the photophobic response of various sulfur and non-sulfur purple bacteria

8/3/2019 the photophobic response of various sulfur and non-sulfur purple bacteria

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Photochemisrry and Phorobiology Vol. SO, N o. 6, pp. 809-815. 1989Printed in Great Britain. All rights reserved 003 1-8hss i~9 03.00+0.00Copyright 0 989 Pergamon Press plc

THE PHOTOPHOBIC RESPONSE OF VARIOUS SULFURAND NONSULFUR PURPLE BACTERIA

E . HUSTEDE,. L I E B E R G E S E L Lnd H. G . SCHLEGEL"Institut fur Mikrobiologie der Georg-August-Universitat, Grisebachstrasse 8, 3.100 Gottingen.W . Germany(Received 14 Apr i l 1989; accepied 97 July 1989)

Abstract-The photopho bic response of 34 strains of nonsulfur and sulfur purple bacteria was examinedwith respect to response-eliciting light intensities. The bacteria were grown in defined synthetic mediaor in Winogradsky columns. Two populat ion methods based on Engelmann's l ight t rap were used todetermine the discrimination thresholds of the bacteria. A single-side irradiation method allowed theest imation of approxim ate values, while the double-side irradiation m ethod provided mo re exact valuesof the discrimination threshold. Sixteen strains belonging to 9 different species exhibited discriminationthresholds between 0.7% and 2.h0/,. Th e motil i ty of the o ther 18 strains proved to be insufficient tomeasure l ight sensit ivit ies with the methods used. T he effect of various environmental factors on thelight sensitivies of Chromatiurn rlinosurn D an d Rhodospirillum rubrum Ha was examined. Themeasurements and observations made in this work recommend strains of Rhodospirillum rubrum an dChromatiurn vinosurn as model organisms for further studieb.

INTRODUCTIONMost motile microorganisms respond to chemicaland physical changes in their environment. Thepho toph obic response is a mo tor response elicitedby light. Photophobic responses in purple bacter iawere f irst described by Engelmann (1882). Heobserved that Bacterium ph otometricum , almost cer-tainly a species of the pu rple sulfur bacter ium genusChromatiurn, abruptly reversed the direction ofmovem ent a f te r a sudden decrease of light intensity.Consequently, the bacter ia accumulated in a l ight-spot projec ted in to a th in layer of cells. Photophobicresponses (formerly called photophobotaxis) aredistinguished from two other reaction types withrespect to l ight. Photokinesis describes the effect ofl ight on the speed of movem ent . The or ienta t ion ofmovement with respect to l ight direction is nowcalled phototaxis (formerly called phototopotaxis)(Hader, 1987; Nultsch and Hader, 1979, 1988).In previous s tudies on the photopho bic responseof purple sulfur bacter ia the signal transduction an dthe light sensitivity were of interest. The actionspec tra for the photophobic response of Chroma-tium vinosum (Duysen s , 1951) and Rhodospirillumrubrum (Molisch, 1907; Manten, 1948; Thomas,1953; Clayton, 1953) coincide with the absorptionspectra of the photosynthetic pigments. Theseresults pointed to the photosynthetic pigments asthe receptors of the photophobic response and ledto the hypothesis that a sudden decrease in thephotosynthetic rate causes the photophobicresponse (Manten, 1948) . La te r , on the basis ofexperiments with Rhodospirillum rubrum ( Ha r a y -ama and I ino, 1976, 1977) and Rhodobactersphaeroides (Armitage and E vans , 1981) the proto n

~~~~~~~~ ~. .~.... ~ ~~ ~~~~ ~*To whom correspondence should be addressed.

