Characterizing Novel Vibrio fischeri Strains: Investigating Biodiversity in the Squid-Vibrio Symbiosis

Nadia C. Ortega*, Zachary J. Houston*, Andrew Cecere, Elijah LaSota, and Tim Miyashiro

Department of Biochemistry and Molecular Microbiology, The Pennsylvania State University, University Park, PA

Introduction The Hawaiian bobtail squid, Euprymna scolopes (Fig.1),

forms a mutualistic symbiosis with Vibrio fischeri. This

marine bacterium colonizes the light organ of E. scolopes,

and the bioluminescence it generates provides the squid with

camouflage from predators through a process known as

counter-illumination.1 In return, the squid provides V. fischeri

with a safe environment and nutrients in the form of free

amino acids and peptides. Within the light organ of the squid

there is significant diversity of V. fischeri strains. What is

the significance of this diversity and what can it teach us

about the relationship between host and symbiont?

Methods Media and culture conditions. V. fischeri was grown in

LBS medium. Conditions for liquid culture incubation were

28° C shaking at 200 RPM.

Motility assays. 3-mL intermediate cultures were grown for

1.5 hours and 5-uL injected into instant ocean + tryptone

(IO-T) motility plates in triplicate. Diameter of rings was

measured every 2 hours.

Luminescence assays. 2-mL intermediate cultures were

grown to OD600=1.0 in the presence and absence of

autoinducer, 3-oxo-C6-HSL, in triplicate. Relative light units

(RLU) were measured in luminometer and corrected by OD

of intermediate cultures.

Squid colonization assays. Introduced naïve, juvenile squid

to inoculum of V. fischeri of about 5,000-CFU/mL. 48 hours

post-inoculation RLU of each squid was measured. The

animals were stored individually in microfuge tubes at -

80°C. CFU levels within squid were determined by

homogenizing them into 700-uL 70% instant ocean and

plating serial dilutions onto LBS agar.

Conclusions Preliminary characterization has shown that these novel

strains of V. fischeri are different from the type strain.

Interestingly, the strains both showed lower luminescence

and slower motility, yet were able to colonize the host and

reached the same cell abundance within the light organ.

References 1. Stabb, E.V. and Visick, K.L. (2013) Vibrio fischeri: A Bioluminescent Light-

Organ Symbiont of the Bobtail Squid Euprymna scolopes. In The Prokaryotes –

Prokaryotic Biology and Symbiotic Associations. Rosenberg, E., DeLong, E.F.,

Stackebrand, E., Lory, S. and Thompson, F. (eds). Berlin Heidelberg: Springer-

Verlag, pp. 497-532.

2. Deloney-Marino, C.R. (2013) Observing Chemotaxis in Vibrio fischeri Using

Soft Agar Assays in an Undergraduate Microbiology Laboratory. J Microbiol

Biol Educ 14: 271-2.

3. Miyashiro T, Ruby EG. Shedding light on bioluminescence regulation in Vibrio

fischeri. Mol Microbiol. 2012;84(5):795-806.

4. Nyholhm, S.V. and McFall-Ngai, M.J. (2004) The winnowing: establishing the

squid-vibrio symbiosis. Nat Rev Microbiol 2: 632-42.

Acknowledgements We thank members of the Miyashiro lab for valuable advice during this study. This

work was supported by the National Institutes of Health grant R00 097032 from the

NIGMS to T.M.

For further information Please e-mail Tim Miyashiro at tim14@psu.edu.

Future directions Do the motility differences in these isolates affect their

ability to colonize the host?

Are these strains equally competitive with the type strain in

host colonization?

What is the genetic basis for the luminescence phenotypes

observed in these isolates? The horizontal transmission exhibited in this symbiosis

allows us to introduce naïve juvenile squid to V. fischeri

cultures. This allows us to compare the colonization abilities

of various strains.4

By measuring luminescence, we were able to determine

whether the squid were colonized (Fig. 5). A positive control

group of juveniles were introduced to an inoculum of ~6800-

CFU/mL ES114 and a negative control was prepared without

introducing any V. fischeri. Juveniles were introduced to an

inoculum of ~600-CFU/mL NAD4 or ZJH4. All three

conditions exhibit luminescence levels 5 orders of magnitude

higher than the aposymbiotic control. These results suggest

that the luminescence of these strains within the light organ

differs.

