Hy­dropho­bic Se­ques­tra­tion

Bio­sur­fac­tants Pro­duced by Mi­crobes Hi­jack Quo­rum Sens­ing in Com­peti­tors

by Kalia Bis­to­las

What do your laun­dry de­ter­gent and hy­dro­car­bon-de­grad­ing ma­rine bac­te­ria have in com­mon? Not much, be­sides sur­fac­tants. These sub­stances lower the sur­face ten­sion be­tween sub­strates, tar­get­ing hy­dropho­bic mol­e­cules like the stain on your fa­vorite shirt or crude pe­tro­leum slicks in the Gulf of Mex­ico. In do­ing so, they serve as de­ter­gents, emul­si­fiers, and dis­per­sants. Hy­dro­car­bon-De­grad­ing Ma­rine Bac­te­ria (HDMB) syn­the­size bio­sur­fac­tants that emul­sify crude pe­tro­leum con­stituents by cap­tur­ing them within am­phiphilic do­mains, al­low­ing the or­gan­isms to use hy­dropho­bic car­bon sub­strates as a prime source of nu­tri­tion. How­ever, bio­sur­fac­tants may do more for HDMBs than pro­vide a tasty, car­bon-rich snack. They can also se­quester hy­dropho­bic com­pounds in­volved in bac­te­r­ial com­mu­ni­ca­tion, as re­ported in the pa­per dis­cussed here.

One of the most ex­cit­ing new ar­eas of mi­cro­bi­ol­ogy is, in fact, how bac­te­ria, both of the same and dif­fer­ent species, com­mu­ni­cate with one an­other. The idea that bac­te­ria just swim around as mute sin­gle cells with lit­tle re­gard for their neigh­bors is fast re­ced­ing. Chief among the de­vices used for talk­ing to each other is quo­rum sens­ing, a way mi­cro­bial cells can "count" the num­ber of other cells in their sur­round­ings to reg­u­late the ex­pres­sion of genes in­volved in such ac­tiv­i­ties as bi­o­lu­mi­nes­cence, toxin pro­duc­tion, or biofilm for­ma­tion. But like com­bat­ants who may give away their po­si­tion by us­ing a ra­dio, their pres­ence can be per­ceived by oth­ers who are keen on dis­rupt­ing such com­mu­ni­ca­tion. This is a com­mon phe­nom­e­non, and many are the ways in which quo­rum sens­ing mol­e­cules can be tam­pered with, in­clud­ing in­hibit­ing their syn­the­sis, in­ac­ti­vat­ing them, or pre­vent­ing their recog­ni­tion. For an ex­ten­sive re­view on this topic, see here. To this list we can now add an­other mech­a­nism, the se­ques­tra­tion of quo­rum sens­ing mol­e­cules by en­cas­ing them in hy­dropho­bic bio­sur­fac­tant mi­celles.

Fig­ure 1. A, C trans­mis­sion elec­tron and epi­flu­o­res­cence mi­croscopy of Co­be­tia grown on suc­ci­nate, B, D TEM and epi­flu­o­res­cence mi­croscopy of Co­be­tia grown on DBT me­dia, il­lus­trat­ing pro­duc­tion of dif­fusible fatty-acid sur­fac­tants (ar­rows). Source

Last year, a group of re­searchers in Chile iso­lated and de­scribed a novel strain of HDMB that pro­duces bio­sur­fac­tants in re­sponse to both abi­otic stim­uli (a spe­cific hy­dro­car­bon) and bi­otic stim­uli (a mi­cro­bial com­peti­tor). Iba­cache-Quiroga and col­leagues ob­tained cul­tures of a bac­terium called Co­be­tia sp. us­ing the sul­fur-rich hy­dro­car­bon diben­zoth­io­phene, or DBT, as the sole source of car­bon and en­ergy (this bac­terium used to be an Arthrobac­ter, then a Halomonas). The re­searchers con­firmed this organism's abil­ity to carry out hy­dro­car­bon me­tab­o­lism by am­pli­fy­ing the gene that en­codes a monooxy­ge­nase en­zyme (DszA) in­volved in DBT degra­da­tion, or "biodesul­fu­r­iza­tion." This en­zyme pro­duces the hy­drotrope, 2‑hydroxylbiphenyl, a po­ten­tial in­ter­me­di­ate in bio­sur­fac­tant pro­duc­tion.

When ex­posed to DBT, cul­tures of this novel Co­be­tia strain sig­nif­i­cantly low­ered the sur­face ten­sion of growth medium. Com­pounds ex­tracted from the cul­ture su­per­natant eas­ily emul­si­fied hexa­de­cane (a hy­dropho­bic, long-chain alkane), sug­gest­ing the pro­duc­tion of bio­sur­fac­tants. Trans­mis­sion elec­tron mi­croscopy (TEM) im­ages show that Co­be­tia grown in DBT had dif­fer­ent outer cell struc­tures than those grown on an al­ter­na­tive growth medium (suc­ci­nate), the DBT-grown bac­te­ria form­ing glob­ules of dif­fusible, sur­fac­tant-like lipid struc­tures on the cell sur­face, in­di­cat­ing that some of the sur­fac­tant re­mained cell-bound.