motive force (PM F) was proposed t o be th e decisivec om pone n t of the sensory transduction chain. I t is ,however , not known how changes in the PMF ac ton the f lagellar motor .Th e f irst quantitative studies on the sensitivity ofpurp le bacter ia to l ight were those of Bud er (1915)studying Thiospirillum jenense. He m e a sur e d thelowest difference of light intensities at a sharp bor-derline between two light fields which could justelicit a respons e of the sw imming bacteria , resultingin their accumulation in the brighter field. Budercalled the percent increment of light intensity 'dis-cr imination threshold' and together with his stu-dent s (S chram mec k, 1934; Schlegel, 1956) measuredthe respective values for Chromatiurn vinosurn,Chrom atium okenii, Thiospirillum jenense a nd Rho-dospirillum rubrum. All these bacter ia were grownin crude culture in Winogradsky columns. Thevalues of the discrimination threshold were in therange f rom 0.9% to 2 .5 %. In the only study apply-ing cells grown in defined medium (Clayton, 1953)the discrimination threshold of R. rubrum wasde te r m ined to b e 2 % . Until 1960 the giant sulfurpurple bacter ia had to be collected in ponds andmaintained in Winogradsky columns. I t was notuntil C. okenii, T. jenense and some other s t ra insand species were grown in enrichment culture andthe growth requirements studied (Schlegel undPfennig, 1961; Pfennig, 1961, 1962) that it wasknown that almost all purple bacter ia can be grownin defined nutr ient solutions. On the basis of theinformation g ather ed within these thir ty years(Pfennig, 1965; Pfennig, 1978) the present investi-gation was star ted. I t a imed to determine discrimi-nation thresholds of the photophobic response of ala rge number of purple bacter ia grown either indefined media or in Winogradsky columns. Twostrains were character ized with respect to the influ-

809


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810 E. HUSTEDEt al.Table 1. Strains of sulfur purple bacteria used throughout this study

Strain ReferenceChromatium vinosum DChromatium vinosum 1611Chromatium vinosum A B G OChromatium vinosum NortenChromatium vinosum HardenbergChromatium minutissimum BN 5711Chromatium purpuratum BN 5500Thiocystis violacea 231 1Chromatium okenii*

ROELOPSON1934), DSM 180N. PFE NNIG,SM 182this workthis workthis workJ . F. I M H O F F ,ers. giftI M H O F Fnd TRUPER1980)N . P F E N N I G ,SM 208this work

Ectothiorhodospira mobilis BN 9903Ectothiorhodospira vacuolata BN 95 12Ectothiorhodospira shaposhnikovii N 1TRUPER1968)I M H O F Ft al . (1981). DSM 21 1 1C H E R N It al. (l969), DSM 243

*This strain did not exist as pure culture but as a monoculture containing about 1% of non-pigmented bacteria.

Table 2. Strains of nonsulfur purple bacteria used throughout this studyStrainsRhodobacter capsulatus K blRhodobacter capsulatus 6950Rhodobacter sphaeroides Si2Rhodobacter sphaeroides ATH 2.4.1Rhodobacter sphaeroides YRhodocyclus gelantinosus 21 50Rhodocyclus tenuis 2761Rhodocyclus tenuis 3661Rhodocyclus tenuis BramwaldRhodocyclus tenuis KiesseeRhodomicrobium vannielii G PRhodopseudomonas acidophila 7050Rhodopseudomonas palustris 1850Rhodopseudomonas palustris BGRhodopseudomonas viridis 2450Rhodospirillum fulvum BramwaldRhodospirillum fulvum ForstRhodospirillum molischianum SRhodospirillum photometricum NTHC132Rhodospirillum rubrum H aRhodospirillum rubrum NortenRhodospirillum rubrum S 1

ReferenceP F E N N I Gnd KL E MME1968). DSM 155R O D E n d G I F F H O R N1983)V A NNI EL 1944), DSM 158REISS-HUSSONt al. (1971). DSM 160N. PFE NNIG,SM 149P F E N N I G1969b), DSM 109N. PFE NNIG,SM 112this workthis workK. S c H M i m , pers. giftP F E N N I G1969a), DSM 137N . P F E N N I G ,SM 126this workN . P F E N N I G ,SM 131this workthis workGIESBERGER1947). DSM 120K . E I M H J E L L E N ,SM 122P F E N N I Gn d K L E M M E1968 ), DSM 107this workV A N NIE L 1944), DSM 467

N . PFENNIG, DSM 152

ence of various environmental factors on the photo-phobic response. A simple method to determine thediscrimination threshold was developed.

