frl( thmj.14f Autoinducer Producing Strains from Natural Environments

Transcription

1 nd MBL Microbil Diversity July 1993 Mrgret I. More frl( thmj.14f O ws isolted which produced light when the utoinducer ws provided by E. colt JM82 + pak2o3. from the lst third of exponentil phse to the beginning of sttionry phse. No non-luminescent strin Proteobcteri. These strins produce utoinducer under both erobic nd nerobic conditions. In three of the isoltes the time coure of utoinducer production ws determined. Autoinducer ws produced complementtion of E. colt JM83 contining lux genes of Vibriofisheri with luxi deletion. We isolted s fculttive nerobic strins, three of them most likely Enterobcteri nd one belonging to the bet ble to produce light if utoinducer of V. fischeri is dded. We screened bout 25 strins for induce bioluminiscence in Vibriofisehert nd/or bcteril strins which contin the lux genes nd re The gol of this study ws to isolte bcteril strins which produce utoinducer-like substnces ble to ABSTRACT Mgdlen Mrtinez- Cnmero Autoinducer Producing Strins from Nturl Environments

2 The compound is N(3-oxo hexnoyl) homoserine lctone. It binds to LuxR protein, which then cts s Meighen, E.A., Dunlp, P.V., 1993). Autoinducer in Vibri fished serves to induce luminescence in this bioluminescent bcterium. trnscriptionl ctivtor of the lux genes including its own gene (Dunlp, P.V., Greenberg, E.P.,1991; 2. OysterPond Solid -3, -4, Sipp. Mt 3, 4, Deep Se Nud -3, -4, Grbge Bech -1, -2, Copepod -2, -3, Oyster Pond -3, -4, Sipp. Fluff 3, 4, Sipp. Beggito -5, -6, 7 14.Sipp. Green Puddle -5, -6, Sipp. Purple Puddle -5, -6, -7 5.Mint(leve extrct) -1, 2, -3 7.Termite 2, 3, 4 2. Prk Avenue 3, -4, -5 1O.Tree trunk -1, -2, Mouth 1, 2, 3 8.BellTower -3, -4, Forest soil 3, 4, 5 3. School Street -3, -4, -5 4.Prk sediment -3, -4, -5 1.CedrSwxnp (Volt Pond) -3, -4, -5 Loction Dilutions Dunlp, MEL, MA. The plsmid pak2o3 crries ll lux genes of Vibrio fisher! except for the deletion of lux I (utoinducer producing gene). The plsmid pak2o3 crries the sme genes but insted hs deletion of luxa (luciferse lph subunit gene). Strins from nturl environments were isolted from 1 freshwter nd 1 mxine hbitts. The smples were diluted in sline or sterile se wter s stted in the tble nd plted onto nutrient gr or PLBS (Pul Dunlp) pltes. Pltes from ll environments were incubted both erobiclly nd nerobiclly (selecting for fculttive nerobes or spore formers). About 4 different colony types were picked from ech inoculum grown under erobic nd nerobic conditions, resulting in bout 8 stins per environment. E. coil JM83 contining pak2i1 nd E. coil JM83 contining pak2o3 were obtined from Pul METHODS we hve screened bout 25 isoltes from nturl environments for the production of n utoinducer like L,1992). In Agrobcterluin tunzefciens similr utoinducer molecule functions in the regultion of Ertvini nd relted species utoinducer regultes the production of the ntibiotic crbpenem (Binton et conjugl trnsfer. re not producing light becuse they only hve one or few components of the lux system. In this study This rises the possibility of the existence of lux genes in different non-luminous bcteri, which substnces s well s for the existence of non-induced lux genes. Similr molecules hve lso been found in other non luminescent bcteri. For exmple in fl INTRODUCTION

3 31 Minoru Soil 3 Minoru Soil Strin Isolted by Environment Rernrks_ t_) 348 Vicky Freshwter Pond 33 Pul Soil 34 Pul Soil Myxobcteriuxn 35 Pul Plnt Azorhizobium 37 Mlein Soil Serrti 38 Mlein Soil Azotobcter 31 Mlem Soil Myxobcterium 311 Ev Freshwter Pond Bcillus 315 Ev Mrshpond 317 Ev Mrshpond 318 Ev Mrshpond 319 Ev Mrshpond 32 Vicky Freshwter Pond 321 vicky Freshwter Pond 324 Vicky Freshwter Pond 325 Vicky Freshwter Pond 326 vicky Freshwter Pond 328 Vicky Freshwter Pond 334 Vicky Freshwter Pond 335 vicky Freshwter Pond 336 vicky Freshwter Pond 338 Vicky Freshwter Pond 34 Vicky Freshwter Pond 341 Vicky Freshwter Pond 345 Vicky Freshwter Pond 346 vicky Freshwter Pond 323 vicky Freshwter Pond 333 vicky Freshwter Pond 343 Vicky Freshwter Pond 36 Mri Soil Myxobcteriusn 314 Ev Freshwter Pond 316 Ev Mrshpond 32 Minoru Freshwter 39 Mlem Soil Myxobcterium 312 Ev Freshwter Pond 313 Ev Freshwter Pond POSITIVE 322 Vicky Freshwter Pond 327 Vicky Freshwter Pond 329 Vicky Freshwter Pond 33 Vicky Freshwter Pond 331 Vicky Freshwter Pond 332 vicky Freshwter Pond 337 vicky Freshwter Pond 339 Vicky Freshwter Pond 342 Vicky Freshwter Pond 344 vicky Freshwter Pond 347 vicky Freshwter Pond ( N Other strins were obtined from course members, who isolted them during the course work:

4 352 Din cve N Sndr Nirzwicld-Buer) were used in omer to identify the isolted strins. The probes were pplied s proposed in her hndout. Rhodrnine lbeled nucleotide probes of vrious big phylogenetic groups (kindly provided by pak211 ( luxfl, which will produce light if complemented with utoinducer. The existence of luciferse All strins were screened for production of utoinducer by stmking them next to E. colt.1m83 + genes ws screened by streking the strins next to the utoinducer producing E. colt 3M 83 + pak2o3. Agrobciertum tumefciens strins A6, NT1 nd C58 were obtined from Ann Mtthysse, U. of. North Crolin, NC. 363 Din cve 372 Alin soil 373 Alin soil 4 Minoru sewter 42 Minoru sewter 43 Minoru sewter 49 Minoru sewter 41 Minoru sewter 413 Vicky sewter 412 Vicky sewter 354 Din cve 355 Din cve 358 Din cve 364 Din cve 366 Din cve 368 Din cve 353 Din cve 356 Din cve 357 Din cve 359 Din cve 36 Din cve 361 Din cve 362 Din cve 365 Din cve 367 Din cve 369 Alin soil 37 Alin soil 371 Alin soil 374 Alin soil 375 Alin soil 376 Alin soil Serrti 41 Minoru sewter 44 Minoru sewter 45 Minoru sewter 46 Minoru sewter 47 Minoru sewter 48 Minoru sewter 411 Minoru sewter 414 Vicky sewter 35 Din cve 351 Din cve 349 Din cve Strin isolted by environment remrks_

5 Q To determine if there is utoinducer production, under nerobic conditions, the strins were grown to turbidity in the nerobic hood, immeditely centrifugted nd the supemtnt ws filter sterilized. E. colt A luxi cells were dded to the now sterile superntnt nd incubted under ertion t room temperture. After one hour light induction ws observed in the drkroom. To study the time course of utoinducer production under erobic conditions, filter sterilized culture superntnt ws ssyed for light induction of E. colt Aluxi t vrious times during the growth curves. RESULTS Screening the bove described strins we found 5 non-luminescent strins producing n utoinducer-like substnce, which induces E. colt JM83 + pak211 to light up: 5 Mintplnt I 9 Forest soil ] isolted on nutrient gr (NA), 92 Forest soil I growth lso on PLBS 94 Forest soil I 313 Fresh wter lke (Ev) growth on NA nd PLBS These strins show the following common chrcteristics: grm-negtive fculttive nerobes motile rods no pigments ctlse positive When hybridized with rhodmine lbeled nucleotide probes, the isoltes could be chrcterized further (the signl ws wek but detectble): 5 Mint plnt J bet - Proteobcteri 9 Forest soil ] Enteric bcteri 92 Forest soil 1 fl? 94 Forest soil ] Enteric bcteri 313 Fresh wter lke (Ev) ] Enteric bcteri When filter sterilized superntnt of nerobiclly incubted cells ws ssyed for utoinducer production, luminescence of E. colt AluxI indicted tht ll S strins mke utoinducer nerobiclly. The erobic controls were lso positive. Generlly strin 5 ws observed to cuse induction of less light thn the other strins. It lso hd slower growth rte under both erobic nd nerobic conditions. To find out when utoinducer is produced during growth, cultures were ssyed t different times s described in Mteril nd Methods.

6 I. B:. OD 6 OD 6 S. = I OD 6 - -L -. I, Ct C., Ci, CD C., U C, C, ) ) ) 4 1 C U C C., Ci,

7 the fct tht Agrobcterium tumefciens utoinducer is slightly different compound, which cn not induce light in Vibriofisheri system. indicte tht utoinducer hs function independent from the energy production pthwy. No Our results indicte tht the production of utoinducer is property of severl different strins found in nture. The five isoltes induce light in both erobic nd nerobic conditions. This might DISCUSSION nm/mi. Autoinducer in strins 313, 5 nd 9 is produced in concentrtions greter thn this s they rech the lst third of the exponentil phse. Autoinducer concentrtion drops below the detection limit s the It is not surprising tht we did not find ny strins which would mke light if complemented with utoinducer production could be detected in the Agrobcteriuni tumefciens strins. This might be due to We re wre tht isolting bcteri on rich medi like nutrient gr or PLBS selects for certin strins The detection limit of utoinducer concentrtion for light induction in the E. coil system is 2-5 cultures rech sttionry phse. Evolutionry, it would hve been difficult for these genes to dopt nother function thn light production. Binton et l. (1992) Gene 116, Americn Society for Microbiology, Wshington, DC. Meighen, E.A., Dunlp, P.V. (1993) Advnces in Microbil Physiology 34, LiTERATURE utoinducer, since this would require the existence of ll Vibrio fisheri lux genes except for luxi. Dunlp, P.V., Greenberg, E.P. (1991) Microbil Cell-Cell Interctions, Edited by Mrtin Dworkin, No non-luminescent strin ws detected which produced light when the utoinducer ws provided by E. coil 3M82 + pak2o3. nd does not give representtion of ll bcteri present in one environment