Claus Härtig

Responses of soil microbial communities to weak electric fields.

Electrokinetically stimulated bioremediation of soils (electro-bioremediation) requires that the application of weak electric fields has no negative effect on the contaminant degrading microbial communities. This study evaluated the hypothesis that weak direct electric current (DC) fields per se do not negatively influence the physiology and composition of soil microbial communities given that secondary electrokinetic phenomena such as soil pH changes and temperatures are minimized. Mildly buffered, water-saturated laboratory mesocosms with agricultural soil were subjected for 34 days to a constant electric field (X=1.4 V cm(-1); J approximately 1.0 mA cm(-2)) and the spatiotemporal changes of soil microbial communities assessed by fingerprints of phospholipids fatty acids (PLFA) and terminal restriction fragment length polymorphisms (T-RFLP) of bacterial 16S rRNA genes. DC-induced electrolysis of the pore water led to pH changes (<1.5 pH units) in the immediate vicinity of the electrodes and concomitant distinct soil microbial community changes. By contrast, DC-treated bulk soil distant to the electrodes showed no pH changes and developed similar PLFA- and T-RFLP-fingerprints as control soil in the absence of DC. Our data suggest that the presence of an electric field, if suitably applied, will not influence the composition and physiology of soil microbial communities and hence not affect their potential to biodegrade contaminants.

Towards a systemic metabolic signature of the arbuscular mycorrhizal interaction

Our experiments addressed systemic metabolic effects in above-ground plant tissue as part of the plant’s response to the arbuscular mycorrhizal (AM) interaction. Due to the physiology of this interaction, we expected effects in the areas of plant mineral nutrition, carbon allocation and stress-related metabolism, but also a notable dependence of respective metabolic changes on environmental conditions and on plant developmental programs. To assess these issues, we analyzed metabolite profiles from mycorrhizal and non-mycorrhizal Lotus japonicus grown under greenhouse conditions at three different time points in the growing season in three different above-ground organs (flowers, sink leaves and source leaves). Statistical analysis of our data revealed a number of significant changes in individual experiments with little overlap between these experiments, indicating the expected impact of external conditions on the plant’s response to AM colonization. Partial least square-discriminant analysis (PLS-DA) nevertheless revealed considerable similarities between the datasets, and loading analysis of the component separating mycorrhizal and non-mycorrhizal plants allowed the defining of a core set of metabolites responsible for this separation. This core set was observed in experiments with and without mycorrhiza-induced growth effects. It corroborated trends already indicated by the significant changes from individual experiments and suggested a negative systemic impact of AM colonization on central catabolic metabolism as well as on amino acid metabolism. In addition, metabolic signals for an increase in stress experienced by plant tissue were recorded in flowers and source leaves.

Adaptation of anaerobically grown Thauera aromatica, Geobacter sulfurreducens and Desulfococcus multivorans to organic solvents on the level of membrane fatty acid composition

The effect of different solvents and pollutants on the cellular fatty acid composition of three bacterial strains: Thauera aromatica, Geobacter sulfurreducens and Desulfococcus multivorans, representatives of diverse predominant anaerobic metabolisms was investigated. As the prevailing adaptive mechanism in cells of T. aromatica and G. sulfurreducens whose cellular fatty acids patterns were dominated by palmitic acid (C16:0) and palmitoleic acid (C16:1cis), the cells reacted by an increase in the degree of saturation of their membrane fatty acids when grown in the presence of sublethal concentrations of the chemicals. Next to palmitic acid C16:0, the fatty acid pattern of D. multivorans was dominated by anteiso‐branched fatty acids which are characteristic for several sulfate‐reducing bacteria. The cells responded to the solvents with an increase in the ratio of straight‐chain saturated (C14:0, C16:0, C18:0) to anteiso‐branched fatty acids (C15:0anteiso, C17:0anteiso, C17:1anteisoΔ9cis). The results show that anaerobic bacteria react with similar mechanisms like aerobic bacteria in order to adapt their membrane to toxic organic solvents. The observed adaptive modifications on the level of membrane fatty acid composition can only be carried out with de novo synthesis of the fatty acids which is strictly related to cell growth. As the growth rates of anaerobic bacteria are generally much lower than in the so far investigated aerobic bacteria, this adaptive response needs more time in anaerobic bacteria. This might be one explanation for the previously observed higher sensitivity of anaerobic bacteria when compared with aerobic ones.

