/10.1128/jb.181.11.3516-3524.1999.

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ABSTRACT We generated random mutations in Synechococcus sp. strain PCC 7942 to look for genes of output pathways in the cyanobacterial circadian system. A derivative of transposon Tn5 was introduced into the chromosomes of reporter strains in which cyanobacterial promoters drive the Vibrio harveyi luxAB genes and produce an oscillation of bioluminescence as a function of circadian gene expression. Among low-amplitude mutants, one mutant, tnp6, had an insertion in a 780-bp open reading frame. The tnp6 mutation produced an altered circadian phasing phenotype in the expression rhythms of psbAI::luxAB,psbAII::luxAB, andkaiA::luxAB but had no or little effect on those of psbAIII::luxAB,purF::luxAB,kaiB::luxAB,rpoD2::luxAB,ndhD::luxAB, andconII::luxAB. This suggests that the interrupted gene in tnp6, named cpmA (circadian phase modifier), is part of a circadian output pathway that regulates the expression rhythms of psbAI, psbAII, andkaiA.

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SHARP, NATASHA J., JOSHUA P. VANDAMM, IAN J. MOLINEUX, and DAVID A. SCHOFIELD. "Rapid Detection of Bacillus anthracis in Complex Food Matrices Using Phage-Mediated Bioluminescence." Journal of Food Protection 78, no. 5 (May 1, 2015): 963–68. http://dx.doi.org/10.4315/0362-028x.jfp-14-534.

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Bacillus anthracis, the causative agent of anthrax, is considered a high-priority agent that may be used in a food-related terrorist attack because it can be contracted by ingestion and it also forms spores with heat and chemical resistance. Thus, novel surveillance methodologies to detect B. anthracis on adulterated foods are important for bioterrorism preparedness. We describe the development of a phage-based bioluminescence assay for the detection of B. anthracis on deliberately contaminated foods. We previously engineered the B. anthracis phage Wβ with genes encoding bacterial luciferase (luxA and luxB) to create a “light-tagged” reporter (Wβ::luxAB) that is able to rapidly detect B. anthracis by transducing a bioluminescent signal response. Here, we investigate the ability of Wβ::luxAB to detect B. anthracis Sterne, an attenuated select agent strain, in inoculated food (ground beef) and milk (2%, baby formula, and half and half) matrices after incubation with spores for 72 h at 4°C as per AOAC testing guidelines. The majority of B. anthracis bacilli remained in spore form, and thus were potentially infectious, within each of the liquid matrices for 14 days. Detection limits were 80 CFU/ml after 7 h of enrichment; sensitivity of detection increased to 8 CFU/ml when enrichment was extended to 16 h. The limit of detection in ground beef was 3.2 × 103 CFU/g after 7 h of enrichment, improving to 3.2 × 102 CFU/g after 16 h. Because the time to result is rapid and minimal processing is required, and because gastrointestinal anthrax can be fatal, the reporter technology displays promise for the protection of our food supply following a deliberate release of this priority pathogen.

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O'Connell, Kevin P., Ann M. Gustafson, M. Deane Lehmann, and Michael F. Thomashow. "Identification of Cold Shock Gene Loci inSinorhizobium meliloti by Using a luxABReporter Transposon." Applied and Environmental Microbiology 66, no. 1 (January 1, 2000): 401–5. http://dx.doi.org/10.1128/aem.66.1.401-405.2000.

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ABSTRACT Using a luxAB reporter transposon, seven mutants ofSinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15°C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the threerrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrnoperons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene,cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15°C.

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Cohen, Michael F., Yasuko Sakihama, Yojiro C. Takagi, Toshio Ichiba, and Hideo Yamasaki. "Synergistic Effect of Deoxyanthocyanins from Symbiotic Fern Azolla spp. on hrmA Gene Induction in the Cyanobacterium Nostoc punctiforme." Molecular Plant-Microbe Interactions® 15, no. 9 (September 2002): 875–82. http://dx.doi.org/10.1094/mpmi.2002.15.9.875.

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The hrmA gene of the N2-fixing cyanobacterium Nostoc punctiforme functions in repressing the formation of transitory motile filaments, termed hormogonia, by plant-associated vegetative filaments. Here, we report that anthocyanins can contribute to induction of hrmA expression. Aqueous extract from fronds of the fern Azolla pinnata, a host of symbiotic Nostoc spp., was found to be a potent inducer of hrmA-luxAB in N. punctiforme strain UCD 328. The hrmA-luxAB inducing activities of A. pinnata, as well as Azolla filiculoides, were positively correlated with levels of frond deoxyanthocyanins. Analyses of the deoxyanthocyanins in frond extracts revealed, in order of predominance, an acetylated glycoside derivative of luteolinidin (m/z 475) and of apigeninidin (m/z 459) and minor amounts of a second luteolinidin derivative. At up to 150 μM, a purified preparation of deoxyanthocyanins only weakly induced hrmA-luxAB on its own, but mixtures with hrmA-luxAB inducers (A. filiculoides extract or the flavonoid naringin) synergistically doubled to tripled their inducing activities. These results suggest that appropriately localized deoxyanthocyanins could function in plant-mediated mechanisms for repressing Nostoc spp. hormogonium formation.

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Gustafson, Ann M., Kevin P. O'Connell, and Michael F. Thomashow. "Regulation ofSinorhizobium meliloti1021rrnA-reporter gene fusions in response to cold shock." Canadian Journal of Microbiology 48, no. 9 (September 1, 2002): 821–30. http://dx.doi.org/10.1139/w02-078.

