New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ

Abstract

Genetically altered or tagged Vibrio fischeri strains can be observed in association with their mutualistic host Euprymna scolopes, providing powerful experimental approaches for studying this symbiosis. Two limitations to such in situ analyses are the lack of suitably stable plasmids and the need for a fluorescent tag that can be used in tandem with green fluorescent protein (GFP). Vectors previously used in V. fischeri contain the p15A replication origin; however, we found that this replicon is not stable during growth in the host and is retained by fewer than 20% of symbionts within a day after infection. In contrast, derivatives of V. fischeri plasmid pES213 were retained by approximately 99% of symbionts even 3 days after infection. We therefore constructed pES213-derived shuttle vectors with a variety of selectable and visual markers. To include a visual tag that can be used in conjunction with GFP, we compared seven variants of the DsRed2 red fluorescent protein (RFP): mRFP1, tdimer2(12), DsRed.T3, DsRed.T4, DsRed.M1, DsRed.T3_S4T, and DsRed.T3(DNT). The last variant was brightest, displaying >20-fold more fluorescence than DsRed2 in V. fischeri. RFP expression did not detectably affect the fitness of V. fischeri, and cells were readily visualized in combination with GFP-expressing cells in mixed infections. Interestingly, even when inocula were dense enough that most E. scolopes hatchlings were infected by two strains, there was little mixing of the strains in the light organ crypts. We also used constitutive RFP in combination with the luxICDABEG promoter driving expression of GFP to visualize the spatial and temporal induction of this bioluminescence operon during symbiotic infection. Our results demonstrate the utility of pES213-based vectors and RFP for in situ experimental approaches in studies of the V. fischeri-E. scolopes symbiosis.

FIG. 1.

Stability of pEVS126 (unshaded bars) and pES213Kn (shaded bars) in V. fischeri after growth in nonselective LBS medium (A) or during squid colonization without kanamycin selection (B). Panel B includes the percent plasmid retention in cells from the inoculum that squid were exposed to and in cells recovered from colonized squid. Error bars (some too small to visualize) indicate standard errors; n = 3 (cultures) in panel A, and n = 8 (squid) at the 24-h, 48-h, and 72-h time points in panel B.

FIG. 2.

High-copy pES213-derivative pVSV104H. (A) Single-base-pair difference (highlighted) between pVSV104 and pVSV104H, which resides within “inverted repeat 2” of the replication origin (12). (B and C) Abundances of pVSV104 and pVSV104 determined by yields from plasmid preparations (B) or real-time PCR (C). Error bars in panels B and C indicate standard errors; n = 5 and n = 6, respectively.

FIG. 3.

Marking strains with pVSV103 to determine strain ratios in mixed cultures. Wild-type strain ES114 and recA mutant EVS510 (12) were marked with pVSV103, which carries lacZ, and mixed in ∼1:1 ratios with unmarked EVS510 or ES114, and the strain ratios were determined during coculturing based on blue/white colony counts on LBS agar containing X-Gal. Relative competitiveness indices represent the strain ratio at each plating divided by the ratio in the inoculum (generation 0) for mixtures of ES114(pVSV103) and ES114 (filled squares), EVS510(pVSV103) and ES114 (filled circles), or EVS510 and ES114(pVSV103) (open circles). The gray line indicates an RCI of 1, which defines no relative competitive defect. The dotted line indicates the calculated RCI values of a strain growing at 90% of the rate of the strain it is competing with.

FIG. 4.

Fluorescence intensities of DsRed variants in V. fischeri. (A and B) Red fluorescence of V. fischeri carrying DsRed alleles when cells were growing in mid-log phase at an OD₅₉₅ of 1 (A) or grown overnight to stationary phase and diluted to an OD₅₉₅ of 1 (B). Data are means with standard errors (n = 3). (C) Sequence upstream of the DsRed.T3(DNT) allele in pVSV207 and pVSV208 and the fluorescence of V. fischeri cells carrying these plasmids during log-phase growth (OD₅₉₅ of 1). The single-base-pair difference is highlighted, the ATG start codon is in boldface, and a consensus ribosome-binding site (RBS) with the most conserved nucleotides underlined is shown for comparison (35).

FIG. 5.

Competition during mixed infection by V. fischeri ES114 derivatives carrying pVSV102 (GFP) or pVSV208 (RFP). Juvenile squid were exposed to a ∼1:1 mixture of the strains at a total concentration of 2,000 to 7,000 CFU ml⁻¹ for 14 h. (A) The relative competitiveness index of ES114(pVSV102), defined as the ratio of ES114(pVSV102) to ES114(pVSV208) in the squid divided by the ratio of these strains in the inoculum, was determined for bacteria in each animal 48 h after inoculation and plotted as a circle. Symbols to the left of the frame indicate that the RCI was <0.01. The average RCI of 1.2 (n = 57) was not significantly different from 1.0 (P > 0.25). (B and C) Epifluorescence images of E. scolopes light organs coinfected with ES114(pVSV102) (green) and ES114(pVSV208) (red), with animal tissue stained with CellTracker Blue (blue). Bars are 100 μm long. The light organs of five typical animals are shown in panel B, with red- and green-tagged cells appearing segregated. The light organ of one animal is shown at two magnifications in panel C, revealing a large pocket of mixed infection by red- and green-tagged cells appearing yellow (indicated by arrows).

FIG. 6.

In situ visualization of lux promoter expression by using pVSV210. (A) Map of pVSV210, which contains a constitutive RFP and has GFP expression driven by the luxICDABE promoter. Stem-loops indicate bidirectional transcriptional terminators flanking the reporter cassette. (B) Schematic representation of one lobe of a two-lobed light organ. Unshaded regions represent internal symbiotic tissues, with three distinct pores leading through ducts to widened antechambers and then into crypts where most symbionts ultimately reside. The three crypt types (29) are designated with numbers. (C) Rate of appearance of red-fluorescent symbiont cells (open symbols) or red- and green-fluorescent cells (filled symbols) in crypt 1 (circles), crypt 2 (squares), and crypt 3 (triangles). The light organs of between three and eight animals were visualized at each time point.