How to Produce NDDBS proteins?

Transcription

1 APPLIED MICROBOLOGY, Nov. 197, p Copyright 197 Americn Society for Microbiology Vol., No. 5 Printed in U.S.A. Growth Responses of Escherichi coli to the Surfctnt Dodecyl Benzene Sulfonte VINCENT A. POLLACK1 AND DEAN A. ANDERSON Deprtment of Microbiology nd Public Helth, Cliforni Stte College, Los Angeles, Los Angeles, Cliforni 93 Received for publiction 7 July 197 When strins of Escherichi coli re grown in broth cultures contining the nionic surfctnt sodium dodecyl benzene sulfonte (NDDBS), they exhibit unique growth responses. After to hr of incubtion, they become slimy nd viscous, nd n ddition of ethnol to the superntnt liquid yields distinctive white, fibrous precipitte. The production of this mteril ws shown to be dependent on the presence of NDDBS in the medium. This precipitte from E. coli ATCC 1133 ws found to contin 1 to 53% protein, 1 to 11% deoxyribonucleic cid, 6.8 to 7.% ribonucleic cid, 15 to 5%o crbohydrte, nd 9% lipid. It is distinctive from nturlly occurring E. coli slimes in severl respects. Our dt suggest tht its formtion is the primry result of the lekge of intrcellulr components into the medium. However, the rte of cell prolifertion indictes prtil but not complete or lethl lysis. A limited utiliztion of NDDBS s crbon source ws lso shown. Anionic surfctnts (detergents) of the lkyl benzene sulfonte type (ABS) were reported by Anderson () to inhibit prtilly the growth of certin grm-negtive bcteri in n ABS-minerl slts gr medium. In ddition, certin sewge isoltes grown in.5% ABS-broth medium developed " considerble mount of slimy mteril" fter 8 hr. Growth inhibition of Escherichi coli by ABS vried with strins. Arkin nd Anderson (Bcteriol Proc., p. 5, 1968) found moderte inhibition of growth of cells, s evidenced by lengthened lg phse nd lowered cell numbers t the sttionry phse, in medi contining the ABS-relted sodium dodecyl benzene sulfonte (NDDBS). Cultures grown in nutrient broth contining.5% NDDBS developed mrked viscosity. Addition of lrge volumes of ethyl lcohol yielded hevy slimy precipitte which included polyscchride component (Molisch rection). This my be tken s generl chrcteristic of E. coli since 63 different strins of this bcterium showed the formtion of similr lcohol-precipitble mteril (APM) when grown with this surfctnt (D. A. Anderson nd I. H. Kornsky, Bcteriol. Proc., p. 16, 1968). Slime production ws stimulted by the NDDBS but not by the unsulfonted dodecyl benzene or the nonsurfctnt sodium benzene sulfonte. I Present ddress: Hylnd Lbortories, Cost Mes, Clif Extending these investigtions, we found tht with E. coli 1133 the APM contined not only polyscchride component but other frctions, indicting tht the production of bcteril slime my represent complex growth response. This pper describes some unique growth responses to NDDBS nd n exmintion of the APM. MATERIALS AND METHODS Bcteri. The bcteril strin used throughout this study ws E. coli B (ATCC 1133) nd ws mintined on nutrient gr (Difco) slnts. The strin grows well on glucose miniml medium nd consistently yields lrge mounts of APM in the presence of surfctnt. Surfctnt. NDDBS ws obtined from Surfct- Co., Inc., Blue Islnd, Ill., under the trde nme "Surco Bio DDBSA." Becuse of its high cidity (ph 1.5), it ws neutrlized before use to ph 7. to 7. with concentrted NOH solution. The surfctnt solution ws then dded to the nutrient broth to give finl concentrtion of.5% by volume nd utoclved. Medi. Nutrient broth (Difco, 8 g/liter) ws used s growth medium for cells incubted with the surfctnt becuse synthetic medi gve low yields of slimy mteril. The bsl slts medium for the sole crbon source experiment contined:. g of MgSO-7H, 1. g of KHPO,.5 g of FeSO -7H,. g of CCl,. g of MnCl-H,.1 g of NMoO,H, nd 1 g of NHC1 per liter of distilled wter. The crbon sources for the experimentl cells were 5. ml 77