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We previously reported that mutants of Sinorhizobium meliloti 1021 carrying luxAB insertions in each of the three 16S rRNA genes exhibited a dramatic ([Formula: see text]28-fold) increase in luminescence following a temperature downshift from 30 to 15°C. These results raised the possibility that the rRNA operons (rrn) of S. meliloti were cold shock loci. In testing this possibility, we found that fusion of the S. meliloti 1021 rrnA promoter to two different reporter genes, luxAB and uidA, resulted in hybrid genes that were transiently upregulated (as measured by transcript accumulation) about four- to sixfold in response to a temperature downshift. These results are consistent with the hypothesis that the rrn promoters are transiently upregulated in response to cold shock. However, much of the apparent cold shock regulation of the initial luxAB insertions was due to an unexpected mechanism: an apparent temperature-dependent inhibition of translation. Specifically, the rrnA sequences from +1 to +172 (relative to the start of transcription) were found to greatly decrease the ability of S. meliloti to translate hybrid rrnluxAB transcripts into active protein at 30°C. This effect, however, was largely eliminated at 15°C. Possible mechanisms for the apparent transient increase in rrnA promoter activity and temperature-dependent inhibition of translation are discussed.Key words: rhizobium, gene expression, luciferase.

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6

Wiles, Siouxsie, Kathryn Ferguson, Martha Stefanidou, Douglas B. Young, and Brian D. Robertson. "Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions." Applied and Environmental Microbiology 71, no. 7 (July 2005): 3427–32. http://dx.doi.org/10.1128/aem.71.7.3427-3432.2005.

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ABSTRACT The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.

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7

Escher, A., D. J. O'Kane, J. Lee, and A. A. Szalay. "Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6528–32. http://dx.doi.org/10.1073/pnas.86.17.6528.

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A 2.2-kilobase-pair (kbp) DNA fragment from Vibrio harveyi contains the luxA and luxB genes separated by a 26-base-pair (bp) intergenic region. The two genes were converted to a single open reading frame by site-specific mutagenesis. A full-length fusion protein is obtained when the new gene is placed under transcriptional control of a T7 promoter in Escherichia coli. Bioluminescence of colonies containing the gene fusion is 0.002% of the wild-type luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] at 37 degrees C. Growth at 23 degrees C results in a greater than 50,000-fold increase in light emission in cells containing fusion protein, whereas only a 3-fold increase in observed with cells containing the luxAB dicistron. Purified fusion protein isolated from E. coli grown at 19 degrees C exists in both monomeric and dimeric forms with specific bioluminescence activities comparable to the heterodimeric wild-type enzyme at 23 degrees C and 37 degrees C. These findings show that the alpha beta fusion polypeptide is functional as a monomer and suggest that its folding is drastically affected at elevated temperature. We hypothesize that the two-subunit bacterial luciferase may have evolved from a monomer as a result of a temperature increase in the environment.

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8

HOSSEINKHANI, Saman, Rose SZITTNER, and Edward A. MEIGHEN. "Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay." Biochemical Journal 385, no. 2 (January 7, 2005): 575–80. http://dx.doi.org/10.1042/bj20040863.

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Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the ‘slow’ X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820–13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175→Gly)] resulted in the ‘slow decay’ X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between ‘slow’ and ‘fast’ luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase–flavin–oxygen intermediate.

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9

Bayer, Thomas, Aileen Becker, Henrik Terholsen, In Jung Kim, Ina Menyes, Saskia Buchwald, Kathleen Balke, Suvi Santala, Steven C. Almo, and Uwe T. Bornscheuer. "LuxAB-Based Microbial Cell Factories for the Sensing, Manufacturing and Transformation of Industrial Aldehydes." Catalysts 11, no. 8 (August 10, 2021): 953. http://dx.doi.org/10.3390/catal11080953.

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The application of genetically encoded biosensors enables the detection of small molecules in living cells and has facilitated the characterization of enzymes, their directed evolution and the engineering of (natural) metabolic pathways. In this work, the LuxAB biosensor system from Photorhabdus luminescens was implemented in Escherichia coli to monitor the enzymatic production of aldehydes from primary alcohols and carboxylic acid substrates. A simple high-throughput assay utilized the bacterial luciferase—previously reported to only accept aliphatic long-chain aldehydes—to detect structurally diverse aldehydes, including aromatic and monoterpene aldehydes. LuxAB was used to screen the substrate scopes of three prokaryotic oxidoreductases: an alcohol dehydrogenase (Pseudomonas putida), a choline oxidase variant (Arthrobacter chlorophenolicus) and a carboxylic acid reductase (Mycobacterium marinum). Consequently, high-value aldehydes such as cinnamaldehyde, citral and citronellal could be produced in vivo in up to 80% yield. Furthermore, the dual role of LuxAB as sensor and monooxygenase, emitting bioluminescence through the oxidation of aldehydes to the corresponding carboxylates, promises implementation in artificial enzyme cascades for the synthesis of carboxylic acids. These findings advance the bio-based detection, preparation and transformation of industrially important aldehydes in living cells.

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10

Wang, Chunxia, Daoben Wang, and Qi Zhou. "Colonization and persistence of a plant growth-promoting bacterium Pseudomonas fluorescens strain CS85, on roots of cotton seedlings." Canadian Journal of Microbiology 50, no. 7 (July 1, 2004): 475–81. http://dx.doi.org/10.1139/w04-040.

