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Physiological properties of the thermotolerant photosynthetic bacterium, Rhodospirillum centenum
FEMS MicrobiologyLetters 67 (1990) 139-144 Published by Elsevier
139
FEMSLE 03871
Physiological properties of the thermotolerant photosynthetic bacterium, Rhodospirillum centenum Rebecca Stadtwald-Demchiek, F. Rudolf Turner, and Howard Gest Phofoayntheti¢ Bacterfa Group. Departmentof Biology, Indiana University. Bloomington. IN. U.S.A.
Received 29 September 1989 Accepted l October 1989 Key words; RhodospiriUum centenum; Photosynthesis; Tbermotolerance; Cysts
1. S U M M A R Y Rhodospirillum centenum nov. sp., isolated from Thermopolis Hot Springs (Wyoming), is a tberrnotolerant non-sulfur purple photosynthetic bacterium that forms desiccation- and heat-resistant cysts under certain nutritional conditions. Phototrophic growth rate is optimal over the temperature range ca. 39-45°C, and the maximum growth temperature is ca. 47°C. The bacterium requires biotin and vitamin B12, and grows readily in synthetic media. Growth rate, however, is markedly stimulated by unknown organic compounds in Soytone and similar preparations from soybeans. Dried cysts of R. centenum show high resistance to heating at 5 5 - 7 5 * C for 48 h, suggesting that cysts provide a mechanism for survival and species dispersal in natural thermal environmerits.
sample (temperature, ca. 5 5 ° C ) collected at the edge of the source pool at Thermopolis Hot Springs, Wyoming [1]. The bacterium was: enriched using a procedure that is selective for anoxygenic photosynthetic bacteria that fix N 2 readily [2], isolated by conventional methods, and given the species name centenum in recognition of the centennial of the first isolation of a purple bacterium in pure culture ( Rhodospirillum rubrum) by Esmarch in 1887 [3]. R. centenum is particularly unusual among known non-sulfur purple photosynthetic bacteria in that it is capable of producing desiccation- and heat-resistant cysts. The present communication describes various properties of R. centenum not detailed in the initial publication on the bacterium [1].
3. MATERIALS A N D M E T H O D S
Rhodospirillum centenum is a novel photosynthetic bacterium, isolated in 1987 from a water
3.1. Bacterial strain The isolation and general characteristics of R. centenum, strain Favinger/Gest, are described in [1]. The organism is available from the American Type Culture Collection (no. 43720).
Correspondence w: H. G~t, Photosynthetic Bacteria Group, Deparament of Biology, Indiana University, Bloomington, IN 47405. U.S.A.
3.Z Media Unless otherwise noted, R. centenum was grown in a medium designated as CENMED, which con-
2. I N T R O D U C T I O N
0378-1097/90/$03.50 © 1990 Fed©rationof European MicrobiologicalSocieties
140 tains per liter of dcionized water: 2.2 g sodium pyruvatc, 0.9 g K2HPO4, 0.6 g KH2PO4, 1 g NH+CI, 5 mg disodium EDTA, 200 m g MgSO+. 7H20, 1 ml trace element solution (see [4]), 75 mg CaCI 2 • 2H20, 2 ml chelated iron solution (prepare by dissolving 1 g F c C I 2 ' 4 H 2 0 and 2 g disodium EDTA in 1 liter deionized water, and adding 3 ml concentrated HCI), 20 # g vitamin B12, 15 btg biotin, 0.5 g Na2S203.5H20; pH adjusted to 6.8 with NaOH. When butyratc was used as the carbon source, pyruvate was omitted from C E N M E D and replaced with 20 mM sodium butyratc.
600
/7-
q00
200
lO0
50
3. 4. Other procedures
Photometric measurements of culture density were made using a Klett-Summerson photometer fitted with a no. 66 filter. In vivo spectra of photosynthetic pigments were determined as described by Sojka et ah [5]. For the Fig. 3 electron micrograph, the cells were treated essentially as described in [6].
