We report on the characterization and target analysis of the small (s)RNA162 in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5′ fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA162 (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA162 is crucial for target interactions. Furthermore, our results indicate that sRNA162-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 5′ end of sRNA162 targets the 5′-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA162 acts as an antisense (as)RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.

Non-coding RNAs are key players in many cellular processes within organisms from all three domains of life. The range and diversity of small RNA functions beyond their involvement in translation and RNA processing was first recognized for eukaryotes and bacteria. Since then, small RNAs were also found to be abundant in archaea. Their functions include the regulation of gene expression and the establishment of immunity against invading mobile genetic elements. This review summarizes our current knowledge about small RNAs used for regulation and defence in archaea.

In spite of the significant impact of biomethylation on the mobility and toxicity of metals and metalloids in the environment, little is known about the biological formation of these methylated metal(loid) compounds. While element-specific methyltransferases have been isolated for arsenic, the striking versatility of methanoarchaea to methylate numerous metal(loid)s, including rare elements like bismuth, is still not understood. Here, we demonstrate that the same metal(loid)s (arsenic, selenium, antimony, tellurium, and bismuth) that are methylated by Methanosarcina mazei in vivo are also methylated by in vitro assays with purified recombinant MtaA, a methyltransferase catalyzing the methyl transfer from methylcobalamin [CH3Cob(III)] to 2-mercaptoethanesulfonic acid (CoM) in methylotrophic methanogenesis. Detailed studies revealed that cob(I)alamin [Cob(I)], formed by MtaA-catalyzed demethylation of CH3Cob(III), is the causative agent for the multimetal(loid) methylation observed. Moreover, Cob(I) is also capable of metal(loid) hydride generation. Global transcriptome profiling of M. mazei cultures exposed to bismuth did not reveal induced methyltransferase systems but upregulated regeneration of methanogenic cofactors in the presence of bismuth. Thus, we conclude that the multimetal(loid) methylation in vivo is attributed to side reactions of CH3Cob(III) with reduced cofactors formed in methanogenesis. The close connection between metal(loid) methylation and methanogenesis explains the general capability of methanoarchaea to methylate metal(loid)s.

We investigated the use of partial collimation on a clinical PET scanner by removing septa from conventional 2D collimators. The goal is to improve noise equivalent count-rates (NEC) compared to 2D and 3D scans for clinically relevant activity concentrations. We evaluated two cases: removing half of the septa (2.5D); and removing two-thirds of the septa (2.7D). System performance was first modeled using the SimSET simulation package, and then measured with the NEMA NU2-2001 count-rate cylinder (20 cm dia., 70 cm long), and 27 cm and 35 cm diameter cylinders of the same length. An image quality phantom was also imaged with the 2.7D collimator. SimSET predicted the relative NEC curves very well, as confirmed by measurements, with 2.5D and 2.7D NEC greater than 2D and 3D NEC in the range of ~5–20 mCi in the phantom. We successfully reconstructed images of the image quality phantom from measured 2.7D data using custom 2.7D normalization. Partial collimation shows promise for optimized clinical imaging in a fixed-collimator system.

We present a simulation study of the effect of different degrees of collimation on countrate performance of a hypothetical PET scanner with LSO crystals. The simulated scanner is loosely based on the geometry of the Siemens Biograph Hi-Rez scanner.

System behavior is studied with a photon tracking simulation package (SimSET).

We investigate the NEMA NU2-2001 count rate and scatter fraction behavior for systems with different amounts of collimation, which is achieved by adding septa to the fully-3D system as in clinical use. We study systems with 2, 5, 11, and 40 septa. The effect of collimation is studied for three patient thicknesses.

The resulting count rate curves for true, scattered, and random coincidences as well as noise equivalent count rates are compared for the different collimation cases. Improved countrate performance with partial collimation is seen. However, except for the largest diameter phantom, the NEC rate increase is seen at higher activities than those used clinically.

The NEC countrate versus activity curves for the LSO systems are also compared to those from a BGO system where partial collimation increases NEC countrate over a clinically relevant activity range.

A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.

