Steven L. Daniel

Old acetogens, new light

Acetogens utilize the acetyl‐CoA Wood‐Ljungdahl pathway as a terminal electron‐accepting, energy‐conserving, CO2‐fixing process. The decades of research to resolve the enzymology of this pathway (1) preceded studies demonstrating that acetogens not only harbor a novel CO2‐fixing pathway, but are also ecologically important, and (2) overshadowed the novel microbiological discoveries of acetogens and acetogenesis. The first acetogen to be isolated, Clostridium aceticum, was reported by Klaas Tammo Wieringa in 1936, but was subsequently lost. The second acetogen to be isolated, Clostridium thermoaceticum, was isolated by Francis Ephraim Fontaine and co‐workers in 1942. C. thermoaceticum became the most extensively studied acetogen and was used to resolve the enzymology of the acetyl‐CoA pathway in the laboratories of Harland Goff Wood and Lars Gerhard Ljungdahl. Although acetogenesis initially intrigued few scientists, this novel process fostered several scientific milestones, including the first 14C‐tracer studies in biology and the discovery that tungsten is a biologically active metal. The acetyl‐CoA pathway is now recognized as a fundamental component of the global carbon cycle and essential to the metabolic potentials of many different prokaryotes. The acetyl‐CoA pathway and variants thereof appear to be important to primary production in certain habitats and may have been the first autotrophic process on earth and important to the evolution of life. The purpose of this article is to (1) pay tribute to those who discovered acetogens and acetogenesis, and to those who resolved the acetyl‐CoA pathway, and (2) highlight the ecology and physiology of acetogens within the framework of their scientific roots.

Utilization of methoxylated aromatic compounds by the acetogen Clostridium thermoaceticum : Expression and specificity of the CO-dependent O-demethylating activity

The aromatic CO-dependent O-demethylating activity of Clostridium thermoaceticum was evaluated. Secondary aromatic substituent groups (-OH, -CO2H, -CH2OH, and -OCH3) were critical to O demethylation. O-demethylating activities and specificities were similar from cells grown at the expense of different methoxylated aromatic compounds; all O-methyl-grown cells catalyzed the same sequential O demethylation of multi-methoxylated compounds, suggesting that a broad specificity O demethylase was involved in O demethylation. In cell-fractionation studies, CO-dependent O demethylation was catalyzed by membrane-associated components.

Acetogenesis coupled to the oxidation of aromatic aldehyde groups

Vanillin cultures of Clostridium formicoaceticum produced higher cell densities than did vanillate cultures. During growth at the expense of vanillin, vanillate was the predominat intermediate formed; 3,4-dihydroxybenzaldehyde was not a significantly detectable intermediate. Acetate and protocatechuate were both produced in equimolar ratio relative to vanillin consumption. 4-Hydroxybenzaldehyde was a growth-supportive aromatic compound for both C. formicoaceticum and Clostridium aceticum (doubling times approximated 5 h), was oxidized stoichiometrically to 4-hydroxybenzoate, and was not appreciably toxic at concentrations up to 15 mM. Acetate was (i) the major reduced end product detected concomitant to growth and to benzaldehyde oxidation and (ii) formed in close approximation to the following stoichiometry: 4 4-hydroxybenzaldehyde + 2CO2+2H2O→4 4-hydroxybenzoate + CH3COOH. We conclude that these two acetogens are capable of benzaldehyde-coupled acetogenesis and growth.

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Abstract. Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [14C]bicarbonate or [14C]oxalate showed that CO2 reduction to acetate and biomass or oxalate oxidation to CO2 was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO2 into biomass than oxalate-grown cells. CO2 conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO2 as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.

