Friends or Foes: When NO means Yes in the Squid-Vibrio symbiosis

By: Linda Xu, Jeanie Lim, and Ha Dang

(*) Title inspired by Davidson et al. 2007

The transmission of symbionts is the key to the maintenance of symbiotic relationships. While many animal-bacteria symbioses are established by the mother passing symbionts to the progeny via horizontal and vertical transfer, some symbiotic relationships need to be established anew every generation through external transfer. One example is the much studied mutualistic relationship between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri. Every newborn squid needs to be infected by the bacteria in order to initiate the symbiotic partnership. This means that the baby squids need to have some discretion as to which bacteria to take up and which to keep out: it is going to be problematic if they end up taking in some pathogenic bacteria as well.

But first, why does E. scolopes need V. fischeri? The bobtail squid has a nocturnal lifestyle: it buries itself in the sand of shallow coastal waters during the day and is only out to hunt for food at night. Its bioluminescent companion provides a light source in the dark, which keeps the squid hidden from the predators’ sight. This may seem counter-intuitive: how does emitting light in the dark protect the squid from being seen? While a squid roams in knee-deep coastal waters, its shadow casted beneath, as a result of the light from the moon and stars above, would be a telltale sign for predators in the deeper water. However, alliance with V. fischeri enables the squid to emit ventral luminescence, creating countershade to camouflage itself, as illustrated in Figure 1 and further explained in this animation.

Figure 1. (A) A schematic drawing showing how E. scolopes uses bioluminescence from V. fischeri to match an illuminated background, such as the ocean surface so as not cast a shadow and avoid predators (of course, the bacteria stay in the squid’s light organ, not in the flashlight!). (B) A photo of Hawaiian bobtail emitting ventral luminescence. (C) This counter-illumination method is used by many other marine lives as well.

In another word, the bacteria keep the squid from being hunted while out hunting! In return, the squid provides the bacteria a residence with generous nutritional supply. However, the temptation of luxurious hospitality necessitates a security system to ward off free riders. The baby squid needs to know how to recognize friends from moochers. Like Mommy always says, don’t open the door to strangers!

In order to ensure that its light organs are free of bacterial infection, the squid installed a security system against microbial invasion. Like many other animals and plants, the bobtail squid uses the notorious antimicrobial agent - nitric oxide (NO) - as a biological arsenal against invading microbes. NO is a small gaseous molecule, easily diffusible and swift to attack. It is highly reactive and able to block off the bacterial heme group, a crucial molecule for microbial respiration. On top of that, many derivatives of NO are radicals, which are highly oxidizing and toxic. Bacteria lacking a defense mechanism will almost immediately experience a halt in growth upon exposure to NO. In the bobtail squid, both the duct leading to the light organs and the deep crypts of the organ are lined with high concentration of NO. Any unprepared bacteria that dare to venture into this no-man’s land will sure to suffer a painful death by this killer gas. NO is brutal and indiscriminate of the bacteria it attacks. It will try to inhibit V. fischeri ‘s respiration just as any other bacteria that crawl into the organs. Yet, it seems, V. fischeri is the only one able to persist in this hazardous environment, unaffected by the bombardment of NO. What then grants the bacteria the exclusive entrance ticket to the squid’s light organ?

V. fischeri (right) is a bioluminescent symbiont found in
the light-sensing organ of the Hawaiian bobtailed squid
(left). Photo by Eric Stabb, University of Georgia.

A study by Wang et al. 2010 (a) may offer an answer to the question above. Scanning through V. fischeri’s genome, Wang and colleagues discovered two genes: one coding for flavohaemoglobin (Hmp) and one for flavorubredoxin (NorV), both of which are proteins that have previously been found to function in bacterial NO defense. The two proteins work in tandem to detoxify NO, even though Hmp plays the major role while NorV assists in anoxic condition. Hmp and NorV are found to be negatively regulated by two proteins, NsrR and NorR, respectively. As NsrR contains a [Fe-S] cluster that can be attacked by NO and NorR can be chemically modified by NO nitrosylation, V. fischeri can sense when a NO attack is taking place and prepare themselves: when NO is not present, both NsrR and NorR are constitutively expressed, blocking the transcription of hmp and norV genes. In contrast, the presence of NO renders both NsrR and NorR nonfunctional and thereby turns on the expression of Hmp (and NorV in anaerobic condition). This way, upon being attacked by NO, V. fischeri dials up the production of Hmp and NorV, creating a chemical defense shield.

Figure 4. The level of oxygen intake is monitored in wild type and mutated V. fischeri strains with or without pretreatment of NO. A halt in oxygen intake is observed in wild type (WT) V. fischeri not pretreated with NO, but not in those pretreated with NO. Mutating nsrR appeared to prevent the halt, while mutating hmp reduced the respiration rate.

