Let There Be Light!

by S. Mar­vin Fried­man

As dis­cussed in a re­cent epic Per­spec­tive on the an­i­mal-mi­crobe sym­bioses (and com­mented in this blog), we are be­gin­ning to un­der­stand the many con­tri­bu­tions made by each part­ner. One of the most widely stud­ied sym­bioses is that of the lu­mi­nous bac­terium Vib­rio fis­cheri and the Hawai­ian Bob­tail squid, Eu­prymna scolopes. In a re­cent pub­li­ca­tion, Heath-Heck­man and cowork­ers de­ter­mined that bac­te­r­ial bi­o­lu­mi­nes­cence reg­u­lates the ex­pres­sion of a squid cir­ca­dian rhythm (cryp­tochrome) gene (cryp­tochromes are a fam­ily of blue light re­cep­tor pro­teins in­volved in cir­ca­dian rhythms of an­i­mals and plants).

Fig. 1: The Hawai­ian Bob­tail Squid. Credit: kahikai.org. Source

Over the past few years, bac­te­r­ial part­ners have been im­pli­cated in their host's cir­ca­dian rhythms. For ex­am­ple, in hu­mans, im­mune com­pe­tence re­quires that such rhythms be in­tact. Nor­mal func­tions of the gut, such as peri­staltic ac­tiv­ity, un­dergo cir­ca­dian rhythms that are likely to be im­pacted by the gut mi­cro­biota. Like­wise, sev­eral con­di­tions, such as obe­sity, di­a­betes, de­pres­sion, and sleep dis­or­ders are as­so­ci­ated with aber­rant cir­ca­dian rhythms and im­bal­ances in the gut mi­cro­biota. Tan­ta­liz­ing though this is, in none of these sys­tems has a di­rect in­ter­ac­tion be­tween sym­biont and host been demon­strated. The first in­stance in­volves the squid and its vib­rio.

The Vib­rio-Squid Busi­ness

V. fis­cheri col­o­nizes deep crypt spaces of the E. scolopes light or­gan lo­cated in the animal's man­tle. Com­plete light or­gan de­vel­op­ment is trig­gered by the symbiont's MAPS (mi­crobe as­so­ci­ated mol­e­c­u­lar pat­terns), namely the lipid A moi­ety of lipopolysac­cha­ride (LPS) and the pep­ti­do­gly­can monomer tra­cheal cy­to­toxin (TCT). The squid is a noc­tur­nal preda­tor and the sym­biont lu­mi­nes­cence peaks at night when the an­i­mal is ac­tive, a tim­ing op­po­site to that of the en­vi­ron­men­tal light cue.

Fig. 2: The cryp­tochromes in the sym­bi­otic or­gan of E. scolopes. (A) The ju­ve­nile an­i­mal. e, eyes; lo, light or­gan, seen through ven­tral man­tle tis­sue. (B) A light mi­cro­graph of a cross sec­tion of the ju­ve­nile light or­gan. The in­te­rior of the or­gan con­tains three ep­ithe­lium-lined crypts (1–3), each har­bor­ing bac­te­ria(b) in the crypt lu­men. Sur­round­ing the light or­gan and con­trol­ling light emis­sion from the or­gan into the en­vi­ron­ment are the ink sac (is) and re­flec­tor ®; hg, hindgut. Source

In­ver­te­brate cryp­tochromes fall into two groups: the mem­bers of one (Cry1) are light ac­ti­vated and lead to degra­da­tion of core clock ma­chin­ery re­pres­sors, whereas the oth­ers (Cry2) are light in­de­pen­dent tran­scrip­tional re­pres­sors of the core clock genes. When these in­ves­ti­ga­tors ex­am­ined the tran­scrip­tional data­bases for the E. scolopes light or­gan, they de­tected ex­pres­sion of two cryp­tochrome genes, des­ig­nated escry1 and escry2. They per­formed real time quan­ti­ta­tive re­verse tran­scrip­tase PCR (qRT­PCR) on ju­ve­nile light or­gans that were col­o­nized with sym­bionts, us­ing four time points over the day/night cy­cle. They com­pared the pat­terns of es­cry ex­pres­sion in the light or­gan to that in the heads of the same ju­ve­nile squids. Lev­els of escry1 and escry2 mR­NAs did not vary sig­nif­i­cantly in the head, but in the light or­gan, escry1 mRNA lev­els alone changed dur­ing the day/night cy­cle. Fur­ther­more, mRNA lev­els peaked in the light or­gan at pe­ri­ods of high light or­gan lu­mi­nes­cence, shifted 6+ hours from that ob­served in the head. An­i­mals caught in the wild re­acted the same way as those raised in the lab. Thus, the pat­tern of escry1 ex­pres­sion is due nei­ther to lab­o­ra­tory con­di­tions nor to a spe­cific life stage. In ad­di­tion, the diel pat­terns of mRNA lev­els in non-col­o­nized squid did not show the vari­a­tion in escry1 mRNA lev­els seen in sym­bi­otic an­i­mals. These data are con­sis­tent with the idea that escry1 ex­pres­sion un­der­goes cy­cles in the light or­gan re­sult­ing from in­duc­tion by light from its sym­bionts.

