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. 2018 Jul 2;84(14):e00603-18.
doi: 10.1128/AEM.00603-18. Print 2018 Jul 15.

An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter

Julie L Stoudenmire  1 , Tara Essock-Burns  2 , Erena N Weathers  1 , Sina Solaimanpour  3 , Jan Mrázek  1 , Eric V Stabb  4
Affiliations

An Iterative, Synthetic Approach To Engineer a High-Performance PhoB-Specific Reporter

Julie L Stoudenmire et al. Appl Environ Microbiol. .

Abstract

Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at 19 additional intervening nucleotide positions for which we did not predict sequence-specific effects, the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri, enabling us to identify bases at particular randomized positions that significantly correlated with high-level "on" or low-level "off" expression. A second round of promoter design rationally constrained 13 additional positions, leading to a reporter with high-level PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized variants of green fluorescent protein (GFP), the latter of which has a half-life of 81 min in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted to a concentration below 10 μM. During symbiotic colonization of its host squid, Euprymna scolopes, the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other members of the Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and reengineering.IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between the "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over 34 billion sequence variants, we identified bases correlated with favorable performance by screening only 83 candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri This promoter design strategy will facilitate the engineering of other regulator-specific reporters.

Keywords: Aliivibrio; photobacterium; synthetic biology.

