Worfd Journal
of Microbioiogy
and Biotechnology
9, 160-163
Effect of carbon monoxide on metabolism and ultrastructure of carboxydobacteria T.G. Volova,* O.A. Guseinov, G.S. Kalacheva, S.E. Medvedeva and A.A. Puzyr Growth of Seliberia carboxydohydrogena was inhibited by CO at 10 to 40% (v/v), resulting in increased substrate utilization and enhanced synthesis of cytochromes and cyclopropane and saturated fatty acids. The bacteria showed increased formation of new membrane structures, with pronounced folding of their cell walls. Key words: Carbon monoxide,
carboxydobacteria,
cytochromes,
The metabolism of carboxydobacteria growing with CO is based on their enzymatic oxidation of CO to CO, and their resistance to the inhibitory effects of CO (Sanjieva & Zavarzin 1971; Nozhevnikova 1974; Meyer & Schlegel 1983). We have found no information on the peculiarities of the ultrastructure of carboxydobacteria or on the effect of CO on the growth, chemical composition and enzymatic activity of the cells. In this paper, we report the results of our studies on the effect of carbon monoxide on the growth kinetics and the peculiarities of metabolism and ultrastructure of carboxydobacteria.
Materials
and Methods
carboxydohydragena Z-1062 (Sanjieva & Zavarzin 1971) was grown autotrophically, in continuous culture in an 8 1 fermenter, containing 2 1 of mineral salts medium (Meyer & Schlegel 1983), in both chemostat and turbidostat regimes (Volova 1980). The dilution rate was 0.05 to 0.40 hK’. The gas mixture Sefiberia
was 10% (v/v)
CO,,
16%
(v/v)
0,.
0 to 40%
(v/v)
CO,
and the
balance was H,. Gas was supplied to the fermenter in continuous flow at 4 vol/vol.min. Analysis of the culture and chemical composition of the cells were estimated by standard procedures. Cells were harvested by centrifugation at 12,000 x g. The respiratory activity of bacteria was determined by measuring the 0, uptake rate of the culture using an oxygen electrode in the fermenter. For enzymatic determinations, harvested washed cells were disrupted ultrasonically and the cell debris removed by
T.G. Volova, O.A. Guseinov, G.S. Kalacheva, S.E. Medvedeva and A.A. Siberian Branch, Krasnoyarsk 660036. Russia; fax: (391 2) 22 25 56. * Corresponding author.
@I 1993 Rapid Communications
160
World joumd
of Oxford
of Microbiology
Ltd
and Biotechnology, Vol 9, 1993
hydrogenase, centrifugation
Seliberia carboxydohydrogena. at 30,000
extracts was determined
x g. Hydrogenase
activity
spectrophotometrically
in the cell-free using Methyl
Viologen as electron acceptor at 600 nm (Gogotov 1979). Cytochrome content was measured spectrophotometrically as described by Probst & Schlegel (1976). For cytological examinations, the cells were fixed in glutaraldehyde and OsO,, dehydrated in an ethanol series and embedded in an Epon 812 and Araldite M mixture. Sections were cut on a ultramicrotome and studied under an electron microscope.
Results
and Discussion
Seliberia carboxydohydrogena Z-1062 could grow autotrophically without CO in the fermenter at a specific growth rate of 0.38 h-l and produced biomass with a protein content of up to 60% dry weight. The average intracellular concentration of lipids was 10% of dry weight. The lipid saturation factor was about 0.68 and the content of the reserve substance, poly-P-hydroxybutyrate, was minimal ( < 0.1% w/w). The hydrogenase activity of the cell-free extracts of carboxydobacteria, grown in CO-free continuous culture at maximum growth rates was 300 pmol H,/min.g of protein. Cytological examinations showed that the ultrastructure of 5’. curborydohydrogena Z-1062 was, in general, typical of other Gram-negative bacteria (Figure la,b). The cells had no inner membrane structures. Small volutin grains (up to five grains per cell section) with sharp boundaries were seen (Figure lb). CO is a respiratory poison and the critical $0 value for S. carboxydohydrogena in the turbidostat was 10% (v/v). Further
increase
of pC0
decreased
the specific
growth
rate;
at 40% (v/v) CO the specific growth rate was only 0.06 h-‘. The inhibition of the specific growth rate by CO was linear
Effect of CO on carboxydobacteria
Figure 1. Morphology and ultrastructure of carboxydobacteria under optimum cultivation conditions (a,b) and on medium with CO (c,d,e,f). om--Outer membrane; n--nucleoid; M--intracellular membrane; V--volutin inclusion; EDI---electron-dense inclusions in nucleoid; etv--electron-transparent vacuoles in cytoplasm. Marker bars represent 0.5 pm.
