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Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO 2 molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-13 C 2 ]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C 1 units derived therefrom and not into other cell compounds.

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent beta-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of beta-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO2. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases.

The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via paracarboxylation of phenol (biological Kolbe-Schmitt carboxylation). In the first step, phenol is converted to phenylphosphate which is then carboxylated to 4-hydroxybenzoate in the second step. Phenylphosphate formation is catalyzed by the novel enzyme phenylphosphate synthase, which was studied. Phenylphosphate synthase consists of three proteins whose genes are located adjacent to each other on the phenol operon and were overproduced in Escherichia coli. The promoter region and operon structure of the phenol gene cluster were Phenol is a natural substrate which is formed from a variety of natural compounds. Phenol arises from tyrosine by tyrosine phenol lyase, but phenol also arises during the degradation of many secondary phenolic plant constituents, notably in the course of the degradation of lignin and phenylpropanoid compounds. Besides phenol, there are many other phenolic compounds, both natural and synthetic ones. Their mineralization proceeds via completely different pathways, depending on whether oxygen is available or not. For instance, groundwater and landfills are free of oxygen. Therefore, anaerobic metabolism of phenolic compounds is of general interest, from both scientific and applied aspects.The initial steps in aerobic phenol metabolism are catalyzed by oxygenases. Phenol is oxidized to catechol (1,2-dihydroxybenzene) by phenol monooxygenases followed by oxygenolytic ring cleavage catalyzed by catechol dioxygenase. Hence, aerobic metabolism of phenol requires molecular oxygen for both ring hydroxylation and ring cleavage. In contrast to aerobic metabolism, anaerobic metabolism cannot rely on oxygen-and oxygenase-dependent steps. Therefore, anaerobic metabolism of phenol and related phenolic compounds promises unprecedented biochemistry. Anaerobic growth of pure cultures on phenol has been shown for sulfate-reducing (5), denitrifying (47,50,51), and iron-reducing (35) bacteria. The list of bacteria growing anaerobically with phenolic compounds is steadily growing (see references in reference 43). In all cases studied, anaerobic growth on phenol requires the presence of CO 2 (50); CO 2 is required as a cosubstrate for phenol carboxylation which results in the formation of 4-hydroxybenzoate. Phenol carboxylation has been known in chemistry for more than 100 years and is referred to as Kolbe-Schmitt carboxylation.Anaerobic phenol metabolism by pure cultures has been studied in some detail only in the denitrifying beta-proteobacterium Thauera aromatica (2, 3, 16, 24, 29-31, 43, 50, 51). It involves two initial steps (Fig.