Lux Reporter Research Articles

Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by β-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the β-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia.

Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab's inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS Mab , a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRS Mtb , is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosR Mab regulon was predicted using bioinformatics based on DosR Mtb motifs however, the role and regulon of DosRS Mab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRS Mab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS Mab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

Combining transposon mutagenesis and reporter genes to identify novel regulators of the topA promoter in Streptomyces

BackgroundIdentifying the regulatory factors that control transcriptional activity is a major challenge of gene expression studies. Here, we describe the application of a novel approach for in vivo identification of regulatory proteins that may directly or indirectly control the transcription of a promoter of interest in Streptomyces.ResultsA method based on the combination of Tn5 minitransposon-driven random mutagenesis and lux reporter genes was applied for the first time for the Streptomyces genus. As a proof of concept, we studied the topA supercoiling-sensitive promoter, whose activity is dependent on unknown regulatory factors. We found that the sco4804 gene product positively influences topA transcription in S. coelicolor, demonstrating SCO4804 as a novel player in the control of chromosome topology in these bacteria.ConclusionsOur approach allows the identification of novel Streptomyces regulators that may be critical for the regulation of gene expression in these antibiotic-producing bacteria.

A quantitative method to assess bacterial adhesion using recombinant bioluminescent Pseudomonas aeruginosa.

Bioluminescence technology has been widely used in the field of medical detection. The bioluminescent lux reporter system provides a non-invasive platform to monitor bacterial growth and expression in real time. This study aimed to establish a method for detecting bacterial adhesion on the surface of materials, including medical devices, by using recombinant bioluminescent Pseudomonas aeruginosa containing a lux reporter. By monitoring the growth and bioluminescent properties of the recombinant PAO1-lux strain, the optimal test conditions for bacterial adhesion detection in vitro were determined to be as follows: an initial inoculation density of 105 to 106 CFU/mL, M9 medium at a pH 6.2, an adhesion time of 6 h, and the collection of adherent bacteria by ultrasonic cleaning. The traditional CFU counting method and the bioluminescence method were compared, and the applicability of the new method was verified by testing the adhesion of bacteria on the surface of various materials. The validated bioluminescent strains could serve as strong candidates to be used as bacterial detection tools in applications such as bacterial adhesion evaluation as well as supplements and alternatives to traditional microbiological testing procedures. In addition, this method has the potential to enable the study of bacterial adhesion on the surface of inanimate objects and living tissues. With the development of this method and its wide applicability, it is expected to become a standard method for the detection of bacterial adhesion and the screening of anti-adhesion materials.

Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe.

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

Different bacterial host-based lux reporter array for fast identification and toxicity indication of multiple metal ions

Although luminescent bacteria-based bioluminescence inhibition assay has been widely used in the toxicity assessment of environmental pollutants, the response of a luminescent bacterium usually lacks specificity to a target analyte. Recently, some specific analyte inductive promoters were fused to the lux genes for the purpose of selective bioluminescent sensing, and suits of specific promoters were fused to lux genes to compose a bioluminescent array sensor for simultaneous identification of multiple analytes. However, specific promoter-based methods still suffer from drawbacks including limited selectivity, slow responding time, expensive to construct different promoters involved plasmids, and laborious to find new promoters. Herein, we proposed a novel strategy to construct a lux reporter array sensor by directly transforming the natural lux genes in different bacterial hosts without the involvement of any specific promoters. Due to the distinct pathways of signal production, the responding time of the current different bacterial host (DBH)-based lux reporter array has nearly an order of magnitude faster than with specific promoter-based methods. The DBH-based lux reporter array was successfully used for simultaneous identification, quantification, and toxicity/bioactivity assessment of multiple metal ions. Obviously, all the chemical synthetic material-based metal ion sensing methods cannot simultaneously achieve analysis and toxicity evaluation. This approach possessed additional advantages of facile construction, easy operation, high selectivity, fast response, and strong adaptability to other analytes. A different bacterial host-based lux reporter array was established for simultaneous analysis and toxicity assessment of multiple metal ions.

The Medicago truncatula Vacuolar iron Transporter-Like proteins VTL4 and VTL8 deliver iron to symbiotic bacteria at different stages of the infection process.

Summary The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule‐specific Vacuolar iron Transporter‐Like (VTL) proteins in Medicago truncatula.Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA‐seq data showing differential expression of VTL4 and VTL8 during early and late infection, respectively.The vtl4 insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly‐arranged VTL4–VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed in vtl4 mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation.These data indicate that VTL8, the closest homologue of SEN1 in Lotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids.