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Figures

FIG 1
FIG 1
Design and screening of synthetic PhoB-dependent promoter variants. (A) Synthetic constructs included four PhoB-binding sites (gray boxes) as well as a −10 promoter element. The −35 promoter region is indicated, although it does not resemble a −35 consensus. Randomized or semirandomized positions are numbered, corresponding to the numbering in panel C. “N” indicates a randomized position (labeled 1 to 3 and 7 to 19). Positions 4 to 6 were restricted to A or T (W). Eighty-three variants of the sequence were cloned upstream of gfp and screened for activity. (B) GFP output for four representative promoter variants. GFP was measured for constructs in ES114 and JLS9 (ΔphoB) grown in FMM medium with a high (378 μM) or low (37.8 μM) added PO4 concentration. Strains with the promoterless parent vector (labeled “None”) show background fluorescence. Values were taken at an OD595 of 1.0 and normalized to red fluorescence. Error bars indicate standard deviations (n = 3). Data from one representative experiment of three are shown. (C) Each variable position (positions 1 to 19) was subjected to a Mann-Whitney U nonparametric test to compare the average GFP output associated with each individual nucleotide (A, T, C, or G) against the average value for constructs with the other nucleotides in that position. Pairs of nucleotides were also compared at each position (A/T versus G/C, C/T versus A/G, G/T versus A/C, and C/G versus A/T). Only comparisons with P values of <0.05 are shown. Values indicate the mean GFP output for all constructs with the nucleotide (or nucleotide pair) at the indicated position minus the mean value for the other constructs. For each of the four strain-medium combinations, nucleotides that correlated with significantly higher or lower GFP levels are indicated by green or red, respectively; *, **, and *** indicate P values of <0.05, <0.01, and < 0.001, respectively. The right column indicates how randomization was further constrained for the next set of variants (round 2). † indicates that the C at position 2 was underrepresented in the screened variants (in only 1 of the 83 variants) and therefore was included in round 2.
FIG 2
FIG 2
Iterative optimization of PhoB-dependent reporter performance. A representative of the best promoters from the first set of semirandomized variants (pEW6AQ) (numbered 4 in Fig. 1B) was compared to an optimal variant (pJLS1088) generated after constraining additional positions, as indicated in Fig. 1C. Reporters were evaluated in the ES114 and ΔphoB strains and were grown in minimal medium amended with high or low concentrations of PO4. The promoterless parent vector pJLS27 (labeled “None”) is included to show the background. Fluorescence values were taken at an OD595 of 1.0, and error bars indicate standard deviations (n = 3). Data show results from one representative experiment of three performed.
FIG 3
FIG 3
A PhoB-dependent reporter is induced upon PO4 depletion in batch culture. (A) GFP/OD595 values and supernatant phosphate levels were measured over time in cultures of wild-type cells containing either the promoterless parent vector (pJLS27) (squares) or the optimized PhoB reporter (pJLS1088) (circles) grown in batch cultures in FMM amended at the outset with 37.8 μM phosphate for a final concentration of 100 μM total phosphate. Filled shapes represent the GFP/OD595 fluorescence, whereas open shapes represent the extracellular PO4 concentration. (B) Growth of the cultures from panel A shown as cell density (OD595) over time. Data from one representative experiment of three are shown.
FIG 4
FIG 4
Response of reporters to low PO4 concentrations in other proteobacteria. Red and green fluorescence from colonies of V. fischeri, V. cholerae, E. coli, S. enterica, or R. pomeroyi carrying a promoterless parent vector (pJLS27 for vibrios or pJLS71 for nonvibrios) or a vector containing the PhoB-activated promoter (pJLS1088 for vibrios or pJLS137 for nonvibrios) are shown. Strains include V. fischeri ES114 and JLS9 (ΔphoB), V. cholerae AC2764 (ΔtcpA) and AC3236 (ΔphoB), E. coli MG1655, S. enterica MS1868, and R. pomeroyi DSS-3. Strains were grown on agar plates with defined medium containing low or high concentrations of added PO4 (see Materials and Methods). Colonies of similar sizes were imaged by using a Nikon Eclipse E600 microscope with a 51005v2 filter, which enabled the simultaneous visualization of both the constitutive red fluorescence and the green fluorescence of the reporter.
FIG 5
FIG 5
Effects of clpX and specific SsrA tags on GFP fluorescence in V. fischeri. Specific fluorescence associated with GFP variants is shown for wild-type strain ES114 or mutants with transposon insertions disrupting clpA, clpX, or clpS. pJLS153 (“stable”) has gfp without an ssrA tag. pJLS150 (*ASV), pJLS151 (*LAA), and pJLS152 (*AAV) all have gfp with a modified ssrA tag, exchanging the last three C-terminal amino acids, as indicated. Strains were grown in SWTO medium with 2 mM IPTG to induce gfp expression. GFP/OD595 values were taken at an OD595 of 1.0, and error bars indicate standard deviations (n = 3). Data from one representative experiment of three performed are shown.
FIG 6
FIG 6
Addition of the *ASV SsrA tag to GFP increases the turnover rate in V. fischeri. ES114 cells carrying pJLS150 (GFP*ASV) (squares) or pJLS153 (parental GFP with no SsrA tag) (circles) were grown in SWTO medium plus 2 mM IPTG or SWTO medium with no inducer to an OD595 of 1.5 before being washed and resuspended in SWTO medium without IPTG. GFP/OD595 values were taken every 15 min for 4 h. Values under noninduced conditions (no IPTG added) were subtracted as the background from values under induced conditions. Error bars indicating standard deviations (n = 3) are smaller than the symbols. Data from one representative experiment of four performed are shown.
FIG 7
FIG 7
Expression of PhoB-dependent reporters in symbiotic V. fischeri cells. (A and B) Juvenile squid were left uninoculated and aposymbiotic (A, left) or were infected with V. fischeri containing either the promoterless vector (pJLS27) (A, right) or the first-round reporter pEW6AQ (B). Light organs (∼250 μm across; outlined with dotted white lines) were visualized by epifluorescence microscopy with a red/green filter at 24 h postinoculation. (C) Confocal microscopic image of a juvenile squid light organ showing one-half of the organ from a juvenile colonized with ES114 cells harboring the second-round PhoB-dependent promoter driving the expression of destabilized GFP (pJLS298). The squid tissue (blue) and the pores, antechambers, and crypts are labeled when visible.
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