Peer Pres­sure In­duces Biofilm Pro­duc­tion

by S. Mar­vin Fried­man

Bac­te­ria are ex­pert cen­sus tak­ers. Us­ing chem­i­cal sig­nals they can rec­og­nize when they are in a crowd, and then they re­spond with ap­pro­pri­ate col­lec­tive be­hav­iors. This cen­sus tak­ing, termed quo­rum sens­ing, ac­ti­vates such di­verse cel­lu­lar processes as bi­o­lu­mi­nes­cence, vir­u­lence fac­tor pro­duc­tion, biofilm for­ma­tion, and sporu­la­tion. Pathogens of­ten con­struct biofilms. The CDC (Cen­ters for Dis­ease Con­trol and Pre­ven­tion) es­ti­mates that about 70% of our bac­te­r­ial In­fec­tions in­volve biofilms, in­clud­ing uri­nary tract in­fec­tions, den­tal caries, catheter in­fec­tions, mid­dle ear in­fec­tions, cys­tic fi­bro­sis, and en­do­cardi­tis. Fur­ther­more, bac­te­ria en­sconced in a biofilm are no­to­ri­ously more re­sis­tant to an­tibi­otics than their free-liv­ing or plank­tonic coun­ter­parts.

Di­a­gram il­lus­trat­ing the de­vel­op­ment of B. sub­tilis into ma­trix-pro­duc­ing can­ni­bals and then spores. At low Spo0A phos­pho­ry­la­tion lev­els, B. sub­tilis ac­ti­vates genes re­quired for the pro­duc­tion of ma­trix and pro­duces two can­ni­bal­ism tox­ins. High phos­pho­ry­la­tion lev­els ini­ti­ate sporu­la­tion. Source

Biofilm for­ma­tion is a com­plex de­vel­op­men­tal process that, it turns out, can be in­flu­enced by chem­i­cal sig­nals pro­duced by nearby cells of other species as well. But first, let me char­ac­ter­ize the play­ers in­volved in biofilm for­ma­tion and the closely linked phe­nom­e­non of sporu­la­tion in Bacil­lus sub­tilis. Sporu­la­tion is an ex­treme mea­sure not to be taken un­less ab­solutely nec­es­sary. The de­ci­sion to take the ten­ta­tive first step into sporu­la­tion is con­trolled by a mas­ter re­sponse reg­u­la­tor called Spo0A. This reg­u­la­tor gov­erns the ex­pres­sion of about 120 genes that in­flu­ence cell com­pe­tence, motil­ity, and an­tibi­otic re­sis­tance, as well as biofilm for­ma­tion and sporu­la­tion. Spo0A it­self is reg­u­lated via phos­pho­ry­la­tion by mem­brane-bound his­ti­dine ki­nases, en­zymes that typ­i­cally act as cel­lu­lar re­cep­tors for sig­nal­ing mol­e­cules in the en­vi­ron­ment. Low lev­els of phos­pho­ry­la­tion of Spo0A cause B. sub­tilis to de­velop into ma­trix-pro­duc­ing (i.e., biofilm form­ing) cells that se­crete two tox­ins: sporu­la­tion-de­lay­ing pro­tein (SDP) and sporu­la­tion killing fac­tor (SKF). They also pro­duce the im­mu­nity pro­teins nec­es­sary for their own sur­vival. The tox­ins kill sus­cep­ti­ble cells of B. sub­tilis and other mi­crobes, thus pro­vid­ing the toxin pro­duc­ers with nu­tri­ents that en­able them to post­pone sporu­la­tion. Such cells have been ap­pro­pri­ately called can­ni­bals, and they are im­por­tant biofilm com­po­nents. When Spo0A phos­pho­ry­la­tion lev­els are high, the genes for ma­trix pro­duc­tion are re­pressed, sporu­la­tion genes are in­duced, and the can­ni­bals progress on to sporu­la­tion.