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Pseudomonas fluorescens CS85, which was previously isolated from the rhizosphere of cotton seedlings, acts as both a plant growth-promoting bacterium and a biocontrol agent against cotton pathogens, including Rhizoctonia solani, Colletotrichum gossypii, Fusarium oxysporum f sp. vasinfectum, and Verticillium dahliae. Strain CS85 was labeled separately with luxAB and gusA. The labeled strains were stably maintained and had high levels of expression of the marker genes, luxAB and gusA, after successive transfers on nonselective medium, long-term preservation, and after recovery from soil. The labeled strains displayed similar biocontrol characteristics (e.g., antibiosis, effects of growth -promotion and disease -control) to the original strain. The labeled strains colonized all surfaces of the young plant root zones, such as roots hairs and lateral roots, although the distribution of the labeled strains on the root surfaces was not uniform. Moreover, the population densities of the labeled strains on the root surface were stably maintained at high levels during the first 2 weeks of plant growth in the native soil, so that about 107–108 CFU/g root were detected, then decreased gradually. Nevertheless, approximately 106 CFU/g root of the labeled strains were observed on the root surfaces 35 d after planting.Key words: plant growth-promoting bacteria, luxAB, gusA, root colonization.

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11

Zhu, Bei Bei, Dao Sheng Wang, Yuan Yuan Cao, Xin Yun Tang, Ri Mao Hua, and Tao Zhong Shi. "Fast Degradation of Chlorpyrifos in Soil by Strain Cupriavidus taiwanensis X1 Labeled with LuxAB." Advanced Materials Research 356-360 (October 2011): 2566–70. http://dx.doi.org/10.4028/www.scientific.net/amr.356-360.2566.

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For researching ecological behaviors of Cupriavidus taiwanensis X1 which has strong hydrolysis activity on chlorpyrifos(CP), reporter gene luxAB was successfully introduced into cells by electroporation. The labeled strain X1-lux with genetic stability and fluorescence was obtained. Both of strain X1 and X1-lux could completely degrade 200 mg/l CP within 12h in minimal salt medium, and experimental results showed that introduction of luxAB did not affect strain X1 growth and degradation on CP. The cells of X1-lux and X1 were inoculated into soil with 500 mg/l CP, the cell concentration and CP residual was detected, and data revealed that strain X1 could absolutely removed CP in 12d and survive in soil. Strain X1 is a potential excellent choice for bioremediation of organophosphorus polluted environments.

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THOUAND, G., P. VACHON, S. LIU, M. DAYRE, and M. W. GRIFFITHS. "Optimization and Validation of a Simple Method Using P22::luxAB Bacteriophage for Rapid Detection of Salmonella enterica Serotypes A, B, and D in Poultry Samples." Journal of Food Protection 71, no. 2 (February 1, 2008): 380–85. http://dx.doi.org/10.4315/0362-028x-71.2.380.

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A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22::luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22::luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 × 109 (±4.3 × 109) PFU/ml of P22, including only 1.4 × 106 (±1 × 106) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 × 102 (±1.75 × 102) CFU Salmonella Typhimurium per ml, but increased to 1.5 × 104 (±1.17 × 104) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 × 103 (±1.5 × 103) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 × 105 (±0.15 × 105) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.

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Sharp, Natasha J., Ian J. Molineux, Martin A. Page, and David A. Schofield. "Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages." Applied and Environmental Microbiology 82, no. 8 (February 12, 2016): 2380–87. http://dx.doi.org/10.1128/aem.03772-15.

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ABSTRACTBacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracisspores in environmental samples. The assay uses an engineeredB. anthracisreporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression ofluxABin an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection ofB. anthracisspores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 105CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 104CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

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Kim, Han-Suk, Sung-Min Kim, Hyun-Jung Lee, Soon-Jung Park, and Kyu-Ho Lee. "Expression of the cpdA Gene, Encoding a 3′,5′-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex." Journal of Bacteriology 191, no. 3 (November 21, 2008): 922–30. http://dx.doi.org/10.1128/jb.01350-08.

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ABSTRACT The intracellular level of cyclic 3′,5′-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium, Vibrio vulnificus. The cpdA gene exists in an operon composed of mutT, yqiB, cpdA, and yqiA, the transcription of which was initiated at position −22 upstream of mutT. A cpdA-null mutant of V. vulnificus contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the cpdA gene was provided in trans, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both cya and crp mutants. In addition, levels of expression of the cpdA::luxAB transcription fusion decreased in cya and crp mutants. The level of expression of cpdA::luxAB in the cya mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of mutT, which includes a putative CRP-binding sequence centered at position −95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the cpdA::luxAB transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of cpdA is activated by the cAMP-CRP complex via direct binding to the regulatory region.

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Gueuné, Hervé, Marie-José Durand, Gérald Thouand, and Michael S. DuBow. "The ygaVP Genes of Escherichia coli Form a Tributyltin-Inducible Operon." Applied and Environmental Microbiology 74, no. 6 (February 1, 2008): 1954–58. http://dx.doi.org/10.1128/aem.02294-07.

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ABSTRACT A tributyltin (TBT) luxAB transcriptional fusion in Escherichia coli revealed that a TBT-activated promoter is located upstream of two cotranscribed orphan genes, ygaV and ygaP. We demonstrate that transcription from the promoter upstream of ygaVP is constitutive in a ygaVP mutant, suggesting that YgaV is an autoregulated, TBT-inducible repressor.