2 78 POLLACK AND ANDERSON APPL. MICROBIOL. of NDDBS (.5%, v/v) per liter nd glucose (.1 g/liter) dded to initite growth. The crbon source for the control cells ws glucose (1 g/liter). Three different medi were used to determine the inherent bility of the strin to produce slime or cpsulr mteril. (A) The medium of Akshi et l. (1) contined per liter: 1 g of peptone,.6 g of yest extrct,. g of KHPO,.1 g of MgSO-7H, nd 1 g of glucose. (B) A modified Akshi medium contined per liter: 8 g of peptone,.6 g of yest extrct,. g of KHPO,.1 g of MgS-7H, nd g of glucose. (C) A medium used for the grdient plte technique (3) contined nutrient gr with % either of glucose or sucrose. To detect the production of extrcellulr deoxyribonuclese, nutrient gr pltes with.% deoxyribonucleic cid (DNA; Difco) were used. Nutient gr nd DNA solutions were utoclved seprtely, cooled slowly, nd combined just prior to dispensing to petri pltes. Culturl conditions. Nonsurfctnt liquid medi (1 liter/-liter Erlenmeyer flsk) for the production of slime mteril were inoculted with loopful of -hr nutrient broth culture. Surfctnt broth medi ( liters/-liter Erlenmeyer flsk) used for the production of the APM received 1-ml -hr broth culture inoculum. Grdient pltes were streked with -hr broth culture by using swbs. All cultures were incubted t 5 C. Recovery of APM. Erlenmeyer flsks ( liter), contining liters of sterile surfctnt broth nd mgnetic stirring br, were plced on mgnetic stirring devices nd incubted with constnt stirring. The temperture of the medi ws kept below 35 C by the judicious use of insultion. After 1 to dys of incubtion, -ml portions were centrifuged in Sorvll refrigerted centrifuge t 13, X g for 3 min. The superntnt liquid ws dded to volumes of cold 95% ethnol. The resulting APM ws recovered by inserting Nichrome wire into the mixture while stirring. This procedure effectively removed ll but the very finest strnds of precipitte. The precipitte ws then wshed three times with cold 95% ethnol, dissolved in wter, lyophilized, weighed, nd stored t 5 C. Viscosity mesurements. Mesurements were mde on.-ml portions of APM solutions nd the superntnt liquids of surfctnt broth cultures in n Ostwld viscometer t 3 C before nd fter tretment with het (boiling wter bth for 15 min), deoxyribonuclese ( Ag in.1 ml), or trypsin (1,Ag in.1 ml). The tubes with dded enzymes were incubted t 3 C for 3 min to insure complete hydrolysis. Anlyticl methods. Protein ws determined by the method of Lowry et l. (19) with crystllized bovine lbumin s the stndrd. DNA ws ssyed by the diphenylmine method (6) with Difco DNA s the stndrd. Ribonucleic cid (RNA) ws determined by the orcinol method fter removl of DNA (11). Totl crbohydrte ws determined by the nthrone method (6) by using glucose s the stndrd. Reducing sugr fter hydrolysis (1 N HCI for 6 hr) ws determined by n dpttion of the Somogyi method (1) with glucose s the stndrd. Hexuronic cid ws determined by the crbzole method of Dische (13) with glucuronic cid s the stndrd. Methyl pentrose ws ssyed by the cysteine hydrochloride method (1) by using fucose s the stndrd. Lipids were mesured by solubility in diethyl ether. A smple (.5 g) ws suspended in 5 ml of ether, shken vigorously, nd then centrifuged t 1, X g for min. The superntnt liquid ws sved, nd the pellet ws wshed twice with ml of ether nd discrded. The lipid-ether solution ws evported to dryness nd weighed. The wter content of lyophilized APM ws determined by drying t 15 C to constnt weight ( hr). Bcteri were counted by the stndrd plte count method. Preprtion of cells for nlysis. After 1,, 3, nd dys of incubtion, cultures grown with nd without surfctnt were centrifuged t 13, X g for 3 min in the cold. The pellets were recovered nd disrupted by sonic tretment (Brnson, Instruments, Inc., Stmford, Conn.). The soniclly treted cells were lyophilized, weighed, nd ssyed for protein, crbohydrte, nd DNA. The superntnt liquids from the surfctnt broth cultures were hrvested for the APM, which ws then ssyed in the sme mnner. No APM ws obtined by dding the superntnt liquids of nutrient broth cultures (without surfctnt) to ethnol. Production of cpsulr mteril. An ttempt ws mde to stimulte norml production of cpsulr or slime mteril (without surfctnt) in medi A nd B. After nd 8 hr, the cultures were exmined microscopiclly for encpsulted cells by the wetfilm Indi ink method (15). The cultures were lso centrifuged to remove the cells, nd the superntnt liquids were dded to volumes of 95% ethnol to determine the presence of APM. The grdient pltes were exmined fter, 8, nd 96 hr for smooth or mucoid colonies (). Detection of extrceuulr deoxyribonuclese. The production of n extrcellulr deoxyribonuclese by this strin ws tested by modified Jefferies et l. (17) method. Nutrient gr pltes contining DNA (.%) were inoculted by stbbing with stright inoculting needle. After 8 hr of incubtion, the pltes were flooded with 5% trichlorocetic cid. Cler zones round the colonies indicte deoxyribonuclese production. RESULTS Utiliztion of the surfctnt. E. coli 1133 ws cpble of cell prolifertion in medium which contined NDDBS s the sole source of crbon (Fig. 1). Cells grown in NDDBS significntly showed longer lg phse thn tht of the control, s evidenced by the vible cell counts ( hr) nd opticl density (OD) redings ( hr). The sttionry phse ws reched in the glucose medium fter 1 hr, wheres tht of the NDDBS cultures ws bout 18 hr. The frequency nd number of cell divisions for the cells in NDDBS ws lower thn the control.

3 VOL., 197 GROWTH RESPONSES OF E.COL79 6.6, /.3 > TIME (hr) FIG. 1. Growth of E. coil 1133 with surfctnt s the sole source of crbon s evidenced by the vible cell count for the culture grown with surfctnt () nd the control culture with glucose (@) nd by the OD t 5 nm for the culture with surfctnt (X) nd with glucose (). The mximum vible cell count ws 3 X 16 cells/ml compred to 17 cells/ml for the control. Comprble cell numbers nd OD redings for both groups, however, indicte growth nd utiliztion of NDDBS s the sole crbon source. The ddition of the superntnt liquids from either cultures (fter hr) to volumes of 95% ethnol did not yield precipitte. Growth of cells in surfctnt broth. The bility of E. coli 1133 to grow in complex medium (nutrient broth) with.5% surfctnt ws tested. A surfctnt concentrtion of.5% (v/v) ws used exclusively becuse lower concentrtion, i.e..1%, did not consistently yield pprecible APM. The growth curves were similr lthough growth of the experimentl cells lgged behind tht of the control. The cell popultion in NDDBS t no time equlled tht of the control, indicting prtil inhibition of cell division by the surfctnt. The turbidity mesurements showed mrked difference between the control nd the experimentl cells. After 8 hr of incubtion, the OD of the NDDBS cultures reched plteu with shrp decline between 1 nd 16 hr, wheres the control showed stedy increse. This sudden decline in OD with no similr loss for the control cells nd the vible cell counts for both groups suggests loss of intrcellulr mteril from the cells in NDDBS (Fig. ). Wet-film Indi ink mounts consistently filed to show encpsulted cells in surfctnt broth cultures, regrdless of the incubtion period. After hr, however, mny cells ppered to be embedded in lrge msses of slimy substnce which ws not penetrted by the ink prticles. The ddition of the superntnt liquid or the whole culture of cells grown without surfctnt to volumes of 95% ethnol did not yield precipitte. The ddition of the superntnt liquids of surfctnt broth cultures to lcohol yielded stringy white precipitte. Composition of the APM. Chemicl nlyses of lyophilized smples of APM showed tht its composition of protein, crbohydrte, nd DNA vried over n incubtion period of 96 hr (Fig. 3). A number of nlyses of smples (Tble 1) from 8-hr cultures in surfctnt showed men composition of 5.8% protein, 1.6% DNA, 7.1% RNA, 17.9% crbohydrte, nd 9.