Effects of Volatile Organic Compounds Synthesized by Bacteria on the Expression from Promoters of the zntA, copA, and arsR Genes Induced in Response to Copper, Zinc, and Arsenic

Heavy metals and arsenic are environmental pollutants and have a negative effect on human health. Volatile organic compounds (VOCs) synthesized by bacteria are able to modulate the growth and metabolism of pro- and eukaryotes, to function as signals for distance communication between organisms, and to influence the action of various stress agents. The effects of sulfur-containing dimethyl disulfide (DMDS) and ketones (2-undecanone, 2-nonanone, 2-heptanone, 2-pentanone, and 2-propanone) on expression from the promoters of the copA, zntA, and arsR genes (PcopA, PzntA, and ParsR) responding to stresses caused by heavy metal ions and arsenic have been studied using lux-biosensors containing inducible promoters of the listed genes and the lux-operon as a reporter. The degree of expression from the respective promoters was determined by the intensity of bioluminescence. The effects of 2-undecanone, 2-nonanone, 2-heptanone, and DMDS led to a lower expression of lux-operon, which is controlled by PcopA and ParsR. The pretreatment of biosensors with these VOCs decreased the induction of expression from promoters PzntA and ParsR under conditions of exposure to ZnSO4 and NaAsO2, respectively, in the presence of CuSO4; this effect was marked only for 2-undecanone. 2-Pentanone increased the expression of lux-reporter from PcopA and PzntA. Pretreatment with 2-pentanone led to a 80% increase in expression of the lux reporter from PzntA compared to the effect of ZnSO4 without 2-pentanone. The effects of VOCs on the expression from promoters of the genes responding to the stresses induced by copper, zinc, and arsenic are multidirectional and depend on VOC structure.

Plant defensin antibacterial mode of action against Pseudomonas species

BackgroundThough many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments.ResultsPlant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments.ConclusionsMtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Evaluation the Use of the lux Reporter System pUC19-luxAB for Rapid and Sensitive Detection of Assimilable Organic Carbon (AOC) in Reverse Osmosis (RO) Feed Water

Evaluation the Use of the lux Reporter System pUC19-luxAB for Rapid and Sensitive Detection of Assimilable Organic Carbon (AOC) in Reverse Osmosis (RO) Feed Water

Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression

BackgroundDolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology.ResultsIn D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5′ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions.ConclusionsBoth of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

ClpP is required for proteolytic regulation of type II toxin-antitoxin systems and persister cell formation in Streptococcus mutans.

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

Molecular and functional evidence of phosphatidylserine synthase in Vibrio parahaemolyticus.

Phosphatidylserine synthase (Pss) catalyzes phosphatidylserine synthesis, which is critical to synthesizing the component of cell membrane. However, few putative pss genes of bacteria have been studied. In this study, it was found that Vibrio parahaemolyticus, a common foodborne pathogen that causes human gastroenteritis, has a type I Pss with two HKD motifs and is a phospholipase D superfamily member. The transcriptional start site of pss was mapped through sequencing and was identified at -37 nucleotides upstream of the start codon. Pss mRNA was found to be expressed mainly during the exponential phase. In addition, the promoter was identified using a lux reporter assay and gel shift assay with an RNA polymerase. To analyze the catalytic activity, a soluble form of His6 -tagged recombinant Pss was overexpressed and purified from Escherichia coli. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry, it was found that Pss can catalyze cytidine diphosphate diacylglycerol and L-serine to form phosphatidylserine. Since Pss is conserved in vibrios, the current study can promote understanding the biosynthesis of phospholipid in Vibrio bacteria that might cause vibriosis. This is the first report of molecular characterization of the pss gene and identification of Pss enzyme activity in V. parahaemolyticus using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

264 - Structural characterization of a newly identified pyocin S8, a proteic antibiotic from Pseudomonas aeruginosa with potential therapeutic applications

Development of alternative antibiotic approaches is urgently required with the emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa (Pa) strains. Pyocins are proteic antibiotics produced by Pa for intraspecies competition for resources. Due to its potency and specificity, these molecules may become ideal candidates as antibiotics for the treatment of Pa infections. Previous reports revealed that H2O2 generated by neutrophils during bacterial infection induces the expression of pyocin genes, indicating a central role of these molecules in host-pathogen interactions. To better understand how pyocins can be used as a potential antimicrobial agent, we characterized a newly identified pyocin S8. Recombinant pyocin S8 displayed potent killing activity against at least 16 MDR isolates, including carbapenemase (SPM-1, GIM-1, VIM-1, IMP-1 and GES-5)-producing Pa strains. We are investigating the role of H2O2 in inducing pyocin gene expression by using a chromosomal promoter S8 fused to a -lux reporter. However, preliminary data indicated that exposure the MDR Pa strains to 1 mM H2O2 for a period of 20 min before the pyocin addition did not improve its killing activity. Next, we determined the crystal structure of C-terminal cytotoxic domain of a mutant E736A pyocin S8 in complex with its immunity protein (S8 DNase E736A-ImS8) at 1.4 A resolution. The Glu736 localizes in DNase active site composed for 32 amino acid H-N-H (His-Asn-His) motif, which coordinates a single divalent metal ion responsible for catalysis. In our structure, we were not able to identify the electronic density of a metal ion, probably due the disorder of metal ion ligands composed by four histidine residues. These results indicate an important function of H-N-H motif for pyocin catalysis, consistent with the loss of killing activity of mutant E736A pyocin S8. Understanding and exploiting the mechanisms by which the pyocin S8 works might represent a promising strategy for future therapeutic applications.

