Sensitive In Situ Monitoring of a Recombinant Bioluminescent
            <i>Yersinia enterocolitica</i>
            Reporter Mutant in Real Time on Camembert Cheese

Maoz, Ariel; Mayr, Robert; Bresolin, Geraldine; Neuhaus, Klaus; Francis, Kevin P.; Scherer, Siegfried

doi:10.1128/aem.68.11.5737-5740.2002

Cited by 25 publications

(14 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This correlates with other experimental data (41) and with the Arrhenius equation predicting a 1.5-to 4-fold reduction in enzyme reaction rate, following a temperature decrease of 10°C, given that the light production by the luciferase genes implies four independent catalytic steps (68). The corrected light emission at 10°C was thus determined as the ratio RLU/OD, multiplied by the factor 12.3.…”

Section: Methodssupporting

confidence: 68%

Transcriptional Analysis of Long-Term Adaptation ofYersinia enterocoliticato Low-Temperature Growth

et al. 2006

Self Cite

View full text Add to dashboard Cite

To analyze the transcriptional response of Yersinia enterocolitica cells to prolonged growth at low temperature, a collection of luxCDABE transposon mutants was cultivated in parallel at optimal (30°C) and suboptimal (10°C) temperatures and screened for enhanced promoter activities during growth until entering stationary phase. Among 5,700 Y. enterocolitica mutants, 42 transcriptional units were identified with strongly enhanced or reduced promoter activity at 10°C compared to 30°C, and changes in their transcriptional levels over time were measured. Green fluorescent protein fusions to 10 promoter regions confirmed the data. The temporal order of induction of the temperature-responsive genes of Y. enterocolitica was deduced, starting with the expression of cold shock genes cspA and cspB and the elevated transcription of a glutamate-aspartate symporter. Subsequently, cold-adapted cells drastically up-regulated genes encoding environmental sensors and regulators, such as UhpABC, ArcA, and methyl-accepting chemotaxis protein I (MCPI). Among the most prominent cold-responsive elements that were transcriptionally induced during growth in early and middle exponential phase are the insecticidal toxin genes tcaA and tcaB, as well as genes involved in flagellar synthesis and chemotaxis. The expression pattern of the lateexponential-to early-stationary-growth phase is dominated by factors involved in biodegradative metabolism, namely, a histidine ammonia lyase, three enzymes responsible for uptake and utilization of glycogen, the urease complex, and a subtilisin-like protease. Double-knockout mutants and complementation studies demonstrate inhibitory effects of MCPI and UhpC on the expression of a putative hemolysin transporter. The data partially delineate the spectrum of gene expression of Y. enterocolitica at environmental temperatures, providing evidence that an as-yet-unknown insect phase is part of the life cycle of this human pathogen.

show abstract

Section: Methodssupporting

confidence: 68%

Transcriptional Analysis of Long-Term Adaptation ofYersinia enterocoliticato Low-Temperature Growth

et al. 2006

Self Cite

View full text Add to dashboard Cite

show abstract

“…Bacterial luciferase (LuxAB) catalyzes the reaction of a longchain aldehyde, reduced flavin mononucleotide, and molecular oxygen, yielding the corresponding carboxylic acid, flavin mononucleotide, water, and light (490 nm). It is functional in many microbes (28), and detection methods are very sensitive and allow in situ measurement in complex environments, including several tissues of living animals (13) and food products (26). Including luciferase as a secondary reporter allowed us to analyze promoter activities in a quantitative and high-throughput manner.…”