% ether-soluble mteril. No seprtion or purifiction of the components ws mde prior to nlysis. Consequently, ssys for hexuronic cid by the crbzole method were inconclusive, nd the vlue obtined for methyl pentose ssyed by the cysteine hydrochloride method my be suspect. Viscosity mesurements. Solutions of APM showed significnt decrese (Tble ) in viscosity fter het nd deoxyribonuclese tretment, s did the control solutions of DNA. tretment hd only slight effect. Het nd deoxyribonuclese tretment effected significnt decrese in the viscosity of the cell-free superntnt liquids of NDDBS cultures (Tble 3). produced little effect. This indicted tht the viscosities of the APM solution nd the superntnt liquids of surfctnt broth cultures cn be ttributed primrily to DNA. Anlysis of cells nd APM. Cells were removed by centrifugtion from 1- to -dy nutrient broth cultures with nd without surfctnt LLi m m 17 u 'I-, -1 w w c TIME (hr) FIG.. Growth of E. coli 1133 in nutrient broth in the presence nd bsence of the surfctnt NDDBS s evidenced by the vible cell count for the culture with surfctnt (U) nd the control culture (-) nd by the OD t 5 nm for the culture with surfctnt (X) nd the control (). i

4 73 u 5 CZ) In POLLACK AND ANDERSON APPL. MICROBIOL. -1 E tn 15 E 1 z z 5 C. I. I- x 1 3 TIME (dys) FIG. 3. Totl proteinl (U), crbohydrte (), DNA (X), nd APM () from cultures growni in surfctnt for dys; conztrol cu/lturies did not yield APM. TABLE 1. Chemicl compositiol of the ext rcellulr A PM from E. coli grow/i in NDDBS Component x x,1-==-----o.s Per cent composition Rnge Menb Protein (6) DNA (5) RNA (3) Totl crbohydrte (5) Reducing sugr () Methyl pentose (1) Lipid (1) Wter (1) APM, lcohol-precipitble mteril; NDDBS, sodium dodecyl benzene sulfonte. Anlyses were mde on APM smples extrcted from the superntnt liquids of E. coli cultures grown in surfctnt for 8 hr. b Numbers in prentheses indicte the number of nlyses mde for ech component. TABLE. Effect of het, deoxyribonuclese, ntd trypsin tretmelits oni the viscosities of solutioils of APM Smple C(mg/m) Tretment Wter control Het DNA.5 Het DNA 1. Het APM 8.5 Het V'iscometer flow time (min) nd nlyzed for protein, crbohydrte, DNA, nd APM (Tble ). In -liter cultures, the totl protein in the surfctnt-grown cells, plus tht in the APM, pproximted tht in the control cells. This pprent conservtion of protein persisted over ll dys, suggesting tht the protein lost from the experimentl cells ws found in the APM. In terms of totl crbohydrte nd DNA, the APM contined greter mounts of these thn the combined contents of experimentl nd control cells. The observtion of greter quntities of crbohydrte nd DNA in the APM thn in both groups of cells suggests either net synthesis of these components in response to the surfctnt or the inbility of the experimentl cells to reutilize the crbo- Before After tret- tretment ment Het tretmnent: smiiiple solutions immersed in boiling wter bth for 15 miin nd then cooled to roomii temperture. tretment: 1 gg in.1 il dded to smple solution, nd then incubted for 3 min t roomii temperture. trettment: 1 eg of trypsin in.1 ml dded to smple solution, nd then incubted for 3 mmiin t roomii temperture. TABLE 3. Effect of het, deoxyriboiiuclese, nd trypsiui tretments on the viscosities of supernitnzt liquids of cultures inzcubted in the bsenice (conitrol) uid preseiice (experimenztl) Qf NDDBS' Smple Wter control Control superntnt liquid Experimentl superntnt liquid Tretment Viscometer flow time (min) After tret- ment Before tretment Het I Het Het Deoxyribonluclese For conditions of tretments, see footnote, Tble. NDDBS, sodium dodecyl benzene sulfonte.