The BceABRS four-component system that is essential for cell envelope stress response is involved in sensing and response to host defence peptides and is required for the biofilm formation and fitness of Streptococcus mutans.

Purpose. Streptococcus mutans is a primary cariogenic pathogen worldwide. In dental biofilms, S. mutans often faces life-threatening insults, such as killing by antimicrobial compounds from competing species and from the host. How such insults affect the physiology and virulence of S. mutans is poorly understood. In this study, we explored this question by investigating the responses of S. mutans strains to several host defence peptides and bacitracin.Methodology. S. mutans UA159 and its isogenic mutants, SmΔbceA, SmΔbceB, SmΔbceR and SmΔbceS, were examined for their antibiotic susceptibility and biofilm formation. The lux reporter strains were constructed to assay the responses of S. mutans to host defence peptides. In addition, the competitive fitness of these mutants against the parent in response to peptide antibiotics was determined in dual-strain mixed cultures.Results. S. mutans UA159 (WT) was generally insensitive to physiological concentrations of α-defensin-1, β-defensin-3, LL-37 and histatin-5, but all of the BceABRS mutants were sensitive to these peptide antibiotics. The response of S. mutans to these peptide antibiotics involved the transcriptional activation of the bceABRS operon itself. Bacitracin or β-defensin-3 at a sub-inhibitory concentration induced biofilm formation in the parent, but not in any of the BceABRS mutants. None of the mutants were able to compete with the parent for persistence in duel-strain cultures in the presence of bacitracin or β-defensin-3.Conclusion. The BceABRS four-component system in S. mutans is involved in sensing, response and resistance to host defence peptides, and is required for the biofilm formation and fitness of S. mutans.

Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system.

Ti and Ri plasmids of pathogenic Agrobacterium strains are stably maintained by the function of a repABC operon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of the Ti2 small RNA and luxAB from Vibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new "Ti/Ri eviction plasmids," each of which carries a repABC from one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacB and in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids into Agrobacterium cells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacB backbone. Furthermore, the highly stable cryptic plasmid pAtC58 in A. tumefaciens C58 was effectively evicted by the introduction of a pBBR1 vector containing the repABC of pAtC58. These results indicate that the set of pBBR1-repABC plasmids is a powerful tool for the removal of stable rhizobial plasmids.

A rapid and reagent-free bioassay for the detection of dioxin-like compounds and other aryl hydrocarbon receptor (AhR) agonists using autobioluminescent yeast.

An autonomously bioluminescent Saccharomyces cerevisiae BLYAhS bioreporter was developed in this study for the simple and rapid detection of dioxin-like compounds (DLCs) and aryl hydrocarbon receptor (AhR) agonists. This recombinant yeast reporter was based on a synthetic bacterial luciferase reporter gene cassette (lux) that can produce the luciferase as well as the enzymes capable of self-synthesizing the requisite substrates for bioluminescent production from endogenous cellular metabolites. As a result, bioluminescent signal production is generated continuously and autonomously without cell lysis or exogenous reagent addition. By linking the expression of the autobioluminescent lux reporter cassette to AhR activation via the use of a dioxin-responsive promoter, the S. cerevisiae BLYAhS bioreporter emitted a bioluminescent signal in response to DLC exposure in a dose-responsive manner. The model dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), could be detected within 4h with a half maximal effective concentration (EC50) of ~ 8.1nM and a lower detection limit of 500pM. The autobioluminescent response of BLYAhS to other AhR agonists, including 2,3,7,8-tetrachlorodibenzofuran (TCDF), polychlorinated bisphenyl congener 126 (PCB-126) and 169 (PCB-169), 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD), benzo[a]pyrene (BaP), and β-naphthoflavone (bNF), were also characterized in this study. The non-destructive and reagent-free nature of the BLYAhS reporter assay facilitated near-continuous, automated signal acquisition without additional hands-on effort and cost, providing a simple and cost-effective method for rapid DLC detection.