Mutation in Shigella flexneri Resulting in Loss of Ability to

Transcription

1 INFECTION AND IMMUNITY, Mv 1974; p Copyright 1974 Americn Society for Microbiology Vol. 9, No. 5 Printed in U.S.A. Muttion in Shigell flexneri Resulting in Loss of Ability to Penetrte HeL Cells nd Loss of Glycerol Kinse Activity ROSALIND KIM AND LAURENCE M. CORWIN Deprtment of Microbiology. Boston University School of Medicine, Boston, Msschusetts Received for publiction 24 Jnury 1974 A colonil vrint of virulent Shigell flexneri 2 hs lost both virulence nd glycerol kinse ctivity. It lso hs severl other ltered chrcteristics: lowered bility to oxidize tricrboxylic cid cycle intermedites, incresed electrophoretic mobility, nd decresed sensitivity to sodium luryl sulfte. Genetic nlysis hs reveled tht the gene governing glycerol kinse ctivity in Shigell hs different chromosoml locus thn tht from Escherichi coli. Furthermore, trnsduction of the Shigell glycerol kinse gene (glp K) into the virulent Shigell strin cn restore the bility to penetrte HeL cells, wheres the gene from E. coli cnnot. About hlf of the glp K mutnts lose this bility, nd only bout hlf of the revertnts of n virulent glp K mutnt regin it. This indictes tht more thn one gene ffects glycerol kinse ctivity in Shigell, only one of which is ssocited with penetrtion. Glycerol kinse ctivity is closely correlted with chnges in electrophoretic mobility, but does not pper to hve ny reltionship to sodium luryl sulfte sensitivity nor to the oxidtion of tricrboxylic cid cycle intermedites. A clssicl method for studying virulence of bcteri is to consider other ltertions ssocited with the muttion from virulence to virulence nd trying to relte them cuslly to the loss of virulence. We hve studied n virulent mutnt of Shigell flexneri 2. This mutnt strin (24570) ws obtined s spontneous opque colonil vrint of the virulent strin (M-42-43) which grows s trnslucent colonies on met extrct gr (9). Opque colonies rise t the reltively frequent rte of one in 101 cells from the virulent strin. This prticulr opque strin hs severl pleiotrophic ltertions ssocited with the muttion. The strin hs lost virulence by virtue of its inbility to penetrte the intestinl epithelil cells (11). In model systems, it is lso unble to cuse kertoconjunctivitis in the guine pig eye (19) nd is unble to penetrte the HeL epithelil cell line (17). In ddition, the virulent strin hs become reltively resistnt to sodium luryl sulfte (SLS) (5), hs three- to fourfold increse in nodic electrophoretic mobility (EM) (6), nd hs n impired bility to utilize certin tricrboxylic cid cycle intermedites (15). Although it is unlikely tht these pleiotropic effects re due to multiple point muttions, it is conceivble tht they could be due to deletion of severl djcent genes. Testing such possibilities by genetic nlyses is difficult due to n inbility to select for strins resistnt to SLS or with n incresed nodic EM. We were fortunte, therefore, to be ble to tke dvntge of the observtion of E. H. LBrec (personl communiction) tht the virulent strin does not ferment glycerol. With such hndle for selection, it ws then possible to directly test by reversion nlyses nd other correltive techniques the reltionship between the pleiotropic chnges nd the bility of orgnisms to penetrte epithelil cells. This pper presents dt to show tht the mutted gene ffecting glycerol utiliztion in the virulent strin reltes to glycerol kinse nd is probbly different gene from the one governing glycerol kinse synthesis in Escherichi coli. It is lso in loction fr distnt from the kcp A muttion which lso governs the bility to penetrte the epithelil cells of the eye nd produce kertoconjunctivitis (10). It will be shown tht loss of glycerol kinse ctivity nd chnges in EM re relted, but no observble correltion of glycerol kinse ctivity with SLS sensitivity or succinte utiliztion ws detected. There is, however, some correltion between glycerol kinse ctivity nd the bility to penetrte HeL cells. The-possibility tht the lck of compete correltion with penetrtion my be relted to polygenic control of glycerol kinse ctivity will be discussed. 916