It seems that our questions has been settled: It is the intricate molecular mechanism to detoxify NO that lets V.fischeri  “talk” to the squid and enter the squid’s light organ.  However, just when the mystery seems to be solved, another puzzle arises. When researchers tried to reenact the battle of V. fischeri against NO outside of the squid, the bacterium was invariably defeated: when V. fischeri were released into an environment with a concentration of NO comparable to that found inside the squid light organs, they still experienced a respiratory arrest (Figure 4). Notably, the halt in oxygen consumption level in wild type (WT) V. fischeri  is ameliorated when the bacteria are first pre-treated with a low concentration of NO. The Δhmp strain cannot produce Hmp and thus still experiences respiratory arrest. The data support the idea that without a mild dose of NO, V. fischeri is unable to counterattack the gas because it does not have enough time to produce enough Hmp before the ambush occurs.  When nsrR, the Hmp inhibitor, is mutated, there is no difference between the pre-treated and non-pretreated bacteria because in the ΔnsrR mutants, Hmp production is always on, and the bacteria have a stock of Hmp ready to function anytime. With hmp transcription inhibited under normal conditions, WT V. fischeri cannot produce enough Hmp in time to respond to the sudden NO rush. The pre-treatment with a low dose of NO acts like a shot of vaccine to V.fischeri, preparing them for a greater NO shock.

Where does V. fischeri then get the “vaccination” in native environment? It turns out that the squid itself is the one to give the bacteria the vaccine shot ahead of time. During the early stage of symbiosis, V. fischeri aggregate in the mucous layer of the squid cilia at the entry to the pore. Within this mucous are vesicles of very dilute NO, not enough to block bacterial respiration but enough to signal V. fischeri’s NsrR and NorR to relieve the repression and start Hmp production. As V. fischeri travels down from the pore into the duct and eventually the crypts of the squid’s light organ, the concentration of Hmp has been constantly building up. This way, when the bacteria enters the highly NO-riched environment in the light organ, they are not taken by surprise. The already sufficient level of Hmp can easily counter the effects of NO and protect V. fischeri as the bacteria establish a stable colony within the light organ.

Figure 5. The role of NO in the initiation of the squid-Vibrio symbiosis.

At this point, one may wonder, if an nsrR mutant can stock up Hmp and be ready to fight NO at anytime without requiring a pre-treatment, why would V.fischeri need this negative regulator of Hmp in the first place? One possible answer is that the ΔnsrR strain is more susceptible to oxidative stress that is present during the establishment of the symbiosis. Much as Hmp is an excellent detoxifier of NO, it can also function as a potent reactive oxygen species (ROS) generator in the highly oxidative environment. Thus a strain that overproduces Hmp such as ΔnsrR is more likely to fall victim to oxidative killing, resulting in reduced fitness. It is also worth noticing that NO also induces the expression of a protein called H-NOX, which subsequently decreases the level of iron uptake in V. fischeri so as to avoid radical formation from the Fenton reactions in the oxidative environment (Wang et al 2010 (b)).

The study illustrates the intricate molecular crosstalk between the Hawaiian bobtail squid E. scolopes and the bioluminescent bacteria V.fischeri during the establishment the symbiotic relationship, which is summarized in Figure 5. In nature, organisms do not communicate via Facebook or email, rather through chemical exchange. When, where, and how much a chemical is expressed in an organism and how its partner responds to this molecule determines the friends or foes status between two species. Indeed, sometimes, the killing “NO” may mean “yes”, as can be seen from the symbiotic relationship between E. scolopes and V. fischeri.

Linda (left), Jeanie (middle) and Ha (right) are Biochemistry majors at Mount Holyoke College

References

Davidson, S.K., Tanya A.K., Renate K., Laura S., and McFall-Ngai M.J. (2004). NO means ‘yes’ in the squid-vibrio symbiosis: Nitric oxide (NO) during the initial stages of a beneficial association. Cellular microbiology. 6(12): 1139-1151.

(a) Wang, Y., Dunn, A. K., Wilneff, J., McFall-Ngai, M. J., Spiro, S., & Ruby, E. G. (2010). Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid–Vibrio symbiosis. Molecular Microbiology. 78(4): 903-915.

(b) Wang, Y., Dufour, Y. S., Carlson, H. K., Donohue, T. J., Marletta, M. A., & Ruby, E. G. (2010). H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by vibrio fischeri. Proceedings of the National Academy of Sciences, 107(18), 8375-8380.

Wang, Y. and Edward G. R. (2011). The roles of NO in microbial symbioses. Cellular Microbiology. 13(4): 518-526

Figure 1A: Adapted from The Hawaiian bobtail squid - when science and nature collide. <https://www.youtube.com/watch?v=KCobcWsYOS8#t=65>. Accessed on November 23, 2014.

Figure 1B: Adapted from Ocean Portal - Smithsonian Museum of Natural History.

<http://ocean.si.edu/sites/default/files/styles/colorbox_full/public/photos/Rev-peppers-ghost-A-B-2-full_1.jpg?itok=_R1X1NKg>. Accessed on November 23, 2014.

Figure 1C: Adapted from Sciblogs. <http://sciblogs.co.nz/infectious-thoughts/files/2013/11/squid-with-bright-glow-view-1.jpg>. Accessed on November 23, 2014.

Figure 2: Illustration by Linda Xu (xu27s@mtholyoke.edu)

Figure 3: Adapted from Joint Genome Institute - United States Department of Energy. <http://jgi.doe.gov/why-sequence-vibrio-fischeri/>. Accessed on November 23, 2014.

Figure 4: Adapted from Wang et al. 2010(a).

Figure 5: Adapted from Wang et al. 2011.