Population dynamics of Vibrio fischeri during infection of Euprymna scolopes

Abstract

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.

FIG. 1.

ID₅₀ of V. fischeri for symbiotic colonization. In two separate experiments juvenile squid were incubated with different numbers of V. fischeri ES114 cells, and the percentage of animals subsequently colonized was determined. The dose at which 50% of the animals became luminescent by 48 h postinoculation, indicated by the dotted lines and black arrow, was determined using the equation y = 0.3735 lnx − 1.794 for experiment 1 (circles) and the equation y = 0.1381 lnx − 0.2459 for experiment 2 (triangles).

FIG. 2.

Kinetics of onset of animal luminescence as a function of V. fischeri dose. Juvenile squid were incubated for 12 h in seawater containing 40 (open diamonds), 580 (closed squares), 36,000 (open triangles), or 800,000 (closed circles) V. fischeri cells, and the development of luminescence by each animal was monitored (n = 12 for each dose). Mean luminescence levels of those animals that became colonized are reported in relative light units. Values below about 10³ units indicate no detectable light production. Error bars depict the standard errors of the means.

FIG. 3.

Site of mini-Tn7 insertions in the V. fischeri genome. PCR products from insertion strains JRM100, JRM101, and JRM102 were sequenced using a primer extending from the left end of the transposon (labeled “L”), and products from strains JRM103 and JRM104 were sequenced from the right end of the transposon (labeled “R”). The resulting sequences were aligned, and the predicted insertion points are indicated.

FIG. 4.

Evidence of the association of V. fischeri cells with host tissue 3 h after inoculation. Three sets of 24 animals were exposed to three different doses of V. fischeri cells: 340, 1,800, and 3,400 per animal. The number of V. fischeri cells (as determined by CFU) associated with light-organ tissue at 3 h postinoculation was determined (left panel) and compared to the percentage of animals that eventually became colonized at these inoculation doses (right panel).

FIG. 5.

Effect of the total dose on the frequency of colonization by one, two, or three strains of V. fischeri from a mixed inoculum. Animals were exposed to the following approximate doses of V. fischeri cells per animal: 500 (open bars; n = 8), 5,000 (stippled bars; n = 15), 16,000 (striped bars; n = 20), and 27,000 (solid bars; n = 14). All doses contained a mixed inoculum of approximately equal proportions of V. fischeri strains ES114, JRM100, and JRM200. The number of different strains detected in each light organ was determined 24 h postinoculation.