Arch Microbiol (1990) 154:550 - 555 Archives of Microbiology Springer-Verlag 1990

New motile anaerobic growing by succinate decarboxylation to propionate

Karin Denger and Bernhard Schink Lehrstuhl Mlkrobiologie I, Eberhard-Karls-Umversit/it, Auf der Morgenstelle 28, W-7400 Tfibingen, Federal Republic of Germany

Received April 25, 1990/Accepted August 14, 1990

Abstract. Three strains of new anaerobic, gram-negative and Pfennig 1981). Some rumen bacteria such as bacteria which grew with succinate as sole source of car- Selenomonas ruminantium (Scheifinger and Wolin 1973) bon and energy were isolated from anoxic marine and or Veillonella alcalescens (Yousten and Delwiche 1961) freshwater mud samples. Cells of the three strains were have been shown to decarboxylate succinate to propi- small, non-spore-forming, motile rods or spirilla. The onate in a reaction not linked to growth. A strictly anaer- guanine-plus-cytosine content of the DNA of strain US2 obic bacterium, Propionigenium modestum, was isolated was 52.6 + 1.0 tool%, of strain Ft2 63.5 _+ 1.4 tool%, and which can grow on the basis of this decarboxylation of strain Ftl 62.6 + 1.0 tool%. Succinate was fermented reaction (Schink and Pfennig 1982b). The reaction se- stoichiometrically to propionate and carbon dioxide. The quence of succinate degradation includes succinyl-CoA, growth yields were 1.2- 2.6 g dry cell mass per tool succi- methylmalonly-CoA, and propionyl-CoA (Hilpert et al. nate degraded. Strains US2 and Ft2 required 0.05% w/v 1984). The small free energy change of the decarboxyla- yeast extract in addition to succinate for reproducible tion reaction (A Go' = -20.5 k J/tool succinate; Thauer et growth. Optimal growth occurred at 30~176 and al. 1977) is converted into a sodium ion gradient which pH 6.8-8.0. Addition of acetate as cosubstrate did not drives synthesis via a sodium ion dependent ATPase stimulate growth with any strain. Strain Ft2 grew only (Hilpert et al, 1984). Propionigenium modestum is a under strictly anaerobic conditions, whereas strains US2 saltwater isolate and requires at least 10 g sodium chlor- and Ftl tolerated oxygen up to 20% in the headspace. ide per liter medium for growth. It was the aim of the Strains US2 and Ft2 grew only with succinate. Strain present study to look for new bacterial strains which Ftl also converted fumarate, aspartate, and sugars to ferment succinate under different conditions. In the pre- propionate and acetate. This strain also oxidized pro- sent paper three new strains conserving energy from suc- pionate with nitrate to acetate. Very low amounts of cinate decarboxylation are described. a c-type cytochrome were detected in propionate plus nitrate- or glucose-grown cells of this strain (0.4 gg x g protein- 1). Moderate activities of avidin-sensitive Materials and methods methylmalonyl-CoA decarboxylase were found in cell- free extracts of all strains. Sources of organisms Key words: Succinate fermentation - Decarboxylation The new strains were isolated from enrichment cultures inoculated energy - Na + bioenergetics - Flagellar motors - Pro- with mud samples from the following sources: Strain US2 from pionate oxidation - Methylmalonyl-CoA marine sediment of the Sippewissett salt marshes, close to Woods Hole, MA, USA; strains Ft2 and Ftl from digested anaerobic sludge of a municipal sewage plant at Marburg-Cappel, FRG. Propionibacterium fi'eudenreichii ssp. shermanii (DSM 20270) and Escherichia coli K]2 (DSM 498) were obtained from the Succinate is an important end product of anaerobic Deutsche Sammlung yon Mikroorganismen, Braunschweig, FRG. fermentations (Gottschalk and Andreesen 1979). Aerobically it is oxidized completely to carbon dioxide in the citric acid cycle. Many sulfate-reducing bacteria can Media and growth conditions oxidize succinate (Widdel 1988), and phototrophic purple All procedures for culuvation and isolation were as previously de- bacteria assimilate succinate as cell carbon source (Biebl scribed (Widdel and Pfennig 1981; Schink and Pfennig 1982a: Schink 1984). The mineral salts medium for isolation and further Offprint requests to K. Denger cultivation was carbonate-buffered (30 mM) and sulfide-reduced 551

