A custom-designed Affymetrix array of the C. crescentus
genome, CauloHI1 (23
), used to quantitate transcript levels upon exposure of Caulobacter
to heavy metals, revealed that several genes were specifically upregulated upon exposure to uranyl nitrate (13
). One of these genes was induced 27.5-fold under uranium stress but was not upregulated in response to other heavy metals in the test screen (13
). We designated this gene urcA
esponse in c
) and selected the urcA
promoter as a candidate to drive uranium reporter constructs. The results of the microarray experiments localized the urcA
+1 transcriptional start site to a 10-bp window (13
) (Fig. ). CC3302Hypp_x_at probe 3 is the most upstream probe in the Affymetrix array that matches the urcA
transcript, placing its +1 site approximately 5 to 15 bp upstream from the end of the immediately adjacent probe 2 (chromosomal position 3552896). A uranium-inducible promoter sequence motif, present in the promoter regions of 11 Caulobacter
genes, has been identified (23
). The urcA
promoter contains two matches to this uranium-specific m_5 motif, located 107 and 55 bp upstream of the putative +1 site (Fig. ). The urcA
transcript overlaps the opposing strand of the CC_3302 gene in the original annotation of the Caulobacter
), but the revised Glimmer (9
) and GeneMark (2
) annotations of the Caulobacter
genome have identified urcA
as the true open reading frame (spanning from chromosomal position 3552927 to position 3553280 on the positive strand), dispensing with the originally annotated CC_3302 gene. The urcA
gene is not essential for viability and is not conserved among other α-proteobacteria.
FIG. 1. urcA promoter region and PurcA reporter schematic diagrams. (A) CC3302Hypp_x_at probe 3 is the most upstream probe in the Affymetrix array that matches the 404-bp urcA transcript (13), placing the +1 transcriptional start site approximately 5 (more ...)
gene encodes a predicted 12.7-kDa protein. The signal sequence prediction tool SignalP (25
) predicted with nearly 100% certainty that UrcA contains an N-terminal signal sequence whose most likely cleavage site is located between residues 30 and 31, and the prokaryotic subcellular protein localization tool SubLoc (14
) predicted with 96% expected accuracy that UrcA is a periplasmic protein. To experimentally test the bioinformatic prediction of UrcA's periplasmic localization, we constructed a xylose-inducible UrcA-mCherry fusion (32
). After xylose induction, the Pxyl urcA-mcherry
strain was imaged by deconvolution microscopy (Fig. ). The fluorescence images are consistent with the hypothesis that UrcA localizes to the cell periphery and to the cell stalk.
FIG. 2. UrcA-mCherry localization. Strain NJH300 was induced with 0.3% xylose for 3 h, and images were obtained by deconvolution microscopy. Phase-contrast and epifluorescence images are shown. The arrows indicate representative cell stalks visible in (more ...)
To determine if the urcA promoter could be a candidate uranium biosensor, we constructed a plasmid-borne LacZ fusion reporter containing 1 kb of the promoter region upstream of the urcA start ATG codon through the first 8 amino acids of UrcA fused to LacZ to create strain NJH199 (Fig. ). The resulting PurcA lacZ reporter strain was exposed to 0, 2.5, or 20 μM uranyl, and the resulting kinetics of β-galactosidase activity was assayed in liquid culture (Fig. ). The PurcA lacZ strain was able to detect the presence of 2.5 μM uranyl, and maximal β-galactosidase activity occurred by 2 h after exposure to either 2.5 or 20 μM uranyl nitrate. To test the sensitivity and specificity of the reporter, the PurcA lacZ strain was exposed to a panel of heavy metals for 2 h and then assayed for β-galactosidase activity (Fig. ). The PurcA lacZ reporter's detection limit for uranyl after 2 h of exposure was about 1.0 μM. The maximum increase in the signal of the PurcA lacZ reporter was 65-fold over the background with 20 μM uranyl. The PurcA lacZ reporter was not stimulated by the presence of lead (150 μM) or chromium (41.6 μM), but it showed cross sensitivity to cadmium at a concentration of 48 μM. In addition to the analysis of the panel of heavy metals, the PurcA lacZ reporter response to nitrate was assayed as a negative control to ensure that the nitrate component of uranyl nitrate salt does not contribute to PurcA lacZ reporter activity.
After our success with the PurcA lacZ reporter, we constructed a reporter strain (NJH371) in which plasmid-borne PurcA drives the expression of UV-excitable green fluorescent protein fluorescence. The PurcA gfpuv reporter construct is shown in Fig. . The time course of GFPuv signal kinetics for PurcA gfpuv after induction with uranyl nitrate is shown in Fig. . The fluorescence activity of the PurcA gfpuv reporter strain reached a maximum between 3 and 4 h after exposure to uranyl, but one-half the maximum activity was observed after about 2 h. It should be noted that as the PurcA gfpuv reporter strain reached a high cell density at 6.5 h (OD660, >0.9), entering stationary phase in the absence of uranium, the basal activity level of PurcA gfpuv began to increase. This result indicates that high-density cultures of the PurcA gfpuv reporter strain should not be used, because they could lead to false positives when samples are probed for the presence of uranium.
The PurcA gfpuv reporter strain was exposed to a panel of heavy metals for 4 h and then assayed for fluorescence activity (Fig. ). The PurcA gfpuv reporter exhibited specificity for uranium, and there was little cross specificity for nitrate (<400 μM), lead (<150 μM), cadmium (<48 μM), or chromium (<41.6 μM). The PurcA gfpuv reporter's detection limit for uranyl after 4 h of exposure was around 0.5 μM. The mean signal increase for the PurcA gfpuv reporter was 4.2-fold over the background with 100 μM uranyl. Despite sizeable standard deviations in reporter fluorescence activity, it should be pointed out that the minimum measured activities of the reporter for uranyl concentrations above 0.5 μM (n = 7) were all greater than the maximum activities measured for nitrate, lead, cadmium, or chromium (n = 3). Interestingly, we did not observe low-level stimulation of the GFPuv reporter by cadmium, in contrast to the LacZ reporter results (Fig. ). An inhibitory effect of 41.6 μM chromium on GFPuv activity was observed for the PurcA gfpuv reporter (Fig. ), but cadmium levels less than 48 μM did not appear to significantly affect PurcA gfpuv reporter activity in the presence of 10 μM uranyl.
Figure demonstrates that the PurcA gfpuv strain distinguished uranium-contaminated groundwater samples (4.2 μM uranium) from uncontaminated groundwater samples (<0.1 μM uranium) collected at the Oak Ridge Field Research Center. Adding 50 μM uranyl nitrate to the uncontaminated water sample yielded comparable photoemission. Using a hand-held UV lamp as the light source, the naked eye alone was sufficient to distinguish PurcA gfpuv reporter strain cultures exposed to the contaminated water (4.2 μM uranium) from cultures exposed to the uncontaminated water (Fig. ), although filtering out the blue region of the spectrum (as shown by isolating the green channel of the RGB image) facilitated discrimination. This key result provides proof of principle that the PurcA gfpuv reporter strain may be used to detect the presence of uranium contamination in real-world water samples, that the reporter's output may be successfully monitored with the naked eye without resorting to a fluorimeter, and that the background chemical composition of the water samples tested does not appear to induce false-positive or -negative results.