Anaerobic benzene degradation was studied with a highly enriched iron-reducing culture (BF) composed of mainly Peptococcaceae-related Gram-positive microorganisms. The proteomes of benzene-, phenol- and benzoate-grown cells of culture BF were compared by SDS-PAGE. A specific benzene-expressed protein band of 60 kDa, which could not be observed during growth on phenol or benzoate, was subjected to N-terminal sequence analysis. The first 31 amino acids revealed that the protein was encoded by ORF 138 in the shotgun sequenced metagenome of culture BF. ORF 138 showed 43% sequence identity to phenylphosphate carboxylase subunit PpcA of Aromatoleum aromaticum strain EbN1. A LC/ESI-MS/MS-based shotgun proteomic analysis revealed other specifically benzene-expressed proteins with encoding genes located adjacent to ORF 138 on the metagenome. The protein products of ORF 137, ORF 139 and ORF 140 showed sequence identities of 37% to phenylphosphate carboxylase PpcD of A. aromaticum strain EbN1, 56% to benzoate-CoA ligase (BamY) of Geobacter metallireducens and 67% to 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX) of A. aromaticum strain EbN1 respectively. These genes are proposed as constituents of a putative benzene degradation gene cluster (∼ 17 kb) composed of carboxylase-related genes. The identified gene sequences suggest that the initial activation reaction in anaerobic benzene degradation is probably a direct carboxylation of benzene to benzoate catalysed by putative anaerobic benzene carboxylase (Abc). The putative Abc probably consists of several subunits, two of which are encoded by ORFs 137 and 138, and belongs to a family of carboxylases including phenylphosphate carboxylase (Ppc) and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase (UbiD/UbiX).

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.The Calvin-Benson-Bassham cycle is the major and most abundant pathway for CO 2 fixation (30). In addition to its essential capacity as the mechanism of primary production in nearly all ecosystems, the Calvin cycle plays a major role in effecting the concentration of atmospheric CO 2 . The Calvin cycle exists in diverse organisms, from bacteria to algae to green plants. The most abundant protein on earth, ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO), catalyzes the first, rate-limiting step in the Calvin cycle (5). RubisCO is a bifunctional enzyme that controls the reduction of CO 2 and the oxygenolysis of ribulose-1,5-bisphophate. Since RubisCO is responsible for the overwhelming amount of carbon fixation by plants, nearly all primary production is linked to the function of this enzyme. RubisCO is a well-studied enzyme because of its extensive agricultural and environmental significance (2, 14, 22).RubisCO exists in multiple natural forms which differ in structure, catalytic property, and O 2 sensitivity (33). Form I RubisCO is a hexadecamer composed of eight large and eight small subunits (L 8 S 8 ) and occurs in photo-and chemoautotrophic organisms. The form II protein consists only of large subunits (L n ), with 25 to 30% amino acid sequence identity to form I (33), and is found in photo-and chemoautotrophs. It is assum...