Bac­te­ria Shine Light On the Squid

Fig. 3: Day/night cy­cle vari­a­tion in E. scolopes cryp­tochrome ex­pres­sion. (A) The ex­pres­sion of escry1 and escry2 in the squid light or­gan and head over four points in the day/night cy­cle as mea­sured by qRT-PCR. Yel­low and black bars de­note the cy­cle of ex­oge­nous light, and the blue and black bars show the cy­cle of bac­te­r­ial light pro­duc­tion in the light or­gan. (B) Ex­pres­sion of escry1 in the light or­gans of ma­ture squid caught in the wild and main­tained in nat­ural light. © escry1 ex­pres­sion over the day/night cy­cle in non­sym­bi­otic light or­gans. Source

Is EsCry1 pro­tein syn­the­sized in close prox­im­ity to the lu­mi­nes­cent bac­te­r­ial part­ner? To find out, the au­thors raised an an­ti­body to a unique pep­tide se­quence on EsCry1. This an­ti­body dis­played cross re­ac­tiv­ity to the cryptep­ithe­lial cells that shel­ter the sym­bi­otic bac­te­ria. The la­bel was de­tected through­out these cells but was con­cen­trated at the api­cal sur­face. So, is bac­te­r­ial lu­mi­nes­cence crit­i­cal for the en­train­ment of escry1 mRNA cy­cling? The level of this mRNA when the squid was col­o­nized by V. fis­cheri mu­tants (lux) de­fec­tive in light pro­duc­tion did not dif­fer sig­nif­i­cantly from that found in non-col­o­nized squids. An­i­mals col­o­nized by the Δlux mu­tants ex­posed to blue light, how­ever, showed an in­crease in escry1 mRNA lev­els sim­i­lar to those found in an­i­mals col­o­nized with wild type bac­te­ria. It should be noted that the cell den­sity reached by the Δlux strain in the light or­gan was only 10% of that of wild type bac­te­ria. Could the fail­ure of the Δlux mu­tant to in­duce ex­pres­sion of escry1 merely re­flect a pop­u­la­tion ef­fect? To an­swer this ques­tion, the re­searchers tested a ly­sine aux­otroph of V. fis­cheri that col­o­nizes the light or­gan to the same ex­tent as the Δlux mu­tant but emits the same level of lu­mi­nes­cence per cell as the wild type. The ly­sine aux­otroph in­duced a much higher ex­pres­sion of escry1 than the Δlux mu­tant, thus in­di­cat­ing that bac­te­r­ial light, not bac­te­r­ial den­sity is a crit­i­cal fac­tor reg­u­lat­ing in­creased ex­pres­sion of escry1.

The in­ves­ti­ga­tors now asked whether light alone could in­duce cy­cling of escry1 ex­pres­sion. Il­lu­mi­nat­ing the light or­gans of non-sym­bi­otic squid with a cy­cle of ex­oge­nous blue light of the same wave­length as that emit­ted by wild type V. fis­cheri re­sulted in no in­crease in escry1 ex­pres­sion. How­ever, ex­po­sure to blue light plus adding the MAMPs lipid A and TCT did in­duce cy­cling. Treat­ment with lipid A or TCT alone did not af­fect lev­els of escry1 mRNA.

Il­lu­mi­nat­ing the Fu­ture

This study clearly im­pli­cates three bac­te­r­ial prod­ucts, namely blue light, lipid A, and a pep­ti­do­gly­can monomer in the cyclic ex­pres­sion of a cryp­tochrome gene in the sym­bi­otic or­gan of the squid. For the first time, a di­rect role of the bac­te­r­ial sym­biont in the cir­ca­dian rhythms of its host has been demon­strated, thus pro­vid­ing im­por­tant in­sights for fu­ture work with the more com­pli­cated mam­malian sys­tems. An emerg­ing hy­poth­e­sis is that mi­cro­biota and host co­op­er­ate to de­velop and main­tain bi­o­log­i­cal rhythms that are es­sen­tial for the home­osta­sis of the sym­bi­otic re­la­tion­ship. These stud­ies bode well for fu­ture work on the mech­a­nism whereby bac­te­r­ial sym­bionts in­flu­ence their host's cir­ca­dian rhythm as well as many of its other cru­cial phys­i­o­log­i­cal ac­tiv­i­ties.

Ref­er­ence

Heath-Heck­man EA, Peyer SM, Whistler CA, Api­cella MA, Gold­man WE, Mc­Fall-Ngai MJ (2013). Bac­te­r­ial bi­o­lu­mi­nes­cence reg­u­lates ex­pres­sion of a host cryp­tochrome gene in the squid-Vib­rio sym­bio­sis MBio. DOI 10.1128/mBio.00167–13

Mar­vin is Pro­fes­sor Emer­i­tus in the De­part­ment of Bi­o­log­i­cal Sci­ences at Hunter Col­lege of CUNY in New York City, and an As­so­ciate Blog­ger for Small Things Con­sid­ered.