MATERIALS AND METHODSBacterial sfrains. The bacteria used throughout thisstudy are listed in Table 1 and Table 2.Media and conditions. The bacteria were cultivated in100 me screw-capped bottle s containing synthe tic mediaor in Winogradsky columns (width 3 cm, height 22 cm).For growth of the nonsulfur purple bacteria the syntheticmedium as described (Pfennig, 1978) was used. Themajority of the purple sulfur bacteria were cu ltivated in amedium of Biebl and Pfennig (1978). Fo r the marinebacterium C. purpuratum 20 g NaCl was added andMgSO, x 7 H,O was increased to 3 g. Chromatium okeniiwas grown in a mediu m as described (Pfennig, 1965 ). Th especies of the genus Ectothiorhodospira were cultivated ina medium of Imhoff (1988).

The Winogradsky columns were prepared as described(Schrammeck, 1934). For the nonsulfur purple bacteriathey contained in the lower third a piece of eggwhite,compost soil and a thin layer of sand and were filled upwith rainwater. Th e columns for the purple sulfur bacteriacontained additionally a piece of gypsum (CaSO,).Th e bacteria in synthetic medium were exposed a t 25Cto continuo us illumination with a 0.4 mWcm-' (1000 Ix)(Osram , 100 W tungsten lamp ). The Winogradsky columnswere placed at a north window with a daylight intensitynot higher than 1500 Ix.Unless otherwise stated , the experim ents described herewere conducted with cells harvested in the middle of theexponential growth phase. The Winogradsky columnsdelivered the best material if the bacteria accumulated asclouds in the water column. After harvest the cells wereeither centrifuged and resuspended in buffers or the bac-terial suspensions were used as such. The cells of R .rubrum Ha were resuspended in 50 mM 4-(2-hydroxy-ethyl)-1-piperazineethanesulphonic acid (Hepes) whichwas adjusted to the required p H with NaO H. M ost experi--


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Photophobic response of purple bacteriaments with Chromatiurn bzinosum D were carried out inSO mM Hepe s buffer. Fo r determin ing fine differences ofdiscrimination thresholds C. Lzinosurn D and al l otherstrains were kept in the growth medium. All experimentswere carried out with about 5 A lox bacter ia ml - l .Preparation of wet mounts. Wet-mounts of bacteriawere prepared to serve as microcuvettes. A drop of th ebacterial suspension and two spa cers (0.2 mm thick)placed on microscope sl ide were covered by a cover sl ipand sur rounded by a mixture of paraffin and vaseline( 1 : 1 ) to avoid air bubbles. Before m easuremen t the sl idewas kept in the dark and prei l luminated for about 30min with 0.32 m W cm - ? (800 Ix) (6 0 W tungsten lamp,Osram). For further detai ls see Results .Measurement of the light sensitivity. For examining thel ight sensi t ivi ty two populat ion methods were used, bothbased on the principle of Engelma nns l ight t rap.(a ) Double-side irradiat ion m ethod (Schlegel, 1956). Fromone side (microsco pe sl ide) the m icrocuvette was i l lumi-nated with the light of L , to provide a homogeneousbackgrou nd intensi ty E , . From the other side the l ight ofL 2 passed a slit (stencil) and proj ected a small light fieldinto the suspension providing the light intensity E 2 . T hemicrocuvette was mounted within a bo x to avoid lightreflexes. Within the light field the total light intensity wasE , + E,. The light intensities were varied by moving thelight sources and calculated on the basis of the squaredistance law. At a given background light intensity, afterexposing the microcuvette for 2 min, that posi t ion ofL , was searched at which visual inspection revealed anaccumulat ion of cells in the light field. The value of th ediscrimination threshold was calculated from E 2 / E l+ E 2 .(b) Single-side irradiat ion metho d. Th is method providedthe homogeneous background intensi ty E , and the l ightintensity E , from the cover slip side only (F ig. l a ) . Diaposi-tive slides with eight light fields of s tepwise decreasedtransparency were projected into the microcuvette for 2min, and that light field was sought which resulted in ajust recognizable accumulation of cells. The relations ofthe light fields and background intensities had previouslybeen measured by means of a 2D gel scanner (C arl Zeiss,Oberkochen) (Kronberg et al., 1983).The method a l lowedscreening for discrimination thresholds by single exposures(Fig. l b ) , but for exact measurements had to be sup-plemented by applying the double-side irradiat ion m ethod.The measurements were carried out with white l ight(double-side irradiation method: 3 W , 12 V , Osram tung-sten lamp; single-side irradiation method: 75 W, 220 V ,Osram tungsten lamp). The l ight intensi t ies were meas-ured with a radiometer (LI-185 with sensor LI-200 S A ,LI-COR, Inc . , Lincoln N E , U S A ) . Unless otherwisestated the exposure t ime was 2 min.Measurement of the effect of temperature on light sensiti-~ i t y . he effect of tempe rature on l ight sensi tivies for C .vinosum D was measured by employing the single-sideirradiat ion meth od and using a waterbath with the requiredtemperature . The microcuvet tes were submerged for 1min in the water before l ight exposure was s tar ted. Theexposure t ime was varied between 2 and 15 min.

