Diversit&auml;t und Abundanz des Ribulose-1,5-bisphosphat Carboxylase/Oxygenase (RubisCO) -Gens <em>cbbL</em> autotropher Bakterien in Agrarb&ouml;den.

Autotrophe Bacteria sind von zentraler Bedeutung f&uuml;r den terrestrischen Kohlenstoffkreislauf, da sie dem an verf&uuml;gbaren organischen Kohlenstoffverbindungen armen Boden Biomasse zuf&uuml;hren und einen Beitrag zur Reduzierung des atmosph&auml;rischen CO2 leisten k&ouml;nnten. Doch w&auml;hrend die autotrophen Prozesse und die daran beteiligten Mikroorganismen in aquatischen Habitaten bereits gut untersucht und verstanden sind, besteht noch erheblicher Forschungsbedarf zur Diversit&auml;t und Abundanz autotropher Bakterienpopulationen in B&ouml;den. In dieser Arbeit sollten zentrale Fragen zur Charakterisierung der autotrophen Gemeinschaften mit Werkzeugen der molekularen mikrobiellen &Ouml;kologie bearbeitet werden. Die meisten Prokaryota, die mit CO2 als einzige Kohlenstoffquelle zu wachsen verm&ouml;gen, fixieren dieses &uuml;ber den Calvin-Benson-Bessham Zyklus. Das Schl&uuml;sselenzym dieses Zykluses ist die Ribulose-1,5-bisphosphat Carboxylase/Oxygenase (RubisCO). Die gro&szlig;e Untereinheit der Form I-RubisCO wird von dem Gen cbbL kodiert, welches phylogenetisch in zwei Hauptentwicklungslinien unterteilt wird: &sbquo;green-like&rsquo; und &sbquo;red-like&rsquo;. Um einen Einblick in die genetische Diversit&auml;t CO2-fixierender Bakterien in unterschiedlich ged&uuml;ngten Agrarb&ouml;den des Dauerd&uuml;ngungsversuchs Ewiger Roggenbau in Halle/Saale zu erlangen, wurde eine auf PCR basierende Methodik entwickelt, die auf der Erfassung des Funktionsgens cbbL zielt. Es wurden Datenbankrecherchen durchgef&uuml;hrt und mittels den anschlie&szlig;enden vergleichenden Sequenzanalysen und phylogenetischen Untersuchungen bekannter cbbL-Sequenzen spezifische Oligonukleotid-Primerpaare konstruiert, die ausgew&auml;hlte cbbL-Sequenzen terrestrischer Bakterien der &sbquo;red-like&rsquo; bzw. der &sbquo;green-like&rsquo; RubisCO-Linien amplifizieren. Mit Hilfe dieser Primer gelang es cbbL-Genbanken anzulegen, die mittels der Restriktions-Fragmentl&auml;ngen-Polymorphismus-(RFLP)-Analyse und Diversit&auml;tindices untersucht und verglichen wurden; ausgew&auml;hlte Sequenzen wurden einer phylogenetischen Zuordnung unterzogen. Mit den entwickelten Primerpaaren konnten in den untersuchten B&ouml;den nur eine geringe Diversit&auml;t an &sbquo;green-like&rsquo; cbbL-Sequenzen festgestellt werden, die phylogenetisch zu den cbbL-Sequenzen von Nitrobacter vulgaris und Nitrobacter winogradskyi nahe verwandt waren. Im Vergleich dazu zeichneten sich die &sbquo;red-like&rsquo; cbbL-Sequenzen aus den B&ouml;den durch eine hohe Diversit&auml;t aus, wobei sie phylogenetisch &uuml;ber die gesamte &sbquo;red-like&rsquo;-Gruppe verteilt waren und sich h&auml;ufig als nur entfernt verwandt zu bekannten cbbL-Sequenzen herausstellten. W&auml;hrend mit der RFLP-Analyse Bodenbehandlungs-spezifische Muster identifiziert wurden, war nach der phylogenetischen Sequenzanalyse keine Cluster-Bildung in Abh&auml;ngigkeit von der Bodenbehandlung zu beobachten. Um den Datensatz an vorhandenen &sbquo;red-like&rsquo; cbbL-Sequenzen zu erweitern, wurden cbbL-Gene aus verschiedenen kultivierten &alpha;- und &beta;-Proteobacteria sowie aus Bakterienisolaten, die in dieser Arbeit aus Boden gewonnen wurden, amplifiziert. Die phylogenetische Sequenzanalyse gruppierte diese cbbL-Sequenzen Taxon-unabh&auml;ngig zu den verschiedenen Clustern des &sbquo;red-like&rsquo;-Baums einschlie&szlig;lich der neuen cbbL-Gencluster aus den Halle-B&ouml;den. Bakterielle Bodenisolate, die als cbbL-positiv identifiziert wurden, konnten basierend auf ihrer 16S rDNA-Sequenz als Organismen der Gram-positiven Gattungen Bacillus, Streptomyces und Arthrobacter klassifiziert werden. Vertreter dieser bakteriellen Gruppen waren bisher nicht als CO2-Fixierer charakterisiert worden. Der physiologische Beweis eines aktiven CO2-fixierenden Metabolismus &uuml;ber RubisCO steht noch aus. Die Ergebnisse der &sbquo;red-like&rsquo; cbbL-Diversit&auml;ts-Studie dienten als Grundlage zur Konstruktion weiterer Oligonukleotide, die in der &bdquo;real-time&ldquo; TaqMan-PCR zur Quantifizierung von &sbquo;red-like&rsquo; cbbL-Genen aus Boden eingesetzt wurden. Dabei wird ersichtlich, dass in den untersuchten Bodenvarianten bis zu 107 cbbL-Genkopien/g Boden enthalten sind. Die unterschiedlichen Bodenbehandlungen scheinen keinen Einfluss auf die Abundanz von &sbquo;red-like&rsquo; cbbL-Genen in B&ouml;den zu nehmen

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture.