Fig­ure 2. Bio­sur­fac­tants shield their hy­dropho­bic tails form­ing spher­i­cal mi­celles. The hy­dropho­bic sec­tions of the sur­fac­tant are close to each other and the hydro­philic head groups face out­wards into the so­lu­tion. These mi­celles cap­ture hy­dropho­bic mol­e­cules such as DBT (used for nu­tri­tion) and mi­cro­bial sig­nal­ing mo­lecules (acyl-HSLs), which in­ter­fere with quo­rum sens­ing. Source

The sur­fac­tant in both the su­per­natant and the outer cell layer was com­posed pri­mar­ily of aliphatic car­bon chains or mix­tures of 3‑hydroxy fatty acids. This caught the re­searchers' at­ten­tion be­cause fatty acids are also known to act as dif­fusible sig­nal­ing mol­e­cules be­tween mi­cro­bial species. In­ter­species com­mu­ni­ca­tion by chem­i­cal (lipid-like) sig­nals par­tic­i­pates in a plethora of in­ter­ac­tions, from co­op­er­a­tive mu­tu­alisms to pre­da­tion. Thus, the au­thors took a closer look at the im­pacts of these bio­sur­fac­tants on quo­rum sens­ing. Sure enough, bio­sur­fac­tant pro­duc­tion was stim­u­lated when Co­be­tia was pre­sented with the po­ten­tial mi­cro­bial com­peti­tors Aeromonas salmoni­cida, Chro­mobac­terium vi­o­laceum, or Agrobac­terium tume­fa­ciens. How­ever, they have yet to iden­tify what ini­ti­ates the bio­sur­fac­tant pro­duc­tion in Co­be­tia, and spec­u­late that syn­the­sis may be stim­u­lated by ex­po­sure to non­spe­cific dif­fusible lipid-like sub­stances.

Could sur­fac­tant pro­duc­tion be used as a se­cret weapon against com­peti­tors, lend­ing Co­be­tia cells a re­turn on their en­ergy in­vest­ment? In­deed, re­sults sug­gested that bio­sur­fac­tant pro­duc­tion gives these cells an ad­van­tage by in­ter­fer­ing with the sig­nal­ing mech­a­nisms of other species, sab­o­tag­ing their de­vel­op­men­tal and as­so­cia­tive be­hav­iors. Sig­nal in­ter­cep­tion oc­curs when fatty acid monomers of sur­fac­tant spon­ta­neously clus­ter to form mi­celles that sol­u­bi­lize the lipophilic sig­nal­ing mol­e­cules. And thus short out cell-to-cell com­mu­ni­ca­tion cir­cuits. Adding pu­ri­fied bio­sur­fac­tant in­hib­ited quo­rum sens­ing in the pur­ple bac­terium, C. vi­o­laceum. This bac­terium uses quo­rum sens­ing to trig­ger the syn­the­sis of vi­o­la­cein, an an­tibi­otic that dou­bles as the pur­ple pig­ment. When the Co­be­tia bio­sur­fac­tant was added, the cells did not be­come pur­ple, be­cause the chem­i­cal sig­nals to trig­ger vi­o­la­cein syn­the­sis were hi­jacked by the Cobetia's mi­celles.

Fig­ure 3. In­hi­bi­tion of quo­rum sens­ing-de­pen­dent phe­notypes by the Co­be­tia sp. bio­sur­fac­tant. A Pur­ple phe­no­type re­sponse of C. vi­o­laceum to dif­fer­ent con­centrations (mg/ml) of bio­sur­fac­tant (BS) pro­duced by Co­be­tia sp.. The pro­duc­tion of the pur­ple pig­ment vi­o­la­cein is un­der quo­rum sens­ing con­trol in C. vio­laceum, there­fore, the loss of this phe­no­type in the pres­ence of BS was as­so­ci­ated with the in­hi­bi­tion of quo­rum sens­ing. At the eval­u­ated con­cen­tra­tions no ef­fect on growth was ob­served. B In­ter­ac­tion of quo­rum sens­ing sig­nals with the bio­sur­fac­tant. HSLs en­riched cell-free su­per­natants of A. salmoni­cida were mixed with the bio­sur­fac­tant and used to in­duce the quo­rum sens­ing pig­mented phe­no­type in the HSL not pro­ducer Co­be­tia strain. A. salmoni­cida was ex­posed to: 1 C. vi­o­laceum; 2 C. vi­o­laceum cell-free su­per­natant; 3 A. salmoni­cida cell-free su­per­natant; 4 un­ex­posed; 5 A. salmoni­cida cell-free su­per­natant mixed with bio­sur­factant; 6 C. vi­o­laceum cell-free su­per­natant mixed with biosur­­factant; 7 A. tume­fa­ciens ex­posed to A. sal­monicida cell-free su­per­natant. C Sur­face ten­sion (ST) of wa­ter at dif­fer­ent con­cen­tra­tions (mg/l in log­a­rith­mic scale) of the BS, and V. an­guil­larum biofilm for­ma­tion at dif­fer­ent con­cen­tra­tions (mg/l). Source