RESULTS

Vario us param eters inf luence l ight sensi tiri ityThe light sensitivit ies depended on various par-amet ers such as growth phase, duration of the darkperiod, intensity of background il lumination andothers. Ch r o m a t i u m v i n o s u m D and R. r u b r u m H awere chosen for detailed studies.Cells from the exponential growth phase exhi-bited higher sensitivities than those taken from lag-

( a ) LIGHT SOURCE

ASPHERICA L LENSEPLANCONVEXLENSE

SLIDE

IMAGING OBJECTIVE

IRIS APERTUR E

811

Figure 1 . Single-side irradiation method. (a )Arrangementand path of race. ( b ) Accum ulations of Chromatiurn L3ino-sum D within light fields after projecting fields of varioustransparencies into the microcuvette.

phase or stationary phase cultures (Fig. 2) . Cellstaken f rom Winogradsky columns exhibi tedmaximum sensitivit ies if the popula t ion of purplebacteria had accumulated as clouds in the waterc o lum n.Pr ior to m easurements bacte r ia were kept for acertain t ime in the dark. The duration of the darkperiod had a significant influence on the bacteria.Cells of C. riinosum D had to be kept in the darkfor 24 h to develop o ptimum light sensitivit ies (Fig.3 ) . Cells of R . r u b r u m H a reached maximum l ightsensitivit ies af ter a dark period of 22 h (da ta notshown) .Th e light sensitivit ies were measured at differentintensities of background il lumination in the rangefrom 4 x 10-4 to 4 mWcm- (1.25-100 Ix).Th e examined nonsulfur purple bacter ia ( R . rubrumNor te n , R. f u l r w n Fors t , R. tenuis Kiessee) exhi-bited almost optimum sensitivit ies at backgroundillumination intensities of 8 K 2 x lo- and4 X lo- mWcm- (20 Ix, 50 Ix and 100 Ix). Allexamined sulfur purple bacteria exhibited maximumsensitivities at 4 mWcm- (2 0 Ix). Only C.purpuratum BN 5500 yielded best results at


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812 E . HUSTEDEf al .

30o I 181

I I I I I I I I0 5 10 15 20 25 30 35

Time (h)Figure 2. The effect of growth phase on light sensitivityof Chromatiurn vinosum D. Number of cellsiml in theculture vessel (0 ) nd discrimination threshold ( 0 ) . h ecells were grown in a defined medium with 0.05% acetate.Before measurements the microcuvettes were kept inthe dark for 24 h and preil luminated for 1 h with0.32 mWcm- (800 Ix).