To determine CFU per animal, we homogenized and plated

the juveniles (Fig. 5). Both natural isolates reach wild-type

abundance within the light organ after 48 hours. These

results suggest that these isolates do not differ in their ability

to colonize the light organ.

Figure 6: V. fischeri luminescence (red) and abundance (black)

within the squid light organ. Both natural isolates show

significantly more cells per squid than the aposymbiotic control.

There is also no significant difference between NAD4, ZJH4, and

the type strain (one-way ANOVA). Averages and standard

deviations are plotted for each condition. Data points represent

animals in the experiment (averages of duplicate CFU counts).

Results To obtain isolates of V. fischeri, we dissected a male adult

squid. Serial dilutions of the homogenized light organ were

plated on LBS and isolated colonies were chosen based on

differing colony morphologies. ZJH4 and NAD4 were

selected for distinct colony sizes and morphologies (Fig. 2).

V. fischeri produces luminescence through a quorum sensing

mechanism (Fig. 4). Each cell secretes a small amount of the

autoinducer, 3-oxo-C6-HSL. At high cell number, the

concentration of this autoinducer is high enough to diffuse

back across the membrane. Through its interaction with

transcription factors, this autoinducer causes changes in gene

expression within the cell, producing luminescence.

To compare the response of these isolates to the autoinducer

(AI), we measured RLU of cultures grown in medium with

and without 3-oxo-C6-HSL.3 We used the type strain, ES114,

as a positive control. The non-luminescent ∆lux strain,

EVS102, does not respond significantly to AI (0.68-fold

response, one-way ANOVA) and functions as a negative

control (Fig 5).

Figure 3: Motility of natural isolates on soft agar plates. ES114

(type strain) swims at 8.0±0.5-mm/hr. NAD4 swims at 2.0±0.5-

mm/hr. ZJH4 swims at 6.0±0.5-mm/hr. One-way ANOVA

analysis shows that the rates of NAD4 and ZJH4 are

significantly different than that of ES114 (p<0.0001).

Figure 4: Luminescence phenotypes of natural isolates. The

response to autoinducer (AI) is significant for the type strain and

ZJH4 (two-way ANOVA). However, the same analysis shows

the response to AI is not significant for the negative control or

NAD4. Shown are the averages of triplicate measurements.

E S 1 1 4 E V S 1 0 2 N AD 4 ZJ H 4

1 0 2

1 0 3

1 0 4

1 0 5

RL

U/A

bs

A I -

A I+

P<0.0001

P>0.05

P<0.0001

P>0.05

Figure 1: (A) Adult male Hawaiian bobtail squid, E. scolopes

(pictured anesthetized in ethanol). (B) The light organ is located

near the ink sac in the squid mantle.

2 cm

A B

Figure 4: The LuxR/3-oxo-C6 complex activates

transcription of the luxI promoter. Positive feedback at this

promoter results in a threshold response to autoinduction.3

Figure 2: The morphologies of the selected natural isolates differ

from the type strain, ES114 (A). NAD4 (B) and ZJH4 (C) are

Vibrio fischeri, but can be differentiated by colony shape and size.

A C

3mm 3mm

B

Objective Our main objective is to characterize different strains of V.

fischeri that originated from the same light organ. This will

yield new data regarding the diversity of V. fischeri strains

associated with wild-caught animals.

To compare motility rates among the isolated strains, we

used soft-agar plates. Injecting these plates with a cell

suspension results in an expanding ring of bacterial growth.2

We measured and plotted the diameters of these rings over

time to obtain motility rates (Fig. 3). The results show that

NAD4 and ZJH4 swim at 25% and 75% the rate of ES114,

respectively. These results suggest that the motility of these

two strains differ within the light organ.

NAD4 shows an insignificant (1.1-fold) response to AI. This

indicates that NAD4 does not luminesce at the same level as

wild type. ZJH4 shows a significant (12-fold) response to AI.

However, this response is significantly lower than the

response of wild-type V. fischeri (27-fold). These results

suggest that these isolates luminesce differently within the

light organ.