(1 mM), and contained 1 ml of selenite-tungstate solution and 1 ml Boehringer, Mannheim, FRG. All ionophores were from Sigma, of trace element solution SL 10 (Widdel et al. 1983) per liter, as well Mtinchen. Gases were obtained from Messer-Griesheim, Darm- as 0.5 ml per liter of 7-vitamins solution (Widdel and Pfennig 1981). stadt, FRG. The pH was adjusted to 7.1 --7.3. Growth experiments were carried out either in 22 ml screw-cap tubes completely filled with medium, or in 120 ml serum bottles filled with 60 ml medium under an atmos- phere of N2/CO2 (90%/10%). All growth experiments were carried Results out at least in duphcates at 30~ unless stated otherwise Em'ichment and isolation

Isolation and characterization Enrichment cultures with freshwater and saltwater me- dium (50 ml) containing 20 mM di-sodium succinate with Pure cultures were obtained by repeated applicatlon of the agar or without 5 mM sodium acetate were inoculated with shake culture method (Pfennig 1978). Purity was checked micro- 5 ml of anoxic mud samples. Turbidity developed within scopically and also by growth tests in complex medium (AC-me- dium, Difco-Laboratories, Detroit, MI, USA). Growth was fol- 1 -2 weeks. In subcultures with the same substrate, tur- lowed by measuring turbidity at 400 nm wavelength with a bidity developed within 2- 5 days and the cell population Spectronic 20 Spectrophotometer (Milton Roy, Rochester, NY, got more and more uniform. After 5-6 transfers, the USA) or in l-ml cuvettes in a spectrophotometer model 100-40 fastest growing cultures were diluted in two subsequent (Hitachi, Tokyo, Japan). Gram staining was carried out according agar shake series each. Three morphologically different to Bartholomew (1962) with Propionibacteriumfreudenreichii and pure cultures growing with succinate as substrate were Escherichia coli as controls. The KOH test according to Gregersen isolated and further characterized. Strain US2 was iso- (1978) was applied in addition. Spore formation was checked after growth in medium with 20% soil extract, 1% Trypton, 0.1% Xylose. lated in saltwater medium whereas strains Ft2 and Ftl and 0.5% MnSO~ according to Hollaus and Sleytr (1972). were freshwater isolates. Two strains (US2 and Ft2) re- Cytochromes were assayed in ceil-free extracts as well as in mem- quired 0.05% yeast extract for growth. brane preparations obtained by uttracentrifugation (1 h at 150000g). Samples were subjected to difference spectroscopy (dithionite-reduced minus air-oxidized) in an Uvikon 860 spectrophotometer (Kontron, Ztirich, Switzerland). The DNA base Morphology and cytological properties composition was determined by thermal denaturation according to De Ley et al. (/970) after extraction described by Marmur (1961). Cells of strain US2 were small, slightly curved rods, 0.2- Escherichia coli strain K12 was taken as reference. 0.3 x 2-3 gm in size (Fig. I a). Cells of strain Ft2 were small spirilla, 0.2 gm x 2-10 gm (Fig. I b). Cells of strain Ftl were short, slightly curved rods, 0.5 x 2- 3 gm in Chemical analyses size, often occurring in pairs (Fig. lc). All strains were very motile in fresh cultures. All strains stained gram- Acetate and propionate were assayed by gas chromatography with negative and the KOH test showed slime formation typi- a Carlo Erba 6000 gas chromatograph (Milano, Italy) as described (Platen and Schink 1987). Succinate was determined as di- cal of gram-negative bacteria. Spore formation was never methylester by capillary gas chromatography (Dehning and Schink observed, neither in defined medium nor in a special 1989). H2 was determined with a thermal conductivity detector and sporulation medium. The guanine-plus-cytosine content a steel column (2 m x 4 ram) packed with 60/80 mesh molecular of the DNA of strain US2 was 52.6 + 1 tool%, of strain sieve (5 A, Serva, Heidelberg, FRG), detector temperature 130~ 63.5 + 1.4 mol%, and of strain Ftl 62.6 +_ 1 mol%. column temperature 50 ~C, carrier gas nitrogen, 78 ml/min. Formation of sulfide from sulfate or sulfur was determined Very low amounts of a cytochrome were detected only half-quantitatively (Cord-Ruwisch 1985). Formation of nitrite from with strain Ftl in crude cell extracts as well as in the nitrate was assayed by azo dye formation with sulfanilic acid and cytoplasmic fraction of propionate plus nitrate- or glu- ~-napthylamine (ProcMzkovfi 1959). cose-grown cells. From the absorption bands at 552 and 420 nm, it was identified as a e-type cytochrome. As- suming a molecular weight of 12000 and an extinction Enzyme assays coefficient for the c~-peak of 19.0 mM-1 cm-1 (Chance and Williams 1955), a cytochrome c content of 0.4 gg Methylmalonyl-CoA decarboxylase activity in crude cell extracts per g protein was calculated. was assayed photometrically at 25~ (modified after Hilpert and Dimroth 1983). The cuvette contained in 1 ml: 100 mM potassium phosphate buffer, pH 7.0, 20raM NaC1. 5 U phosphotrans- acetylase, and 0.1 mM methylmalonyl-CoA. Control experiments Growth conditions and physiology were carried out with crude cell extract preincubated for 15 rain at room temperature with 0.1 nag avidin or with 0.1 mg avidin pre- All strains fermented succinate stoichiometrically to pro- treated with 0.1 mg biotin. Protein in crude cell extracts was determined with CuSO4 in pionate (Table 1) and carbon dioxide (not determined). alkaline solution (Zamenhoff 1957). The growth rates with 20 mM succinate were 0.045 h-1 (td = 15 h) with strain US2, 0.025 h -1 (td = 28 h) with strain Ft2, and 0.05 h-1 (td = 14 h) with strain Ftl. Opti- Chemicals mal growth occurred at 30-37~ and pH 6.8- 8.0. The temperature limits were 24 and 45 ~ C, the pH range was All chemicals were of reagent grade quality and obtained from 6.3- 8.4. Phosphate concentrations up to 50 mM did not Fluka, Neu-Ulm; Merck, Darmstadt; Sigma, Mfinchen, and influence growth. Optimal growth occurred with succi- 552