4. RESULTS A N D DISCUSSION 4.1. Nmritiou of R, ceuteuum R. cemenum grows as an anaerobic phototroph
or, alternatively, as a heterotrophic aerobe in darkness. Biotin and vitamin B12 are required for growth in synthetic media, for example, in C E N M E D (carbon source, pyruvate) [1], and growth is markedly stimulated by supplementation with Soytone and other preparations from
CENMED + S0yt0ne CENMED inciner~l~ed Soytone
3.3. Incubation conditions
Except for dark/aerobic growth and experiments dealing with temperature-dependent growth characteristics, cultures were incubated in a light cabinet maintained at ca. 37°C. Saturating illumination was provided by banks of incandescent Lumiline lamps. For determination of optim u m temperature range and maximum temperature of growth, cultures (in completely-filled screw cap tubes of 17 ml capacity) were incubated in glass-sided water baths and illuminated with the light sources indicated. Dark/aerobic cultures were grown at a temperature of 37°C.
• o
~ ~
~-
,~
,'
Time (hours)
Fig. I. Effect of Soyton¢ on growth of R. cetne~lRm. The cultures were grown photouophically at 37°C. Where indirated, CENMED medium was supplen~nted with 0.4~ Soytone (0) or the equivalent amount of the inorganicconstituents of Soytone (~; residue from oxidation of Soytone in a Parr bomb).
soybeans. This effect is illustrated in Fig. 1. Numerous organic compounds, of various kinds, have been tested in the hope of identifying specific growth stimulants, thus far without success. Little or no effect was found with 20 m M acetate, xylose, mannitoL glucose, fructose, galactos¢; Casamino acids (Difco); the amino acid pools specified by Juni et aL [7]; yeast extract; tryptone; lipoi¢ acid, or various other vitamins required by different species of non-sulfur purple bacteria. To confirm that the active components of Soytone are organic, we conducted tests with the inorganic residue of Soytone oxidized with 100% 02 in a Parr bomb. The inorganic residue had no effect on the rate of photosynthetic growth in C E N M E D medium (see Fig. 1). It is noteworthy that R. cemenum, in contrast to most other non-sulfur purple bacteria, is unable to use malate or other C4 dicarboxylic acids as carbon sources,
141
i
i
i
i
875
d _o
.?
z
I
500
i
I
i
/
i
t
i
600 70o 800 Wavelength (nm) Fig. 2. Absorptionspectrumof R. cenrenum cells suspendedin 30% bovinesel~m albumin. 4.2. Photosynthetic pigments The in vivo absorption spectrum of R. centenum cells grown phototrophically is shown in Fig. 2. Absorbaney peaks are observed in the infra-red region at 800 and 875 nm, at 587 nm, and at wavelengths absorbed by carotenoids. The spectrum is very simlar to that of R. rubrum [8], indicating that the major photopigments are bacteriochlorophyll a and carotenoids of the "normal spirilloxanthin series'. Electron micrographs of thin sections of R. centenum disclosed that the photosynthetic membranes of this organism are present in the cell periphery in the form of concentric lamellae (Fig. 3). A similar membrane configuration has been observed in R. salexigeos [9}; other Rhod,~spirillum species contain vesicular or other types of membrane assemblies. The formation of photopigmen[s in typical non-sulfur purple bacteria is subject to regulation by molecular oxygen. Ordinarily, 02 inhibits pigment synthesis, and this is readily observed by incubating agar Petri dish cultures aerobically in darkness after a period of anaerobic growth in the light. During the dark/aerobic incubation, growth
continues (for example, in Rhodobacter capsulatus or sphaeroides) and due to the 02 effect, a nonpigmented 'halo" of growth develops around the pigmented center of each colony. With R. cemenum, however, the colonies enlarge during the dark/aerobic incubation, but halos do not develop. This observation indicates the absence of stringent Oz-regulation of photopigment synthesis in R. cemenum. 4+3. Optimum and maximum growth temperatures The dependence of photosynthetic growth rate of R. centenum on temperature is shown in Fig. 4. It can be seen that growth rate is optimal over the range ca. 39-4,~ o C, and then declines rapidly with further increase in temperature. The maximum temperature permitting growth is approximately 47°C. Similar results were recently observed by Resnick and Madigan |10] with a P,hodopseudomonas strain isolated from a New Mexico hot spring microbial mat. Thus, it is evident that non-sulfur purple bacteria capable of growing at
Fig. 3. Electronmicr~p'aphof ultralhin section of R. cenlenum; markerbar, 0.5 ~*m.
