Imperfect Asymmetry of Life: Earth Microbial Communities Prefer D-Lactate but Can Use L-Lactate Also

1. Introduction

T he notion that, on a given planet, biological organisms can take up and metabolize only one of the two enantiomers of a chiral nutrient has been in existence for some time (Halpern et al., 1966; Kelley et al., 1975). Without full, experimental validation, this method was used on Mars to look for evidence for biological activity. It began with the labeled release experiment (LR), one of the biological tests conducted by the Viking mission. The LR was specifically designed to detect heterotrophic metabolism. A radioactive nutrient broth containing ¹⁴C-labeled glycolate, formate, lactate, alanine, and glycine was added to soil, while the headspace above the sample was monitored for appearance of radioactive gas that would result from degradation of the added nutrients (Levin and Straat, 1976). For the two chiral compounds, lactate and alanine, both dextrorotatory (D) and levorotary (L) enantiomers were supplied, justifiably, in case life was enantiomerically biased. The LR test was positive, but its interpretation was complicated by the fact that no native soil organic carbon could be detected by the gas chromatograph–mass spectrometer (GCMS) aboard the Viking landers, an instrument with a sensitivity of parts per billion. The absence of organic carbon implied the absence of organisms. Hence, the LR result may be abiotic in nature: martian soil may contain inorganic, photochemical oxidants such as peroxide and superoxide (Klein, 1977, 1978). The abiotic hypothesis would explain both the LR and GCMS results. The oxidants, together with direct light destruction, would have destroyed the organics from four billion years of meteoric infall and from a possible early biota. The oxidants also would have reacted with the LR nutrients, which would have resulted in a false positive. Not all researchers agree with this interpretation, however. Some argued that the LR and GCMS results can be reconciled in a biological explanation: a martian bacterial population small enough to escape detection by the GCMS but large enough to be registered in the more sensitive LR (Glavin et al., 2001). More recently, Houtkooper and Schulze-Makuch (2007) also raised the possibility of martian organisms that use hydrogen peroxide as antifreeze. In a failed attempt to resolve the debate, the Mars Oxidant experiment (MOx) aboard the Russian Mars ’96 mission was designed to measure the redox potential of Mars with a series of redox-sensitive thin films (McKay et al., 1998). Included in the MOx was a pair of amino acid enantiomers, D- and L-cysteine. The MOx team assumed that inorganic oxidants would attack both enantiomers, while biological agents would destroy only one of them. Recently, Navarro-González et al. (2003) assumed that L-glucose and D-alanine are biologically incorrect enantiomers on Earth and used them to measure chemical reactivity in the Atacama Desert. Where this measurement accounted for all reactivity, the soil was considered to be Mars-like and chemically oxidizing.

An effort to test nutrient stereospecificity was recently initiated in our laboratory. Substrate stereospecificity on the enzyme level is well established. For example, Milton and colleagues (1992) showed that the native, L-form of the HIV-1 protease, composed of L-amino acids, interacts only with the L-form peptide substrate (composed of L-amino acids), not with the D-form. The reverse is true if the enzyme is in the D-form, synthesized from D-amino acids. It binds the D-form, not the L-form, of the substrate. Our objective is twofold: (1) to verify that what is observed with enzymes can be extrapolated to whole-cell physiology and (2) to determine how different compounds might differ in stereoselectivity. Our initial data (Sun et al., 2009), and data obtained independently by Warmflash et al. (2009), indicate that glucose is stereospecific. Representatives of all three known life-forms on Earth (Bacteria, Archaea, Eukarya) utilize D-glucose but not L-glucose.

The chiral biology of lactate, the subject of the present study, is more complicated. First, lactate formation is not homochiral. In most lactic acid bacteria, fermentation of carbohydrates yields a mixture of D- and L-isomers, although not necessarily in equal quantity (see review by Garvie, 1980 and references therein). Given that both isomers are available on Earth, natural selection would be expected to favor microorganisms that use both isomers over those that use one isomer exclusively. Indeed, some bacteria appear to be dual users and possess two versions of the lactate dehydrogenase, one specific for the D-isomer and one for the L-isomer. These enzymes are required to oxidize lactate to pyruvate, which is achiral and can be further oxidized to carbon dioxide via the tricarboxylic acid cycle (Garvie, 1980). How widespread such dual users are is unknown, but they are common at least among the medically or economically significant bacteria that have been studied. To complicate the matter further, some bacteria also possess a lactate racemase, which catalyzes the stereo-optical conversion of lactate from one enantiomeric configuration to another (Dennis and Kaplan, 1963; Hiyama et al., 1968; Stetter and Kandler, 1973; Hino and Kuroda, 1993; Goffin et al., 2005). These bacteria would be DL-lactate users even if they have only one type of the lactate dehydrogenase.

Therefore, there is a need for critical evaluation of lactate with respect to stereospecificity. This need cannot be met by the data available in the literature, which is qualitative or semiquantitative and limited to a small number of species of bacteria (Oren and Gurevich, 1995; Traudt and Kleinberg, 1996; Evans and Martin, 1997; Duncan et al., 2004; Goffin, 2004). We assessed stereospecificity in lactate with carefully controlled experiments based on natural soil microbial communities. We also studied individual bacteria, archaea, and eukaryotic fungi to compare different life-forms and to compare cultured and natural cells. Our results indicate that, unlike glucose, lactate is not a stereospecific substrate.