0.51 , , , , , , ]0 5 10 15 20 25 30Time (h)

Figure 3. The effect of storage time in the dark on lightsensitivity of C . vinosum D . The cells were grown in adefined medium with 0.05% acetate and were harvestedin the middle of the exponential growth phase.2 x lo-* mWcm-* (50 Ix) background illuminationintensity (data not shown).Cells of R. rubrum Ha were resuspended in50 mM Hepes buffers pH 6.0,50 m M MOPS bufferspH 6.5, in 100 mM phosphate buffers pH 6.0 or infresh growth medium to examine the influence ofthe kind of buffers o n light sensitivities. Th ere w asno significant difference between the light sensitivi-ties measured in synthetic buffers and those meas-ured in synthetic cultu re mediu m; the discriminationthresholds amounted to 0.8-1.1%.The light-sensitivity of C. vinosum D was testedin 10, 50 and 100 mM solu t ions of Tris-HC1 buffer,pH 7 .5 ; Hepes buffer , pH 7 .2 ; KNa-phosphatebuffer, pH 7.35 and Mops buffer, pH 7.2. 50 m MHepes buffer was the best sui ted. After a darkperiod of 24 h cells of C. vinosum D showed adiscrimination threshold of 1 .2%. Therefore thesensitivity in a synthetic buffer com es close t o thatin the cul ture medium.

PH

Figure 4. The effect of the p H value on light sensitivityof Rhodospirillum rubrum Ha. The cells were harvestedin the middle of the exponential growth phase and wereresuspended in 50 m M HEPES buffer at various pHvalues. The microcuvettes were stored in the dark for 28 hand preilluminated for 30 min with 0.31, mWcm- (800 1x1.

Ascorbate(mM)Figure 5. The effect of different concentrations of ascorh-ate on light sensitivity of Chromatiurn tinosum D . Cellswere grown in defined medium with 0.05% acetate andharvested in the late stationary growth phase. The rnicro-cuvettes were stored in the dark for 23 h.

Th e effect of the p H value o n light sensitivity wasmeasured in 50 mM Hepes buffer. Rhodospirillumrubrum Ha showed maximum sensitivity betweenp H 6 and p H 7 (Fig. 4).Motility was impaired whenthe pH was below pH 6 or above pH 7. The l ightsensitivity of C. vinosum D was opt imal at pH 7.2(data no t shown) .Furthermore, the effect of the presence of thereducing agent ascorbate, which removes oxygen,was examined. The light sensitivity of C. vinosumD was measured with various concentrat ions ofascorbate in the microcuvette. Ascorbate can notbe utilized as carb on or energy source by C. vinosumD (Thiele, 1966). The addition of ascorbateenhanced sensitivity (Fig. 5). No respiration-fig-ures occurred in the microcuvettes when the con-centration of ascorbate exceeded 20 mM. With R .rubrum H a the influence of ascorbate on light sensi-tivity was examined especially with regard to thedurat ion of th e dark period. Cel ls which w ere resus-


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Photophobic response of purple bacteria 813pended in ascorbate-containing (0.05%) growthmedium exhibited maximum sensitivity (0.8%)aftera dark period of 7 h. The discrimination thresholdincreased to 10% when the dark per iod wasexten ded to 24 h. P rovided th at the cells were resus-pended in an ascorbate-free medium, the discrimi-nation threshold was 8.1% after a dark period of7 h. A n extension of the dark per iod to 24 h broughtabout an increase of light sensitivity reaching athreshold of 2.5%.Rhodospirillum rubrum shows th e sam e sensitivi-t ies a t 10 and a t 25C (Clay ton, 1953). In this studythe light sensitivity of C. vinosum D was measuredaccording to the single irradiation method at tem-pera tures be tween 5 and 35C. Within this intervalsensitivity proved to be constant (data not shown).Th e e f fect of various carbo n sources on light sen-sitivity was stu died . Whe n R. rubrum H a was grownwith 0.1% of succinate, alanine or acetat e as carbonsource the succinate medium proved to be the bestsuited medium to provide highly motile cells and toachieve high sensitivities. Chromatium vinosum Dwas grown autotrophically and heterotrophicallywith 0.05% of acetate, pyruvate, malate, succinateor fumara te as ca rbon source . Mot i l i ty and l ightsensitivity were almost equal under all conditionsexamined. A cetate was chosen as carbon source forfurther experiments because it resulted in fastergrowth than other ca rbon sources .Discrimination thresholds of t!arious strains ofpurple bacteria grown in synthetic medium andin Winogradsky columns