142 the lower fringe of the thermophilic temperature range occur in natural thermal waters. 4.4. Heat resistance o f R. centenum cysts
During dark/aerobic growth in media containing butyrate as the carbon source, R. centenum produces thick-walled cysts (Fig. 5), The formation of cysts appears to be directly eorre]ated with the formation of high levels of poly-/~-hydroxybutyrate (of the order of 30% of the dry weight) [1]. We have found that aerobic (dark) growth on butyrate agar slants consistently leads to abundant cyst formation, and such preparations were used to test cyst thermostability. Cysts are not formed to a significant extent in R. centenum cultures grown photosynthetically on butyrate (plus bicarbonate), and cells from cultures of this kind are designated as "vegetative'. The alternative cell forms were filtered onto appropriate discs and dried for one day at 37°C. The discs were then incubated at 5 5 ° C for various lengths of time, and then placed on an agar growth medium that permitted rapid anaerobic phototrophic growth of survivors (in Gas Pak jars, at ca. 30-33°C). Even after 36-48 h of heating, discs of cysts showed extensive growth in the survival test. With vegeta-
0,3
i
t
l
Fig. 5. Scanning electron micrograph of glular~dehydc-fixcd critical point.dried cysls of R. cenlenum; marker bar, l Fro.
tire cells, on the other hand, a negligible fraction of the cells survived 12 h of heating, Almost identical results were observed in other experiments with heating temperatures of 6 5 ° C and 75°C.
i
0.2 5, C O N C L U D I N G R E M A R K S
o Q 0.1
I
30
35
40
Temperature
45
I
50
I'C)
Fig. 4. Photonophic growth rate of R ¢enlenum as a function of lemperalure. Cells were grown in CENMED synthetic rn~ dium,
The descriptions of non-sulfur purple bacteria in volume 3 of Ikrgey's Manual of Systematic Bacteriology (published in 1989) indicate that practically all of these organisms have growth optima between 30 and 35 o C, or at lower temperature, The only exceptions noted are Rhodospirillure salexigens ( 4 0 ° C ) and Rhodospirillum saliaarum (42°C). The presence of viable nonsulfur purple bacteria in hot spring waters at temperatures of 55 to 0 0 ° C [1,11] and the ability of some pure culture isolates to grow at 4 4 ° C [12] or 4 7 ° C [10 and this paper], however, indicate
143 t h a t there is a p h y s i o l o g i c a l class of these o r g a n i s m s that is m o r e t h e r m o t o l e r a n t t h a n the typical m e s o p h i l c s that h a v e b e e n used extensively for r e s e a r c h o n bacterial p h o t o s y n t h e s i s m e c h a n i s m s . C a r e f u l e x a m i n a t i o n o f n e w isolates f r o m h o t s p r i n g s c a n b ¢ expt,,,ted to disclose a d d i t i o n a l e x a m p l e s o f t h e r m o t o l e r a n t s t r a i n s useful for further studies.
ACKNOWLEDGEMENTS T h i s research w a s s u p p o r t e d by g r a n t D M B 8415291 f r o m t h e U . S . N a t i o n a l Science F o u n d a tion. W e t h a n k Jeffrey Favinger for technical assistance.
REFERENCES lit Favinger, J., Stadtwald, R. and Gest. H. (1989) A. v. Leeuwenhoek J. Microbiol. 55, 291-296.
[2] Gest, H., Favinger, J.L and Madigan, M.T. (1985) FEMS Microbiol. Ecol. 31.317-322. [3l Esmarch, E. (1887) Centralbl. Bacteriol. u. Parasitenkunde 1. 225-230. 14[ Fry. B.. Gesl, H. and Hayes. J.M. {1984) FEMS Microbiol. Len. 22, 283-287. [5] Sojka, GA,, Freeze, H.H. and Gest. H. (1970) Arch. Biochem. Biophys. 136. 578-580. 16] GCSL H. and Favinger. J.L. (1983) Arch. Microbiol. 136, 11-16. [71 Juni, E., Heym, G.A. and Bradley, [LA. (1984) J. Baeteriol. 160. 958-965. [8] BicbL H. and Drcws. G. (1969) Zealr. BakL Parasil. lnfektionskr, u. Hyg. 123. 425-452. [9] Drews, G. (1981) Arch. Microbiol. 130, 325-327. [10J Resnick, S.M. and Madigan. M.T. (1989) FEMS Miclrobiol. Len. 65. 165-170. [11] Gorlenko, V.M.. Kompantseva. E.L and lh.,chkova, N.N. ~1985) Mikrobiologiya 54, 848-853; English translation in Microbiolosy, March 1986, pp. 681-685. [121 Mangels, L.A., Favinger, J.L., Madigan, M.T. and Gest, H. (1986) FEMS MicrobioL LetL 36, 99-104.
