FNR-mediated regulation of bioluminescence and anaerobic respiration in the light-organ symbiont Vibrio fischeri

Abstract

Vibrio fischeri induces both anaerobic respiration and bioluminescence during symbiotic infection. In many bacteria, the oxygen-sensitive regulator FNR activates anaerobic respiration, and a preliminary study using the light-generating lux genes from V. fischeri MJ1 cloned in Escherichia coli suggested that FNR stimulates bioluminescence. To test for FNR-mediated regulation of bioluminescence and anaerobic respiration in V. fischeri, we generated fnr mutants of V. fischeri strains MJ1 and ES114. In both strains, FNR was required for normal fumarate- and nitrate-dependent respiration. However, contrary to the report in transgenic E. coli, FNR mediated the repression of lux. ArcA represses bioluminescence, and P(arcA)-lacZ reporters showed reduced expression in fnr mutants, suggesting a possible indirect effect of FNR on bioluminescence via arcA. Finally, the fnr mutant of ES114 was not impaired in colonization of its host squid, Euprymna scolopes. This study extends the characterization of FNR to the Vibrionaceae and underscores the importance of studying lux regulation in its native background.

FIG. 1

Genomic context and function of fnr in V. fischeri. (A) Gene arrangement around fnr in V. fischeri ES114. Numbers represent the corresponding VF#### ORF designation. Stem-loop icons indicate the positions of Rho-independent transcriptional terminators predicted using TransTermHP (Kingsford, et al., 2007), with a confidence score of 100 in each case. “#aa” indicates the number of amino acids encoded by each ORF. VF1308 and VF1309 (black arrows) indicate ORFs with similarity to the N and C termini of E. coli FNR, respectively. The striped arrow shows the complete fnr based on our sequence revision. (B) The predicted Rho-independent transcriptional terminator between fnr and VF1310. (C) Growth of V. fischeri MJ1, fnr mutant EVS601, and restored fnr⁺ strain JB27 along with ES114, fnr mutant JB1, and restored fnr⁺ strain JB2 on defined medium with glycerol and fumarate, incubated in anaerobic jars at 28°C. (D) E. coli MC4100 and fnr mutant PC2 with vector pDMA5 or pJLB6, which contains the V. fischeri ES114 fnr, grown on M9 medium with glycerol and nitrate in anaerobic jars at 37°C.

FIG. 2

Luminescence per OD₅₉₅ of fnr mutants. In Panels A and B, specific luminescence is shown at different culture densities for V. fischeri ES114 (solid diamonds), ES114 fnr mutant JB1 (empty diamonds), MJ1 (solid squares), MJ1 fnr mutant EVS601 (empty squares), and dark ΔluxCDABEG mutant EVS102 (solid triangles) grown in batch cultures that were; (A) aerobic (50 ml medium in 250-ml flask) or (B) anaerobic (20 ml medium in 165-ml bottles with anaerobic headspace) at 24°C with shaking (200 rpm). ES114, JB1, and EVS102 were omitted from panel B, because luminescence was not detected above background for these strains under these conditions. Bars in panel B indicate standard deviation (n=5). Error bars were omitted in panel A, because they were generally smaller than (and never extended above) the data symbols. For Panel C, ES114 (wild type), JB22 (lacI^q P_A1/34-lux), and their respective fnr mutants (represented by hatched bars) JB1 and ANS25, respectively (Table 1), were grown under anaerobic conditions. “AI” indicates supplementation with 140 nM 3-oxo-C6-HSL autoinducer, and “IPTG” indicates isopropyl-β-D-thiogalactoside was added to 2 mM to induce luxCDABEG expression in strains containing lacI^q P_A1/34-lux. Data is the average peak luminescence per OD₅₉₅ with standard deviation (n=2). Asterisks indicate that the fnr mutant was significantly (p<0.01) brighter than the corresponding isogenic fnr-positive strain. Other comparisons were not significant (p>0.05).

FIG. 3

FNR-mediated regulation of arcA promoter-lacZ reporters. LacZ reporter activity expressed in Miller units for (Panel A) ES114 derivatives ANS23 (ΔarcA∷lacZ) and ANS24 (ΔarcA∷lacZ Δfnr∷tmpR), or (Panel B) the MJ1 derivatives JB28 (ΔarcA∷lacZ) and JB29 (ΔarcA∷lacZ Δfnr∷tmpR). Culture conditions (aerobic or anaerobic) are as described in the Figure 2 legend. Averages with standard deviation are indicated (n=3). The LacZ reporter activity shown is approximately 100-fold above the background determined using strains ES114 and JB1, which lack the ΔarcA∷lacZ allele.

FIG. 4

Colonization of E. scolopes by fnr mutant and wild type. (A) Average symbiotic luminescence in E. scolopes hatchlings inoculated with ES114 (solid diamonds), or the fnr mutant JB1 (empty diamonds), (n=14). Control squid receiving no V. fischeri inoculum (empty squares) did not yield any bioluminescence (n=4). Bars above graph indicate periods of ambient light (empty bar) and darkness (solid bar). (B) Average colonization levels in CFU V. fischeri per squid 36 h after inoculation with ES114 (solid bar) or JB1 (hatched bar). Treatments are not significantly different (p=0.7). Bars indicate standard deviation (n=14 for ES114 and 13 for JB1). (C) Competitiveness of JB1 when presented in a mixed (~1:1) inoculum with wild type and recovered from squid after 48 h. Each symbol represents the RCI determined from one squid, defined as the ratio of JB1:ES114 in the squid divided by the ratio in the inoculum. Combined data from three experiments is presented. The dashed line represents equal competitiveness and in this case is also the mean RCI (n=60).