Green Fluorescent Protein Labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for Safety-Related Studies

Ma, Li; Zhang, Guodong; Doyle, Michael P.

doi:10.1371/journal.pone.0018083

Cited by 65 publications

(59 citation statements)

References 26 publications

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“…The plasmids pyycGc and plisKc were cloned into chemically competent E. coli NEB5␣ cells (New England BioLabs) and transformed into the conjugation donor E. coli HB101 (Biological Resource Centre of the Institut Pasteur, Paris, France), which contains the helper plasmid pRK24. Transformants were then conjugated into recipient L. monocytogenes strains according to the method of Ma et al (38), and the strains carrying the pPL2 constructs were selected by chloramphenicol (25 g/ml) on ALOA agar (Lab M, Ltd., Lancashire, United Kingdom) at 37°C. Integration of pPL2 into each recipient L. monocytogenes strain was confirmed by PCR using primers NC16 and PL95 (37), and the presence of the insert was confirmed using gene-specific primers.…”

Section: Complementationmentioning

confidence: 99%

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Pöntinen

Markkula

Lindström

et al. 2015

Appl Environ Microbiol

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Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ⌬resE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature. Listeria monocytogenes is a low-GϩC, Gram-positive, nonspore-forming coccobacillus ubiquitously found in the environment (1, 2). It has been isolated from foods and food-processing premises, where it has been confirmed to be able to persist for several years (3-7). L. monocytogenes is the causative agent of listeriosis, with a reported annual incidence of approximately 0.3 case per 100,000 population in the United States and 0.2 to 0.8 case per 100,000 population in Europe (8-10). Although relatively rare, listeriosis is nevertheless a severe foodborne disease with a mortality of up to 20 to 30% (1,8,11,12). L. monocytogenes surmounts a wide range of stress conditions and is able to grow at temperatures as low as Ϫ0.4°C (13). This sets critical challenges for the modern food industry, since L. monocytogenes is consequently able to grow in ready-to-eat foods with a long shelf-life and in products stored at refrigeration temperatures. Thus, it is crucial to study and elucidate the factors enhancing the cold tolerance of L. monocytogenes. Two-component regulatory signaling systems (TCSs) are among the major systems that aid bacteria in adapting to many arduous stress factors encountered in nature and during food processing. A typical TCS is constituted of a transmembrane sensor histidine kinase (HK) and a cognate cytoplasmic response regulator (RR) (14-17). The signaling pathway of the TCS is based on protein phosphorylation. In the simplest form of signal transduction, the sensor domain of the HK detects a specific stimulus or stress factor in cells or in their environment. This leads to the autophosphorylation of a specific His residue in the HK dimerization domain. The cognate RR then ca...

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Section: Complementationmentioning

confidence: 99%

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Pöntinen

Markkula

Lindström

et al. 2015

Appl Environ Microbiol

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“…Externally added genes commonly affect the growth rate of the host 20,32 although the magnitude of the effect varies between organisms. 33 The relationship between the number of lacI genes added and the length of the cell cycle was approximately linear. As the fraction of the total cell protein comprised of LacI was approximately linear with the number of lacI genes added, there is an approximately linear relationship between the fraction of total cell protein comprising LacI and the cell cycle length.…”

Section: Discussionmentioning

confidence: 97%

Towards a whole-cell modeling approach for synthetic biology

Purcell

Jain

Karr

et al. 2013

Chaos: An Interdisciplinary Journal of Nonlinear Science

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Despite rapid advances over the last decade, synthetic biology lacks the predictive tools needed to enable rational design. Unlike established engineering disciplines, the engineering of synthetic gene circuits still relies heavily on experimental trial-and-error, a time-consuming and inefficient process that slows down the biological design cycle. This reliance on experimental tuning is because current modeling approaches are unable to make reliable predictions about the in vivo behavior of synthetic circuits. A major reason for this lack of predictability is that current models view circuits in isolation, ignoring the vast number of complex cellular processes that impinge on the dynamics of the synthetic circuit and vice versa. To address this problem, we present a modeling approach for the design of synthetic circuits in the context of cellular networks. Using the recently published whole-cell model of Mycoplasma genitalium, we examined the effect of adding genes into the host genome. We also investigated how codon usage correlates with gene expression and find agreement with existing experimental results. Finally, we successfully implemented a synthetic Goodwin oscillator in the whole-cell model. We provide an updated software framework for the whole-cell model that lays the foundation for the integration of wholecell models with synthetic gene circuit models. This software framework is made freely available to the community to enable future extensions. We envision that this approach will be critical to transforming the field of synthetic biology into a rational and predictive engineering discipline. Synthetic biology aims to engineer synthetic genetic circuits to endow cells and organisms with the ability to address new applications, including the production of drugs and industrial products, the design of new therapeutics for human diseases, and the study of basic biological processes. Despite significant advances in the field over the last decade, the synthetic biology design cycle has been hampered by the lack of predictive models. In contrast, more established engineering disciplines, such as civil and electrical engineering can rely on rational design by using models that can capture the behavior of real-world systems with a high degree of accuracy. This is currently not the case for synthetic biology, as models are generally only able to make qualitative predictions about system behavior. As a result, producing a functional engineered biological system usually requires extensive manual tuning by trial-and-error, a timeconsuming process that significantly slows the biological design process. The lack of predictive power in current models is partly because these models account for only the synthetic circuits themselves, but not other ongoing processes in the cells in which they reside. However, the complex dynamics of synthetic circuits may affect the host cell and vice versa, leading to divergence between current models and experimental results. Recently, a whole-cell model of a small and simple bacteri...

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“…For live cell imaging, GFP expression in bacteria is used extensively and expression systems are available for many different bacterial species, including clinically relevant strains of Salmonella, Streptococcus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli O157:H7 [41,[44][45][46][47]. Plasmid based gene expression is a well-established and long-used method in microbiology.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

et al. 2013

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Bacterial adhesion and biofilm growth can cause severe biomaterial-related infections and failure of medical implants. To assess the antifouling properties of engineered coatings, advanced approaches are needed for in situ monitoring of bacterial viability and growth kinetics as the bacteria colonize a surface. Here, we present an optimized protocol for optical real-time quantification of bacterial viability. To stain living bacteria, we replaced the commonly used fluorescent dye SYTO ® 9 with endogenously expressed eGFP, as SYTO ® 9 inhibited bacterial growth. With the addition of nontoxic concentrations of propidium iodide (PI) to the culture medium, the fraction of live and dead bacteria could be continuously monitored by fluorescence microscopy as demonstrated here using GFP expressing Escherichia coli as model organism. The viability of bacteria was thereby monitored on untreated and bioactive dimethyloctadecyl [3-(trimethoxysilyl)propyl]ammonium chloride (DMOAC)-coated glass substrates over several hours. Pre-adsorption of the antimicrobial surfaces with serum proteins, which mimics typical protein adsorption to biomaterial surfaces upon contact with host body fluids, completely blocked the antimicrobial activity of the DMOAC surfaces as we observed the recovery of bacterial growth. Hence, this optimized eGFP/PI viability assay provides a protocol for unperturbed in situ monitoring of bacterial viability and colonization on engineered biomaterial surfaces with single-bacteria sensitivity under physiologically relevant conditions.

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Green Fluorescent Protein Labeling of Listeria, Salmonella, and Escherichia coli O157:H7 for Safety-Related Studies

Cited by 65 publications

References 26 publications

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Towards a whole-cell modeling approach for synthetic biology

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

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