139
FEMSLE 03871
Physiological properties of the thermotolerant photosynthetic bacterium, Rhodospirillum centenum Rebecca Stadtwald-Demchiek, F. Rudolf Turner, and Howard Gest Phofoayntheti¢ Bacterfa Group. Departmentof Biology, Indiana University. Bloomington. IN. U.S.A.
Received 29 September 1989 Accepted l October 1989 Key words; RhodospiriUum centenum; Photosynthesis; Tbermotolerance; Cysts
1. S U M M A R Y Rhodospirillum centenum nov. sp., isolated from Thermopolis Hot Springs (Wyoming), is a tberrnotolerant non-sulfur purple photosynthetic bacterium that forms desiccation- and heat-resistant cysts under certain nutritional conditions. Phototrophic growth rate is optimal over the temperature range ca. 39-45°C, and the maximum growth temperature is ca. 47°C. The bacterium requires biotin and vitamin B12, and grows readily in synthetic media. Growth rate, however, is markedly stimulated by unknown organic compounds in Soytone and similar preparations from soybeans. Dried cysts of R. centenum show high resistance to heating at 5 5 - 7 5 * C for 48 h, suggesting that cysts provide a mechanism for survival and species dispersal in natural thermal environmerits.
sample (temperature, ca. 5 5 ° C ) collected at the edge of the source pool at Thermopolis Hot Springs, Wyoming [1]. The bacterium was: enriched using a procedure that is selective for anoxygenic photosynthetic bacteria that fix N 2 readily [2], isolated by conventional methods, and given the species name centenum in recognition of the centennial of the first isolation of a purple bacterium in pure culture ( Rhodospirillum rubrum) by Esmarch in 1887 [3]. R. centenum is particularly unusual among known non-sulfur purple photosynthetic bacteria in that it is capable of producing desiccation- and heat-resistant cysts. The present communication describes various properties of R. centenum not detailed in the initial publication on the bacterium [1].
3. MATERIALS A N D M E T H O D S
Rhodospirillum centenum is a novel photosynthetic bacterium, isolated in 1987 from a water
3.1. Bacterial strain The isolation and general characteristics of R. centenum, strain Favinger/Gest, are described in [1]. The organism is available from the American Type Culture Collection (no. 43720).
Correspondence w: H. G~t, Photosynthetic Bacteria Group, Deparament of Biology, Indiana University, Bloomington, IN 47405. U.S.A.
3.Z Media Unless otherwise noted, R. centenum was grown in a medium designated as CENMED, which con-
2. I N T R O D U C T I O N
0378-1097/90/$03.50 © 1990 Fed©rationof European MicrobiologicalSocieties
140 tains per liter of dcionized water: 2.2 g sodium pyruvatc, 0.9 g K2HPO4, 0.6 g KH2PO4, 1 g NH+CI, 5 mg disodium EDTA, 200 m g MgSO+. 7H20, 1 ml trace element solution (see [4]), 75 mg CaCI 2 • 2H20, 2 ml chelated iron solution (prepare by dissolving 1 g F c C I 2 ' 4 H 2 0 and 2 g disodium EDTA in 1 liter deionized water, and adding 3 ml concentrated HCI), 20 # g vitamin B12, 15 btg biotin, 0.5 g Na2S203.5H20; pH adjusted to 6.8 with NaOH. When butyratc was used as the carbon source, pyruvate was omitted from C E N M E D and replaced with 20 mM sodium butyratc.
600
/7-
q00
200
lO0
50
3. 4. Other procedures
Photometric measurements of culture density were made using a Klett-Summerson photometer fitted with a no. 66 filter. In vivo spectra of photosynthetic pigments were determined as described by Sojka et ah [5]. For the Fig. 3 electron micrograph, the cells were treated essentially as described in [6].
4. RESULTS A N D DISCUSSION 4.1. Nmritiou of R, ceuteuum R. cemenum grows as an anaerobic phototroph
or, alternatively, as a heterotrophic aerobe in darkness. Biotin and vitamin B12 are required for growth in synthetic media, for example, in C E N M E D (carbon source, pyruvate) [1], and growth is markedly stimulated by supplementation with Soytone and other preparations from
CENMED + S0yt0ne CENMED inciner~l~ed Soytone
3.3. Incubation conditions
Except for dark/aerobic growth and experiments dealing with temperature-dependent growth characteristics, cultures were incubated in a light cabinet maintained at ca. 37°C. Saturating illumination was provided by banks of incandescent Lumiline lamps. For determination of optim u m temperature range and maximum temperature of growth, cultures (in completely-filled screw cap tubes of 17 ml capacity) were incubated in glass-sided water baths and illuminated with the light sources indicated. Dark/aerobic cultures were grown at a temperature of 37°C.