World Journalof Microbiology and Bio~echnology, Val 9, 199J
161
T.G. Volova et al. Table
1. Composition
Specific growih rate W’)
co concentration (% v/v) 0 10 20 30 40
Values ND-Not
l
of carboxydobacteria
of four separate
varying
Biomass
0.38 0.36 0.23 0.16 0.06
are means determined.
under
experiments
concentrations yield
(g/g)
World Journal
of Microbiology
Chemical
culture.’ cell
compositlon
Yh
Yo2
Protein
Lipld
2.0 1.7 1.4 1.0 0.7
0.26 0.16 0.13 0.09 0.04
56.7 59.5 58.0 59.2 60.2
9.1 11.0 10.0 11.0 ND
yielding
consistent
up to 25% (v/v) CO but at higher concentrations the nature of the inhibition changed qualitatively and the cells showed multiple changes. CO acted on carboxydobacterial cell respiration. The respiratory activity of carboxydobacteria, grown at non-inhibiting CO concentrations (< 10% v/v), was 0.2 to 0.35 pmol O,/min.mg dry weight, with endogenous respiration equal to 0.02 pmol O,/min.mg dry weight. With CO up to 30% (v/v), the respiration activity decreased to 0.120 pmol O,/min.mg dry weight. With increasing inhibitor concenkations, the specific consumption of H, and 0, to synthesize the biomass grew and the efficiency of their utilization decreased (see Table I). Examination of the bacterial chemical composition also enabled the CO effect to be quantified (see Table I). The ratio of nitrogen-containing and reserve components in carboxydobacteria did not change significanfly under growth inhibition by CO. Considerable inhibition of carboxydobacteria growth by high CO concentrations (10 to 40% v/v) did not decrease the cell protein content or lead to significant accumulation of poly$-hydroxybutyrate. The total cell content of lipids remained about 10% (w/w), but the composition of the fatty acids changed somewhat (see Table 2); the bacteria produced more saturated and cyclopropane fatty acids instead of unsaturated fatty acids at high CO concentrations. This effect probably protects cell structures from the damaging effect of the inhibitor. The hydrogenase activity of S. carboxydohydrogena was maximal at 20% (v/v) CO, (see Table 3). With increasing concentrations of CO, cytochromes a and b (but not c) increased (see Table 3). Cells grown without CO showed little evidence of an inner membrane structure. However, in the presence of CO, inner membranes were clearly formed (Figure Ic,e). Characteristically for CO-inhibited carboxydobacteria, electron-transparent vacuoles were formed in the cytoplasm (Figure If). The presence of CO in the cultural medium also resulted in changes in the cell surface profile. Compared with the cells grown under optimal conditions, which had a
162
of CO in turbidostat
and Biotechnology. Vol 9, 1993
results.
Standard
deviations
(% of dry weight) Poly-fi-hydroxybulyrate Trace 0.2 ND 2.2 1.2
did not exceed
10% of values
given
smooth outer membrane, cells grown with CO had numerous deep folds in their outer membranes (Figure Ia,d,e). The consequent increase in cell surface area seems to create more favourable conditions for the cells to consume the substrate. Thus, the carboxydobacteria S. carboxydohydrogena, having a CO-resistant hydrogenase, were specifically resistant to CO. The bacteria enhanced their production of cytochromes and enlarged the area of their membranes at high CO concentrations. As a result, the bacteria used more energy to convert substrate into biomass and although they continued to grow at 40% (v/v) CO, biomass yield was greatly diminished.
Table 2. Composiiion of fatty acids carboxydobacteria grown in varying continuous culture.* Fatty
acidt
CO concentration 0
14:o 15:o 15:l 16:0 16:l 17:o cyc17:o 18:0 18:l cyc19:o Ratio
(w/w)
of lipids (% w/w) CO concentrations
0.6 0.3 0.8 35.6 40.5 Trace 2.2 1.1 17.6
of in
(% v/v)
10
20
30
None 0.5 Trace 30.7 35.1 Trace 8.1 2.7 22.5 0.4
None 0.4 Trace 34.4 36.9 Trace 9.5 2.2 16.6 Trace
None 0.7 Trace 33.7 36.0 Trace 9.5 1.8 17.9 0.4
acids 0.86
0.87
of saturated to unsaturated 0.68 0.74
fatty
‘Values are means of four separate experiments yielding consistent results. Standard deviations did not exceed 5% of values given. t The numbers before and after the colon indicate, respectively, the number of carbon atoms and double bonds. Cyc-cyclopropane fatty acid.