The mi­cro­colony co­cul­ture screens re­veal in­ter­species in­ter­ac­tions be­tween B. sub­tilis and soil or­gan­isms. Rep­re­sen­ta­tive im­ages of mi­cro­colony co­cul­ture screens of the B. sub­tilis ma­trix re­porter (PtapA-yfp) strain with four dif­fer­ent soils. Bright-field im­ages were over­laid with flu­o­res­cent im­ages false-col­ored green. (Bar = 1 mm.) Flu­o­res­cent B. sub­tilis re­porter colonies (ar­row­heads) are present in each field of view. Pu­ta­tive in­ducer colonies (ar­rows) are the non­flu­o­res­cent colonies in close prox­im­ity to the flu­o­res­cent re­porter colonies. Source

One of the his­ti­dine ki­nases that plays an im­por­tant role in main­tain­ing the can­ni­bal sub­pop­u­la­tion, KinC, is it­self ac­ti­vated by the self-pro­duced sur­factin, an an­timi­cro­bial lipopep­tide. Small mol­e­cules pro­duced by other mi­crobes can also in­flu­ence biofilm for­ma­tion in B. sub­tilis by­ac­ti­vat­ing KinC. For ex­am­ple, nisin, an an­timi­cro­bial syn­the­sized by Lac­to­coc­cus lac­tis, in­duces a hy­per­can­ni­bal­ism phe­no­type in B. sub­tilis. The au­thors of the pa­per I will now dis­cuss de­cided to pur­sue this fur­ther and in­ves­ti­gate if other soil mi­crobes could trig­ger the dif­fer­en­ti­a­tion of B. sub­tilis into ma­trix-pro­duc­ing can­ni­bals.

Sev­eral B. sub­tilis re­porter strains were con­structed for use in these stud­ies in which the ypf (yel­low flu­o­res­cent pro­tein) marker gene was linked to pro­mot­ers spe­cific for ma­trix pro­duc­tion and can­ni­bal­ism. These pro­mot­ers in­cluded those for the tapA operon (codes for one of the ma­trix com­po­nents) and the skf gene (codes for the sporu­la­tion-killing fac­tor). When these spe­cific pro­mot­ers are ac­tive, the yfp gene will be co-tran­scribed and the cells will ex­hibit a yel­low flu­o­res­cence.

The au­thors de­vised a screen to iden­tify other pos­si­ble soil bac­te­ria that, when grow­ing nearby, could trig­ger ma­trix for­ma­tion in B. sub­tilis. Un­der their as­say con­di­tions, B. sub­tilis by it­self does not pro­duce ma­trix. They co-cul­tured the tapA re­porter strain on agar plates with soil sam­ples gath­ered from var­i­ous sites and used flu­o­res­cence mi­croscopy to iden­tify ma­trix-in­duc­ing, non­flu­o­res­cent colonies ad­ja­cent to flu­o­resc­ing B. sub­tilis re­porter colonies. These ma­trix-in­duc­ing clones were then sub­jected to a sec­ond screen, this time em­ploy­ing the can­ni­bal­ism re­porter strain (Pskf-ytp). All colonies that ac­ti­vated ma­trix pro­duc­tion also ac­ti­vated can­ni­bal­ism.

Max­i­mum-like­li­hood phy­lo­ge­netic tree of the or­gan­isms iden­ti­fied as ma­trix in­duc­ers. The num­ber of se­quenced rep­re­sen­ta­tives within each group is in­di­cated on the right. Source

16S rRNA analy­sis showed that the ma­jor­ity of the ma­trix-in­duc­ing iso­lates be­longed to the genus Bacil­lus, the only ex­cep­tion be­ing Pseudomonas mon­teilli. In­ter­est­ingly, none of the 64 iso­lates an­a­lyzed were B. sub­tilis strains. They then tested 14 phy­lo­ge­net­i­cally di­verse Bacil­lus lab­o­ra­tory strains and found that the ma­jor­ity ac­ti­vated the B. sub­tilis ma­trix and can­ni­bal­ism re­porters. In con­trast, only 2 of 13 closely re­lated B. sub­tilis strains did so. Thus, B. sub­tilis pro­duces ma­trix in re­sponse to dif­fer­ent Bacil­lus species more of­ten than it does in re­sponse to B. sub­tilis strains.

Next the au­thors in­ves­ti­gated the role of the sen­sor his­ti­dine ki­nases in ma­trix (Pskf-yfp) in­duc­tion by four species us­ing wild-type and ki­nase-dele­tion B. sub­tilis strains. In­duc­tion by B. mega­terium and B. cereus was re­duced or abol­ished in the ab­sence of kinD, as ex­pected if in­duc­tion was by a ki­nase-de­pen­dent Spo0A mech­a­nism. None of the other his­ti­dine ki­nases were re­quired by any strain. In­duc­tion by B. val­lis­mor­tis and B. at­rophaeus did not re­quire any of the ki­nases.