The quo­rum sens­ing sig­nals that elicit vi­o­la­cein pro­duc­tion are acyl-ho­moser­ine lac­tones (acyl-HSL), which are also pro­duced by the fish pathogen A. salmoni­cida. The in­ves­ti­ga­tors found that com­bin­ing A. salmoni­cida (which pro­duces the acyl-HSL sig­nal for pig­ment pro­duc­tion) and a mu­tated strain of the pig­ment pro­duc­ing C. vi­o­laceum that could not make its own acyl-HSLs, gen­er­ated pur­ple cul­tures. Adding sur­fac­tants from Co­be­tia in­hib­ited pig­men­ta­tion syn­the­sis in C. vi­o­laceum — no pur­ple phe­no­type was seen. Thus, the bio­sur­fac­tant pro­duced by Co­be­tia al­tered a competitor's phe­no­type by mod­u­lat­ing its chem­i­cal sig­nal­ing path­ways.

In or­der to test the lim­its of Cobetia's bi­o­log­i­cal in­ter­fer­ence, the au­thors added the sur­fac­tant to cul­tures of the salmon pathogen, V. an­guil­larum. Whereas Cobetia's bio­sur­fac­tant didn't im­pair growth of V. an­guil­larum, it dis­abled its abil­ity to or­ga­nize into biofilms. So, does Co­be­tia mod­ify the path­o­genic be­hav­ior of A. salmoni­cida? When they mea­sured the ac­tiv­ity of quo­rum sens­ing-de­pen­dent vir­u­lence genes (glyc­erophos­pho­lipid cho­les­terol acyl­trans­ferase GCAT and aerolysin), they found that tran­scrip­tion in both loci came to a screech­ing halt when A. salmoni­cida were grown in the pres­ence of pu­ri­fied bio­sur­fac­tant. By cul­tur­ing Co­be­tia and A. salmoni­cida to­gether, Co­be­tia ef­fec­tively sab­o­tages the com­mu­ni­ca­tion sys­tem of A. salmoni­cida, se­cur­ing a com­pet­i­tive ad­van­tage and ren­der­ing A. salmoni­cida non-path­o­genic.

Bio­sur­fac­tant pro­duc­tion in the pres­ence of both abi­otic and bi­otic stim­uli is im­por­tant to both nu­tri­ent as­sim­i­la­tion and neu­tral­iz­ing com­peti­tors. Fatty acid sur­fac­tants act as dif­fusible mes­sen­gers, ca­pa­ble of in­ter­fer­ing with biofilm pro­duc­tion and the ex­pres­sion of vir­u­lence genes. This mod­u­la­tion of quo­rum sens­ing be­tween mi­crobes may be a promis­ing ap­proach to pathogen man­age­ment. By hi­jack­ing chem­i­cal sig­nals be­tween pathogens, it may be pos­si­ble to sup­press vir­u­lence and quell or­ga­nized at­tacks on the host or­gan­ism, side­step­ping many of the dif­fi­cul­ties as­so­ci­ated with an­tibi­otic re­sis­tance. Thus, it helps to know what lan­guage these or­gan­isms speak, in or­der to learn about their cell-to-cell con­ver­sa­tions.

 

Ref­er­ence

Iba­cache-Quiroga C, Ojeda J, Es­pinoza-Ver­gara G, Oliv­ero P, Cuel­lar M, Di­na­marca MA. 2013. The hy­dro­car­bon-de­grad­ing ma­rine bac­terium Co­be­tia sp. strain MM1IDA2H‑1 pro­duces a bio­sur­fac­tant that in­ter­feres with quo­rum sens­ing of fish pathogens by sig­nal hi­jack­ing. Mi­cro­bial Biotech­nol­ogy, 6, 394–405. PMID 23279885

 

Kalia Bis­to­las was a mem­ber of a mi­cro­bi­ol­ogy class taught by Shana Gof­fredi at Oc­ci­den­tal Col­lege where stu­dents write a blog piece pat­terned on Small Things Con­sid­ered. She is cur­rently a grad­u­ate stu­dent at Cor­nell.