Inhibition of Vitamin K2 and Carotenoid Synthesis

Transcription

1 JOURNAL OF BACTRIOLOGY, Sept. 197, p Cpyright a 197 American Sciety fr Micrbilgy Vl. 13, N. 3 Printed in U.S.A. Inhibitin f Vitamin K2 and Cartenid Synthesis in Staphylcccus aureus by Diphenylamine RAY K. HAMMOND AND DAVID C. WHIT Bichemistry Department, University f Kentucky Medical Center, Lexingtn, Kentucky 56 Received fr publicatin 26 January 197 Diphenylamine at cncentratins hich did nt effect the grth rate inhibited the synthesis f vitamin K2 in bth anaerbic and aerbic cultures by abut 5%. At this cncentratin, diphenylamine inhibited the synthesis f the cyclic cartenids b-cartene and the rubixanthins 25 t 35 % anaerbically and 6 t 9% aerbically. The inhibitin f synthesis f cyclic cartenids and vitamin K2 by diphenylamine had n detectable effect n the frmatin f the membrane-bund electrn transprt system. Diphenylamine (DPA) inhibits the synthesis f cartenid pigments in several micrrganisms at cncentratins hich d nt affect the grth rate (12, 17). Depending n the cncentratin and the rganism, DPA inhibits (12) r des nt inhibit (13) sterid synthesis r affects () r des nt affect (13) fatty acid synthesis. DPA des nt affect the synthesis f triglyceride in yeast (17). There is a marked inhibitin f benquinne frmatin (12, 18) and a smehat less severe inhibitin f naphthquinne frmatin in a number f micrrganisms (16). ven thugh synthesis f varius lipid cmpnents f the membrane can be inhibited, the ttal mass f the membrane frmed by cells grn in DPA appears t be nrmal (15). In vie f the pssible invlvement f membrane lipids in the frmatin f the electrn transprt system f Staphylcccus aureus (5), the effect f DPA n the synthesis f lipids and the frmatin f the membrane-bund electrn transprt system ere examined. MATRIALS AND MTHODS Materiais. DPA (Fisher Chemical C., St. Luis, M.) as used ithut further purificatin. Other materials ere as described (7, 9, 22). Grth f S. aureus U-71. The media, culture cnditins, and harvesting prcedures have been described (9). Fr anaerbic grth, the medium as sparged ith dexygenated nitrgen beginning just after autclaving and cntinuing thrughut the experiment (5, 22). The frmatin f the electrn transprt system as induced by aerating anaerbic cultures gring expnentially as described (5). The bacterial density as determined by the absrbancy at 75 nm (22). DPA as disslved in dimethyl sulfxide and added t the culture, s that the final cncentratin f DPA as 7,uM and dimethyl sulfxide as.% (v/v). xtractin f the lipids. The lipids ere extracted by the Bligh and Dyer prcedure (3) as mdified (9). A prtin f the lipid extract as sapnified, and the cartenids ere separated chrmatgraphically and assayed spectrphtmetrically (9). A secnd prtin f the lipid extract as purified by thin-layer chrmatgraphy, and the vitamin K2 as assayed spectrphtmetrically (7, 8). In lipids frm bacteria grn ith DPA, the vitamin K2 fractin islated frm the thin-layer plates as applied t a secnd Silica Gel G thin-layer plate and chrmatgraphed ith isctane-chlrfrm (2:1, v/v). The vitamin K2 band (RF value.3 t.) as recvered and had n spectral evidence f DPA cntaminatin. Nne f the purified cartenid fractins shed spectral evidence f DPA cntaminatin. Phsphlipids ere analyed as described previusly (22). Assay f the electrn transprt system. Cytchrmes ere measured by difference spectrscpy, xygen utiliatin as measured ith the plargraph in the presence f L-lactate, and prtheme as measured by the reduced pyridine hemchrme as described previusly (22). RSULTS Isprenid lipids f S. aureus. S. aureus U-71 has been shn t cntain the cartenids phytene, c-cartene, B-cartene, phytfluenl, a phytfluenl-like cartenid, rubixanthin, and three rubixanthin-like cartenids as the majr cmpnents (9). Vitamin Ka isprenlgues ith side chains cntaining er t nine isprene units have been identified in this strain f S. aureus (7, 8). In cells grn fr six t eight divisins in the presence f 5,uc f mevalnate-2-1c per 5 ml, 5% f the lipid 1C as recvered in the vitamin K2 and the nine cartenids. Inhibitin f grth by DPA. DPA inhibits grth abve 18,AM (Table 1). At a DPA cncentratin f 7,uM, the final yield f cells as re- 611