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Onai, Kiyoshi, Megumi Morishita, Shino Itoh, Kazuhisa Okamoto, and Masahiro Ishiura. "Circadian Rhythms in the Thermophilic Cyanobacterium Thermosynechococcus elongatus: Compensation of Period Length over a Wide Temperature Range." Journal of Bacteriology 186, no. 15 (August 1, 2004): 4972–77. http://dx.doi.org/10.1128/jb.186.15.4972-4977.2004.

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ABSTRACT Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T. elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene. A promoter region of the psbA1 gene of T. elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T. elongatus. The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60°C). Theses results indicate that T. elongatus has the circadian clock that is widely temperature compensated.

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Riedel, Kathrin, Thomas Ohnesorg, Karen A. Krogfelt, Thomas S. Hansen, Kenji Omori, Michael Givskov, and Leo Eberl. "N-Acyl-l-Homoserine Lactone-Mediated Regulation of the Lip Secretion System inSerratia liquefaciens MG1." Journal of Bacteriology 183, no. 5 (March 1, 2001): 1805–9. http://dx.doi.org/10.1128/jb.183.5.1805-1809.2001.

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ABSTRACT The analysis of Serratia liquefaciens MG1 ′luxAB insertion mutants that are responsive toN-butanoyl-l-homoserine lactone revealed that expression of lipB is controlled by the swrquorum-sensing system. LipB is part of the Lip exporter, a type I secretion system, which is responsible for the secretion of extracellular lipase, metalloprotease, and S-layer protein.

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Rosseels, Valérie, Virginie Roupie, Denise Zinniel, Raúl G. Barletta, and Kris Huygen. "Development of Luminescent Mycobacterium avium subsp. paratuberculosis for Rapid Screening of Vaccine Candidates in Mice." Infection and Immunity 74, no. 6 (June 2006): 3684–86. http://dx.doi.org/10.1128/iai.01521-05.

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ABSTRACT Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.

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Bachmann, Herwig, Filipe Santos, Michiel Kleerebezem, and Johan E. T. van Hylckama Vlieg. "Luciferase Detection during Stationary Phase in Lactococcus lactis." Applied and Environmental Microbiology 73, no. 14 (May 18, 2007): 4704–6. http://dx.doi.org/10.1128/aem.02807-06.

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ABSTRACT The luminescence signal of luxAB-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of Lactococcus lactis, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.

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Lee, Hyun-Jung, Kyung-Je Park, Ah Young Lee, Sung Goo Park, Byoung Chul Park, Kyu-Ho Lee, and Soon-Jung Park. "Regulation of fur Expression by RpoS and Fur in Vibrio vulnificus." Journal of Bacteriology 185, no. 19 (October 1, 2003): 5891–96. http://dx.doi.org/10.1128/jb.185.19.5891-5896.2003.

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ABSTRACT In a proteomic analysis of rpoS-deficient Vibrio vulnificus versus the wild type, one of the down-regulated proteins in the rpoS mutant strain was identified as a Fur protein, a ferric uptake regulator. The expression of a fur::luxAB fusion was significantly influenced by sigma factor S, the rpoS gene product, and positively regulated by Fur under iron-limited conditions.

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Koch, Birgit, Tommy H. Nielsen, Dan Sørensen, Jens Bo Andersen, Carsten Christophersen, Søren Molin, Michael Givskov, Jan Sørensen, and Ole Nybroe. "Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System." Applied and Environmental Microbiology 68, no. 9 (September 2002): 4509–16. http://dx.doi.org/10.1128/aem.68.9.4509-4516.2002.

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ABSTRACT Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.

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Kretzer, Jan, Mathias Schmelcher, and Martin Loessner. "Ultrasensitive and Fast Diagnostics of Viable Listeria Cells by CBD Magnetic Separation Combined with A511::luxAB Detection." Viruses 10, no. 11 (November 13, 2018): 626. http://dx.doi.org/10.3390/v10110626.

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The genus Listeria includes foodborne pathogens that cause life-threatening infections in those at risk, and sensitive and specific methods for detection of these bacteria are needed. Based on their unrivaled host specificity and ability to discriminate viable cells, bacteriophages represent an ideal toolbox for the development of such methods. Here, the authors describe an ultrasensitive diagnostic protocol for Listeria by combining two phage-based strategies: (1) specific capture and concentration of target cells by magnetic separation, harnessing cell wall-binding domains from Listeria phage endolysins (CBD-MS); and (2) highly sensitive detection using an adaptation of the A511::luxAB bioluminescent reporter phage assay in a microwell plate format. The combined assay enabled direct detection of approximately 100 bacteria per ml of pure culture with genus-level specificity in less than 6 h. For contaminated foods, the procedure included a 16 h selective enrichment step, followed by CBD-MS separation and A511::luxAB detection. It was able to consistently detect extremely low numbers (0.1 to 1.0 cfu/g) of viable Listeria cells, in a total assay time of less than 22 h. These results demonstrate the superiority of this phage-based assay to standard culture-based diagnostic protocols for the detection of viable bacteria, with respect to both sensitivity and speed.

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Lee, Hyun-Jung, So Hyun Bang, Kyu-Ho Lee, and Soon-Jung Park. "Positive Regulation of fur Gene Expression via Direct Interaction of Fur in a Pathogenic Bacterium, Vibrio vulnificus." Journal of Bacteriology 189, no. 7 (January 19, 2007): 2629–36. http://dx.doi.org/10.1128/jb.01791-06.