• o
~ ~
~-
,~
,'
Time (hours)
Fig. I. Effect of Soyton¢ on growth of R. cetne~lRm. The cultures were grown photouophically at 37°C. Where indirated, CENMED medium was supplen~nted with 0.4~ Soytone (0) or the equivalent amount of the inorganicconstituents of Soytone (~; residue from oxidation of Soytone in a Parr bomb).
soybeans. This effect is illustrated in Fig. 1. Numerous organic compounds, of various kinds, have been tested in the hope of identifying specific growth stimulants, thus far without success. Little or no effect was found with 20 m M acetate, xylose, mannitoL glucose, fructose, galactos¢; Casamino acids (Difco); the amino acid pools specified by Juni et aL [7]; yeast extract; tryptone; lipoi¢ acid, or various other vitamins required by different species of non-sulfur purple bacteria. To confirm that the active components of Soytone are organic, we conducted tests with the inorganic residue of Soytone oxidized with 100% 02 in a Parr bomb. The inorganic residue had no effect on the rate of photosynthetic growth in C E N M E D medium (see Fig. 1). It is noteworthy that R. cemenum, in contrast to most other non-sulfur purple bacteria, is unable to use malate or other C4 dicarboxylic acids as carbon sources,
141
i
i
i
i
875
d _o
.?
z
I
500
i
I
i
/
i
t
i
600 70o 800 Wavelength (nm) Fig. 2. Absorptionspectrumof R. cenrenum cells suspendedin 30% bovinesel~m albumin. 4.2. Photosynthetic pigments The in vivo absorption spectrum of R. centenum cells grown phototrophically is shown in Fig. 2. Absorbaney peaks are observed in the infra-red region at 800 and 875 nm, at 587 nm, and at wavelengths absorbed by carotenoids. The spectrum is very simlar to that of R. rubrum [8], indicating that the major photopigments are bacteriochlorophyll a and carotenoids of the "normal spirilloxanthin series'. Electron micrographs of thin sections of R. centenum disclosed that the photosynthetic membranes of this organism are present in the cell periphery in the form of concentric lamellae (Fig. 3). A similar membrane configuration has been observed in R. salexigeos [9}; other Rhod,~spirillum species contain vesicular or other types of membrane assemblies. The formation of photopigmen[s in typical non-sulfur purple bacteria is subject to regulation by molecular oxygen. Ordinarily, 02 inhibits pigment synthesis, and this is readily observed by incubating agar Petri dish cultures aerobically in darkness after a period of anaerobic growth in the light. During the dark/aerobic incubation, growth
continues (for example, in Rhodobacter capsulatus or sphaeroides) and due to the 02 effect, a nonpigmented 'halo" of growth develops around the pigmented center of each colony. With R. cemenum, however, the colonies enlarge during the dark/aerobic incubation, but halos do not develop. This observation indicates the absence of stringent Oz-regulation of photopigment synthesis in R. cemenum. 4+3. Optimum and maximum growth temperatures The dependence of photosynthetic growth rate of R. centenum on temperature is shown in Fig. 4. It can be seen that growth rate is optimal over the range ca. 39-4,~ o C, and then declines rapidly with further increase in temperature. The maximum temperature permitting growth is approximately 47°C. Similar results were recently observed by Resnick and Madigan |10] with a P,hodopseudomonas strain isolated from a New Mexico hot spring microbial mat. Thus, it is evident that non-sulfur purple bacteria capable of growing at
Fig. 3. Electronmicr~p'aphof ultralhin section of R. cenlenum; markerbar, 0.5 ~*m.