2 612 HAMMOND AND WHIT J. BACTRIOL. TABL 1. Grth yield fs. aureus U-71 grn ith DPA DPA cncn& Grth yieldb Clr f cellsc.1 Orange 37.2 Orange White White _d a DPA as added in dimethyl sulfxide ith the final dimethyl sulfxide cncentratin.% (v/v). Cultures ere grn in 25-ml rlenmeyer flasks fr 18 hr at 37 C (1). b Bacterial density as measured as absrbancy at 75 nm in 13-mm diameter rund test tubes (22). When the absrbancy as greater than.6, the cultures ere diluted 1 t 1 ith ater befre measurement. c Clr f the bacterial pellet after centrifugatin at, X g fr 1 min. d N clr detected. duced by less than 1%, and the grth rate as nt affected. In cells grn ith 7,UM DPA, apprximately 2% f the DPA added t the culture as recvered in the lipid extract. The remainder as in the medium after the cells ere harvested by centrifugatin. Inhibitin f cartenid synthesis by DPA. In the anaerbic grth cycle, DPA had n effect n the grth rate r the synthesis f phytene, phytfluenl, the phytfluenl-like pigment r c-cartene (Fig. 1). There as a threefld inhibitin f the level f the trace cyclic cartenids (5-cartene and the rubixanthins) early in statinary phase. By late statinary phase, the cyclic cartenid cncentratin as depressed by 2% in cells grn ith DPA. The cyclic cartenids represent such a small prtin f the ttal cartenids in anaerbic cells that the ttal cartenid as depressed by less than 1% by the end f the grth cycle. Oxygen markedly stimulates the synthesis f cartenids in S. aureus (9). When an anaerbic, expnentially gring culture as aerated, a rapid synthesis f the cyclic cartenids, 5-cartene, and the rubixanthins ccurred (Fig. 2). At the nset f aeratin, there as a sharp decrease in the cntent f phytene and the phytfluenls that as flled by a marked increase in the phytfluenls (Fig. 2). When DPA as added at the nset f aeratin, there as an initial decrease in phytene as in the cntrl, but the phytene cncentratin never recvered (Fig. 3). The synthesis f the ther cartenids as markedly inhibited. After hr f aerbic grth, the cntrl culture cntained 156 nmles f cartenid per g (dry eight), f hich the phytene represented 35 nmles, the 5-cartene 22 nmles, and the rubixanthins 72 nmles. In the presence f DPA after hr f aerbic grth, the cells cntained 6 nmles f cartenid per g (dry eight), f hich phytene represented 22 nmles, 5-cartene 1.1 nmles, and the rubixanthins 1.5 nmles (Fig. 2, 3). Inhibitin f vitamin K2 frmatin. During the anaerbic grth cycle, the presence f 7,uM DPA reduced the vitamin K2 level by abut half ithut affecting the grth rate (Fig. ). When air as added t lgarithmically gring cells, there as a stimulatin f grth and vitamin K2 frmatin. Additin f air and DPA shed n effect n grth, but the final level f vitamin K2 achieved (1.1,Amles per g, dry eight) as again abut half that fund in lgarithmically gring aerbic cells (2. /Amles per g dry eight). In this strain f S. aureus, bth anaerbic and aerbic cultures achieved the same level f vitamin K2 in the statinary phase (2. t 2.,umles per g dry eight). I.- 3: r Ia, m I :L t a II 1,2,3a -Zd 1 DCAROTN FIG. 1. Cartenid levels in S. aureus U-71 during anaerbic grth in the presence f 7 um DPA. Bacteria ere grn anaerbically (5, 9) as illustrated in the ler graph. Slid symbls represent grth ith DPA and pen symbls ithut DPA. The cells ere harvested, the lipid as extracted and sapnified, the cartenids ere separated chrmatgraphically, and the pigments ere assayed spectrphtmetrically (9). The numerals 1, 2, 3, and refer t rubixanthin and the three rubixanthin-like cartenids (9); 5 refers t a phytfluenl-like cartenid (9).