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ABSTRACT In pathogenic bacteria, the ability to acquire iron, which is mainly regulated by the ferric uptake regulator (Fur), is essential to maintain growth as well as its virulence. In Vibrio vulnificus, a human pathogen causing gastroenteritis and septicemia, fur gene expression is positively regulated by Fur when the iron concentration is limited (H.-J. Lee et al., J. Bacteriol. 185:5891-5896, 2003). Footprinting analysis revealed that an upstream region of the fur gene was protected by the Fur protein from DNase I under iron-depleted conditions. The protected region, from −142 to −106 relative to the transcription start site of the fur gene, contains distinct AT-rich repeats. Mutagenesis of this repeated sequence resulted in abolishment of binding by Fur. To confirm the role of this cis-acting element in Fur-mediated control of its own gene in vivo, fur expression was monitored in V. vulnificus strains using a transcriptional fusion containing the mutagenized Fur-binding site (fur mt::luxAB). Expression of fur mt::luxAB showed that it was not regulated by Fur and was not influenced by iron concentration. Therefore, this study demonstrates that V. vulnificus Fur acts as a positive regulator under iron-limited conditions by direct interaction with the fur upstream region.

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Mudge, SR, WR Lewis-Henderson, and RG Birch. "Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants." Functional Plant Biology 23, no. 1 (1996): 75. http://dx.doi.org/10.1071/pp9960075.

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Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

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Moses, Sarit, Moshe Aftalion, Emanuelle Mamroud, Shahar Rotem, and Ida Steinberger-Levy. "Reporter-Phage-Based Detection and Antibiotic Susceptibility Testing of Yersinia pestis for a Rapid Plague Outbreak Response." Microorganisms 9, no. 6 (June 11, 2021): 1278. http://dx.doi.org/10.3390/microorganisms9061278.

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Pneumonic plague is a lethal infectious disease caused by Yersinia pestis, a Tier-1 biothreat agent. Antibiotic treatment can save infected patients; however, therapy should begin within 24 h of symptom onset. As some Y. pestis strains showed an antibiotic resistance phenotype, an antibiotic susceptibility test (AST) must be performed. Performing the Clinical and Laboratory Standards Institute (CLSI)-recommended standard process, which includes bacterial isolation, enumeration and microdilution testing, lasts several days. Thus, rapid AST must be developed. As previously published, the Y. pestis-specific reporter phage ϕA1122::luxAB can serve for rapid identification and AST (ID-AST). Herein, we demonstrate the ability to use ϕA1122::luxAB to determine minimal inhibitory concentration (MIC) values and antibiotic susceptibility categories for various Y. pestis therapeutic antibiotics. We confirmed the assay by testing several nonvirulent Y. pestis isolates with reduced susceptibility to doxycycline or ciprofloxacin. Moreover, the assay can be performed directly on positive human blood cultures. Furthermore, as Y. pestis may naturally or deliberately be spread in the environment, we demonstrate the compatibility of this direct method for this scenario. This direct phage-based ID-AST shortens the time needed for standard AST to less than a day, enabling rapid and correct treatment, which may also prevent the spread of the disease.

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Riccillo, Pablo M., Cecilia I. Muglia, Frans J. de Bruijn, Andrew J. Roe, Ian R. Booth, and O. Mario Aguilar. "Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1748–53. http://dx.doi.org/10.1128/jb.182.6.1748-1753.2000.

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ABSTRACT The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance.Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.

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Iizumi, Taro, Masahiro Mizumoto, and Kanji Nakamura. "A Bioluminescence Assay Using Nitrosomonas europaeafor Rapid and Sensitive Detection of Nitrification Inhibitors." Applied and Environmental Microbiology 64, no. 10 (October 1, 1998): 3656–62. http://dx.doi.org/10.1128/aem.64.10.3656-3662.1998.

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ABSTRACT An expression vector for the luxAB genes, derived fromVibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2 − production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.

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Nelson, Eric J., Hege S. Tunsjø, Pat M. Fidopiastis, Henning Sørum, and Edward G. Ruby. "A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence." Applied and Environmental Microbiology 73, no. 6 (February 2, 2007): 1825–33. http://dx.doi.org/10.1128/aem.02255-06.

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ABSTRACT 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.

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Gillor, Osnat, Ayelet Harush, Ora Hadas, Anton F. Post, and Shimshon Belkin. "A Synechococcus PglnA::luxAB Fusion for Estimation of Nitrogen Bioavailability to Freshwater Cyanobacteria." Applied and Environmental Microbiology 69, no. 3 (March 2003): 1465–74. http://dx.doi.org/10.1128/aem.69.3.1465-1474.2003.

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ABSTRACT In contrast to extensive studies of phosphorus, widely considered the main nutrient limiting phytoplankton biomass in freshwater ecosystems, there have been few studies on the role of nitrogen in controlling phytoplankton populations. This situation may be due partly to the complexity in estimating its utilization and bioavailability. In an attempt to provide a novel tool for this purpose, we fused the promoter of the glutamine synthetase-encoding gene, P glnA, from Synechococcus sp. strain PCC7942 to the luxAB luciferase-encoding genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus chromosome to yield the reporter strain GSL. Light emission by this strain was dependent upon ambient nitrogen concentrations. The linear response range of the emitted luminescence was 1 mM to 1 μM for the inorganic nitrogen species tested (ammonium, nitrate, and nitrite) and 10- to 50-fold lower for glutamine and urea. When water samples collected from along a depth profile in Lake Kinneret (Israel) were exposed to the reporter strain, the bioluminescence of the reporter strain mirrored the total dissolved nitrogen concentrations determined for the same samples and was shown to be a sensitive indicator of the concentration of bioavailable nitrogen.

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Nair, Usha, Colleen Thomas, and Susan S. Golden. "Functional Elements of the Strong psbAIPromoter of Synechococcus elongatus PCC 7942." Journal of Bacteriology 183, no. 5 (March 1, 2001): 1740–47. http://dx.doi.org/10.1128/jb.183.5.1740-1747.2001.

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ABSTRACT The psbAI gene of the cyanobacteriumSynechococcus elongatus PCC 7942 is one of threepsbA genes that encode a critical photosystem II reaction center protein, D1. Regulation of the gene family in response to changes in the light environment is complex, occurs at transcriptional and posttranscriptional levels, and results in an interchange of two different forms of D1 in the membrane. Expression of psbAIis downregulated under high-intensity light (high light) in contrast to induction of the other two family members. We show that, in addition to a known accelerated degradation of the psbAI message, promoter activity decreases upon exposure to high light. Unlike the other psbA genes, additional sequences upstream of thepsbAI −35 element are required for expression. Mutagenizing the atypical psbAI −10 element from TCTCCT to TATAAT increased the magnitude of expression from bothpsbAI::lacZ andpsbAI::luxAB fusions but did not affect downregulation under high light. Inactivation of group 2 sigma factor genes rpoD2 and sigC, in both wild-type and −10-element mutagenized backgrounds, resulted in elevatedpsbAI::luxAB expression but did not alter the response to high light. The results are consistent with redundancy of promoter recognition among cyanobacterial group 2 sigma factors. Electrophoretic mobility shift assays showed that the DNA sequence corresponding to the untranslated leader of thepsbAI message binds one or more proteins from an S. elongatus extract. The corresponding region of psbAIIefficiently competed for this binding activity, suggesting a shared regulatory factor among these disparately regulated genes.

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Andrey, Diego O., Adriana Renzoni, Antoinette Monod, Daniel P. Lew, Ambrose L. Cheung, and William L. Kelley. "Control of the Staphylococcus aureus Toxic Shock tst Promoter by the Global Regulator SarA." Journal of Bacteriology 192, no. 22 (September 24, 2010): 6077–85. http://dx.doi.org/10.1128/jb.00146-10.

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ABSTRACT The Staphylococcus aureus SarA global regulator controls the expression of numerous virulence genes, often in conjunction with the agr quorum-sensing system and its effector RNA, RNAIII. In the present study, we have examined the role of both SarA and RNAIII on the regulation of the promoter of tst, encoding staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1). In vitro DNA-protein interaction studies with purified SarA using gel shift and DNase I protection assays revealed one strong SarA binding site and evidence for a weaker site nearby within the minimal 400-bp promoter region upstream of tst. In vivo analysis of tst promoter activation using a ptst -luxAB reporter inserted in the chromosome revealed partial but not complete loss of tst expression in a Δhld-RNAIII strain. In contrast, disruption of sarA abrogated tst expression. No significant tst expression was found for the double Δhld-RNAIII-ΔsarA mutant. Introduction of a plasmid containing cloned hld-RNAIII driven by a non-agr-dependent promoter, pHU , into isogenic parental wild-type or ΔsarA strains showed comparable levels of RNAIII detected by quantitative reverse transcription-PCR (qRT-PCR) but a two-log10 reduction in ptst-luxAB reporter expression in the ΔsarA strain, arguing that RNAIII levels alone are not strictly determinant for tst expression. Collectively, our results indicate that SarA binds directly to the tst promoter and that SarA plays a significant and direct role in the expression of tst.

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32

Justus, Tamara, and Susan M. Thomas. "P XIV A.9 Bioluminescent detection of DNA damage via umuC1-luxAB gene fusion." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 379, no. 1 (September 1997): S125. http://dx.doi.org/10.1016/s0027-5107(97)83047-3.

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Jacobs, Myra, Philip J. Hill, and Gordon S. A. B. Stewart. "Highly bioluminescent Bacillus subtilis obtained through high-level expression of a luxAB fusion gene." Molecular and General Genetics MGG 230, no. 1-2 (November 1991): 251–56. http://dx.doi.org/10.1007/bf00290675.

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Cohen, Michael F., and John C. Meeks. "A Hormogonium Regulating Locus, hrmUA, of the Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and its Response to an Extract of a Symbiotic Plant Partner Anthoceros punctatus." Molecular Plant-Microbe Interactions® 10, no. 2 (March 1997): 280–89. http://dx.doi.org/10.1094/mpmi.1997.10.2.280.

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Transposon-generated mutant strain UCD 328 of Nostoc punctiforme strain ATCC 29133 has a phenotype of an increased sensitivity to a hormogonium-inducing factor exuded by a symbiotic plant partner, Anthoceros punctatus, and an initial increased hormogonium-dependent infection of the plant. Sequence analysis showed that the transposition site in strain UCD 328 lies within a 1,251-bp open reading frame (ORF), designated hrmA, that displays no significant similarity to known database sequences. A second, 837-bp ORF (hrmU) ends 2 bp 5′ from the start of hrmA and has the signature sequences belonging to a family of NAD(P)H-dependent oxidoreductases. Strains having insertional mutations in hrmU or hrmA reproduce the strain UCD 328 phenotype. Transcriptional fusions of luxAB to hrmU or hrmA show an 8- to 10-fold peak increase in luciferase activity 13 to 20 h after the start of incubation in the presence of an aqueous extract of A. punctatus. A promoter induced by the extract was deduced to be between 2.0 to 3.4 kb from the translational start of hrmU. A multicopy plasmid that contains hrmUA within a 6.2-kb fragment conferred an increased infection phenotype on wild-type N. punctiforme 29133. This plasmid and another plasmid containing 4.4 kb of DNA 5′ of the transposition site prevented extract-dependent induction of hrmA-luxAB transcription in strain UCD 328, implicating titration of some trans-activator(s) by the cloned fragments. We hypothesize a role for hrmUA in the inhibition of hormogonium formation by the metabolism of an unknown hormogonium-regulating metabolite.

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35

Boyanapalli, Ramakrishna, George S. Bullerjahn, Christa Pohl, Peter L. Croot, Philip W. Boyd, and R. Michael L. McKay. "Luminescent Whole-Cell Cyanobacterial Bioreporter for Measuring Fe Availability in Diverse Marine Environments." Applied and Environmental Microbiology 73, no. 3 (December 8, 2006): 1019–24. http://dx.doi.org/10.1128/aem.01670-06.

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ABSTRACT A Synechococcus sp. strain PCC 7002 Fe bioreporter was constructed containing the isiAB promoter fused to the Vibrio harveyi luxAB genes. Bioreporter luminescence was characterized with respect to the free ferric ion concentration in trace metal-buffered synthetic medium. The applicability of the Fe bioreporter to assess Fe availability in the natural environment was tested by using samples collected from the Baltic Sea and from the high-nutrient, low-chlorophyll subarctic Pacific Ocean. Parallel assessment of dissolved Fe and bioreporter response confirmed that direct chemical measurements of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton.

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Justus, Tamara, and Susan M. Thomas. "Construction of a umuC′–luxAB plasmid for the detection of mutagenic DNA repair via luminescence." Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 398, no. 1-2 (February 1998): 131–41. http://dx.doi.org/10.1016/s0027-5107(97)00215-7.

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Loessner, M. J., M. Rudolf, and S. Scherer. "Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods." Applied and environmental microbiology 63, no. 8 (1997): 2961–65. http://dx.doi.org/10.1128/aem.63.8.2961-2965.1997.

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Trzebiatowski, Jodi R., Daniel M. Ragatz, and Frans J. de Bruijn. "Isolation and Regulation of Sinorhizobium meliloti 1021 Loci Induced by Oxygen Limitation." Applied and Environmental Microbiology 67, no. 8 (August 1, 2001): 3728–31. http://dx.doi.org/10.1128/aem.67.8.3728-3731.2001.

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ABSTRACT Eleven Sinorhizobium meliloti 1021 loci whose expression was induced under low oxygen concentrations were identified in a collection of 5,000 strains carrying Tn5-1063 (luxAB) transcriptional reporter gene fusions. The 11 Tn5-1063-tagged loci were cloned and characterized. The dependence of the expression of the tagged loci on the FixL/FixJ oxygen-sensing two-component regulatory system was examined. Three of the loci were found to be dependent uponfixL and fixJ for their expression, while one locus showed a partial dependence. The remaining seven loci showedfixL- and fixJ-independent induction of expression in response to oxygen limitation. This suggests that inS. meliloti, additional regulatory system(s) exist that respond either directly or indirectly to oxygen limitation conditions.

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Milcamps, A., D. M. Ragatz, P. Lim, K. A. Berger, and F. J. de Bruijin. "Isolation of carbon- and nitrogen-deprivation-induced loci of Sinorhizobium meliloti 1021 by Tn5-luxAB mutagenesis." Microbiology 144, no. 11 (November 1, 1998): 3205–18. http://dx.doi.org/10.1099/00221287-144-11-3205.

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40

Loessner, M. J., C. E. Rees, G. S. Stewart, and S. Scherer. "Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells." Applied and environmental microbiology 62, no. 4 (1996): 1133–40. http://dx.doi.org/10.1128/aem.62.4.1133-1140.1996.

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41

Bozcal, Elif, Melih Dagdeviren, Atac Uzel, and Mikael Skurnik. "LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo." PLOS ONE 12, no. 2 (February 24, 2017): e0172877. http://dx.doi.org/10.1371/journal.pone.0172877.

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42

Blouin, K., S. G. Walker, J. Smit, and R. Turner. "Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes." Applied and environmental microbiology 62, no. 6 (1996): 2013–21. http://dx.doi.org/10.1128/aem.62.6.2013-2021.1996.

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Unge, A., and J. Jansson. "Monitoring population size, activity, and distribution of gfp-luxAB-tagged Pseudomonas fluorescens SBW25 during colonization of wheat." Microbial Ecology 41, no. 4 (May 2001): 290–300. http://dx.doi.org/10.1007/s002480000047.

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Unge, Annika, Riccardo Tombolini, Lars Mølbak, and Janet K. Jansson. "Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dualgfp-luxAB Marker System." Applied and Environmental Microbiology 65, no. 2 (February 1, 1999): 813–21. http://dx.doi.org/10.1128/aem.65.2.813-821.1999.

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ABSTRACT A dual marker system was developed for simultaneous quantification of bacterial cell numbers and their activity with the luxABand gfp genes, encoding bacterial luciferase and green fluorescent protein (GFP), respectively. The bioluminescence phenotype of the luxAB biomarker is dependent on cellular energy status. Since cellular metabolism requires energy, bioluminescence output is directly related to the metabolic activity of the cells. By contrast, GFP fluorescence has no energy requirement. Therefore, by combining these two biomarkers, total cell number and metabolic activity of a specific marked cell population could be monitored simultaneously. Two different bacterial strains, Escherichia coli DH5α and Pseudomonas fluorescens SBW25, were chromosomally tagged with the dual marker cassette, and the cells were monitored under different conditions by flow cytometry, plate counting, and luminometry. During log-phase growth, the luciferase activity was proportional to the number of GFP-fluorescent cells and culturable cells. Upon entrance into stationary phase or during starvation, luciferase activity decreased due to a decrease in cellular metabolic activity of the population, but the number of GFP-fluorescing cells and culturable cells remained relatively stable. In addition, we optimized a procedure for extraction of bacterial cells from soil, allowing GFP-tagged bacteria in soil samples to be quantitated by flow cytometry. After 30 days of incubation of P. fluorescensSBW25::gfp/lux in soil, the cells were still maintained at high population densities, as determined by GFP fluorescence, but there was a slow decline in luciferase activity, implicating nutrient limitation. In conclusion, the dual marker system allowed simultaneous monitoring of the metabolic activity and cell number of a specific bacterial population and is a promising tool for monitoring of specific bacteria in situ in environmental samples.

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Kunert, Anja, Martin Hagemann, and Norbert Erdmann. "Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB." Journal of Microbiological Methods 41, no. 3 (August 2000): 185–94. http://dx.doi.org/10.1016/s0167-7012(00)00162-7.

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Kaniga, K., M. P. Sory, I. Delor, C. Saegerman, J. N. Limet, and G. R. Cornelis. "Monitoring of Yersinia enterocolitica in murine and bovine feces on the basis of the chromosomally integrated luxAB marker gene." Applied and Environmental Microbiology 58, no. 3 (1992): 1024–26. http://dx.doi.org/10.1128/aem.58.3.1024-1026.1992.

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Guzzo, Angelina, and Michael S. DuBow. "A luxAB transcriptional fusion to the cryptic celF gene of Escherichia coli displays increased luminescence in the presence of nickel." Molecular and General Genetics MGG 242, no. 4 (February 1994): 455–60. http://dx.doi.org/10.1007/bf00281796.

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Zhang, Tianyu, William R. Bishai, Jacques H. Grosset, and Eric L. Nuermberger. "Rapid Assessment of Antibacterial Activity against Mycobacterium ulcerans by Using Recombinant Luminescent Strains." Antimicrobial Agents and Chemotherapy 54, no. 7 (April 26, 2010): 2806–13. http://dx.doi.org/10.1128/aac.00400-10.

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ABSTRACT Mycobacterium ulcerans causes Buruli ulcer, an emerging infectious disease for which antimicrobial therapy has only recently proven to be beneficial. The discovery and development of new drugs against M. ulcerans are severely impeded by its very slow growth. Recombinant bioluminescent strains have proven useful in drug development for other mycobacterial infections, but the ability of such strains to discriminate bacteriostatic from bactericidal activity has not been well demonstrated. We engineered recombinant M. ulcerans strains to express luxAB from Vibrio harveyi. In drug susceptibility tests employing a wide range of antimicrobial agents and concentrations, the relative light unit (RLU) count measured in real time was a reliable surrogate marker for CFU counts available 3 months later, indicating utility for the rapid determination of drug susceptibility and discrimination of bacteriostatic and bactericidal effects. A second important finding of this study is that the addition of subinhibitory concentrations of the ATP-binding cassette transporter inhibitor reserpine increases the susceptibility of M. ulcerans to tetracycline and erythromycin, indicating that drug efflux may explain at least part of the intrinsic resistance of M. ulcerans to these agents.

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Zhu, Jinsong, Karin Jäger, Todd Black, Kelly Zarka, Olga Koksharova, and C. Peter Wolk. "HcwA, an Autolysin, Is Required for Heterocyst Maturation in Anabaena sp. Strain PCC 7120." Journal of Bacteriology 183, no. 23 (December 1, 2001): 6841–51. http://dx.doi.org/10.1128/jb.183.23.6841-6851.2001.

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ABSTRACT In many filamentous cyanobacteria, vegetative cells can differentiate into heterocysts, cells that are specialized for aerobic fixation of N2. Synthesis of the heterocyst envelope polysaccharide is dependent on the gene hepA inAnabaena sp. strain PCC 7120. In search of genes that are involved in the regulation of hepA, we transposon mutagenized strain DR1069, which bears a chromosomalhepA::luxAB fusion. One resulting mutant, designated HNL3, grows normally in medium with nitrate and shows poor induction of hepA in response to nitrogen deprivation. In HNL3, transposon Tn5-1058 is inserted within gene hcwA, a constitutively expressed open reading frame whose predicted product resemblesN-acetylmuramoyl-l-alanine amidases. Reconstruction of the mutation confirmed that the mutant phenotype resulted from the insertion of the transposon. The induction ofhepA in HNL3 is partially restored upon recombination of HNL3 with plasmid-borne, wild-type hcwA. Moreover, HcwA expressed in Escherichia coli exhibits wall-lytic activity. These results suggest that the degradation, or possibly reconstruction, of the cell peptidoglycan layer is a prerequisite for heterocyst maturation.

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Riccillo, Pablo M., Monica M. Collavino, Daniel H. Grasso, Reg England, Frans J. de Bruijn, and O. Mario Aguilar. "A guaB Mutant Strain of Rhizobium tropici CIAT899 Pleiotropically Defective in Thermal Tolerance and Symbiosis." Molecular Plant-Microbe Interactions® 13, no. 11 (November 2000): 1228–36. http://dx.doi.org/10.1094/mpmi.2000.13.11.1228.

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Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using Tn5-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.

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Journal articles on the topic 'LuxAB'

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