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum. Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites

Identification of new enzymes potentially involved in anaerobic naphthalene degradation by the sulfate-reducing enrichment culture N47.

The sulfate-reducing highly enriched culture N47 is capable to anaerobically degrade naphthalene, 2-methylnaphthalene, and 2-naphthoic acid. A proteogenomic investigation was performed to elucidate the initial activation reaction of anaerobic naphthalene degradation. This lead to the identification of an alpha-subunit of a carboxylase protein that was two-fold up-regulated in naphthalene-grown cells compared to 2-methylnaphthalene-grown cells. The putative naphthalene carboxylase subunit showed 48% similarity to the anaerobic benzene carboxylase from an iron-reducing, benzene-degrading culture and 45% to alpha-subunit of phenylphosphate carboxylase of Aromatoleum aromaticum EbN1. A gene for the beta-subunit of putative naphthalene carboxylase was located nearby on the genome and was expressed with naphthalene. Similar to anaerobic benzene carboxylase, there were no genes for gamma- and delta-subunits of a putative carboxylase protein located on the genome which excludes participation in degradation of phenolic compounds. The genes identified for putative naphthalene carboxylase subunits showed only weak similarity to 4-hydroxybenzoate decarboxylase excluding ATP-independent carboxylation. Several ORFs were identified that possibly encode a 2-naphthoate-CoA ligase, which is obligate for activation before the subsequent ring reduction by naphthoyl-CoA reductase. One of these ligases was exclusively expressed on naphthalene and 2-naphthoic acid and might be the responsible naphthoate-CoA-ligase

Microbial hydrocarbon degradation at coal gasification plants.

Almost every bigger city harbors a former coal gasification plant, where sometimes huge amounts of contaminants such as tar oil have leaked into the subsurface. This continuous release of monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, xylene (BTEX), as well as polycyclic aromatic hydrocarbons (PAH) such as naphthalene has resulted in considerable contaminant plumes. After decades of deposition, contaminant dissolution from the source is often in equilibrium with biodegradation processes. However, owing to slow dissolution and limited attenuation potentials, these sites are expected to remain impacted for hundreds and thousands of years. Today, novel means allow assessing biodegradation at former gasification sites by, e.g., metabolite analysis, fingerprinting of substrate spectra, or mass balances based on electron acceptor depletion. Biodegradation can even be quantified by stable isotope fractionation analysis. Also, knowledge on aerobic microbial hydrocarbon-degrading microorganisms is quite elaborate, which helps to understand degradation in unsaturated zones. However, these heavily contaminated aquifers usually turn anoxic, and apart from the degradation of toluene, ethylbenzene, and methylnaphthalene, the biochemistry of most anaerobic degradation pathways is still elusive. Hence, the controls of in situ biodegradation processes are still poorly understood. Recently, it has become apparent that the spatial separation of electron acceptors and contaminants in contaminant plumes caused by limited mixing in aquifers may be one of the most important factors limiting biodegradation in these systems. Electron acceptors are depleted in the center of plumes, restricting degradation activities to the fringe zones, where electron acceptors and contaminants meet in steep geochemical counter-gradients. Microbial communities in water and sediment samples from gasification plants generally exhibit abundance orders of magnitude higher than in uncontaminated references. At the same time, total community composition can be similarly diverse, albeit significant structural distinctions have been reported for the populations found in strongly or less impacted zones. The occurrence of both typical and uncultured degradation key-players seems to be correlated with zones of contamination and the prevailing biogeochemical conditions. Especially in degradation hot-spots at plume fringes, the abundance of specific hydrocarbon degraders can be surprisingly high. We highlight that substantial research efforts still need to be devoted to a better understanding of key aromatics and hydrocarbon degraders under iron- and sulfate-reducing, and methanogenic conditions, as well as to the biogeochemical and ecological controls of their activity in contaminated subsurface systems

Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria.

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

Quantification of bacterial RubisCO genes in soils by cbbL targeted real-time PCR.

Soils harbor a high diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit coding genes (cbbL). Real-time PCR was used to quantify this gene in differently managed agricultural soils and soil microhabitats. We developed primers and a TaqMan probe that target the &quot;red-like&quot; RubisCO gene cbbL. Primers and probe were developed based on cbbL sequences of selected bacterial pure cultures and of environmental clones. The amount of cbbL copies in the investigated soils were detected in the range of 6.8x10(6) to 3.4x10(7) &quot;red-like&quot; cbbL copies/g soil. The cbbL genes could be located entirely in the clay and silt fraction, while the coarse sand fractions revealed no detectable level of bacterial RubisCO genes. These results indicate that bacteria with RubisCO coding genes are numerous and widespread in soils, however the functional implication of this gene in soils is not yet clear

Identification of enzymes involved in anaerobic benzene degradation by a strictly anaerobic iron-reducing enrichment culture.

Summary 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 ( approximately 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)