3 VOL. 13, 197 DPA INHIBITION OF LIPID SYNTHSIS 613 Phsphlipid cncentratin. Anaerbically gring S. aureus in the expnential phase cntained abut 3,umles f phsphlipid per g (dry eight). The phsphlipids increase t 5,umles per g (dry eight) in the anaerbic statinary phase. The additin f 7 FM DPA t the culture had n effect n the ttal phsphlipid cntent f anaerbically gring cells. When anaerbically gring cells in the expnential grth phase ere aerated, the ttal phsphlipid increased rapidly t abut 5,umles per g (dry eight) and as maintained at this level until the rganism entered the statinary grth phase. When DPA as added at the time f aeratin, the phsphlipid increased t 5,imles per g (dry eight) and as maintained at that level. DPA at 7 FM had little effect n the phsphlipid cncentratin f S. aureus. Frmatin f the electrn transprt system. The sitch frm anaerbic t aerbic grth caused the inductin f the membrane-bund electrn transprt system (5). When 7 Mm DPA as added at the nset f aerbic grth, there as n 1- ) Il 1 PHYTOFLUNOL -i T 62.3 LO u 1 TTAL t1, GROWTH -I 1 I I ~-:.1 x- x PHYTOFLUNOL LO GROWTH 3~ \ O 2 2 OURS FIG. 2. Cartenid levels during the sitch frm anaerbic t aerbic grth. xpnentially gring cells incubated anaerbically ere aerated at the time indicated ( T ) ith 6 liters fair per min per 8 liters f medium as described (5). Samples ere ithdran and analyed as in Fig t FIG. 3. Cartenid levels during the sitch frm anaerbic t aerbic grth in the presence f DPA. In an experiment like that illustrated in Fig. 2, DPA (7 JAM) as added at the time f aeratin ( T ), and the cartenids ere assayed as in Fig. 1. detectable change in the rate f grth, the synthesis f prtheme, r the appearance f enymatically reducible cytchrmes b,, and a (Fig. 5). The final levels f these pigments ere similar t thse in the cntrl experiments. There as n detectable difference in the rate f xygen utiliatin in the presence f L-lactate r in the critical xygen cncentratin beteen cntrl and cells grn ith DPA. The critical xygen cncentratin is a sensitive measure f the functin f the electrn transprt system (5, 2). DISCUSSION DPA had multiple effects n the t majr isprenid lipids in S. aureus U-71 at 7,uM, a cncentratin hich had n effect n bacterial grth. Vitamin K2 bisynthesis as depressed by abut 5% bth anaerbically and aerbically (Fig. ). This strain f S. aureus as unusual in that it frmed as much vitamin K2 in the anaerbic statinary phase as it did hen gring aerbically. Usually anaerbically gring S. aureus frms very little vitamin K2 (2).

