Diversity and abundance of Crenarchaeota in terrestrial habitats studied by 16S RNA surveys and real time PCR

Novel phylogenetic lineages of as yet uncultivated crenarchaeota have been frequently detected in low to moderate-temperature, marine and terrestrial environments. In order to gain a more comprehensive view on the distribution and diversity of Crenarchaeota in moderate habitats, we have studied 18 different terrestrial and freshwater samples by 16S rDNA-based phylogenetic surveys. In seven different soil samples of diverse geographic areas in Europe (forest, grassland, ruderal) and Asia (permafrost, ruderal) as well as in two microbial mats, we have consistently found one particular lineage of crenarchaeota. The diversity of Crenarchaeota in freshwater sediments was considerably higher with respresentative 16S rDNA sequences distributed over four different groups within the moderate crenarchaeota. Systematic analysis of a 16S rDNA universal library from a sandy ecosystem containing 800 clones exclusively revealed the presence of the soil-specific crenarchaeotal cluster. With primers specific for non-thermophilic crenarchaeota we established a rapid method to quantify archaeal 16S rDNA in real time PCR. The relative abundance of crenarchaeotal rDNA was 0.5-3% in the bulk soil sample and only 0.16% in the rhizosphere of the sandy ecosystem. A nearby agricultural setting yielded a relative abundance of 0.17% crenarchaeotal rDNA. In total our data suggest that soil crenarchaeota represent a stable and specific component of the microbiota in terrestrial habitats.     

<Emphasis Type="Italic">Xanthium italicum</Emphasis>, <Emphasis Type="Italic">Xanthium strumarium</Emphasis> and <Emphasis Type="Italic">Arctium lappa</Emphasis> as new hosts for <Emphasis Type="Italic">Diaporthe helianthi</Emphasis>

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.     

The enzymes of oxalate metabolism: unexpected structures and mechanisms

Oxalate degrading enzymes have a number of potential applications, including medical diagnosis and treatments for hyperoxaluria and other oxalate-related diseases, the production of transgenic plants for human consumption, and bioremediation of the environment. This review seeks to provide a brief overview of current knowledge regarding the major classes of enzymes and related proteins that are employed in plants, fungi, and bacteria to convert oxalate into CO(2) and/or formate. Not only do these enzymes employ intriguing chemical strategies for cleaving the chemically unreactive C-C bond in oxalate, but they also offer the prospect of providing new insights into the molecular processes that underpin the evolution of biological catalysts.     

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.     

Diversity of green-like and red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils

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.     

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

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).     

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.     

EPR spectroscopic characterization of the manganese center and a free radical in the oxalate decarboxylase reaction: identification of a tyrosyl radical during turnover

Several molecular mechanisms for cleavage of the oxalate carbon-carbon bond by manganese-dependent oxalate decarboxylase have recently been proposed involving high oxidation states of manganese. We have examined the oxalate decarboxylase from Bacillus subtilis by electron paramagnetic resonance in perpendicular and parallel polarization configurations to test for the presence of such species in the resting state and during enzymatic turnover. Simulation and the position of the half-field Mn(II) line suggest a nearly octahedral metal geometry in the resting state. No spectroscopic signature for Mn(III) or Mn(IV) is seen in parallel mode EPR for samples frozen during turnover, consistent either with a large zero-field splitting in the oxidized metal center or undetectable levels of these putative high-valent intermediates in the steady state. A narrow, featureless g = 2.0 species was also observed in perpendicular mode in the presence of substrate, enzyme, and dioxygen. Additional splittings in the signal envelope became apparent when spectra were taken at higher temperatures. Isotopic editing resulted in an altered line shape only when tyrosine residues of the enzyme were specifically deuterated. Spectral processing confirmed multiple splittings with isotopically neutral enzyme that collapsed to a single prominent splitting in the deuterated enzyme. These results are consistent with formation of an enzyme-based tyrosyl radical upon oxalate exposure. Modestly enhanced relaxation relative to abiological tyrosyl radicals was observed, but site-directed mutagenesis indicated that conserved tyrosine residues in the active site do not host the unpaired spin. Potential roles for manganese and a peripheral tyrosyl radical during steady-state turnover are discussed.     

Non-phototrophic CO 2 fixation by soil microorganisms

Although soils are generally known to be a net source of CO₂ due to microbial respiration, CO₂ fixation may also be an important process. The non-phototrophic fixation of CO₂ was investigated in a tracer experiment with ¹⁴CO₂ in order to obtain information about the extent and the mechanisms of this process. Soils were incubated for up to 91 days in the dark. In three independent incubation experiments, a significant transfer of radioactivity from ¹⁴CO₂ to soil organic matter was observed. The process was related to microbial activity and could be enhanced by the addition of readily available substrates such as acetate. CO₂ fixation exhibited biphasic kinetics and was linearly related to respiration during the first phase of incubation (about 20–40 days). The fixation amounted to 3–5% of the net respiration. After this phase, the CO₂ fixation decreased to 1–2% of the respiration. The amount of carbon fixed by an agricultural soil corresponded to 0.05% of the organic carbon present in the soil at the beginning of the experiment, and virtually all of the fixed CO₂ was converted to organic compounds. Many autotrophic and heterotrophic biochemical processes result in the fixation of CO₂. However, the enhancement of the fixation by addition of readily available substrates and the linear correlation with respiration suggested that the process is mainly driven by aerobic heterotrophic microorganisms. We conclude that heterotrophic CO₂ fixation represents a significant factor of microbial activity in soils.     

Effects of Insulin Therapy on Myocardial Lipid Content and Cardiac Geometry in Patients with Type-2 Diabetes Mellitus

Aims/Hypothesis

Recent evidence suggests a link between myocardial steatosis and diabetic cardiomyopathy. Insulin, as a lipogenic and growth-promoting hormone, might stimulate intramyocardial lipid (MYCL) deposition and hypertrophy. Therefore, the aim of the present study was to investigate the short-term effects of insulin therapy (IT) on myocardial lipid content and morphology in patients with T2DM.

Methods

Eighteen patients with T2DM were recruited (age 56±2 years; HbA1c: 10.5±0.4%). In 10 patients with insufficient glucose control under oral medication IT was initiated due to secondary failure of oral glucose lowering therapy (IT-group), while 8 individuals did not require additional insulin substitution (OT-group). In order to assess MYCL and intrahepatic lipid (IHLC) content as well as cardiac geometry and function magnetic resonance spectroscopy (MRS) and imaging (MRI) examinations were performed at baseline (IT and OT) and 10 days after initiation of IT. Follow up measurements took place 181±49 days after IT.

Results

Interestingly, basal MYCLs were 50% lower in IT- compared to OT-group (0.41±0.12 vs. 0.80±0.11% of water signal; p = 0.034). After 10 days of IT, an acute 80%-rise in MYCL (p = 0.008) was observed, while IHLC did not change. Likewise, myocardial mass (+13%; p = 0.004), wall thickness in end-diastole (+13%; p = 0.030) and concentricity, an index of cardiac remodeling, increased (+28%; p = 0.026). In the long-term MYCL returned to baseline, while IHCL significantly decreased (−31%; p = 0.000). No acute changes in systolic left ventricular function were observed.

Conclusions/Interpretation

The initiation of IT in patients with T2DM was followed by an acute rise in MYCL concentration and myocardial mass.