Effect of CO on carboxydobacferia
Table 3. Concentration of cytochromes at various CO concentrations.’ co concentration (% v/v)
Cytochrome
0 10 20 30 40
0.60 0.60 0.66 0.60 0.61
*Values
and
concentration
are
means
of four
(nmollmg
0.05 0.06 0.03 0.03 0.04 separate
0.45 0.50 0.54 0.57 0.69
* f * & f
of carboxydobacteria
(plmol
C
0.05 0.05 0.03 0.03 0.03
0.09 0.09 0.09 0.12 0.09
experiments
yielding
References G ogotov,
activity
protein)
b
a k f f f +
enzymatic
I.N. 1979 Hydrogenases of microorganisms. Uspekhi Mikrobiologii 14, 3-27. (In Russian.) Meyer, 0. & Schlegel, H. 1983 Biology of aerobic carbon monoxide-oxidizing bacteria. Annd Review of Microbiology 3 7, 277-310. Nozhevnikova, A.N. 1974 Carboxydobacteria (bacteria, oxidizing carbon monoxide). PhD Thesis. Institute of Microbiology, USSR Academy of Sciences, Moscow. (In Russian.) Probst, I. & Schlegel, H. 1976 Respiratory components and oxidase
f 0.01 + 0.01 * 0.01 * 0.01
consistent
Activity of hydrogenase H,/mln mg protein) 300 300 500 140 120
* 0.01
grown
* f f + *
40 40 40 20 20
results.
activities in Ah&genes eutraph~, Biachimicu et Biophysicu Ada 440, 412-428. Sanjieva, E.U. & Zavarzin, G.A. 1971 Bacteria oxidizing carbon monoxide. Dokludy Akudemii Nauk SSSR 196, 9X-959. (In Russian.) Volova, T.G. 1980 Autotrophic continuous cultivation of Seliberiu curboxydohydrogena. Microbiologiyu 49, 20-24. (In Russian.)
(Received in revised form 8 August 1992)
World Jowml
of Microbiology
1992; accepted 12 August
and Biotechnology. Vol 9, 1993
163
of Microbioiogy
and Biotechnology
9, 160-163
Effect of carbon monoxide on metabolism and ultrastructure of carboxydobacteria T.G. Volova,* O.A. Guseinov, G.S. Kalacheva, S.E. Medvedeva and A.A. Puzyr Growth of Seliberia carboxydohydrogena was inhibited by CO at 10 to 40% (v/v), resulting in increased substrate utilization and enhanced synthesis of cytochromes and cyclopropane and saturated fatty acids. The bacteria showed increased formation of new membrane structures, with pronounced folding of their cell walls. Key words: Carbon monoxide,
carboxydobacteria,
cytochromes,
The metabolism of carboxydobacteria growing with CO is based on their enzymatic oxidation of CO to CO, and their resistance to the inhibitory effects of CO (Sanjieva & Zavarzin 1971; Nozhevnikova 1974; Meyer & Schlegel 1983). We have found no information on the peculiarities of the ultrastructure of carboxydobacteria or on the effect of CO on the growth, chemical composition and enzymatic activity of the cells. In this paper, we report the results of our studies on the effect of carbon monoxide on the growth kinetics and the peculiarities of metabolism and ultrastructure of carboxydobacteria.
Materials
and Methods
carboxydohydragena Z-1062 (Sanjieva & Zavarzin 1971) was grown autotrophically, in continuous culture in an 8 1 fermenter, containing 2 1 of mineral salts medium (Meyer & Schlegel 1983), in both chemostat and turbidostat regimes (Volova 1980). The dilution rate was 0.05 to 0.40 hK’. The gas mixture Sefiberia
was 10% (v/v)
CO,,
16%
(v/v)
0,.
0 to 40%
(v/v)
CO,
and the
balance was H,. Gas was supplied to the fermenter in continuous flow at 4 vol/vol.min. Analysis of the culture and chemical composition of the cells were estimated by standard procedures. Cells were harvested by centrifugation at 12,000 x g. The respiratory activity of bacteria was determined by measuring the 0, uptake rate of the culture using an oxygen electrode in the fermenter. For enzymatic determinations, harvested washed cells were disrupted ultrasonically and the cell debris removed by
T.G. Volova, O.A. Guseinov, G.S. Kalacheva, S.E. Medvedeva and A.A. Siberian Branch, Krasnoyarsk 660036. Russia; fax: (391 2) 22 25 56. * Corresponding author.