2 VOL. 9, 1974 MUTATION IN S. FLEXNERI 917 MATERIALS AND METHODS Bcteril strins. The strins used in this study re chrcterized in Tble 1. S. flexneri 2 strin is colonil vrint which rose spontneously from virulent strin M Both strins were kindly provided by S. Forml. The glycerol-positive strins re spontneous revertnts of strin These rose t rte of one in 104 cells. The M glycerol-negtive mutnts were obtined by using mutgenic procedure which will be described subsequently. The E. coli K-12 strin W1895 is n HfrC (met-). Strin 27p, n E. coli K-12 strin with promotor muttion in the glycerol kinse gene, ws kindly provided by D. Richey. Strin 76p is n E. coli K-12 strin which lcks glycerol kinse. Growth of cells nd preprtion of extrcts. The miniml medium used for growth contined, per liter of distilled wter: K2HPO4 (10.5 g), KH,PO4 (4.5 g), MgCl2 (0.05 g), nd (NH4)2SO4 (1 g). Crbon sources were dded to give finl concentrtion of 0.02 M glycerol nd 0.04 M DL--glycerophosphte (DL-- GP). Csein cid hydrolyste (Difco, vitmin-free) ws dded to miniml medium to give finl concentrtion of 1%. Agr pltes hd, in ddition, 15 g of gr per liter. Luri broth supplemented with 0.2% glucose nd 5 mm CC12 (5) ws used s the growth medium for the bcteri when the electrophoretic mobility or SLS sensitivity ws tested. Nutrient broth (Difco) ws used s the growth medium for studying biologicl oxidtion. Growth ws mesured in Busch & Lomb Spectronic 20 spectrophotometer t wvelength of 625 nm. The cultures were incubted t 37 C on rotry shker operted t 200 rpm. The cells were hrvested in the logrithmic phse, pelleted t 0 C, wshed with cold 1% NCl solution, nd resuspended in 0.1 M tris(hydroxymethyl)minomethne-hydrochloride TABLE 1. Orgnism Genotype of strins used Relevnt mrkers rh glpk Shigell flexneri 25 M _ _ - M rhl + + Hfr 256C _ + glp K revertnts (10)d _ + M glpk mutnts (10)d _ Escherichi coli K-12 W DR 1 27p p + Abbrevitions: rh, rhmnose; glpk, glycerol kinse; +, synthesized or utilized; -, not synthesized or utilized. " All of the S. flexneri strins required nicotinic nd sprtic cid t 5 nd 20 jg/ml, respectively. cderivtive of conjugtion between E. coli Hfr, P4x6, nd M (22). d ( ), Number of mutnts. buffer, ph 7.4. The suspended cells were disrupted by four tretments in 50-W sonictor (Het Systems Ultrsonics, Inc., model W185) for totl of 80 s t 0 C. The resulting suspension ws centrifuged t 30,000 x g for 20 min t 0 C, nd the superntnt cell-free extrct ws used for enzyme ssys. Glycerol kinse ssy. Glycerol kinse ctivity ws ssyed by mesuring the formtion of ['4C IL-- GP from ["C ]glycerol s described by Bermn nd Lin (2). Protein concentrtions were determined ccording to the method of Lowry et l. (18). Interrupted mting. Crosses between donor S. flexneri strin M-256, n Hfr strin formed by hving received the F-lc portion of E. coli K-12 strin P4x-6, nd S. flexneri 2 strin were performed s described previously (22). Selections were mde on miniml glycerol or miniml rhmnose pltes supplemented with 5,g of sprtic cid per ml. Trnsduction experiments. The conditions used for performing trnsductions hve been reported in nother pper (5). Microelectrophoresis. EM ws mesured by the method of Corwin nd Tlevi (6). Lysis by SLS. Lysis studies were performed s described by Corwin et l. (5). Oxygen Uptke. Oxidtion mesurements were crried out essentilly s described by Umbriet et l. (24) by using the conventionl Wrburg technique. The cells were grown in nutrient broth to sttionry phse. HeL cell ssy. The HeL cell monolyers were mintined in milk dilution bottles in medium consisting of miniml essentil medium (8), with Hnks blnced slt solution (13), supplemented with 10% fetl clf serum, 20,gmol of glutmine per ml, 100 U of penicillin G per ml, nd 100 ug of streptomycin per ml. The cover-slip culture technique ws used to prepre monolyers for penetrtion experiments (17). Mutngenesis nd penicillin selection. M cells were grown in 10 ml