Nine strains of Chromatiaceae were grown inWinogradsky columns and in synthetic medium tomeasure l ight sensitivity (Table 3) . Minimum dis-cr imination thresholds of cells grown in syntheticmedium ranged f rom 0.7% (Chromatium okenii) to1.4% (Chromatium vinosum 1611). Afte r growth inWinogradsky columns all strains except C. okeniiexhibited discrimination thresholds of 2.5% or2.6%. With C. okenii 0 . 7% wa s m e a sure d . Thus ,this strain had the highest sensitivity of all strains

examined in this study, both af ter growth in syn-the t ic medium a nd in a W inogradsky column.Measurements with R. rubrum Nor ten and R.f u l w m Forst showed that neither growth in syn-thetic medium nor growth in Winogradsky columnsprovide a clear advantage with respect to motilityor light sensitivity. The discrimination thresholds ofthe highly motile strains of nonsulfur purple bacteriaamoun ted to va lues be tween 0.8% (R . rubrum H a )and 2 .4% (Rhodospirillum photometricumNTHC132) (Table 4) .The cul tures of 18 strains (e .g. all examined spe-cies of the genera Ectothiorhodospira, Rhodopseu-domonas and Rhodobacter) contained a consider-able number of non-motile cells. Therefore theirdiscrimination thresholds could not be determined.Photophobic reactions of those species, which didnot accum ulaie in the l ight f ield, were dem onst ratedby microscopic inspection.The light sensitivities of the highly motile strainsof purple bac te r ia could be demons tra ted impress-ively by the single irradiation method when a black-and-white picture was projected into a micro-cuve t te . Af te r 5 min exposure, the bacter ia repro-duced the picture exactly (Fig. 6).

DISCUSSIONThe compara t ive s tudy of 34 strains of purplebacteria provided information ( i) on th e aptitude ofthese strains to investigate the mechanism of thephotophobic response, ( i i) on the effect of the

growth o n assay m edia o n the discrimination thres-hold and ( ii i) on the advantages of a new methodto estimate l ight sensitivit ies. ( i) The search forbacterial strains, which are as well or better suitedto investigate the mechanism of the photophobicresponse than those known before, did not revealoutstanding strains. Rhodospirillum rubrum and Rb.sphaeroides were applied as model organisms instudies which aimed at elucidating the molecularmechanisms of phototactic signal transduction(Throm 1968; Harayama and I ino, 1977; Armitageet al . , 1985). (ii) The medium in which the cells areTable 3. Minimum discrimination thresholds of sulfur purple bacteria after growthin synlhetic medium an d in Winogradsky columns

StrainChromatiurn tinosum DChromatium tlinosum 1611Chromatium minutissimum BN 57 1 1Chromatium purpuratum BN, 5500Chromatiurn vinosum A B G OChromatium vinosum NortenChromatiurn tinosurn HardenbergChromatium okeniiThiocysfis iiolaceu 23 11

~ ~~ ~ ~ ~~ ~-.._ ~~~~

Discrimination threshold ( YO )Synthetic growth Winogradsky~. . ~~~~~~~~~~~~~ ~~~ ~~~~

co u m nedium~~~~~~~~~ ~~ ~ ~

0.8 2.51.I 2.6- 2. 51.3 2.60.9 2.51 o 1.50.9 2.50.7 0 .7- 2.5


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814 E. HUSTEDEr al.Table 1. Minimum discrimination thresholds of nonsulfur purple bacteria

DiscriminationStrain threshold ( YO )~~~~ ~. ~~ ~~~

Rhodocyclus reriuis Kiessee 1.6Rhodocyclus renuis Bramwald 1.8Rhodospirillum fulvum Bramwald 1 .?IRhodospirillum fulvum Fors t 0.9:kRhodospirillum photometricum N T H C 132 2.4Rhodospirillum rubrum Ha 0.8Rhodospirillum rubrum Norten 0 . Y~~~~~~ ~~ .Measurements were conducted in the synthetic growth medium.*Grown in a Winogradsky column.Cultures of the fol lowing strains contained a considerable number of non-motile cel ls. Therefore their discrimination thresholds could n o t bedetermined: Rhodobacrer capsulatus K b l , Rhodobacter cupsulatus 6950,Rhodobacrer sphaeroides ATH 2.4.1 ., Rhodobacrer sphaeroides Y . Rho-docyclus gelatinosus 2150, Rhodocyclus tenuis 2761. Rhodocvclus reiiitis366 1 , Rhodomicrobium rmnielii GP, Rhodopseudomonus acidophilu7050. Rhodopseudom otias pulusrris 1850, Rhodopseudoinonas pulusrris

BG, Rhodopseudomonas viridis 2450, Rhodospirillum molischianuni S ,Rhodospirillum rubrum S1

Figure 6. Cells of C. vinosum D reproduced a halftonepicture.The sl ide was projected into the microcuvette. T hepicture was taken after 5 min exposure t ime.