@I 1993 Rapid Communications
160
World joumd
of Oxford
of Microbiology
Ltd
and Biotechnology, Vol 9, 1993
hydrogenase, centrifugation
Seliberia carboxydohydrogena. at 30,000
extracts was determined
x g. Hydrogenase
activity
spectrophotometrically
in the cell-free using Methyl
Viologen as electron acceptor at 600 nm (Gogotov 1979). Cytochrome content was measured spectrophotometrically as described by Probst & Schlegel (1976). For cytological examinations, the cells were fixed in glutaraldehyde and OsO,, dehydrated in an ethanol series and embedded in an Epon 812 and Araldite M mixture. Sections were cut on a ultramicrotome and studied under an electron microscope.
Results
and Discussion
Seliberia carboxydohydrogena Z-1062 could grow autotrophically without CO in the fermenter at a specific growth rate of 0.38 h-l and produced biomass with a protein content of up to 60% dry weight. The average intracellular concentration of lipids was 10% of dry weight. The lipid saturation factor was about 0.68 and the content of the reserve substance, poly-P-hydroxybutyrate, was minimal ( < 0.1% w/w). The hydrogenase activity of the cell-free extracts of carboxydobacteria, grown in CO-free continuous culture at maximum growth rates was 300 pmol H,/min.g of protein. Cytological examinations showed that the ultrastructure of 5’. curborydohydrogena Z-1062 was, in general, typical of other Gram-negative bacteria (Figure la,b). The cells had no inner membrane structures. Small volutin grains (up to five grains per cell section) with sharp boundaries were seen (Figure lb). CO is a respiratory poison and the critical $0 value for S. carboxydohydrogena in the turbidostat was 10% (v/v). Further
increase
of pC0
decreased
the specific
growth
rate;
at 40% (v/v) CO the specific growth rate was only 0.06 h-‘. The inhibition of the specific growth rate by CO was linear
Effect of CO on carboxydobacteria
Figure 1. Morphology and ultrastructure of carboxydobacteria under optimum cultivation conditions (a,b) and on medium with CO (c,d,e,f). om--Outer membrane; n--nucleoid; M--intracellular membrane; V--volutin inclusion; EDI---electron-dense inclusions in nucleoid; etv--electron-transparent vacuoles in cytoplasm. Marker bars represent 0.5 pm.
World Journalof Microbiology and Bio~echnology, Val 9, 199J
161
T.G. Volova et al. Table
1. Composition
Specific growih rate W’)
co concentration (% v/v) 0 10 20 30 40
Values ND-Not
l
of carboxydobacteria
of four separate
varying
Biomass
0.38 0.36 0.23 0.16 0.06
are means determined.
under
experiments
concentrations yield
(g/g)
World Journal
of Microbiology
Chemical
culture.’ cell
compositlon
Yh
Yo2
Protein
Lipld
2.0 1.7 1.4 1.0 0.7
0.26 0.16 0.13 0.09 0.04
56.7 59.5 58.0 59.2 60.2
9.1 11.0 10.0 11.0 ND
yielding
consistent
up to 25% (v/v) CO but at higher concentrations the nature of the inhibition changed qualitatively and the cells showed multiple changes. CO acted on carboxydobacterial cell respiration. The respiratory activity of carboxydobacteria, grown at non-inhibiting CO concentrations (< 10% v/v), was 0.2 to 0.35 pmol O,/min.mg dry weight, with endogenous respiration equal to 0.02 pmol O,/min.mg dry weight. With CO up to 30% (v/v), the respiration activity decreased to 0.120 pmol O,/min.mg dry weight. With increasing inhibitor concenkations, the specific consumption of H, and 0, to synthesize the biomass grew and the efficiency of their utilization decreased (see Table I). Examination of the bacterial chemical composition also enabled the CO effect to be quantified (see Table I). The ratio of nitrogen-containing and reserve components in carboxydobacteria did not change significanfly under growth inhibition by CO. Considerable inhibition of carboxydobacteria growth by high CO concentrations (10 to 40% v/v) did not decrease the cell protein content or lead to significant accumulation of poly$-hydroxybutyrate. The total cell content of lipids remained about 10% (w/w), but the composition of the fatty acids changed somewhat (see Table 2); the bacteria produced more saturated and cyclopropane fatty acids instead of unsaturated fatty acids at high CO concentrations. This effect probably protects cell structures from the damaging effect of the inhibitor. The hydrogenase activity of S. carboxydohydrogena was maximal at 20% (v/v) CO, (see Table 3). With increasing concentrations of CO, cytochromes a and b (but not c) increased (see Table 3). Cells grown without CO showed little evidence of an inner membrane structure. However, in the presence of CO, inner membranes were clearly formed (Figure Ic,e). Characteristically for CO-inhibited carboxydobacteria, electron-transparent vacuoles were formed in the cytoplasm (Figure If). The presence of CO in the cultural medium also resulted in changes in the cell surface profile. Compared with the cells grown under optimal conditions, which had a
162
of CO in turbidostat
and Biotechnology. Vol 9, 1993
results.