2. Materials and Methods

3. Results

All five bacteria consumed D- and L-lactate. In each culture, quantities of the two isomers decreased simultaneously from an initial concentration of about 1 mM to around 0.1 mM, beyond which no further decline was observed (Fig. 1). These DL-lactate users include Gram-positive species Micrococcus luteus, Bacillus simplex, and Arthrobacter globiformis as well as Gram-negative species Pseudomonas fluorescens and Ochrobactrum anthropi.

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FIG. 1.

Consumption of D-lactate (filled symbol) and L-lactate (open symbol) by laboratory-grown bacteria and archaea. Only archaeon Natrialba asiatica utilized lactate in a stereospecific manner (using D-lactate but not L-lactate). All five bacteria used the two enantiomers indiscriminately.

The archaeon Natrialba asiatica used D-lactate but not L-lactate. Four other archaea, including Halostagnicola sp., Natronobacterium sp., Haloarcula mukohataei, and Picrophilus oshimae, were also studied. None of them consumed lactate under our experimental conditions.

Soil, lake water, and sediment samples consumed the two isomers in an asynchronous manner (Fig. 2). D-lactate consumption started either immediately or after only a short time period. In contrast, L-lactate consumption started after a relatively longer preparation phase. The onset differential between the two isomers ranged from one hour in soil Mojave #1 to five hours in soil Mojave #2, soil Australia #2, LV lake, and LA lake.

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FIG. 2.

Consumption of D-lactate (filled symbol) and L-lactate (open symbol) in soil, sediment, and lake samples. In all samples, active D-lactate consumption began sooner than did L-lactate consumption. cfu, colony-forming units.

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4. Discussion

Our data indicate that lactate is not a stereospecific substrate. The fact that six randomly selected bacteria all use L-lactate as well as D-lactate (Fig. 1) suggests that dual users are common in the domain Bacteria. While some microorganisms do take up lactate in a stereospecific manner, they do not always favor the same enantiomer. Hence, while the archaeon Natrialba asiatica is a D-lactate user (Fig. 1), the literature contains many examples of L-lactate bacteria (Garvie, 1980; Traudt and Kleinberg, 1996). Although complex, eukaryotic organisms are less relevant to the astrobiology of Mars, most invertebrates, plants, and animals metabolize only L-lactate (Cristescu, 2008; Passarella et al., 2008). Our knowledge about D-, L-, and DL-lactate users, especially among the domain Archaea, is incomplete and perhaps even inaccurate. This much is clear: lactate is not a stereospecific substrate as previously understood.

At the same time, lactate is not a stereo-indiscriminate substrate either. All ten soil-, lake-, and sediment-borne microbial communities studied displayed a preference for D-lactate; active consumption of D-lactate started consistently and significantly sooner than L-lactate (Fig. 2). Presumably, D-lactate dehydrogenase activity was constitutive or readily induced. In contrast, the enzymes for metabolizing L-lactate—the lactate racemase, the L-lactate dehydrogenase, or both—were relatively slower to induce. Ultimately, this implies that lactate production in the studied environments is biased toward the D-form. The fact that the bias is consistent in different habitats sampled across different continents is a strong indication that what we observed in these experiments may be a general characteristic of microbial communities on Earth.

The chiral biology of lactate is, therefore, different from that of glucose. In the latter case, chirality appears to be an insurmountable barrier to life. Consequently, sugar metabolism on Earth is confined to the dextrorotary side of the barrier, with no capability to access L-glucose (Sun et al., 2009; Warmflash et al., 2009). We refer to this situation as perfect asymmetry. Lactate, in contrast, is a case of imperfect asymmetry: life operates on both sides of the chiral barrier with unequal efficiencies. On Earth, D-lactate appears to be the primary isomer and L-lactate the secondary isomer. Apparently, for lactate, the chirality barrier has been breached more than once in the biological history of Earth, as the lactate racemase arose multiple times (Le Bras and Garel, 1991; Taguchi and Ohta, 1991; Kochhar et al., 1992). This explains the widespread presence of dual users among different microbial groups. Remarkably, these organisms are able to access both D- and L-lactate, but they do not make both D- and L-enzymes. As mentioned earlier, D- and L-enzymes are identical proteins that differ only in the enantiomeric configuration of the constituent amino acids. As shown with HIV-1 protease, D-enzymes only bind D-substrates, while L-enzymes only bind L-substrates (Milton et al., 1992). D-lactate and L-lactate dehydrogenases are both L-enzymes, composed of L-amino acids.

Lactate still may be considered in planetary chiral life detection, although the signal from a lactate experiment would be subtle compared to the signal from a glucose experiment. Instead of consuming only one of the two enantiomers supplied, a positive biological response would manifest as a difference in the consumption onset between the two enantiomers. Simultaneous onsets between the two reactions would be abiotic in nature. Constructing an in situ experiment with sufficient precision to distinguish the two outcomes would be an engineering challenge. If the challenge could be overcome, enantiomerically selective consumption of lactate could be deployed on Mars as part of a chiral LR. Such an effort is timely in light of the recent evidence for methanogenesis on Mars (Formisano et al., 2004) and the question with regard to its origin, whether it is biological or thermogenic. Methanogenic archaea alone cannot metabolize lactate. In syntrophic associations with bacteria, however, they can (Yang and Tang, 1991). Methanogens typically utilize simple carbon compounds such as formate, propanol, and acetate, which are achiral and of no value for chiral experiments. However, if methanogens are sustained on Mars, there has to be a supply of the substrates, either from a geochemical source or a biota. The latter question is amenable to a sugar-based, chiral life-detection experiment.