In Klebsiella pneumoniae nitrogen fixation is tightly controlled in response to ammonium and molecular oxygen by the NifL/NifA regulatory system. Under repressing conditions, NifL inhibits the nif-specific transcriptional activator NifA by direct protein-protein interaction, whereas under anaerobic and nitrogen-limited conditions sequestration of reduced NifL to the cytoplasmic membrane impairs inhibition of cytoplasmic NifA by NifL. We report here on a genetic screen to identify amino acids of NifL essential for sequestration to the cytoplasmic membrane under nitrogen-fixing conditions. Overall, 11,500 mutated nifL genes of three independently generated pools were screened for those conferring a Nif− phenotype. Based on the respective amino acid changes of nonfunctional derivatives obtained in the screen, and taking structural data into account as well, several point mutations were introduced into nifL by site-directed mutagenesis. The majority of amino acid changes resulting in a significant nif gene inhibition were located in the N-terminal domain (N46D, Q57L, Q64R, N67S, N69S, R80C, and W87G) and the Q-linker (K271E). Further analyses demonstrated that positions N69, R80, and W87 are essential for binding the FAD cofactor, whereas primarily Q64 and N46, but also Q57 and N67, appear to be crucial for direct membrane contact of NifL under oxygen and nitrogen limitation. Based on these findings, we propose that those four amino acids most likely located on the protein surface, as well as the presence of the FAD cofactor, are crucial for the correct overall protein conformation and respective surface charge, allowing NifL sequestration to the cytoplasmic membrane under derepressing conditions.

Objectives

We have added a block detector model to the Simulation System for Emission Tomography (SimSET) software version 2.9.

Methods

The new model simulates the detector system as a collection of right rectangular boxes and allows for very flexible positioning of these boxes. This model allows users to simulate typical block-based cylindrical tomographs, pixelated positron emission mammography (PEM) detectors, and many more imaginative tomograph designs. We have tested the block detector software against analytically derived results and against SimSET simulations of dual-headed and cylindrical detector tomographs. We have also compared experimental and simulated sensitivities for a General Electric DSTE PET for 3 different phantom diameters in 2d and 3d acquisition modes.

Results

The tests against analytically derived results and against simulations were validated both statistically using the t-test and visually by comparing profiles through the sinograms. Within the limits of statistical fluctuation, the new software passed all tests. In comparisons with data from the PET scanner, the simulation showed better agreement than previous SimSET releases, but still showed substantially increased coincidence sensitivity.

Conclusions

We believe the increased sensitivity is a result of the very simple default models used for energy resolution and scintillation light collection, and the lack of any livetime correction. The new release provides a user-modifiable function where all these factors can be realistically modeled for a given tomograph. The SimSET software, including source code, remains in the public domain.

Background

Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation.

Principal Findings

We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean.

Significance

This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles.

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.

We present the derivation of a live-time model for predicting count rates in computer simulations of PET scanners. Computer models are frequently used to investigate new PET scanner configurations, but they typically do not account for the count losses caused by scanner-specific electronics and processing. The live-time fraction depends strongly on the photon flux incident on the detector. We modeled the live-time of a clinical PET scanner by relating measured and simulated single photon fluxes. Our model used data from a specific scanner, but the approach is generally applicable.

We applied the live-time model to partial collimation on a PET scanner; in particular, a scanner with septa positioned between every third detector ring (“2.7D” acquisition mode). The photon flux was measured and simulated for conventional acquisition modes (2D, 3D), and simulated for partial collimation (2.7D). These data were used in the model to predict live-time for partial collimation. The model was then validated against measurements in 2.7D mode. At low activity the model was very accurate at predicting the live-time fraction. Over-estimation of count-rates by the simulations lead to an uncertainly in the live-model. The uncertainty increased with activity concentration, reaching 0.9% and 2.2% at 20 kBq/mL for singles and coincidence live-time, respectively. When applied to 2.7D mode, the model predicted coincidence live-time accurate to 2.2% and 10% at 5 kBq/mL and 20 kBq/mL in the phantom, respectively. The 2.7D singles-counting live-time was predicted to within 0.2% of the measured value for up to 20 kBq/mL in the phantom.

Partially collimated PET systems have less collimation than conventional 2-D systems and have been shown to offer count rate improvements over 2-D and 3-D systems. Despite this potential, previous efforts have not established image-based improvements with partial collimation and have not customized the reconstruction method for partially collimated data. This work presents an image reconstruction method tailored for partially collimated data. Simulated and measured sensitivity patterns are presented and provide a basis for modification of a fully 3-D reconstruction technique. The proposed method uses a measured normalization correction term to account for the unique sensitivity to true events. This work also proposes a modified scatter correction based on simulated data. Measured image quality data supports the use of the normalization correction term for true events, and suggests that the modified scatter correction is unnecessary.