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

Tertiary alcohols, such as tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA) and higher homologues, are only slowly degraded microbially. The conversion of TBA seems to proceed via hydroxylation to 2-methylpropan-1,2-diol, which is further oxidized to 2-hydroxyisobutyric acid. By analogy, a branched pathway is expected for the degradation of TAA, as this molecule possesses several potential hydroxylation sites. In Aquincola tertiaricarbonis L108 and Methylibium petroleiphilum PM1, a likely candidate catalyst for hydroxylations is the putative tertiary alcohol monooxygenase MdpJ. However, by comparing metabolite accumulations in wild-type strains of L108 and PM1 and in two mdpJ knockout mutants of strain L108, we could clearly show that MdpJ is not hydroxylating TAA to diols but functions as a desaturase, resulting in the formation of the hemiterpene 2-methyl-3-buten-2-ol. The latter is further processed via the hemiterpenes prenol, prenal, and 3-methylcrotonic acid. Likewise, 3-methyl-3-pentanol is degraded via 3-methyl-1-penten-3-ol. Wild-type strain L108 and mdpJ knockout mutants formed isoamylene and isoprene from TAA and 2-methyl-3-buten-2-ol, respectively. It is likely that this dehydratase activity is catalyzed by a not-yet-characterized enzyme postulated for the isomerization of 2-methyl-3-buten-2-ol and prenol. The vitamin requirements of strain L108 growing on TAA and the occurrence of 3-methylcrotonic acid as a metabolite indicate that TAA and hemiterpene degradation are linked with the catabolic route of the amino acid leucine, including an involvement of the biotin-dependent 3-methylcrotonyl coenzyme A (3-methylcrotonyl-CoA) carboxylase LiuBD. Evolutionary aspects of favored desaturase versus hydroxylation pathways for TAA conversion and the possible role of MdpJ in the degradation of higher tertiary alcohols are discussed.

Synthesis of Short-Chain Diols and Unsaturated Alcohols from Secondary Alcohol Substrates by the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

ABSTRACT The Rieske nonheme mononuclear iron oxygenase MdpJ of the fuel oxygenate-degrading bacterial strain Aquincola tertiaricarbonis L108 has been described to attack short-chain tertiary alcohols via hydroxylation and desaturation reactions. Here, we demonstrate that also short-chain secondary alcohols can be transformed by MdpJ. Wild-type cells of strain L108 converted 2-propanol and 2-butanol to 1,2-propanediol and 3-buten-2-ol, respectively, whereas an mdpJ knockout mutant did not show such activity. In addition, wild-type cells converted 3-methyl-2-butanol and 3-pentanol to the corresponding desaturation products 3-methyl-3-buten-2-ol and 1-penten-3-ol, respectively. The enzymatic hydroxylation of 2-propanol resulted in an enantiomeric excess of about 70% for the (R)-enantiomer, indicating that this reaction was favored. Likewise, desaturation of (R)-2-butanol to 3-buten-2-ol was about 2.3-fold faster than conversion of the (S)-enantiomer. The biotechnological potential of MdpJ for the synthesis of enantiopure short-chain alcohols and diols as building block chemicals is discussed.

Impact of membrane fatty acid composition on the uncoupling sensitivity of the energy conservation of Comamonas testosteroni ATCC 17454

The fatty acid composition of pyruvate-grown Comamonas testosteroni ATCC 17454 was analyzed after growth at 30 and 20°C and after half-maximum growth inhibition caused by different membrane-active chemicals at 30°C. Palmitic acid (16:0), palmitoleic acid (16:1 ω7c) and vaccenic acid (18:1 ω7c) were the dominant fatty acids. At 20°C, the proportion of palmitic acid decreased and those of palmitoleic and vaccenic acid increased. Saturation degree was also lowered when half-maximum growth inhibition was caused by 4-chlorosalicylic acid, 2,4-dichlorophenoxyacetic acid and 2,4-dinitrophenol and, to a lesser extent, in the presence of 2,4-dichlorophenol, phenol and ethanol. It appeared that the dissociated forms of the former group of chemicals were preferentially incorporated near the head group region of the lipid bilayer, thereby somewhat extending the outer region of the membranes, and that the increased amount of bent, unsaturated fatty acids helped to maintain membrane integrity. Irrespective of how the decrease of the saturation degree was triggered, it caused electron transport phosphorylation (adenosine triphosphate synthesis driven by n-hexanol oxidation) to become more sensitive to uncoupling. Apparently, the viscosity and phase stability of the cytoplasmic membrane of C. testosteroni were maintained at the price of a reduced protection against energy toxicity.