Fig. 1 a-c. Phase contrast photomicrographs of strain US2, Ft2 and Ftl. Bar equals 10 I.tm for all panels

Table 1. Fermentation stoichiometries and growth yields of the Strains US2 and Ft2 were specialized for succinate strains US2, Ft2 and Ftl with succinate as substrate fermentation only and did not ferment any other organic substrates including other dicarboxylic acids, sugars, lac- Strain Sub- Cell Sub- Pro- Elec- Molar strate dry strate pionate tron growth tate, pyruvate, glycerol, ethanol, ethylene glycol, acetoin, degra- mass assimi- formed reco- yield and 2,3-butandiol. H2 or formate was not oxidized, ded a formed b lated c very nitrate, sulfate, thiosulfate, sulfite, or sulfur was not [gmol] [mg] [gmol] [Ixmol] [%] [g/mol] reduced. Unlike these strains, strain Ftl exhibited a broader US2 1000 1.23 14.5 1010 102 1.2 metabolic versatility. It fermented fumarate, aspartate, Ft2 795 2.04 24.1 745 97 2.6 Ftl 925 2.25 26.6 890 99 2.4 and sugars to propionate and acetate (Table 2). Weak growth occurred with malate, pyruvate, or glycerol as Experiments were carried out in 120-ml serum bottles filled half substrates. with medium under N2/CO2 (90%/10%) gas mixture and sealed. The amount of fermentation products agreed with Strains US2 and Ft2 required 0.05% yeast extract in addition to complete conversion of the substrates according to the succinate for growth following equations: a Substrate degraded was calculated from the difference of substrate concentration at the beginning and the end of the experiment succinate 2- + H20 ~ propionate- + HCO3 b Cell dry mass was calculated via ceil turbidity using the conversion AG; = 20.5 kJ/mol succinate factors 0.10D4o0 = 49 mg dry mass per liter (US2), 0.10D~oo = 3 fumarate 2 - + 6 H20 ~ 2 propionate- + acetate- 37 mg dry mass per liter (Ft2), 0.10D4o0 = 30 mg dry mass per liter (Ftl), which were obtained by direct gravimetric determination + 4 HCO3 + H + in 500 ml cultures grown ruth succinate AG~) = - 81.1 kJ/mol c Assimilation of substrates into cell material was calculated using fumarate the formula ~C4H703) for cell material, and the following assimi- 3 aspartate- + 6 H20 ~ 2 propionate- + acetate- lation equation for succinate: 17 succinate 2- + 32 H20 ~ 14 + 4 HCO3 ~C4H703) -- 12 HCO3 + 22 OH-; thus, 11.8 gmol succinate was + 3 HN~- + H + degraded to form 1 mg cell material AGo' = - 51.3 kJ/mol aspartate 3 glucose/fructose ~ 4 propionate- + 2 acetate- + 2 HCO3 + 8 H + nate at concentrations of 20 to 40 mM. The saltwater AG~ = - 308.1 kJ/mol isolate US2 grew in medium containing 0.1 -2.0% NaC1 hexose (w/v) and 0.015-0.3% MgC12 x 6 H20 (w/v), respective- ly, but growth ceased after 3-4 transfers in freshwater 4 xylose/arabinose ~ 4 propionate- + 3 acetate- medium (containing 0.1% NaC1 and 0.015% MgC12). + 2 HC03 + 9 H + AGb = - 264.0 kJ/mol pen- The freshwater isolates Ft2 and Ftl tolerated NaC1 up to tose. 1% (w/v) and MgC12 up to 0.15% (w/v). Strain Ft2 grew only under strictly anaerobic con- Addition of hemin at concentrations up to 100 ~tg/1 did ditions, whereas strains US2 and Ftl tolerated traces of not enhance growth yields with fumarate or aspartate. In oxygen as visualized in oxygen gradient tube cultures. growth experiments with 20 mM succinate plus 2 - 8 mM Growth of strain Ftl was possible even under air in a sodium nitrate, growth of strain Ftl was enhanced by phosphate-buffered medium with succinate as substrate. nitrate (Fig. 2). Nitrate was reduced stoichiometrically to Addition of acetate as cosubstrate did not stimulate nitrite. Figure 3 shows that also propionate could be growth with any strain. oxidized to acetate while nitrate was reduced to nitrite. 553 Table 2. Fermentation stoichiometries and growth yields of strata Ftl