Section: Vol 74 2008 High-throughput Analysis Of In Vivo-expressed mentioning

confidence: 99%

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Bachmann

Kleerebezem

Vlieg

2008

Appl Environ Microbiol

View full text Add to dashboard Cite

Understanding the functional response of bacteria to their natural environment is one of the current challenges in microbiology. Over the past decades several techniques have been developed to study gene expression in complex natural habitats. Most of these methods, however, are laborious, and validation of results under in situ conditions is cumbersome. Here we report the improvement of the recombinase-based in vivo expression technology (R-IVET) by the implementation of two additional reporter genes. The first one is an ␣-galactosidase gene (melA), which facilitates the rapid identification of in vivo-induced genes. Second, the bacterial luciferase genes (luxAB) are transcriptionally coupled to the resolvase gene, which allows rapid validation and characterization of in vivo-induced genes. The system is implemented and validated in the industrially important lactic acid bacterium Lactococcus lactis. We demonstrate the applicability of the advanced R-IVET system by the identification and validation of lactococcal promoter elements that are induced in minimal medium compared to the commonly used rich laboratory medium M17. R-IVET screening led to the identification of 19 promoters that predominantly control expression of genes involved in amino acid and nucleotide metabolism and in transport functions. Furthermore, the luciferase allows high-resolution transcription analysis and enabled the identification of complex medium constituents and specific molecules involved in promoter control. Rapid target validation exemplifies the high-throughput potential of the extended R-IVET system. The system can be applied to other bacterial species, provided that the reporter genes used are functional in the organism of interest.Understanding the responses of microorganisms in their natural environments is one of the big challenges of the postgenomic era. Complex niches such as soil, decaying plant material, and the (mammalian) gastrointestinal tract show a rich bacterial diversity and complex microbial activity patterns. Sampling complexity and environmental dynamics hamper comprehensive transcriptional analysis of a specific microorganism residing in such a complex environment. To overcome these challenges, a number of tools to study in vivo gene expression were developed during the past decades (25). One of the most powerful technologies is termed in vivo expression technology (IVET) and is based on transcriptional fusions of genomic DNA fragments to a selectable reporter gene (32). Recombinase-based IVET (R-IVET) is an IVET variation that employs a resolvase as the primary reporter gene. Upon expression of the resolvase, a chromosomally located marker, situated between two recombination sites, is excised from the genome (1). This irreversible step "records" in situ promoter activity and acts as a promoter-trap system, allowing the accumulation of promoter activity detection over extended in situ incubation periods. Moreover, the system is able to detect very low and transient gene expression in complex environmental condi...

show abstract

“…Pseudomonas aeruginosa (XEN5) and Proteus mirabilis (XEN44) that had been engineered to be stably bioluminescent by transformation with a transposon containing the entire Photorhabdus luminescens lux operon [45] were a kind gift from Xenogen Corp. (Alameda, CA).…”

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection

et al. 2006

View full text Add to dashboard Cite

Background and Objective: To compare the effectiveness of antimicrobial photodynamic therapy (PDT), standard endodontic treatment and the combined treatment to eliminate bacterial biofilms present in infected root canals. Study Design/Materials and Methods: Ten singlerooted freshly extracted human teeth were inoculated with stable bioluminescent Gram-negative bacteria, Proteus mirabilis and Pseudomonas aeruginosa to form 3-day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify bacterial burdens. PDT employed a conjugate between polyethylenimine and chlorin(e6) as the photosensitizer (PS) and 660-nm diode laser light delivered into the root canal via a 200-m fiber, and this was compared and combined with standard endodontic treatment using mechanical debridement and antiseptic irrigation. Results: Endodontic therapy alone reduced bacterial bioluminescence by 90% while PDT alone reduced bioluminescence by 95%. The combination reduced bioluminescence by > 98%, and importantly the bacterial regrowth observed 24 hours after treatment was much less for the combination (P < 0.0005) than for either single treatment. Conclusions: Bioluminescence imaging is an efficient way to monitor endodontic therapy. Antimicrobial PDT may have a role to play in optimized endodontic therapy. Lasers Surg.

show abstract

Sensitive In Situ Monitoring of a Recombinant Bioluminescent Yersinia enterocolitica Reporter Mutant in Real Time on Camembert Cheese

Cited by 25 publications

References 24 publications

Transcriptional Analysis of Long-Term Adaptation ofYersinia enterocoliticato Low-Temperature Growth

Transcriptional Analysis of Long-Term Adaptation ofYersinia enterocoliticato Low-Temperature Growth

High-Throughput Identification and Validation of In Situ-Expressed Genes of Lactococcus lactis

Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection

Contact Info

Product

Resources

About