142 the lower fringe of the thermophilic temperature range occur in natural thermal waters. 4.4. Heat resistance o f R. centenum cysts
During dark/aerobic growth in media containing butyrate as the carbon source, R. centenum produces thick-walled cysts (Fig. 5), The formation of cysts appears to be directly eorre]ated with the formation of high levels of poly-/~-hydroxybutyrate (of the order of 30% of the dry weight) [1]. We have found that aerobic (dark) growth on butyrate agar slants consistently leads to abundant cyst formation, and such preparations were used to test cyst thermostability. Cysts are not formed to a significant extent in R. centenum cultures grown photosynthetically on butyrate (plus bicarbonate), and cells from cultures of this kind are designated as "vegetative'. The alternative cell forms were filtered onto appropriate discs and dried for one day at 37°C. The discs were then incubated at 5 5 ° C for various lengths of time, and then placed on an agar growth medium that permitted rapid anaerobic phototrophic growth of survivors (in Gas Pak jars, at ca. 30-33°C). Even after 36-48 h of heating, discs of cysts showed extensive growth in the survival test. With vegeta-
0,3
i
t
l
Fig. 5. Scanning electron micrograph of glular~dehydc-fixcd critical point.dried cysls of R. cenlenum; marker bar, l Fro.
tire cells, on the other hand, a negligible fraction of the cells survived 12 h of heating, Almost identical results were observed in other experiments with heating temperatures of 6 5 ° C and 75°C.
i
0.2 5, C O N C L U D I N G R E M A R K S
o Q 0.1
I
30
35
40
Temperature
45
I
50
I'C)
Fig. 4. Photonophic growth rate of R ¢enlenum as a function of lemperalure. Cells were grown in CENMED synthetic rn~ dium,
The descriptions of non-sulfur purple bacteria in volume 3 of Ikrgey's Manual of Systematic Bacteriology (published in 1989) indicate that practically all of these organisms have growth optima between 30 and 35 o C, or at lower temperature, The only exceptions noted are Rhodospirillure salexigens ( 4 0 ° C ) and Rhodospirillum saliaarum (42°C). The presence of viable nonsulfur purple bacteria in hot spring waters at temperatures of 55 to 0 0 ° C [1,11] and the ability of some pure culture isolates to grow at 4 4 ° C [12] or 4 7 ° C [10 and this paper], however, indicate
143 t h a t there is a p h y s i o l o g i c a l class of these o r g a n i s m s that is m o r e t h e r m o t o l e r a n t t h a n the typical m e s o p h i l c s that h a v e b e e n used extensively for r e s e a r c h o n bacterial p h o t o s y n t h e s i s m e c h a n i s m s . C a r e f u l e x a m i n a t i o n o f n e w isolates f r o m h o t s p r i n g s c a n b ¢ expt,,,ted to disclose a d d i t i o n a l e x a m p l e s o f t h e r m o t o l e r a n t s t r a i n s useful for further studies.
ACKNOWLEDGEMENTS T h i s research w a s s u p p o r t e d by g r a n t D M B 8415291 f r o m t h e U . S . N a t i o n a l Science F o u n d a tion. W e t h a n k Jeffrey Favinger for technical assistance.
REFERENCES lit Favinger, J., Stadtwald, R. and Gest. H. (1989) A. v. Leeuwenhoek J. Microbiol. 55, 291-296.
[2] Gest, H., Favinger, J.L and Madigan, M.T. (1985) FEMS Microbiol. Ecol. 31.317-322. [3l Esmarch, E. (1887) Centralbl. Bacteriol. u. Parasitenkunde 1. 225-230. 14[ Fry. B.. Gesl, H. and Hayes. J.M. {1984) FEMS Microbiol. Len. 22, 283-287. [5] Sojka, GA,, Freeze, H.H. and Gest. H. (1970) Arch. Biochem. Biophys. 136. 578-580. 16] GCSL H. and Favinger. J.L. (1983) Arch. Microbiol. 136, 11-16. [71 Juni, E., Heym, G.A. and Bradley, [LA. (1984) J. Baeteriol. 160. 958-965. [8] BicbL H. and Drcws. G. (1969) Zealr. BakL Parasil. lnfektionskr, u. Hyg. 123. 425-452. [9] Drews, G. (1981) Arch. Microbiol. 130, 325-327. [10J Resnick, S.M. and Madigan. M.T. (1989) FEMS Miclrobiol. Len. 65. 165-170. [11] Gorlenko, V.M.. Kompantseva. E.L and lh.,chkova, N.N. ~1985) Mikrobiologiya 54, 848-853; English translation in Microbiolosy, March 1986, pp. 681-685. [121 Mangels, L.A., Favinger, J.L., Madigan, M.T. and Gest, H. (1986) FEMS MicrobioL LetL 36, 99-104.