of Luri broth to log phse (5 x 10' to 8 x 101 cells per ml). The cells were wshed twice in sline nd resuspended in 0.5 ml of 0.1 M citrte buffer (ph 5.6). To the suspension 0.1 ml of N-methyl-N'-nitro-N'-nitrosogunidine (1 mg/ml) mde freshly ws dded (1). The mixture ws incubted for 20 min t 37 C. The celis were then dded to 4.5 ml of Luri broth nd grown for 3 h to llow the muttion to be expressed. A 5-ml mount of cells ws wshed twice nd grown in 200 ml of miniml glucose for six genertions to exclude uxotrophs. Penicillin selections were done three times in miniml glycerol to enrich for gip K- cells by using finl concentrtion of 2,000 U of penicillin per ml nd llowing the cells to incubte with the penicillin t 37 C for 90 min (12). The cells were then plted on miniml DL--GP pltes, nd the colonies tht rose were tested for their bility to grow on miniml glycerol. Glycerol-positive revertnts of strin (glp K-) were isolted by plting exponentil cells on miniml glycerol pltes. The colonies were restreked on miniml glycerol pltes for purifiction. RESULTS Growth chrcteristics of S. flexneri. Viru-

3 918 KIM AND CORWIN INFECT. IMMUNITY lent S. flexneri strin M grows either on glycerol or L-c-GP s crbon source, wheres its virulent mutnt, strin 24570, cn onlv utilize L--GP. Strin 24570, fter long lg phse, grew lmost s well s its virulent prent (Tble 2). These growth chrcteristics suggested tht muttion hd occurred in the glycerol kinse gene (glp K) which codes for the first enzyme in the pthwy required for the dissimiltion of glycerol (16). Glycerol kinse ctivity of S. flexneri nd E. coli. The ctivity of glycerol kinse ws mesured in strins M-42-4,3 nd As expected, strin showed no ctivity whether induced with glycerol or L--GP (Tble 3). Strin M possessed glycerol kinse ctivity, but this ws found to be lower (between 43 nd 47'% ) thn tht found in wild-type E. coli strin W1485. Km nd Vmx. To determine whether the lower ctivity of the Shigell enzyme ws due to different enzyme thn tht of' E. coli or to lowered quntity of' the sme enzyme, or both, the Km nd Vmx of glycerol kinse of extrcts of these two strins were mesured. The Km of glycerol kinse of' the Shigell strin ws 2 / 1-' M, wheres tht of the E. coli strin ws 1.5 x 10-' M (Tble 4). These differences were not considered to be significnt. The Vmx in these two strins, on the other hnd, differed considerbly. Since these dt were obtined from crude extrcts, the difference in Vmx could hve been due to either lower levels of enzyme or different enzyme. It hs been observed tht the Shigell glycerol kinse formed zone of precipittion with specific ntiserum prepred ginst the crystlline E. coli glycerol kinse (21). If the two enzymes re indeed the sme or very similr, then it seems likely tht E. coli produces lmost twice s much glycerol kinse s Shigell. Mpping. Due to the close genetic homology between Shigell nd E. coli (3), we determined TABLE 2. Growth medium Genertion times of S. flexneri strins M nd Genertion time (min)" M Miniml glycerol 72 NGc Miniml L--GP Growth medi contined 0.05% csein hydrolyste plus 0.02 M glycerol or 0.04 M L--GP. b Slope of the stright line portion of the semilogrithmic curve of opticl density t 625 nm versus time. c NG, No growth. TABLE 3. Glycerol kinse ctivity of S. flexneri nd E. coli strins Strin Inducer Sp ct glycerol kinse S. flexneri M None 13 Glycerol 68 L--GP None 0 Glycerol 0 L--GP 0 E. coli W 1485 None 10 Glycerol 129 L--GP 90 Cells were grown for 6 to 10 genertions s described in Mterils nd Methods in 1% csein hydrolyste ± inducer (0.02 M glycerol or 0.04 M L--GP). Specific ctivity is expressed s nnomoles of L--GP formed per minute per milligrm of protein. TABLE 4. Comprison of Vmx nd Km vlues of glycerol kinse from S. flexneri nd E. coli Strin Crbon Km v source (M I to 5) S. flexneri 2 M Glycerol E. coli K-12 W1485 Glycerol All mesurements were mde by Bermn nd Lin's method (2). 'Expressed s nnomoles per minute per milligrm of protein. whether the Shigell glp K gene mpped t the sme chromosoml site s in E. coli. Since rhmnose (rh) mps next to glp K in E. coli t 76 min of the chromosome (7), rhmnose utiliztion ws used s cotrnsducible mrker. Although S. flexneri 2 is rh negtive, it cn be reverted by mutgens. The rh locus in Shigell is locted t the sme genetic position s in E. coli (S. Flkow, personl communiction). Phge P,vir ws used to trnsduce the glp K nd rh genes from virulent Shigell strin M (rh+ glp Kt ) into virulent strin (rh- glp K-). Selection ws mde for glp K+ or rh+ trnsductnts. Of 300 glp K+ trnsductnts, ll were found to hve remined rh- (Tble 5). When rh ws the selected mrker, ll of' the trnsductnts remined glp K-. This indicted tht rh nd glp K were not cotrnsducible in Shigell. The trnsduction between wild-type E. coli K-12 strin W1485 (rh+ glp K+) nd S. flexneri strin (rhglp K-) ws used to verify tht rh nd glp K were indeed cotrnsducible from E. coli (Tble 5). Further evidence indicting tht rh nd glp K in Shigell were not cotrnsducible ws the

4 VOL. 9, MUTATION IN S. FLEXNERI 919 TABLE 5. Trnsduction of the glpk mrker Inheritnce of unselected Donor Recipient Selected mrker scored mrkers M (rh+) glpk M (rh+) rh E. coli K glpk M (rh-) E. coli (glpk-) glpk 20 0 Except s otherwise indicted, the genotypes of the strins re s follows: S. flexneri 2 strin M is glpk+ nd strin is glpk-. The rh+ M is revertnt of M which is normlly rh-. E. coli glpk- ws obtined from D. Richey. trnsduction by P,vir grown on Shigell strin M (rh- glp K+) into E. coli strin DR 1 (rhl glp K-). All 20 glp K+ trnsductnts remined rh+ (Tble 5). Conjugtion studies were then used to get n pproximte loction of the glp K locus in Shigell. The mle donor used ws S. flexneri strin 256 (glp K+), n Hfr strin formed by hving received the F-lc portion of E. coli K-12 strin P4x-6. Hfr (22). As fr s could be determined by genetic nlyses for other selected mrkers, Hfr 256 retined only the F-lc region of the E. coli donor genome nd otherwise exhibited the chrcteristics of typicl S. flexneri 2 strin. Furthermore, crosses with E. coli recipients showed mting polrity similr to tht of the P4x-6 strin from which it ws derived, i.e., pro-thr-rg-his-gl-lc-f (22). In our hnds, the thr mrker could be trnsferred in 8 to 11 min, or bout 3 to 6 min erlier thn the results obtined by Schneider nd Flkow (22), who studied cross between Hfr 256 nd n E. coli recipient strin. Our mting used thr, rg, his multiuxotrophic derivtive of S. flexneri 2 s recipient for Hfr 256. We lso observed shllow initil slope, followed by shrp upwrd inflection. The ccurcy of extrpolting this shllow slope to the bsciss ws limited, nd the time of entry vlues re the rnge of three experiments selecting for threonine. The results of n interrupted mting study using Hfr 256 nd glp K re shown in Fig. 1. The glp K+ locus ppers to be trnsferred t bout 40 to 45 min from the origin. This plces the Shigell glp K t pproximtely 55 to 60 min (Fig. 2) on the Tylor mp (23). Although this experiment ws repeted four times with essentilly the sme result, it is pprent tht the sctter ws significnt t low recombinnt levels. This ws probbly due to the reltively high rte of reversion of to glp K+, which mde the controls nd experimentl vlues vrible. Therefore, these dt re presented, not to indicte the precise loction of the E 0 z 4 z 0 cr y 200H 1oo0 0 t glpk -O TIME (MIN) FIG. 1. Mrker trnsfer in cross between Hfr Shigell flexneri M-256 nd F- S. flexneri Selection for glp K+ recombinnts ws mde fter pproprite dilutions. S. flexneri strin M-256 is glp K+, nd strin is glp K-. Shigell glp K, but rther to support the trnsduction dt indicting tht the site of the Shigell glp K is not identicl with E. coli t 76 min on the mp nd is fr distnt from the kcp A locus close to pur E (11). Other experiments to define more precisely the Shigell glp K site hve been unsuccessful. Cotrnsduction of glp K with thymine or mltose mrkers, which re in the vicinity of glp K on the chromosome, hs not been found. Selection of the mltose mrker ws bsed on the work of Forml et l. (10), showing tht virulence could be restored to n virulent S. flexneri 5 mutnt by conjugl trnsfer of genes ssocited with the ml region. If the Shigell glp K is ner ml, it nevertheless is not cotrnsduced with it. Reltion of glp K to pleiotropic ltertions of To scertin whether the presence or bsence of the Shigell glp K gene is relted geneticlly to the other ltertions ttributble rh

5 920 KIM AND CORWIN INFECT. IMMUNITY gip A' 'Nglp T FIG. 2. Prtil mp of Escherichi coli. Pertinent mrkers on the genetic mp of E. coli re ccording to Tylor (25). to the muttion from M to 24570, dditionl glp K mutnts were obtined from the virulent strin. In ddition, number of glp K+ revertnts were isolted from s well s from one of the new glp K mutnts of M which proved virulent. These revertnts rose spontneously t the rte of one in 105 cells. These strins were tested to see whether their electrophoretic mobility, biologicl oxidtion, nd virulence differed from those of the prent strins. If the muttion from M to is simple point muttion, then it should be ble to revert, nd the ltered chrcteristics due to the muttion should revert bck to the originl stte. If the muttion is deletion of severl genes, no true reversion should tke plce, lthough suppressor muttions might ffect one or more of the phenotypic ltertions. The possibility of simultneous muttions, lthough usully unlikely, must be considered in S. flexneri 2. To those working with this orgnism, the discovery of high rtes of muttion of certin genes or phenotypes is not uncommon. For exmple, the colonil vrition from trnsluscent to opque occurs t rte of one in 104 cells. These could rise, of course, by muttions t different loci. The ml muttion in Shigell nd the glp K gene lso mutte t high frequency. Thus, the reminder of the results section will be concerned with the genetic reltionships of the pleiotropic ltertions in the virulent strin. Microelectrophoresis. The EM of the virulent strin M nd its virulent mutnt hve been extensively studied in this lbortory (6). It hs been shown tht under similr conditions of growth (Luri broth with 0.2% glucose nd 5 mm CCl2), strin M showed n EM of 0.56,um per s per V per cm, wheres strin ws much more negtive with vlue of 1.87,um per s per V per cm (6). The EM of glp K+ revertnts s well s the M glp K- mutnts nd its glp K+ revertnts were mesured (Tble 6). Also given re the vlues for trnsductnts which received the glp K gene from E. coli strins 1895 or 27p. These dt show correltion between the presence of glycerol kinse nd the EM in these Shigell strins. If cell ws glp K+, the EM vlues were less thn 0.6,um per s per V per cm; if the cell ws glp K-, the EM ws more negtive, with vlues rnging from 1.2 to 2.0,um per s per V per cm. This reltionship did not hold in the E. coli strins tested. They hd very high negtive EM (over 3.0 jm per s per V per cm) whether glp K- or glp K+. The Shigell trnsductnts which received the glp K gene from E. coli donors ll possessed low EM, indicting tht the high chrge of E. coli ws not due to gene in the region of glp K. The chrge of E. coli ws probbly due to nother fctor, such s fimbrie, which crries negtive chrge (14). Lysis by SLS. The sensitivity of S. flexneri nd E. coli to SLS hve been studied, nd t TABLE 6. Electrophoretic mobility nd glycerol kinse Strin EM type glpk Shigell flexneri 2 M Low Intermedite glpk+ revertnts Low + (10)b M glpk mutnts (8) Intermedite M I8 glpk+ rever- Low + tnts (7)c Escherichi coli K-12 Hfr C strin W1895 High + 27pd High + 76p glpk mutnt High glpk+ trnsductntse (W1895) x (2) Low + (27p) x (4) Low + EM is expressed in micrometers per second per volt per centimeter. Vlues re divided into three types: low, <0.6; intermedite, 1.2 to 2.0; nd high, >3.0. 'Numbers in prentheses indicte the number of strins tested. cthese revertnts re from 18, nother virulent glpk mutnt of M d Strin 27p is promotor mutnt which increses the glycerol kinse ctivity twofold. It ws obtined from D. Richey nd E. C. C. Lin, s ws 76p. e Strin in the prenthesis is the donor strin in phge P,vir trnsduction.

6 VOL. 9, 1974 MUTATION IN S. FLEXNERI 921 lest three genes re known to determine this chrcteristic. One gene ner the lctose operon ws demonstrted to govern sensitivity to SLS in E. coli (20). The two other genes re cotrnsducible with the rbinose operon (5). It hs been shown tht the virulent S. flexneri strin M nd its virulent mutnt, strin 24570, differed in their reltive resistnce to the nionic detergent SLS. The virulent mutnt is fr less sensitive to SLS thn its virulent prent (Tble 7). E. coli K-12 is much more sensitive to SLS thn both of these Shigell strins (5). The percentge of lysis per 10 min of the virulent strin rnged from 0 to 20%; the virulent strin hd lysis of 25 to 50%, nd E. coli strin W-1895 lysed 50 to 75%. Bsed on these vlues, sensitivity to SLS cn be divided into three clsses: resistnt, intermedite, nd sensitive. To estblish whether SLS sensitivity ws relted to the muttion which mde virulent strin incpble of utilizing glycerol s crbon source, the different mutnts were tested. The glp K+ revertnts remined resistnt to SLS s its prent, but out of nine M glp K- mutnts tested, two remined intermedite like M-42-43, nd seven becme sensitive to SLS. No glp K+ trnsductnts from n M to cross regined the SLS lysis chrcteristics of the M Thus, the glp K gene does not ffect SLS lysis chrcteristics. Biologicl oxidtion. Virulent Shigell strin M hs been shown to oxidize succinte, fumrte, nd mlte t fster rte thn its virulent mutnt. This hs been shown in whole cells nd in cell-free extrcts (15). When the glycerol kinse mutnts derived from these strins were studied, the oxygen uptke by these strins resembled tht of the prents (Tble 8). For exmple, strin hs n uptke of 3.5 Aliters of 02 per 109 cells per 10 min, nd its glp K+ revertnts hve rte rnging from 1.6 to 3.6,uliters of 02 per 109 cells per 10 min. Strin M hs rte of 18.6 TABLE 7. Sensitivity to sodium lurvl sulfte Strin Lysis clss S. flexneri M I S. flexneri R E. coli W1895 S M glpk mutnts (9)b 2I + 7 S glpk+ revertnts (10) 10 R Abbrevitions: S, sensitive (>55%); I, intermedite (25 to 50%); R, resistnt (0 to 20%). b Numbers in prentheses represent number of strins tested. TABLE 8. Succinte oxidtion by sttionry-phse cells of S. flexneri 2 Strin Oxygen uptke" M M glpk (8)b 8.6 to 16.0c glpk glpk+ revertnts (10) 1.6 to 3.6c Cells were grown in nutrient broth without glucose nd with vigorous shking. Uptke is expressed s uliters of oxygen per 109 cells per 10 min. 'Numbers in prentheses represent number of strins tested. c Rnge of vlues obtined.,uliters of 02 per 109 cells per 10 min, nd its M glp K- mutnts hve rte rnging from 8.6 to 16.0 pliters of 02 per 109 cells per 10 min. Thus, there ppers to be no direct reltionship between glp K nd the oxidtion of these tricrboxylic cid cycle intermedites. Virulence. The bility of virulent strins to penetrte HeL cells (4) nd to cuse kertoconjunctivitis correltes well with the bility to invde the intestinl epithelil cells of strved guine pigs nd monkeys (17). Using the method of LBrec et l. (17), we verified the fct tht virulent S. flexneri strin did not invde or multiply within HeL cells. In contrst, virulent S. flexneri strin M hevily infected the HeL cells, with most of the bcteri ppering in the cytoplsm. By using glycerol kinse s the mrker to distinguish between the prent strins, mutnts, nd their derivtive strins, penetrtion of HeL cells by these strins ws tested. "Virulence" will be used in the nrrow sense to describe this bility to penetrte HeL cells. Tble 9 shows tht 50'% of the glp K+ revertnts derived from virulent strin regined the bility to penetrte HeL cells. Similrly, 44% of the glp K- strins derived from virulent strin M (glp K+) vi mutgenesis were incpble of penetrtion. One of these, virulent M glp K- strin I8, ws ble to revert to glp K+, nd ll four colonies tested hd regined virulence s mesured by HeL cell penetrtion. Conjugnts receiving the glp K mrker from E. coli K-12 strins 27p or W1895 were not cpble of mking the virulent strin regin virulence. Trnsductnts receiving the glp K mrker from virulent S. flexneri strin M showed tht 39% hd been converted from virulent (24570) to virulent strins (Tble 10). Thus, there is prtil correltion between the glp K phenotype nd penetrtion. This puzzling result seems to indi-