5 VOL., 197 GROWTH RESPONSES OF E. COLI 731 TABLE. Totl proteini, crbohydrte, nid DNA of cells nd APM (experimentl) grown in the presence of NDDBS nd cells (conztrol) the bsence of NDDBS Component Protein Crbohydrte DNA Incu- 1,-+,-rv period (dys) Cells Experimentl APM Totl Control cells Grown in liters of broth culture. APM, lcohol-precipitble mteril; NDDBS, sodium dodecyl benzene sulfonte. hydrte nd DNA relesed into the medium by the ction of the surfctnt. Production of n extrcellulr deoxyribonuclese. The bility of this strin to produce n extrcellulr deoxyribonuclese ws tested to determine its potentil for the reutiliztion of DNA lost from the cells in surfctnt broth. In severl experiments, no deoxyribonuclese formtion ws found. Production of cpsulr or slime mteril. E. coli 1133 is nonmucoid t 5 nd 37 C on nutrient gr. Exmintion of cells in medi A nd B by the wet-film Indi ink method filed to show encpsulted cells fter nd 8 hr (Tble 5). The superntnt liquids of these cultures did not yield APM. The grdient pltes contining glucose or sucrose did not show smooth or mucoid colonies fter hr of incubtion; good growth ws noted. However, 11 smooth colonies were found fter 8 nd 96 hr on pltes contining glucose. These were locted t the highest concentrtion of sugr (%) on the pltes, indicting tht minute portion of the popultion is cpble of the production of cpsulr or slime mteril under suitble conditions on solid, but not in liquid, medi. DISCUSSION This investigtion indictes tht E. coli 1133 is cpble of utiliztion of the surfctnt NDDBS s the sole source of crbon. If, s hs been suggested (5, 7, 9), the lkyl portion of the NDDBS molecule is broken down by the enzymes responsible for the hydrolysis of ftty cids (,B-oxidtion), our results re in greement with those workers who hve found tht the degrdtive system for ftty cids is functionl nd inducible in E. coli (, 31). E. coli is cpble of growing in complex medium, such s nutrient broth, in the presence of.5% NDDBS nd the growth (cell division) ws shown to be prtilly inhibited by the surfctnt. A loss of OD for the cells in the surfctnt medium ws noted fter 1 to 15 hr, without concomitnt drop in the vible cell count. If OD redings my be ssumed to be roughly proportionl to the mounts of intrcellulr mteril, this decline suggests loss of intrcellulr constituents. The known disruptive effect of nionic surfctnts on the osmotic function of bcteril cells () tends to support our view tht one of the effects of NDDBS in this system is to cuse the lekge of intrcellulr components into the medium (1). The ppernce of APM ws found to occur to hr fter inocultion into nutrient broth contining.5%76 NDDBS. Microscopic exmintion of the cultures filed to show encpsulted cells. The APM my thus be more ppropritely termed bcteril slime, since mny TABLE 5. Productiont of cpsulr or slime mteril by E. coli strinl 1133 Incubtion Grdient pltes period (hr) Medium A AMedium B Glucose Sucrose No cpsules No cpsules Rough colonies Rough colonies No APMb No APM 8 No cpsules No cpsules Smooth colonies Rough colonies No APM No APM 96 I A few mucoid Rough colonies colonies Broth culture exmined for encpsulted cells by wet-film Indi ink method. bsuperntnt liquid from culture dded to volumes of 95'% ethnol nd noted for ppernce of lcohol-precipitble mteril (APM).

6 73 POLLACK AND ANDERSON APPL. MICROBIOL. cells were seen to be embedded in lrge msses of this mteril. The production of surfctntstimulted slime hs been reported (37; V. Arkin nd D. A. Anderson, Bcteriol Proc., p. 5, 1968) in different strin of E. coli lthough the mteril ws not chrcterized chemiclly. The evidence presented herein, however, seems to indicte tht the APM from surfctnt broth cultures my not be clssified s "typicl" E. coli slime for the following resons. (i) The extrcellulr APM contins reltively high nucleic cid content (1. to 11.3% DNA, 6.8 to 7.