B. sub­tilis pos­sesses a Spo0A-de­pen­dent im­mu­nity to some Bacil­lus species. Shown are B. mega­terium and B. val­lis­mor­tis colonies over­laid with em­bed­ded agar lawns of ei­ther wild-type (WT) or ΔSpo0A B. sub­tilis. B. mega­terium does not kill ei­ther B. sub­tilis strain; B. val­lis­mor­tis kills the Spo0A dele­tion mu­tant strain but not wild-type B. sub­tilis. Source

The au­thors pos­tu­lated that this ki­nase-in­de­pen­dent in­duc­tion could be due to a de­crease of the non-ma­trix pro­duc­ers and the con­comi­tant in­crease of the ma­trix-pro­duc­ing can­ni­bal sub­pop­u­la­tion. This could come about if the ma­trix-in­duc­ing colonies were elab­o­rat­ing a toxin that was sim­i­lar to B. sub­tilis's own can­ni­bal toxin. Such a toxin would be ef­fec­tive against non-ma­trix pro­duc­ing cells but the can­ni­bal cells would be re­sis­tant. They tested this no­tion by grow­ing colonies of ma­trix-in­duc­ing strains on agar plates and then over­lay­ing with ei­ther wild-type B. sub­tilis or an spo0A dele­tion mu­tant. The pop­u­la­tion of spo0A mu­tants would con­tain no re­sis­tant can­ni­bals. Af­ter growth, the over­lays were ex­am­ined for the ex­tent of killing as mea­sured by the di­am­e­ter of the halo. Some Bacil­lus species, such as B. mega­terium, pro­duced no halo with ei­ther strain, whereas B. val­lis­mor­tis pro­duced a much larger killing zone on the spo0A mu­tant strain than on the wild-type strain. Thus, B. sub­tilis pos­sesses a spo0A-de­pen­dent im­mu­nity to these strains, per­haps via re­sis­tance to a can­ni­bal­is­tic toxin. These re­sults are con­sis­tent with the hy­poth­e­sis pre­sented. How­ever, dif­fer­en­tial in­hi­bi­tion be­tween non-can­ni­bals and can­ni­bals has not been demon­strated, and al­ter­na­tive ex­pla­na­tions can be en­vi­sioned. Thus, val­i­da­tion of the au­thors' pro­posal will re­quire fur­ther study.

Phy­lo­ge­netic analy­sis of the ma­trix in­duc­ing strains found that those that in­duced via an Spo0A-spe­cific, can­ni­bal­is­tic killing fell into a group closely re­lated to B. sub­tilis, whereas those that uti­lized the KinD-de­pen­dent mech­a­nism were more dis­tantly re­lated. Two species, B. cereus and B. licheni­formis, in­duced ma­trix via both mech­a­nisms. It is of in­ter­est here that KinD has re­cently been cat­e­go­rized as a 'check­point' pro­tein that reg­u­lates the pro­gres­sion from ma­trix pro­duc­tion to sporu­la­tion.

Three ap­proaches were used to see if ma­trix in­duc­tion is ef­fected by com­pounds se­creted by the in­duc­ing strains. In two of the ex­per­i­ments, the ma­trix-in­duc­ing strain was grown in agar, then the cells were phys­i­cally re­moved by one of two tech­niques, and the 'con­di­tioned' cell-free agar was over­laid with a lawn of a re­porter strain of B. sub­tilis. Sim­i­larly, the third ex­per­i­ment grew the re­porter strain in a con­di­tioned medium in which a ma­trix-in­duc­ing strain had been grown. In all three cases, flu­o­res­cence was in­duced in the re­porter cells. Al­though the chem­i­cal iden­tity of the ma­trix-in­duc­ing mol­e­cules is still un­known, these re­sults clearly show that ma­trix in­duc­tion is me­di­ated by ex­tra­cel­lu­lar sig­nals.

What are we to make of this? Are can­ni­bal­ism and biofilm for­ma­tion ac­tiv­i­ties of im­port in the real world of soil? If so, who comes out ahead, the biofilm mak­ers that avoid pre­ma­ture sporu­la­tion? But if this is the case, why doesn't "win­ner take all?" And how come there are so many dif­fer­ent kinds of fairly closely re­lated Bacil­lus bac­te­ria in soil? As is true for many phe­nom­ena stud­ied in the lab, the way this all plays out in the real world re­mains to be more fully elu­ci­dated.

 

Ref­er­ence

Shank EA, Klepac-Ceraj V, Col­lado-Tor­res L, Pow­ers GE, Losick R, Kolter R (2011). In­ter­species in­ter­ac­tions that re­sult in Bacil­lus sub­tilis form­ing biofilms are me­di­ated mainly by mem­bers of its own genus. PNAS USA, 108 (48). PMID 22074846

 

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.