Standard
deviations
(% of dry weight) Poly-fi-hydroxybulyrate Trace 0.2 ND 2.2 1.2
did not exceed
10% of values
given
smooth outer membrane, cells grown with CO had numerous deep folds in their outer membranes (Figure Ia,d,e). The consequent increase in cell surface area seems to create more favourable conditions for the cells to consume the substrate. Thus, the carboxydobacteria S. carboxydohydrogena, having a CO-resistant hydrogenase, were specifically resistant to CO. The bacteria enhanced their production of cytochromes and enlarged the area of their membranes at high CO concentrations. As a result, the bacteria used more energy to convert substrate into biomass and although they continued to grow at 40% (v/v) CO, biomass yield was greatly diminished.
Table 2. Composiiion of fatty acids carboxydobacteria grown in varying continuous culture.* Fatty
acidt
CO concentration 0
14:o 15:o 15:l 16:0 16:l 17:o cyc17:o 18:0 18:l cyc19:o Ratio
(w/w)
of lipids (% w/w) CO concentrations
0.6 0.3 0.8 35.6 40.5 Trace 2.2 1.1 17.6
of in
(% v/v)
10
20
30
None 0.5 Trace 30.7 35.1 Trace 8.1 2.7 22.5 0.4
None 0.4 Trace 34.4 36.9 Trace 9.5 2.2 16.6 Trace
None 0.7 Trace 33.7 36.0 Trace 9.5 1.8 17.9 0.4
acids 0.86
0.87
of saturated to unsaturated 0.68 0.74
fatty
‘Values are means of four separate experiments yielding consistent results. Standard deviations did not exceed 5% of values given. t The numbers before and after the colon indicate, respectively, the number of carbon atoms and double bonds. Cyc-cyclopropane fatty acid.
Effect of CO on carboxydobacferia
Table 3. Concentration of cytochromes at various CO concentrations.’ co concentration (% v/v)
Cytochrome
0 10 20 30 40
0.60 0.60 0.66 0.60 0.61
*Values
and
concentration
are
means
of four
(nmollmg
0.05 0.06 0.03 0.03 0.04 separate
0.45 0.50 0.54 0.57 0.69
* f * & f
of carboxydobacteria
(plmol
C
0.05 0.05 0.03 0.03 0.03
0.09 0.09 0.09 0.12 0.09
experiments
yielding
References G ogotov,
activity
protein)
b
a k f f f +
enzymatic
I.N. 1979 Hydrogenases of microorganisms. Uspekhi Mikrobiologii 14, 3-27. (In Russian.) Meyer, 0. & Schlegel, H. 1983 Biology of aerobic carbon monoxide-oxidizing bacteria. Annd Review of Microbiology 3 7, 277-310. Nozhevnikova, A.N. 1974 Carboxydobacteria (bacteria, oxidizing carbon monoxide). PhD Thesis. Institute of Microbiology, USSR Academy of Sciences, Moscow. (In Russian.) Probst, I. & Schlegel, H. 1976 Respiratory components and oxidase
f 0.01 + 0.01 * 0.01 * 0.01
consistent
Activity of hydrogenase H,/mln mg protein) 300 300 500 140 120
* 0.01
grown
* f f + *
40 40 40 20 20
results.
activities in Ah&genes eutraph~, Biachimicu et Biophysicu Ada 440, 412-428. Sanjieva, E.U. & Zavarzin, G.A. 1971 Bacteria oxidizing carbon monoxide. Dokludy Akudemii Nauk SSSR 196, 9X-959. (In Russian.) Volova, T.G. 1980 Autotrophic continuous cultivation of Seliberiu curboxydohydrogena. Microbiologiyu 49, 20-24. (In Russian.)
(Received in revised form 8 August 1992)
World Jowml
of Microbiology
1992; accepted 12 August
and Biotechnology. Vol 9, 1993
163
