Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the -10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.     

Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.     

A Simple Screen to Identify Promoters Conferring High Levels of Phenotypic Noise

Genetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host–pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression.     

A high-throughput approach to promoter study using green fluorescent protein

Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic. By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP. In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates. Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis. In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats. Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose. Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates. They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time. These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format.     

Reporter genes lucFF, luxCDABE, gfp, and dsred have different characteristics in whole-cell bacterial sensors

The selection of a genetic reporter can be difficult because of the wide range of genes available. In order to reduce the selection, we compared the performance of different reporter genes: firefly luciferase (Photinus pyralis lucFF), bacterial luciferase operon (Photorhabdus luminescens luxCDABE), green fluorescent protein (Aequorea victoria gfp), and red fluorescent protein (Discosoma sp. dsred) in whole-cell bacterial sensors. Escherichia coli sensor bacteria were engineered to contain a reporter plasmid that carries the reporter gene under the control of mercury- (mer from Tn21) or arsenite- (ars from R773) responsive regulatory units. Characteristics of the strains were studied by using different arsenite or mercury concentrations and incubation times. The lowest detectable concentration of analytes and the fastest responses were achieved with lucFF or luxCDABE as reporter genes. The fluorescent proteins, GFP and DsRed, gave responses at higher analyte concentrations and after significantly longer incubation times. The results indicate that luciferases are better reporters in whole-cell sensor bacteria.     

Direct detection and quantification of horizontal gene transfer by using flow cytometry and gfp as a reporter gene

A new cultivation-independent method for studying conjugal gene transfer between bacteria was evaluated. The method was based on direct detection and enumeration of donor and transconjugant bacterial cells by flow cytometry. Specific detection of transconjugants was obtained by using a conjugative plasmid tagged with a reporter gene (gfp) encoding green fluorescent protein. A chromosomal encoded repressor (lacI(ql)) repressed expression of GFP in the donor bacteria. Enumeration of the donor cells was performed after induction of GFP expression by the addition of inducer isopropyl-thio-beta-D-galactoside (IPTG). The method presented here provided simple and precise quantification of horizontal gene transfer between both Escherichia coli and Pseudomonas putida strains.     

Quantitative analysis of gene expression with an improved green fluorescent protein. p6.

The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities. We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+. Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli. The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo. However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity. This effect is probably due to the higher absorbance of cells containing GFP+. Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene. Possibilities of using GFP variants beyond this range are discussed.     

Development of a monitoring vector for Leuconostoc mesenteroides using the green fluorescent protein gene

The vector pCW5 with plasmid pC7, originally isolated in Lactobacillus paraplantarum C7 derived from kimchi, was constructed using a p32 strong promoter, the pC7 replicon, and green fluorescent protein (GFP) as the reporter. The constructed vector was transformed into E. coli and Leuconostoc mesenteroides, and GFP expression detected using a Western blot analysis. GFP fluorescence was recognized in E. coli and Leuconostoc mesenteroides using a confocal microscope. In addition, GFP fluorescence was also clearly detected in several industrially important lactic acid bacteria (LAB), including Lactobacillus bulgaricus, Lactobacillus paraplantarum, and Lactobacillus plantarum. Thus, pCW5 was shown to be effective for Leuconostoc mesenteroides when using GFP as the reporter, and it can also be used as a broad-host-range vector for other lactic acid bacteria.     

Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria.

We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3). Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.     

不同转化条件对3种农杆菌GFP基因在本氏烟草中瞬时表达的影响

以本氏烟草（Nicotiana benthamiana）为植物材料,分析了不同农杆菌菌株（LBA4404菌株、EHA105菌株、GV3101菌株）、菌液浓度以及侵染时间在瞬时转化过程中对报告基因GFP荧光表达量的影响。结果显示,不同的农杆菌菌株瞬时表达外源基因的最适浓度和时间均有所不同：LBA4404菌株在菌悬液OD₆₀₀值为0.8时所介导的瞬时表达效率最高;而EHA105和GV3101菌株在菌悬液OD₆₀₀值为0.6时可达到最高瞬时表达效率。LBA4404菌株所介导的瞬时表达在农杆菌注射后第2天时表达量最高,而EHA105和GV3101菌株所介导的瞬时表达在农杆菌注射后第4天时表达量最高。不同菌株间比较分析表明,LBA4404菌株所介导的瞬时表达效率最高。上述结果表明,农杆菌菌株以及浓度和侵染时间等转化条件均是影响瞬时表达效率的重要因素。     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.