% RNA). Such high levels of nucleic cid hve not been reported in detiled nlyses of the slime or cpsulr mteril of E. coli (1, 3,, 18, 5). (ii) The viscosity of liquid cultures nd queous solutions of slime nd cpsulr mteril of E. coli is due to the polyscchride component (1, 5). The viscosity of surfctnt broth cultures nd queous solutions of APM is ttributed here to the DNA component. (iii) The viscosity of solutions of APM is gretly decresed by het tretment, wheres the polyscchride of E. coli (18) is unffected by tretment in the sme mnner. (iv) This strin (ATCC 1133) does not form slime in liquid medi designed for its production. In spite of the rguments presented bove, the possibility of the production of highmoleculr-weight polyscchride in response to NDDBS cnnot be disregrded, since significnt polyscchride component (15.9 to 5.%) ws detected. Chemicl nlysis demonstrted the presence of smll quntity (.6%) of methyl pentose. The presence of methyl pentose (fucose) in the extrcellulr polyscchride mteril of bcterium ws first shown by Norris et l. (3) with Lctobcillus bifidus nd hs been tken s n indictor of slime mteril by Wilkinson et l. (3) nd Kng nd Mrkovitz (18). Further, nucleic cids hve been found s extrcellulr mteril in some bcteri. Certin hlophilic orgnisms (Microcossus hlodenitrificns, Vibrio costicolus, nd Pseudomons slinri) produce viscous extrcellulr mteril, consisting predomintely of DNA, when incubted in suboptiml concentrtions of slt (8). Cmpbell et l. (7) observed DNA content of 8% in cultures of Micrococcus sodonensis. Ctlin nd Cunninghm (1) noted the ccumultion of extrcellulr DNA by vrious nonhlophilic bcteri, including Micrococcus, Stphylococcus, nd P. eruginos. The sme results hve been reported (9) for P. fluorescens, Alcligenes feclis, S. ureus, nd S. epidermidis. The extrcellulr ccumultion of DNA ws demonstrted under conditions which were designed to minimize extrcellulr deoxyribonuclese ctivity (19). Since this strin of E. coli filed to produce n extrcellulr deoxyribonuclese, the ccumultion of DNA shown in the prllel nlyses of cells nd AMP from surfctnt broth cultures my be explined. Egon (16) found tht the extrcellulr slime of P. eruginos strin 6 contined mnnose (1 to 6%), DNA (1 to 1%), RNA (3 to 1%), nd protein ( to 3%). Crson nd Egon (8) found tht the extrcellulr mteril from the sme bcteril strin contined DNA which rose spontneously from n intrcellulr origin owing to cellulr disintegrtion. They concluded tht the production of DNA slime nd polyscchride ( mnnn) were two individul phenomen. We suggest tht the extrcellulr APM from E. coli cultures incubted with surfctnt is likewise consequence of two seprte phenomen: portions of this mteril result from the lekge of intrcellulr components (DNA, RNA, nd protein) due to the ction of the surfctnt, nd the high-moleculr-weight polyscchride my be synthetic result of the brekdown of portions of the surfctnt molecule. ACKNOWLEDGMENT This investigtion ws supported by Federl Wter Pollution Control Administrtion grnt no. WP LITERATURE CITED 1. Akshi, S., H. Goto, M. Sski, A. Tsuym, Y. Si, nd T. Kuno Chemicl, immunochemicl nd electronmicroscopicl studies on the polyscchride produced by mucous strin of E. coli. Bull. Soc. Chem. Biol. 7: Anderson, D. A Growth responses of certin bcteri to ABS nd other surfctnts. Air Wter Pollut. Int. J. 1: Anderson, E. S., nd A. H. Rogers Slime polys.cchrides of the Enterobctericee. Nture (London) 198: Beiser, S. M., nd B. D. Dvis Mucoid mutnts of Escherichi coli. J. Bcteriol. 7: Bogn, R. H., nd C. N. Swyer Biochemicl degrdtion of synthetic detergents. I. Studies on the reltion between chemicl structure nd biochemicl oxidtion. Sewge Ind. Wstes 8: Burton, K. A A study of the conditions nd mechnism of the diphenylmine rection for the colorimetric estimtion of deoxyribonucleic cid. Biochem. J. 6: Cmpbell, J. N., J. B. Evns, J. J. Perry, nd C. F. Niven, Jr An extrcellulr mteril elborted by Micrococcus sodonensis. J. Bcteriol. 8: Crson, K. J., nd R. G. Egon Cellulr disintegrtion with concomitnt relese of slime nd production of extrcellulr mnnn by Pseudomons eruginos: two seprte phenomen. Cn. J. Microbiol. 1: Ctlin, B. W Extrcellulr deoxyribonucleic cid of bcteri nd deoxyribonuclese inhibitor. Science 1: Ctlin, B. W., nd L. S. Cunninghm Studies of extrcellulr bcteril deoxyribonucleic cids. J. Gen. Microbiol. 19:5-539.

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