The haem-uptake gene cluster in Vibrio fischeri is regulated by Fur and contributes to symbiotic colonization

Abstract

Although it is accepted that bacteria-colonizing host tissues are commonly faced with iron-limiting conditions and that pathogenic bacteria often utilize iron from host-derived haem-based compounds, the mechanisms of iron acquisition by beneficial symbiotic bacteria are less clear. The bacterium Vibrio fischeri mutualistically colonizes the light organ of the squid Euprymna scolopes. Genome sequence analysis of V. fischeri revealed a putative haem-uptake gene cluster, and through mutant analysis we confirmed this cluster is important for haemin use by V. fischeri in culture. LacZ reporter assays demonstrated Fur-dependent transcriptional regulation of cluster promoter activity in culture. GFP-based reporter assays revealed that gene cluster promoter activity is induced in symbiotic V. fischeri as early as 14 h post inoculation, although colonization assays with the haem uptake mutant suggested an inability to uptake haem does not begin to limit colonization until later stages of the symbiosis. Our data indicate that the squid light organ is a low iron environment and that haem-based sources of iron are used by symbiotic V. fischeri cells. These findings provide important additional information on the availability of iron during symbiotic colonization of E. scolopes by V. fischeri, as well as the role of haem uptake in non-pathogenic host-microbe interactions.

Figure 1

Organization of the putative heme uptake gene cluster in V. fischeri ES114. A. The region corresponds to open reading frames VF_1220 to VF_1228 (left to right). Comparisons of these gene products to known heme uptake proteins are described in Table 2. Flanking open reading frames (VF_1219, hypothetical protein and VF_1229, VgrG) are depicted by white arrows. B. Representation of the DNA fragments cloned to generate the reporter plasmids. Arrows indicate the approximate region of the putative promoter for each construct.

Figure 2

The putative heme uptake genes are necessary for growth of V. fischeri with hemin as the sole iron source. Cultures of wild type and AKD910 (ΔVF_1220-1228) were grown in mineral salts mediumcontaining iron chelator, with and without hemin added. Optical density was measured over time for wild type grown without hemin (open squares), wild type grown with hemin (filled squares), AKD910 grown without hemin (open triangles), and AKD910 grown with hemin (filled triangles). Error bars represent standard error of the mean.

Figure 3

Heme-uptake promoter activity is induced in response to low iron concentrations and this effect is mediated through Fur. pAKD912 (P_{VF_1225}-lacZ) or pAKD914 (P_{VF_1226}-lacZ) (see Fig. 1B) was introduced into A. wild-type V. fischeri, or B. a strain lacking fur (YLW111). White bars indicate strains containing pAKD912, while shaded bars indicate strains containing pAKD914. Where indicated, iron chelator and/or hemin were added to the culture medium. A student’s t-test was used to determine whether the addition of hemin to cultures containing iron chelator significantly influenced the Miller unit values for each reporter in the wild-type background (p < 0.05). Although the trend of a decrease in promoter activity in response to hemin was consistent in all experiments, the difference was not consistently significant, and was not significant in the experiment shown.

Figure 4

Heme uptake gene cluster promoter activity is induced during symbiotic colonization, and GFP expression patterns are similar to those observed in culture under low iron conditions. A–D. Aposymbiotic juvenile E. scolopes were inoculated with either wild-type V. fischeri containing the control plasmid pVSV209 where RFP is expressed constitutively and there is no promoter to drive gfp expression (A and B), or wild-type V. fischeri containing pAKD911, where RFP is expressed constitutively and gfp expression is under the control of the promoter associated with the VF_1225-VF_1220 gene cluster (C and D). Epifluorescence images of individual juvenile squid light organs 28 h post-inoculation were taken using either a filter to visualize RFP (A and C) or GFP (B and D). Numbers of bacteria per light organ were determined by plating and, for these images, were 2.76 × 10⁵ and 3.31 × 10⁵ for V. fischeri containing pVSV209 and pAKD911, respectively. Similar results were observed at 14 and 48 h post-inoculation (data not shown). E–H. Microscopic images of individual V. fischeri cells containing pAKD911 either captured using regular light (E and G) or using a filter to visualize GFP (F and H). Cells were grown in LBS medium either without (E and F) or with (G and H) iron chelator. Corresponding to the β-galactosidase reporter assays (Fig. 3), GFP expression is detectable only when the iron chelator is present in the culture medium. Scale bar indicates approximately 100 μm for A–D and approximately 5 μm for E–H.

Figure 5

Heme uptake and/or utilization contributes to symbiotic colonization. A. Graphical representation of colonization competitiveness in the combined 94 and 100 h assays. Each symbol represents a RCI value from one animal. The dashed line indicates the average RCI value (0.33) for all animals in the experiment. The animals represented by open symbols were clonally colonized by wild-type V. fischeri. B. Numerical data for all time points assayed. For the ~ 24 hour and ~ 96 h time points, data was combined from two independent experiments. Significance was calculated using a student’s t-test.