Substrate Substrate Cell dry Substrate Products formed [p.mol] Electron Molar growth degraded mass formed assimilateda recovery yield [pmol] [rag] [pmol] propionate acetate [%] [g/tool] Fumarate 220 1.85 25.3 129 64 99 8.4 Aspartate 220 1.91 26.2 140 62 105 8.7 Glucose 220 4.28 29.1 240 ] l 9 95 19.5 Fructose 220 4.60 31.3 253 116 99 20.9 Xylose 220 3.69 30.3 194 145 103 16.8 Arabinose 220 3.69 30.3 184 130 96 t 6.8

Experiments were carried out in 22-ml screw-cap tubes filled completely with medium. Calculations of substrate degradation and cell dry mass formation as in Table ] The following assimilation equations were used for calculataon: 17 fumarate 2- + 42 H20 --+ 12(C4H703) + 20 HCO3 + 14 OH 17 aspartate- + 42 HzO ~ 12(C4H703) -/- 20 HCO3- -F /4 H20 -F 17 NH3 + 3H + 17 glucose/fructose + 6 OH- --+ 24(C4H703) + 6 HCO;- + 18 H20 17 xylose/arabinose + 5 OH --+ 20(C4H703) + 5 HCOj + 15 HzO

present study, showing that the ability of Propionigenh~m 2 modestum to grow by decarboxylation of succinate to propionate was not an entirely unique phenomenon. ~-- -85- o 0,8 i E E Moreover, termitida and Sporomusa malonica were recently shown to grow by succinate decarboxyla- I -,-) -O tion as well (Breznak et al. 1988: Dehning et al. 1989), D 0.4 {3 _ ~ -.-~ 0 and also a thermophilic bacterium was isolated which O can grow by succinate fermentation (Stares and Zehnder, pers. communication). Two of our new isolates as well as 0.0 0 -0 the Sporomusa species and the thermophilic isolate are I I I I I 0 2 z 6 8 freshwater organisms. Our medium contained only 0.1% NaC1 (w/v) and 0.015% MgC12 x 6H20 (w/v) showing Nitrote [mM] that enhanced NaC1 concentrations are not a necessary Fig. 2. Growth of strain Ftl with 20 mM succlnate in the absence prerequisit of energy-conserving succinate fermentation. and presence of nitrate. Experiments were performed at 30~ in Molar growth yields of ] .2 to 2.6 g dry cell mass per mol 22-ml screw-cap tubes. Symbols: cell density, [] acetate, nitrite. succinate were calculated which are in the same range as OD4oo: optical density at 400 nm the one determined for Propionigenium modestum (2.4 g dry cell mass per tool succinate). These values correlate No other inorganic electron acceptor such as sulfate, with the small free energy change of succinate de- thiosulfate, sulfite, or sulfur was reduced; neither H2 nor carboxylation (AGo' = 20.6 kJ/mol succinate). The yield formate was oxidized by strain Ftl. differences observed may be due to different efficiencies of the energy-conserving systems involved in these new isolates. Enzyme activities Strains US2 and Ft2 were specialized on succinate fermentation only and did not grow with any other sub- The key enzyme of succinate fermentation, methyl- strate tested. Unlike these two specialists, strain Ftl malonyl-CoA-decarboxylase, was assayed in French- showed a broader substrate spectrum and fermented also press cell extracts prepared from succinate-grown cells of fumarate, aspartate, glucose, fructose, arabinose: and xy- all three strains. With strains US2 and Ftl, an activity of lose to acetate and propionate. Weak growth occurred 0.33 U/rag protein was detected. If extracts were preincu- also with malate, pyruvate, and glycerol as substrates. bated with avidin, only 25% of this activity was found. Growth yields with fumarate and aspartate were higher Avidin preincubated with biotin had no inhibitory effect. than with succinate because additional energy can be With strain Ft2, the methylmalonyl-CoA decarboxylase conserved by the acetate-kinase reaction. However, these activity was 0.25 U/mg protein; this activity was only growth yields (8.4- 8.7 g/tool) are small if compared to slightly inhibited by avidin. those observed with Propionibacterium sp. with lactate (10.2-12.9 g/tool; Stouthamer ] 980), and this difference Discussion is even more expressed for growth with glucose (19.5- 20.9 vs. 50-70 g/tool). These comparably low growth Physiology yields suggest that strain Ftl does not conserve energy by electron transport in fumarate reduction, similar to Three new strains of succinate-fermenting bacteria were other recently isolated gram-negative propionate- enriched and isolated from anoxic mud samples in the forming bacteria (Schink and Pfennig 1982b; Schink et 554