4 61 HAMMOND AND WHIT J. BACTRIOL. c ' A A- / l grth (Fig. 1). Phytene synthesis as inhibited slightly during aerbic grth (Fig. 3), as in ther strains f S. aureus (19). The mst marked effect f DPA as n the aerbic synthesis f the cyclic cartenids (Fig. 3). The aerbic synthesis f 5-cartene as inhibited 2-fld and the rubixanthins 5-fld by DPA (Fig. 2, 3). Abut 2% f the DPA as recvered in the lipid extract (3,umles per g, dry eight). This is abut the same cncentratin f DPA accumulated in the membranes f Bacillus megaterium (16). This DPA cncentratin apprximates the vitamin K2 cntent f the membrane and is LO W Q8 X..6. I i a.2a IL Is FIG.. Vitamin K2 levels during anaerbic and aerbic grth. Circles indicate bacterial densities (upper graphs) and ttal vitamin K2 (ler graphs) in S. aureus U-71 grn anaerbically in the absence f DPA () r in the presence f 7 pm DPA (@). Grth (A, upper curve) and vitamin K2 (A, ler curve) f cells grn anaerbically and then aerated ( I ) as in Fig. 2. DPA (7.m) as added at the initiatin f aeratin. Frm studies f the incrpratin and turnver, the pathays f cartenid metablism in S. aureus U-71 appear t fll the usual sequence f phytene desaturatin thrugh t-cartene, cycliatin t b-cartene, and hydrxylatin t frm the rubixanthins ith an alternate pathay f hydratin t phytfluenl, flled by cycliatin t the rubixanthins (9). The hydrxylatin f 5-cartene t frm rubixanthins appears t invlve mlecular xygen and a mixed xidase system and is respnsible fr the majr prtin f the rubixanthin (1). The anaerbic pathay invlves hydrxylatin t the phytfluenls and functins t a limited extent during bth anaerbic and aerbic grth (9, 1). Phytene, the initial -carbn cartenid, accumulates in many micrrganisms in the presence f DPA (6, 11, 1, 16). In anaerbically gring S. aureus, DPA had little effect n the frmatin f phytene, the phytfluenls, r c-cartene (Fig. 1). c-cartene synthesis as inhibited 33% and the rubixanthin synthesis by 22% in the presence f DPA during anaerbic 1 ad r IL 2J , 2 'I -I 2 FIG. 5. Frmatin f the membrane-bund electrn transprt system in the sitch frm anaerbic t aerbic grth. Aeratin as initiated at indicated time ( i, upper curve) f anaerbically gring cultures as in Fig. 2. Prtheme as assayed as the reduced-minusxidied pyridine hemchrme (5). Cytchrme xidase as estimated frm the reduced-minus-reduced COsaturated difference spectra and cytchrmes b + and a ere estimated frm the reduced-minus-xidied difference spectrum (5). Cells ere reduced in the presence f 2 mm sdium L-lactate. i c cr (L,

5 VOL. 13, 197 DPA INHIBITION OF LIPID SYNTHSIS 615 times the highest level f cartenids fund in this strain f S. aureus. The inductin f the. electrn transprt system after aerating anaerbic cultures required abut 2 hr (Fig. 5). In this perid, there as at least a 2-fld increase in b-cartene and the rubixanthins (Fig. 2). When DPA as added at the nset f aerbic grth, the ttal cartenid and vitamin K2 level as depressed 5%, and the synthesis f the r,ubixanthins as depressed sixfld in the 2-hr perid. This inhibitin by DPA ccurred ithut any detectable change in the frmatin r functin f the electrn transprt system under these cnditins f grth (Fig. 5). DPA had n effect n the frmatin f cytchrme a hich suppsedly cntains an isprenid side chain n its heme. A fully frmed electrn transprt system cntains 9 nmles f prtheme per g (dry\> eight) distributed beteen cytchrme b and cytchrme xidase. In the presence f DPA, the ttal cartenid cncentratin as depressed frm 15 nmles t 6 nmles per g, dry eight (rubixanthin frm 75 t 1.1 nmles per g, dry eight) and the vitamin K2 frm 2. t 1.1,mles per g (dry eight). These levels ere apparently adequate fr functin at 37 C. The levels f DPA used in this study ere cnsiderably bel thse reprted by Baker (1) t inhibit electrn transprt. S. aureus has a high level f vitamin K2 relative t the