7 922 KIM AND CORWIN INFECT. IMMUNITY TABLE 9. HeL cell infection by strins of S. flexneri 2 Strin glpk Penetrtion M /1 M glpk mutnts - 5/9 M I8, glpk - 0/1 18 glpk+ revertnts + 4/ glpk - 0/ glpk+ revertnts + 5/10 Expressed s number strins tested. TABLE 10. of virulent strins/totl HeL Cell penetrtion by S. flexneri Method Donor Recipient Penetrtion' Trnsduction 27p /4 Trnsduction M /18 Conjugtion W /5 E. coli strins 27p nd W1895 nd S. flexneri strin re virulent; S. flexneri strin M is virulent. 'Expressed s number of virulent strin/totl strins tested. cte tht there my be two different genes ffecting glp K, one of which is relted to HeL cell penetrtion nd the other not. DISCUSSION It hs been demonstrted tht the virulent S. flexneri 2 strin M hs mutted to n virulent strin, 24570, which no longer ferments glycerol, due to loss of glycerol kinse (glp K) ctivity. Reversion of the mutnt to glp K+ restores the bility to penetrte HeL cells to only hlf of the revertnts. Furthermore, selection of dditionl glp K mutnts of M only results in loss of virulence in hlf of these strins. The strins re clssified s virulent if they cn penetrte HeL cells. One conclusion from such results might be tht more thn one gene ffects glycerol kinse ctivity in Shigell, but only one is involved with the bility of the bcteri to penetrte HeL cells. The first clue leding to this hypothesis ws the fct tht virulent S. flexneri produced pink colonies on 1% glycerol-mcconkey gr, wheres E. coli strins formed drk-red colonies. This indicted t the very lest difference in the quntity of glycerol kinse produced. Richey nd Lin (21) hve presented evidence compring the enzyme from our strins of Shigell with tht from E. coli indicting tht the enzymes re essentilly identicl. The Shigell enzyme rects with ntiserum prepred ginst the crystlline E. coli enzyme by using the Ouchterlony double-diffusion technique, is sensitive to fructose diphosphte, nd hs nerly identicl Km vlues. Thus, it would seem tht there might be difference in the level of derepression of glp K in the two species, lthough comprison of turnover numbers of both enzymes ws not possible in the bsence of purified preprtion of the Shigell enzyme. The evidence presented in this pper is not consistent with the simple explntion of difference in derepression of the synthesis of the enzyme. First, the glp K genes of the two gener pper to be t different loci on the chromosome. The E. coli glp K gene cotrnsduces with rh, wheres the Shigell glp K gene does not. Interrupted mting studies lso plces the rh nd glp K genes in Shigell prt. It does not pper tht this is simply mtter of trnsloction of genes. When the Shigell glp K is introduced into E. coli, it does not cotrnsduce with rh. Perhps more importnt distinction between the genes in the different strins is their reltion to virulence. Trnsduction of the glp K+ gene from E. coli into the virulent Shigell strin (producing drk-red colonies on glycerol-mcconkey gr) does not restore virulence. Trnsduction of glp K from the virulent Shigell into does give rise to virulent trnsductnts, but not lwys. Similrly, only some glp K+ revertnts of the virulent strin regin virulence, nd only some glp K mutnts of the virulent Shigell lose virulence. Thus, there is n indiction tht t lest two genes ffecting glycerol kinse ctivity exist. The complete correltion of glycerol kinse ctivity with chnges in electrophoretic mobility of Shigell remins unexplined. This correltion is observed whether the enzyme in Shigell is from Shigell or donted from E. coli. The three- to fourfold increse in negtive chrge upon the loss of the enzyme ctivity my be due to the necessity of the enzyme for the synthesis of certin cell envelope components, i.e., phospholipids, but this cnnot relte to virulence per se since only the gene in Shigell hs ny correltion with virulence. Rther, it would seem tht perhps some specific cell wll stte my ffect glycerol kinse ctivity nd once the enzyme is present it cuses the ltertion in electrophoretic mobility. The muttion of the virulent M to ws selected on the bsis of n ltertion in colonil morphology, not the bility to ferment glycerol. The lck of correltion of succinte oxidtion nd SLS sensitivity to glycerol kinse does not necessrily men tht these chrcteristics might not correlte, t lest prtilly, with colonil morphology. Thus, if pr-