We present a simulation study of the global count-rate performance of a positron emission tomography (PET) scanner with different levels of partial collimation to maximize the noise equivalent count rate for whole-body PET imaging. We achieve partial collimation by removing different numbers of septal rings from the standard 2-D septa set for the GE Advance PET scanner. System behavior is studied with a photon tracking simulation package, which we modify to enable the production of random coincidences. The simulations are validated with measured data taken in 2-D and fully 3-D acquisition mode on a GE Advance system using the National Electrical Manufacturers Association NU-2 count-rate phantom with two sets of annular sleeves to expand the diameter to 27 and 35 cm. For all diameters and in 2-D and fully 3-D mode, there is good agreement between measurements and simulations. All studies use the three phantom diameters to evaluate the effect of patient thickness for each amount of collimation. Optimized system parameters, such as maximum ring difference for single slice rebinning, are determined for the five partially collimated systems considered. The resulting global count rates for true, scattered, and random coincidences, the noise equivalent count (NEC) rates, and the scatter fractions for different levels of collimation are compared along with the results from the conventional 2-D and fully 3-D modes. Improved statistical data quality relative to both 2-D and fully 3-D data is found with the partially collimated systems, particularly when one-half or two-thirds of the septal rings are removed. An increase in NEC rates of as much as 50% is found for clinically relevant activities between 5–10 mCi (184–370 MBq). Scatter fractions for the partially collimated systems are intermediate between the 2-D and fully 3-D numbers. Many factors that affect image quality have not been considered in this paper. However, the significant increase in statistical data quality warrants further investigation of the impact of partial collimation on clinical whole-body PET imaging.

The methanogenic archaeon Methanosarcina mazei strain Gö1 uses versatile carbon sources and is able to fix molecular nitrogen with methanol as carbon and energy sources. Here, we demonstrate that when growing on trimethylamine (TMA), nitrogen fixation does not occur, indicating that ammonium released during TMA degradation is sufficient to serve as a nitrogen source and represses nif gene induction. We further report on the transcriptional regulation of soluble methyltransferases, which catalyze the initial step of methylamine consumption by methanogenesis, in response to different carbon and nitrogen sources. Unexpectedly, we obtained conclusive evidence that transcription of the mtmB2C2 operon, encoding a monomethylamine (MMA) methyltransferase and its corresponding corrinoid protein, is highly increased under nitrogen limitation when methanol serves as a carbon source. In contrast, transcription of the homologous mtmB1C1 operon is not affected by the nitrogen source but appears to be increased when TMA is the sole carbon and energy source. In general, transcription of operons encoding dimethylamine (DMA) and TMA methyltransferases and methylcobalamine:coenzyme M methyltransferases is not regulated in response to the nitrogen source. However, in all cases transcription of one of the homologous operons or genes is increased by TMA or its degradation products DMA and MMA.

The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon.

Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M. mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of E. coli GlnK. M. mazei GlnK1 is encoded in the glnK1-amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E. coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure. M. mazei glnK1 was cloned and overexpressed in E. coli, and GlnK1 was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E. coli GlnK. First, GlnK1 from M. mazei is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between M. mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed. Because M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by NifL in response to molecular oxygen and combined nitrogen. We recently showed that K. pneumoniae NifL is a flavoprotein, which apparently senses oxygen through a redox-sensitive, conformational change. We have now studied the oxygen regulation of NifL activity in Escherichia coli and K. pneumoniae strains by monitoring its inhibition of NifA-mediated expression of K. pneumoniae ø(nifH′-′lacZ) fusions in different genetic backgrounds. Strains of both organisms carrying fnr null mutations failed to release NifL inhibition of NifA transcriptional activity under oxygen limitation: nif induction was similar to the induction under aerobic conditions. When the transcriptional regulator Fnr was synthesized from a plasmid, it was able to complement, i.e., to relieve NifL inhibition in the fnr mutant backgrounds. Hence, Fnr appears to be involved, directly or indirectly, in NifL-dependent oxygen regulation of nif gene expression in K. pneumoniae. The data indicate that in the absence of Fnr, NifL apparently does not receive the signal for anaerobiosis. We therefore hypothesize that in the absence of oxygen, Fnr, as the primary oxygen sensor, activates transcription of a gene or genes whose product or products function to relieve NifL inhibition by reducing the flavin adenine dinucleotide cofactor under oxygen-limiting conditions.

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.

Environmental DNA libraries from three different soil samples were constructed. The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80%. The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization. Thirty-six positive E. coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype. These E. coli clones were studied further. All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts. Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase. Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3. The deduced orf1 gene product showed similarities to members of the DedA family of proteins. The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases. The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.