A core set of metabolite sink/source ratios indicative for plant organ productivity in Lotus japonicus

Plant growth is an important process in physiological as well as ecological respect and a number of metabolic parameters (elemental ratios as well as steady-state levels of individual metabolites) have been demonstrated to reflect this process on the whole plant level. Since plant growth is highly localized and is the result of a complex interplay of metabolic activities in sink and source organs, we propose that ratios in metabolite levels of sink and source organs are particularly well suited to characterize this process. To demonstrate such a connection, we studied organ-specific metabolite ratios from Lotus japonicus treated with mineral nutrients, salt stress or arbuscular mycorrhizal fungi. The plants were displaying a wide range of biomass and of flower/biomass ratios. In the analysis of our data we looked for correlations between shifts in sink/source metabolite ratios and plant productivity (biomass accumulated at the time of harvest). In addition we correlated shifts in metabolite ratios comparing competing generative and vegetative sink organs with shifts in productivity of the two organs (changes in flower/biomass ratios). In our analyses we observed clear shifts of carbohydrates and of compounds connected to nitrogen metabolism in favour of sink organs of particularly high productivity. These shifts were in agreement with general differences in metabolite steady-state levels when comparing sink and source organs. Our findings suggest that differentiation of sink and source organs during sampling for metabolomic experiments substantially increases the amount of information obtained from such experiments.

Evaluation of FT-IR spectroscopy as a tool to quantify bacteria in binary mixed cultures

Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A calibration procedure based on Principal Component Regression was developed for estimating the ratio of the bacterial species. Data for method calibration were gained from pure cultures and artificially assembled communities of known ratios of the two member populations. Moreover, to account for physiological variability, FT-IR measurements were performed with organisms sampled at different growth phases. Measurements and data analyses were subsequently applied to growing mixed cultures revealing that growth of R. ruber was almost completely suppressed in co-culture with P. putida. Population ratios obtained by fatty acid analysis as an independent reference method were in high agreement with the FT-IR derived ratios.

Rhizosphere Protists Change Metabolite Profiles in Zea mays

Plant growth and productivity depend on the interactions of the plant with the associated rhizosphere microbes. Rhizosphere protists play a significant role in this respect: considerable efforts have been made in the past to reveal the impact of protist-bacteria interactions on the remobilization of essential nutrients for plant uptake, or the grazing induced changes on plant-growth promoting bacteria and the root-architecture. However, the metabolic responses of plants to the presence of protists or to protist-bacteria interactions in the rhizosphere have not yet been analyzed. Here we studied in controlled laboratory experiments the impact of bacterivorous protists in the rhizosphere on maize plant growth parameters and the bacterial community composition. Beyond that we investigated the induction of plant biochemical responses by separately analyzing above- and below-ground metabolite profiles of maize plants incubated either with a soil bacterial inoculum or with a mixture of soil bacteria and bacterivorous protists. Significantly distinct leaf and root metabolite profiles were obtained from plants which grew in the presence of protists. These profiles showed decreased levels of a considerable number of metabolites typical for the plant stress reaction, such as polyols, a number of carbohydrates and metabolites connected to phenolic metabolism. We assume that this decrease in plant stress is connected to the grazing induced shifts in rhizosphere bacterial communities as shown by distinct T-RFLP community profiles. Protist grazing had a clear effect on the overall bacterial community composition, richness and evenness in our microcosms. Given the competition of plant resource allocation to either defense or growth, we propose that a reduction in plant stress levels caused directly or indirectly by protists may be an additional reason for corresponding positive effects on plant growth.