Bidirectional usage of ferulate by the acetogen Peptostreptococcus productus U-1 : CO2 and aromatic acrylate groups as competing electron acceptors

The influence of CO2 on the ability of Peptostreptococcus) productus U-1 (ATCC 35244) to use an aromatic acrylate group as an energy-conserving electron acceptor during O-methyl-dependent growth was examined. Ferulate (a methoxylated phenylacrylate), unlike hydroferulate (a methoxylated phenylpropionate), supported growth under CO2-limited conditions. Two phases occurred during ferulate utilization in CO2-limited cultures. In phase I (maximum growth), O-methyl-derived reductant was coupled mainly to acrylate group reduction, and acetate synthesis (CO2 as reductant sink) was minimal. In phase II, acetate synthesis increased, but cell yields in this phase were much less than in phase I. In CO2-enriched cultures, distinct phases were not observed; reductant was coupled equally to CO2 and acrylate group reduction. Under CO2-enriched conditions, O-methyl and acrylate groups were incompletely metabolized, and molar growth yields were significantly lower compared to CO2-limited conditions. Resting cell studies indicated that O-demethylase and aromatic acrylate oxidoreductase activities were induced by ferulate. These findings demonstrated that P. productus U-1 can use the aromatic acrylate oxidoreductase system as a sole, energy-conserving, electron-accepting process, but is not able to prevent the simultaneous use of the bioenergetically less favourable acetyl-CoA pathway during O-methyldependent growth.

Nitrite as an energy-conserving electron sink for the acetogenic bacterium Moorella thermoacetica

Nitrite served as an energy-conserving electron acceptor for the acetogenic bacterium Moorella thermoacetica. Growth occurred in an undefined (0.1% yeast extract) medium containing 20 mM glyoxylate and 5 mM nitrite and was essentially equivalent to that observed in the absence of nitrite. In the presence of nitrite, acetate (the normal product of glyoxylate-derived acetogenesis) was not detected during growth. Instead, growth was coupled to nitrite dissimilation to ammonium, and acetogenesis was limited to the stationary phase. Furthermore, membranes from glyoxylate-grown cells under nitrite-dissimilating conditions were deficient in the b-type cytochrome that is typically found in the membranes of acetogenic cells. Unlike glyoxylate, other acetogenic substrates (fructose, oxalate, glycolate, vanillin, and hydrogen) were not growth supportive in the undefined medium containing nitrite, and glyoxylate-dependent growth did not occur in a nitrite-supplemented, basal (without yeast extract) medium. Glyoxylate-dependent growth by Moorella thermoautotrophica was not observed in the undefined medium containing nitrite.

Acetogenesis: Reality in the Laboratory, Uncertainty Elsewhere

This chapter focuses on recent work in our research group that further extends our awareness of the diverse metabolic potentials of acetogens and, consequently, broadens our uncertainty in making accurate predictions of the role acetogens actually play at the ecosystem level (i.e., “elsewhere” per the title of this chapter). Without debating what ecosystems are, acetogens are difficult to study in their natural habitat. This difficulty stems largely from the fact that the main product we think they make (i.e., acetate) is not easily assessed (a gaseous product minimizes this complication) and likely turns over rapidly in vivo. Likewise, many of the substrates they may consume are also problematic to assess. In addition, approaches such as the [3H]thymidine incorporation method to assess the productivity of acetogens may greatly underestimate their magnitude (Winding, 1992; Wellsbury et al., 1993). Thus, although enrichment and physiological studies have been somewhat elegant in recent years relative to defining acetogenic potentials in the laboratory, comparatively little is known about what they really do “elsewhere” (as emphasized in Chapter 7). Clearly, native ecosystems such as forests have little in common with test-tube cultures. In the present chapter and those that follow in Part IV these realities and uncertainties are addressed.