8 VOL. 9, 1974 ticulr cell wll muttion which results in colonil morphology chnges lso cuses loss of glycerol kinse in Shigell, then the ltered chrcteristics of might correlte with glycerol kinse. Interestingly, we hve obtined glp K mutnt of M-42-43, I8, which upon reversion to glp K+ did restore HeL cell penetrtion in four out of four cses. ACKNOWLEDGMENTS Roslind Kim ws predoctorl fellow supported by f'unds from the Ntionl Defense Eduction Act. This investigtion ws supported by the U.S. Army Medicl Reserch nd Development Commnd. Deprtment of the Army, reserch contrct no. DADA M-8146, under the sponsorship of' the Commission on Enteric Infections of the Armed Forces Epidemiologicl Bord. LITERATURE CITED 1. Adelberg. E. A.. M. Mndel, nd G. C. C. Chen Optiml conditions for mutgenesis by N-methyl-N'- nitro-n-nitrosogunidine in Escherichi coli. Biochem. Biophvs. Res. Commun. 18: Bermn, M., nd E. C. C. Lin Glvcerol-specific revertnts of phosphoenolvpyruvte phosphotrnsferse mutnt: suppression by the desensitiztion of glycerol kinse to feedbck inhibition. J. Bcteriol. 105: Brenner. D. J.. G. R. Fnning. F..J. Skermn. nd S. Flkow Polynucleotide sequence divergence mong strins of Escherichi coli nd closelv relted orgnisms. J. Bcteriol. 109: Clbi, In vitro interction of Shigell flexneri with leukocvtes nd HeL cells. J. Infect. Dis. 122: Corwin, L. M., S. W. Rothmn. R. Kim. nd L. A. Tlevi Mechnisms nd genetics of resistnce to sodium luryl sulfte in strins of Shigell nd Escherichi coli. Infect. Immunity 4: Corwin, L. M., nd L. A. Tlevi Muttion in Shigell flexneri 2 resulting in incresed electrophoretic mobility. Infect. Immunity 5: Cozzrelli. N. R., nd E. C. C. Lin Chromosoml loction of the structurl gene for glycerol kinse in Escherichi coli. J. Bcteriol. 91: Egle, H The minimum vitmin requirements of' the L nd HeL cells in tissue culture, the production of specific vitmin deficiencies, nd their cure. J. Exp. Med. 102: MUTATION IN S. FLEXNERI Flkow. S.. H. Schneider. L. S. Bron. nd S. B. Forml Virulence of Escherichi-Shigell genetic hvbrid for the guine pig. J. Bcteriol. 86: Forml. S. B.. P. Gemski. Jr., L. S. Bron, nd E. H. LBrec Chromosoml locus which controls the bilitv of Shigell flexneri to evoke kertoconjunctivitis. Infect. Immunitv 3: Forml. S. B.. E. H. LBrec, A. Plmer, nd S. Flkow Protection of monkeys ginst experimentl shigellosis with ttenuted vccines. J. Bcteriol. 90: Gorini, L., nd H. Kufmn Selecting bcteril mutnts by the penicillin method. Science 131: Hnks, J. H.. nd R. B. Wllce Reltion of oxygen nd temperture in the preservtion of tissues bv refrigertion. Proc. Soc. Exp. Biol. Med. 71: Hrris. J. O., nd R. M. Kline Electrophoresis of motile bcteri. J. Bcteriol. 72: Kim, R.. nd L. M. Corwin Fctors ffecting virulence of Shigell flexneri: virulent strin with ltered metbolism of succinte, fumrte, nd mlte. Infect. Immunitv 7: Koch, J. P., S. Hyshi, nd E. C. C. Lin The control of dissimiltion of glycerol nd L--glycerophosphte in Escherichi coli. J. Biol. Chem. 239: LBrec, E. H., H. Schneider, T. J. Mgnni, nd S. B. Forml Epithelil cell penetrtion s n essentil step in the pthogenesis of bcillry dysentery. J. Bcteriol. 88: Lowrv. 0. H., N. J. Rosebrough. A. L. Frr. nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. Biol. Chem. 193: Mckel, D. C., L. F. Lngley, nd L. A. Venice The use of the guine pig conjunctive s n experimentl model for the study of virulence of Shigell orgnisms. Amer. J. Hygiene 73: Nkmur. M Genetic determintion of resistnce to criflvine, phenethyl lcohol, nd sodium dodecyl sulfte in Escherichi coli. J. Bcteriol. 96: Richey, D. P., nd E. C. C. Lin Importnce of fcilitted diffusion for effective utiliztion of glvcerol by Escherichi coli. J. Bcteriol. 112: Schneider, H., nd S Flkow Chrcteriztion of n Hfr strin of Shigell flexneri. J. Bcteriol. 88: Tylor, A. L Current linkge mp of Escherichi coli. Bcteriol. Rev. 34: Umbreit. W. W., R. H. Burris, nd J. F. Stuffer Mnometric techniques nd tissue metbolism, 7th ed. Burgess Publishing Co., Minnepolis.