suspended to assay the discrimination threshold isof relatively low importance o n bacterial sensitivity.Cells taken from clouds in Winogradsky columns,considered grown under quasi-natural conditions,do not show higher sensitivities than cells grown insynthetic media and resuspended in various syn-thetic buffer solutions. The fact that light sensitivityin synthetic buffer solutions was found t o be as high( R . r u b r u m H a ) or only little lower (C . vinosumD ) than in the growth medium indicates that nocompound of the growth medium is indispensiblefor the mechanism of photophobic reactions. ( i i i)The single side irradiation method, developed inthis study, has the advantage t hat a single exposure

leads to an approximate value for estimation ofl ight sensitivity. However neither the photographicmethods used to prepare slides with l ight spots ofstepwise different transparencies no r the scanningappara tus t o m easure the t ransparenc ies of fe red theaccuracy required. T he estimations provided by thelatter method consequently have to be sup-plem ented by data obtai ned by Buder 's double-sideirradiation me tho d, which provides highly accuratevalues but requires a whole series of experimentsto o bta in on e s ingle va lue .The discrimination thresholds measured in thiss tudy were lower than those measured in formerstudies. The f irst determination of thresholdsyielded 3% as maxim um sensitivity for C. vinosumand R . rubrum (Schrammeck, 1934). Th e minimumthresholds measured until now were 0.8% fo r C.vinosum and 1% fo r R. rubrum; 2 % were de te r -mined for C. okenii and 2.5% were measured withThiospirillum jenense. For the study performed in1948/50 (Schlegel, 1956) the fo ur species were actu -ally grown in Winogradsky columns. The firstmeasurements with cells of R. r u b r u m grown in asimple synthetic medium brought abou t a minimumdiscrimination threshold of 2 % (Clayton, 1953). Inthe present study advanced growth conditions andimproved conditions of measurement were applied.Strains belonging to four different species exhibiteddiscrimination thresholds below 1% ( C h r o m a t i u mrinosum O.8%, C h r o m a t i u m okenii 0.7'/0, R h o d o -s p i r i l l u m r u b r u m Q.8%, R h o d o s p i r i l l u m f u l v u m0.9% ). M ost of th e oth er suff iciently motile strainsshowed sensitivit ies between 1 and 2 % . Astonish-ingly, the light sensitivities of the highly motilestrains taken from culture collections and of thoserecently isolated from their habitats were not differ-ent. P robably discrimination thresholds between 0.7a nd 1.0% represent the maximum light sensitivityof purple bacter ia .


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Photophobic response of purple bacteria 815Th e elucidation of the photophobic response andthe signal transduction of the purple bacteriarequires concentration of research on a single strain.On the basis of the measurements and observationsmade in this study, R . rubrum an d C . vinosum Dare the organisms of choice to function as models.Due to their growth properties, high motility andhigh light sensitivity they ap pear to be the approp ri-ate species to study the photophobic response b ymeans of population methods.

Acknowledgements-This work was supported by theDeutsche Forschungsgemeinschaft and the Fonds derChemischen Industrie. We wish to thank G.v. Minniger-o d e , G . Beuermann, V . Neuhoff and D . Taube for the iradvice and Karin Schmidt , F. Giffhorn and J. Imhoff forproviding bacterial strains.

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the photophobic response of various sulfur and non-sulfur purple bacteria

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