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Jessica Mendez, Desirée Cascales, Ana I Garcia-Torrico, Jose A Guijarro
Yersinia ruckeri is a bacterium causing fish infection processes at temperatures below the optimum for growth. A derivative Tn5 transposon was used to construct a library of Y. ruckeri mutants with transcriptional fusions between the interrupted genes and the promoterless luxCDABE and lacZY operons. In vitro analysis of β-galactosidase activity allowed the identification of 168 clones having higher expression at 18°C than at 28°C. Among the interrupted genes a SAM-dependent methyltransferase, a diguanylated cyclase, three genes involved in legionaminic acid synthesis and three transcriptional regulators were defined...
2018: Frontiers in Microbiology
J Zhang, J R Liang, R Duan, S Qin, M Xiao, H Q Jing, X Wang
Objective: In this study, we analyze the regulation mechanisms of the expression of ampD in AmpC β-lactamase and the regulation mechanism of β-N-acetylglucosaminidase (NagZ) in Yersinia enterocolitica . Methods: We construct the mutation strains of Yersinia enterocolitica AmpD (AmpD1-3) gene ( ampD 1-3), Low-Molecular-Mass Penicillin-Binding Proteins (LMM PBPs) gene ( pbp 4, pbp 5 a , pbp 5 b , pbp 7), NagZ gene ( nagZ ), and ampR gene by deleting and complementing genes, and induce them by cefoxitin. We determined the activity of AmpC β-lactamase activity (U) of mutant strains (basal level and induce level) by using cephalothiophene hydrolysis method, the promoter activity of AmpC β-lactamase ((relative light unit (RLU)) was detected by the luxCDABE reporter system, and the activity of β-N-acetylglucosaminidase (nmol/L) was determined by by using 4-nitrophenyl N-acetyl-β-D-glucosaminide as the chromogenic substrate...
June 6, 2018: Zhonghua Yu Fang Yi Xue za Zhi [Chinese Journal of Preventive Medicine]
Saba Imdad, Akhilesh Kumar Chaurasia, Kyeong Kyu Kim
Antimicrobial resistance (AMR) in pathogens is the result of indiscriminate use of antibiotics and consequent metabolic/genetic modulation to evolve survival strategies and clonal-selection in AMR strains. As an alternative to antibiotic treatment, antivirulence strategies are being developed, not only to combat bacterial pathogenesis, but also to avoid emerging antibiotic resistance. Vibrio vulnificus is a foodborne pathogen that causes gastroenteritis, necrotizing wound infections, and sepsis with a high rate of mortality...
2018: Frontiers in Cellular and Infection Microbiology
Yingjie Zhou, Johan Memelink, Huub J M Linthorst
Salicylic acid (SA) is an essential hormone for development and induced defense against biotrophic pathogens in plants. The formation of SA mainly derives from chorismate via demonstrated isochorismate synthase (ICS) and presumed isochorismate pyruvate lyase (IPL)-mediated steps in Arabidopsis thaliana, but so far no plant enzyme displaying IPL activity has been identified. Here, we developed an E. coli SA biosensor to screen for IPL activity based on the SalR regulator/salA promoter combination from Acinetobacter sp ADP1, to control the expression of the reporter luxCDABE...
May 23, 2018: Molecular Genetics and Genomics: MGG
Marzena Matejczyk, Grzegorz Świderski, Renata Świsłocka, Stanisław Józef Rosochacki, Włodzimierz Lewandowski
The most important problems of anti-cancer therapy include the toxicity of the drugs applied to healthy cells and the multi-drug cells resistance to chemotherapeutics. One of the most commonly used anticancer drugs is doxorubicin (DOX) used to treat certain leukemias and non-Hodgkin's lymphomas, as well as bladder, breast, stomach, lung, ovarian, thyroid, multiple myeloma and other cancers. Preliminary studies showed that metal complex with DOX improve its cytostatic activity with changes in their molecular structure and distribution of electrons, resulting in a substantial change of its biological activity (including antitumor activity)...
July 2018: Journal of Trace Elements in Medicine and Biology
J Hurtado-Gallego, K Martín-Betancor, I Rodea-Palomares, F Leganés, R Rosal, F Fernández-Piñas
This work describes the construction of two novel self-luminescent bioreporter strains of the cyanobacterium Nostoc sp. PCC 7120 by fusing the promoter region of the sodA and sodB genes (encoding the superoxide dismutases MnSod and FeSod, respectively) to luxCDABE from Photorhabdus luminescens aimed at detecting pollutants that generate reactive oxygen species (ROS), particularly O2 - . Bioreporters were tested against methyl viologen (MV) as the inducer of superoxide anion (O2 - ). Both bioreporters were specific for O2 - and Limits of detection (LODs) and Maximum Permissive Concentrations (MPCs) were calculated: Nostoc sp...
June 2018: Chemosphere
Christine M Hustmyer, Chelsea A Simpson, Stephen G Olney, Douglas B Rusch, Matthew L Bochman, Julia C van Kessel
Experimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating the construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of the luxCDABE bioluminescence operon and the betIBA-proXWV osmotic stress operon in Vibrio harveyi We describe a new method called <u>r</u>apid <u>a</u>rbitrary PCR <u>i</u>nsertion <u>l</u>ibraries (RAIL) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries...
June 1, 2018: Journal of Bacteriology
Zhisong Cui, Xiao Luan, Huichao Jiang, Qian Li, Guangfei Xu, Chengjun Sun, Li Zheng, Yizhi Song, Paul A Davison, Wei E Huang
A toxicity biosensor Acinetobacter baylyi Tox2 was constructed with the host strain A. baylyi ADP1 harboring a new and medium-copy-number plasmid pWH1274_lux, and was applied to detect the cytotoxicity of heavy metal contaminated seawater. The gene cassette luxCDABE was controlled by constitutively expressed promoter Ptet on pWH1274_lux and the bioluminescence intensity of the biosensor reduces in proportional to the concentrations of toxic compounds. A. baylyi Tox2 exhibits tolerance to salinity, hence it is applicable to seawater samples...
June 2018: Chemosphere
Olga E Melkina, Vasilii S Koval, Alexander A Ivanov, Alexei L Zhuze, Gennadii B Zavilgelsky
DNA sequence-specific fluorescent dimeric bisbenzimidazoles DBP(n) and DBPA(n), noncovalently interacting with A-T pairs in the minor groove of double-stranded DNA were used for studying and monitoring the expression of histone-like H-NS-dependent promoters. Histone-like H-NS selectively binds to AT-rich segments of DNA and silences a large number of genes in bacterial chromosomes. The H-NS-dependent promoters of Quorum Sensing (QS)-regulated lux operons of the marine bacteria mesophilic Aliivibrio fischeri, psychrophilic Aliivibrio logei were used...
March 2018: Microbiological Research
O E Melkina, I I Goryanin, G B Zavilgelsky
The effects of histone-like protein H-NS on transcription of promoters of the Quorum Sensing regulated operons from marine luminescent mesophilic bacterium Aliivibrio fischeri and psychrophilic Aliivibrio logei, as well as from pathogenic Pseudomonas aeruginosa, are studied. In the present work, the plasmids carrying DNA fragments with the promoters Pr1f (upstream of the luxICDABEG operon from A. fischeri), Pr1l (upstream of the luxCDABEG operon from A. logei), Pr2l (upstream of luxI gene from A. logei), PluxCf (upstream of luxC gene from A...
February 2017: Genetika
Yazhou Zhou, Jiyuan Zhou, Yuxin Ji, Lu Li, Yafang Tan, Guang Tian, Ruifu Yang, Xiaoyi Wang
Yersinia pestis has evolved from Yersinia pseudotuberculosis serotype O:1b. A typical Y. pestis contains three plasmids: pCD1, pMT1 and pPCP1. However, some isolates only harbor pCD1 (pCD1+ -mutant). Y. pestis and Y. pseudotuberculosis share a common plasmid (pCD1 or pYV), but little is known about whether Y. pseudotuberculosis exhibited plague-inducing potential before it was evolved into Y. pestis. Here, the luxCDABE::Tn5::kan was integrated into the chromosome of the pCD1+ -mutant, Y. pseudotuberculosis or Escherichia coli K12 to construct stable bioluminescent strains for investigation of their dissemination in mice by bioluminescence imaging technology...
March 2018: Microbes and Infection
Chang Liu, Chuchu Li, Yuhuang Chen, Huijing Hao, Junrong Liang, Ran Duan, Zhaoke Guo, Jing Zhang, Zhongzhi Zhao, Huaiqi Jing, Xin Wang, Shihe Shao
Yersinia enterocolitica encodes a chromosomal AmpC β-lactamase under the regulation of the classical ampR-ampC system. To obtain a further understanding to the role of low-molecular-mass penicillin-binding proteins (LMM PBPs) including PBP4, PBP5, PBP6, and PBP7, as well as NagZ and AmpR in ampC regulation of Y. enterocolitica , series of single/multiple mutant strains were systematically constructed and the ampC expression levels were determined by luxCDABE reporter system, reverse transcription-PCR (RT-PCR) and β-lactamase activity test...
2017: Frontiers in Cellular and Infection Microbiology
Bo Jiang, Guanghe Li, Yi Xing, Dayi Zhang, Jianli Jia, Zhisong Cui, Xiao Luan, Hui Tang
Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013...
October 2017: Chemosphere
Wolfram Volkwein, Christopher Maier, Ralph Krafczyk, Kirsten Jung, Jürgen Lassak
The analysis of the function of essential genes in vivo depends on the ability to experimentally modulate levels of their protein products. Current methods to address this are based on transcriptional or post-transcriptional regulation of mRNAs, but approaches based on the exploitation of translation regulation have so far been neglected. Here we describe a toolbox, based on amber suppression in the presence of Nε -acetyl-l-lysine (AcK), for translational tuning of protein output. We chose the highly sensitive luminescence system LuxCDABE as a reporter and incorporated a UAG stop codon into the gene for the reductase subunit LuxC...
October 20, 2017: ACS Synthetic Biology
Jinwoo Kim, Minsik Kim, Seongmi Kim, Sangryeol Ryu
Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen...
August 2, 2017: International Journal of Food Microbiology
Angela Langer, Adriana Moldovan, Christian Harmath, Susan A Joyce, David J Clarke, Ralf Heermann
Phenotypic heterogeneity in microbial communities enables genetically identical organisms to behave differently even under the same environmental conditions. Photorhabdus luminescens, a bioluminescent Gram-negative bacterium, contains a complex life cycle, which involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic cell types, designated as the primary (1°) and secondary (2°) cells. The 1° cells are bioluminescent, pigmented and can support nematode growth and development...
2017: PloS One
Teunke van Rossum, Aleksandra Muras, Marco J J Baur, Sjoerd C A Creutzburg, John van der Oost, Servé W M Kengen
The use of bioreporters in high-throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants...
May 2017: Microbial Biotechnology
Bo Fu, Qiaolian Wu, Minyan Dang, Dangdang Bai, Qiao Guo, Lixin Shen, Kangmin Duan
New antimicrobial agents are urgently needed to treat infections caused by drug-resistant pathogens and by pathogens capable of persisting in biofilms. The aim of this study was to identify traditional Chinese herbs that could inhibit biofilm formation of Pseudomonas aeruginosa, an important human pathogen that causes serious and difficult-to-treat infections in humans. A luxCDABE-based reporter system was constructed to monitor the expression of six key biofilm-associated genes in P. aeruginosa. The reporters were used to screen a library of 36 herb extracts for inhibitory properties against these genes...
2017: BioMed Research International
Shiv Bharadwaj, Robert J Mitchell, Anjum Qureshi, Javed H Niazi
Electronic-cigarettes (e-cigarette) are widely used as an alternative to traditional cigarettes but their safety is not well established. Herein, we demonstrate and validate an analytical method to discriminate the deleterious effects of e-cigarette refills (e-juice) and soluble e-juice aerosol (SEA) by employing stress-specific bioluminescent recombinant bacterial cells (RBCs) as whole-cell biosensors. These RBCs carry luxCDABE-operon tightly controlled by promoters that specifically induced to DNA damage (recA), superoxide radicals (sodA), heavy metals (copA) and membrane damage (oprF)...
April 15, 2017: Biosensors & Bioelectronics
Leon H Oosterik, Huruma N Tuntufye, Jessica Tsonos, Tom Luyten, Sam Noppen, Sandra Liekens, Rob Lavigne, Patrick Butaye, Bruno M Goddeeris
Avian pathogenic Escherichia coli (APEC) are responsible for significant economic losses in the poultry industry. In this study, a model for investigating the pathogenesis of APEC infections was established. APEC strain CH2 (O78) was marked with the luciferase operon (luxCDABE) using a Tn7 transposon and tissues of experimentally infected chickens were analysed for a correlation between the bioluminescent signal and the number of bacteria. Transposition of the lux operon into the chromosome of the APEC isolate did not affect sensitivity to lytic bacteriophages and there was no effect on virulence in an intratracheal infection model in 1-day-old chicks, although results with a subcutaneous infection model were inconclusive...
October 2016: Veterinary Journal
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