I--1 5-- Campylobacter with 29- 38 tool% is considerably lower E t...a than that of strain Ftl and therefore an assignment to this genus is not possible. Sporomusa species ferment 0J r-a 3-,-) -@:r- succinate, however, they form spores, which none of the o -o E new isolates does. There are similarities between the new 0 GJ 0 isolates and the rumen bacterium SeIenomonas rumi- 0,2 o -4 2(1= nantium with respect to gram-staining reaction, DNA D 0 L base ratio, and degradation of succinate, but Selenomonas 0,4 t 0 - 3 .~ ruminantium cannot grow with succinate. The taxonomic r Z 0 assignment of these new isolates will be treated in a later communication. Strains US2 and Ftl were deposited with 0,0 - 0 the Deutsche Sammlung von Mikroorganismen, Braun- I I I I k 0 3 6 9 D_ schweig, under the numbers 5848 and 5849, respectively. Nitrote [mM] Fig. 3. Growth of strain Ftl with 3 mM propionate in the absence Acknowledgements. The authors thank Elke Steppohn and Fituat and presence of nitrate. Experimental conditions as described in Yildiz for help in enrichment and isolation of the new strains, and Fig. 2. Symbols: Cell density, A propionate, [] acetate, nitrite. Rolf Thauer and Peter Dimroth for helpful discussions concerning 0D4oo: optical density at 400 nm the energetics of our new isolates. al. 1987). This view is further supported by the obvious References lack of significant amounts of a b-type cytochrome. Growth of strain Ftl with succinate was enhanced by Bartholomew JM (1962) Variables influencing results, and the pre- cise definition of steps in Gram staining as a means of stan- addition of nitrate which was reduced stoichiometrically dardizing the results obtained. Stare Technol 37:139- 155 to nitrite. Also propionate, the end product of fermenta- B]ebl H, Pfennig N (1981) Isolation of members of the family tive metabolism, could be oxidized to acetate and carbon Rhodospirillaceae. 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J Biol Chem 217:395 - succinate fermentation is driven directly by a sodium 4O7 Cord-Ruwisch R (1985) A quick method for the determination of ion gradient (Laubinger and Dimroth 1989) which is dissolved and precipitated sulfides in cultures of sulfate-reduc- generated upon decarboxylation of methylmalonyl-CoA ing bacteria. J Microbiol Methods 4:33-36 (Hilpert et al. 1984). With our new isolates, enzyme activi- Dehning I, Schink B (1989) Malonomonas rubra gen. nov. sp. nov., ties of the methylmalonlyl-CoA-decarboxylase could be a microaerotolerant anaerobic bacterium growing by detected which were inhibited by avidin indicating a bio- decarboxylation of malonate. Arch Microbiol 151 : 427 - 433 tin enzyme as described for Veillonella alcalescens Dehning I, Stieb M. Schink B (1989) Sporomusa malonica sp. nov., a homoacetogenic bacterium growing by decarboxylation of (Hilpert and Dimroth 1983) and Propionigenium mod- malonate or succinate. Arch Microbiol 151 : 421 -- 426 estum. 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