Vibrio fischeri outer membrane protein OmpU plays a role in normal symbiotic colonization

Abstract

The nascent light-emitting organ of newly hatched juveniles of the Hawaiian sepiolid squid Euprymna scolopes is specifically colonized by cells of Vibrio fischeri that are obtained from the ambient seawater. The mechanisms that promote this specific, cooperative colonization are likely to require a number of bacterial and host-derived factors and activities, only some of which have been described to date. A characteristic of many host-pathogen associations is the presence of bacterial mechanisms that allow attachment to specific tissues. These mechanisms have been well characterized and often involve bacterial fimbriae or outer membrane proteins (OMPs) that act as adhesins, the expression of which has been linked to virulence regulators such as ToxR in Vibrio cholerae. Analogous or even homologous mechanisms are probably operative in the initiation and persistence of cooperative bacterial associations, although considerably less is known about them. We report the presence in V. fischeri of ompU, a gene encoding a 32.5-kDa protein homolog of two other OMPs, OmpU of V. cholerae (50.8% amino acid sequence identity) and OmpL of Photobacterium profundum (45.5% identity). A null mutation introduced into the V. fischeri ompU resulted in the loss of an OMP with an estimated molecular mass of about 34 kDa; genetic complementation of the mutant strain with a DNA fragment containing only the ompU gene restored the production of this protein. The expression of the V. fischeri OmpU was not significantly affected by either (i) iron or phosphate limitation or (ii) a mutation that renders V. fischeri defective in the synthesis of a homolog of the OMP-regulatory protein ToxR. The ompU mutant grew normally in complex nutrient media but was more susceptible to growth inhibition in the presence of either anionic detergents or the antimicrobial peptide protamine sulfate. Interestingly, colonization experiments showed that the ompU null mutant initiated a symbiotic association with juvenile light organ tissue with only about 60% of the effectiveness of the parent strain. When colonization did occur, it proceeded more slowly and resulted in an approximately fourfold-smaller bacterial population. Surprisingly, there was no evidence that in a mixed infection with its parent, the ompU-defective strain had a competitive disadvantage, suggesting that the presence of the parent strain provided a shared compensatory activity. Thus, the OmpU protein appears to play a role in the normal process by which V. fischeri initiates its colonization of the nascent light organ of juvenile squids.

FIG. 1

SDS-polyacrylamide gel of OMPs isolated from cells of V. fischeri strain ESR1 grown under different culture conditions. Lane 1, cells grown in nutrient medium SWT (7.2 μg of protein loaded); lane 2, cells grown in SWT under iron limitation (6.5 μg of protein loaded); lane 3, cells grown in a minimal medium under phosphate limitation (8.0 μg of protein loaded). Molecular mass standards (stds) are indicated. The arrow indicates the 34-kDa band.

FIG. 2

Inhibition of light organ colonization by V. fischeri cells treated with an antiserum directed against P. profundum OmpL, a homolog of V. fischeri OmpU. Groups of between 7 and 10 newly hatched, uncolonized juveniles of E. scolopes were individually inoculated by placing them for 3 h in 5 ml of seawater containing approximately 10⁴ cells of V. fischeri ESR1 that were either untreated (●) or treated with OmpL antiserum (▪) or preimmune serum (□). Control animals (○) were incubated in seawater without added V. fischeri cells. The data shown are representative of those obtained in four separate experiments.

FIG. 3

Alignment of deduced amino acid sequence encoded by the V. fischeri (VFIS) ompU gene with those of V. cholerae (VCHO) ompU and P. profundum (PPRO) ompL genes. Residues that are identical to those in the V. fischeri sequence are indicated by an asterisk (*), and gaps that were inserted to optimize alignment are indicated by periods.

FIG. 4

Two-step scheme for constructing an insertional mutation in the V. fischeri ompU gene. In step 1, a Cm^r cassette is used to replace an internal portion of the ompU gene in pFA3, resulting in pFA5. In step 2, to facilitate screening of recombinants, a Tp^r cassette is also added to pFA5, yielding pFA8. This plasmid was then introduced into V. fischeri to produce an ompU mutation by double recombination. Abbreviations: lacZ, β-galactosidase gene; ColE1 ori, ColE1 origin of replication; F1 ori, F1 origin of replication; Ap^r, ampicillin resistance gene; Cm^r, chloramphenicol resistance gene; Kn^r, kanamycin resistance gene; Tp^r, trimethoprim resistance gene.

FIG. 5

SDS-polyacrylamide gel of proteins isolated from different strains of V. fischeri. Cultures were grown at room temperature in SWT medium for 14 to 18 h with shaking. Lane 1, KR-tox1 (toxR omp⁺); lane 2, ESR1 (parent strain, ompU⁺); lane 3, OM3 (ompU gene replacement mutant); lane 4, OM3 pFA9 (OM3 complemented with pFA9, ompU⁺). Positions of molecular mass standards (stds) are indicated. The arrow indicates the 34-kDa OmpU protein.

FIG. 6

Effects of bile, protamine, and SDS on the growth of strains OM3 and ESR1. Cultures of the ompU mutant strain OM3 (hatched bars) or its parent, ESR1 (solid bars), were inoculated into LBS medium containing different concentrations of bovine bile, protamine sulfate, or SDS. (A) After 24 h of growth at 28°C, dilutions of the bile-containing cultures were spread on LBS agar medium, and the numbers of CFU in the cultures were calculated. The value of each bar is the average of two separate experiments. Similarly, LBS cultures containing either protamine sulfate (B) or SDS (C) were incubated for 24 h at 28°C, and the resulting relative cell density was estimated spectrophotometrically. Cultures of strain OM3 carrying either a wild-type copy of ompU on pFA9 (gray bars) or the vector control, pVO8 (open bars), were also tested on SDS-containing LBS medium. Error bars indicate standard errors of the means.

FIG. 7

Colonization efficiency of V. fischeri ompU mutant strain. Groups of newly hatched, uncolonized juvenile squids were placed into seawater containing between 700 and 1,500 cells of either the ompU mutant, OM3, or its parent strain, ESR1, per ml for 3 h. In each experiment the OM3 inoculum was determined to have at least as many cells as the ESR1 inoculum. Starting 14 h after inoculation and continuing at different intervals, the animals were assayed for the production of luminescence, an indication of successful colonization. The percentage of animals in the OM3-inoculated group (○) and the ESR1-inoculated group (●) that produced luminescence was determined. Data points are the means of three separate experiments, and error bars indicate the standard errors of the means.