Oxalate metabolism by the acetogenic bacterium Moorella thermoacetica

Whole-cell and cell-extract experiments were performed to study the mechanism of oxalate metabolism in the acetogenic bacterium Moorella thermoacetica. In short-term, whole-cell assays, oxalate consumption was low unless cell suspensions were supplemented with CO(2), KNO(3), or Na(2)S(2)O(3). Cell extracts catalyzed the oxalate-dependent reduction of benzyl viologen. Oxalate consumption occurred concomitant to benzyl viologen reduction; when benzyl viologen was omitted, oxalate was not appreciably consumed. Based on benzyl viologen reduction, specific activities of extracts averaged 0.6 micromol oxalate oxidized min(-1) mg protein(-1). Extracts also catalyzed the formate-dependent reduction of NADP(+); however, oxalate-dependent reduction of NADP(+) was negligible. Oxalate- or formate-dependent reduction of NAD(+) was not observed. Addition of coenzyme A (CoA), acetyl-CoA, or succinyl-CoA to the assay had a minimal effect on the oxalate-dependent reduction of benzyl viologen. These results suggest that oxalate metabolism by M. thermoacetica requires a utilizable electron acceptor and that CoA-level intermediates are not involved.

Bidirectional transformation of aromatic aldehydes by Desulfovibrio desulfuricans under nitrate‐dissimilating conditions

M. PAREKH, H.L. DRAKE AND S.L. DANIEL 1996. Desulfovibrio desulfuricans ATCC 27774 was screened for reactivity against aromatic compounds during lactate‐dependent, nitrate‐dissimilating growth. Only aromatic aldehydes (benzaldehyde, 2‐hydroxybenzaldehyde, 3‐hydroxybenzaldehyde, 4‐hydroxybenzaldehyde, vanillin, iso‐vanillin and o‐vanillin) were reactive and, with the exception of 2‐hydroxybenzaldehyde, were stimulatory to lactate‐dependent growth. Aromatic aldehydes were transformed to their corresponding benzoate and benzyl alcohol derivatives, with the ratio of benzoate‐to‐benzyl alcohol derivatives being dependent upon lactate availability. In presence of lactate, aromatic aldehydes were primarily reduced to their corresponding benzyl alcohol derivatives; in the absence of lactate, aromatic aldehydes were mainly oxidized to their corresponding benzoate derivatives. In the absence of nitrate, 3‐hydroxybenzaldehyde was neither reduced nor oxidized. These results indicate that D. desulfuricans is competent in the bidirectional transformation of aromatic aldehydes under nitrate‐dissimilating conditions and that the direction of transformation (i.e. reduction or oxidation) is regulated by reductant availability.

Evaluation of glyphosate-tolerant soybean cultivars for resistance to bacterial pustule

Xanthomonas axonopodis pv. glycines causes bacterial pustule of soybean, which is a common disease in many soybean-growing areas of the world and is controlled by a single recessive gene (rxp gene) commonly found in many conventional glyphosate-sensitive soybean cultivars. Since glyphosate-tolerant cultivars are commonly planted today, there has been no information about whether these new cultivars have bacterial pustule resistance. The goal of this study was to screen glyphosate-tolerant soybean cultivars for resistance to X. axonopodis pv. glycines. Three experiments were completed to evaluate resistance. In experiment 1, 525 commercial glyphosate-tolerant cultivars from 2001 were inoculated with X. axonopodis pv. glycines strain UIUC-1. Following inoculation, many of the cultivars were resistant (developed no detectable pustule symptoms) although 152 (~29%) developed bacterial pustule. In experiment 2, the aggressiveness of three strains (UIUC-1, UIUC-2, and ATCC 17915) of X. axonopodis pv. glycines were compared on three bacterial pustule-susceptible, glyphosate-tolerant cultivars. One strain (UIUC-1) was less aggressive than the other two (UIUC-2 and ATCC 17915) on all three cultivars examined. In experiment 3, 45 cultivars from 2005 (all different from 2001) were inoculated with X. axonopodis pv. glycines ATCC 17915. A range of disease severities developed with five cultivars (11%) having disease severity ratings as high as or higher than those on a susceptible check cultivar. Overall, these results suggested that resistance to bacterial pustule occurs in glyphosate-tolerant soybean cultivars, but not at 100% frequency, which means bacterial pustule outbreaks could occur when a susceptible cultivar is planted and conditions are conducive for bacterial pustule development.