cytchrme cntent. In Haemphilus parainfluenae, the mlar rati beteen 2-demethyl vitamin K2 and cytchrme b is maintained at 15 t 1, even thugh the cytchrme b cntent f the membrane can vary ver a ide range (21). The vitamin K2 t cytchrme b rati f S. aureus is 5 t 1 in the aerbic expnential phase r 25 t 1 hen grn ith DPA. The high rati beteen vitamin K2 and the cytchrme b and the fact that vitamin K2 as frmed during anaerbic grth in hich n functinal cytchrmes ere frmed suggested that vitamin K2 might have functins in the membrane ther than in electrn transprt. ACKNOWLDGMNTS This investigatin as supprted by cntract (73) ith the Agricultural Research Service, U.S. Department f Agriculture, administered by the astern Utiliatin and Develpment Divisin, Philadelphia, Pa., and by Public Health Service grant GM-1285 frm the Institute f General Medical Sciences. R. K. Hammnd as supprted by grant 5-T1-GM-126 frm the Natinal Institute f General Medical Sciences t the Department f Bichemistry, University f Kentucky Medical Center. LITRATUR CITD 1. Baker, J Diphenylamine inhibitin f electrn transprt in plant mitchndria. Arch. Bichem. Biphys. 13: Bishp, D. H. L., K. D. Pandya, and H. K. King Ubiquinne and vitamin K in bacteria. Bichem. J. 83: Bligh,. G., and W. J. Dyer A rapid methd f ttal lipid extractin and purificatin. Can. J. Bichem. Physil. 37: Ch, K. Y., W. A. Crpe, and M. R. J. Saltn ffect f diphenylamine n the fatty acid cmpsitin f sme bacterial lipids. Bichem. J. 93:26c-28c. 5. Frerman, F.., and D. C. White Membrane lipid changes during frmatin f a functinal electrn transprt system in Staphylcccus aureus. J. Bacteril. 9: Gdin, T. W., M. Jamikrn, and J. S. Willmer Studies n cartengenesis. 7. Further bservatins cncerning the actin f diphenylamine in inhibiting the synthesis f ft-cartene in Phycmyces blakesleeanus. Bichem. J. 53: Hammnd, R. K., and D. C. White Separatin f vitamin K2 isprenlgues by reversed phase thin layer chrmatgraphy. J. Chrmatgr. 5: Hammnd, R. K., and D. C. White Frmatin f vitamin K2 isprenlgues by Staphylcccus aureus. J. Bacteril. 1: Hammnd, R. K., and D. C. White Cartenid frmatin by Staphylcccus aureus. J. Bacteril. 13: Hammnd, R. K., and D. C. White Inhibitin f cartenid hydrxylatin in Staphylccus aureus by mixedfunctin xidase inhibitrs. J. Bacteril. 13: Jensen, S. L., G. Chen-Baire, T.. M. Nakayama, and R. Y. Stanier The path f cartenid synthesis in a phtsynthetic bacterium. Bichim. Biphys. Acta 29: Kakutani, Y Detectin f sme isprenids and the influence f diphenylamine n the bisynthesis f isprenid by Sprblmyces shibatanus. J. Bichem. (Tky) 61: Olsn, J. A., and H. Kniley, Jr The effect f diphenylamine n cartenid, sterl and fatty acid synthesis in Phycmyces blakesleeanus. Arch. Bichem. Biphys. 97: Rilling, H. C A study f inhibitin f cartenid synthesis. Arch. Bichem. Biphys. 11: Saltn, M. R. J., and A. F. M. thisham-ud-din The lcaliatin f cytchrmes and cartenids in islated bacterial membranes and envelpes. Aust. J. xp. Bil. Med. Sci. 3: Saltn, M. R. J., and M. D. Schmitt ffects f diphenylamine n cartenids and menaquinnes in bacterial membranes. Bichim. Biphys. Acta 135: Slechta, L.,. Gabriel, and. Hffmann-Ostenhf Separatin f cartenid synthesis frm fat synthesis in Rh6dtrula gracilis by diphenylamine. Nature (Lndn) 181: Sugimura, T., and H. Rudney The effect f aerbisis and diphenylamine n the cntent f ubiquinne in Rhdspirillum rubrum. Bichim. Biphys. Acta 62: Suue, G Studies n cartengenesis by Micrcccus pygenes var aureus. J. Bichem. (Tky) 6: White, D. C Factrs affecting the affinity fr xygen f cytchrme xidases in Haemphilus parainfluenae. J. Bil. Chem. 238: White, D. C Synthesis f 2-dimethyl vitamin K2 and the cytchrme system in Haemphilus. J. Bacteril White, D. C., and F.. Frerman xtractin, characteriatin, and cellular lcaliatin f the lipids f Staphylcccus aureus. J. Bacteril. 9: