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1.  Biofilm formation of mucosa-associated methanoarchaeal strains 
Although in nature most microorganisms are known to occur predominantly in consortia or biofilms, data on archaeal biofilm formation are in general scarce. Here, the ability of three methanoarchaeal strains, Methanobrevibacter smithii and Methanosphaera stadtmanae, which form part of the human gut microbiota, and the Methanosarcina mazei strain Gö1 to grow on different surfaces and form biofilms was investigated. All three strains adhered to the substrate mica and grew predominantly as bilayers on its surface as demonstrated by confocal laser scanning microscopy analyses, though the formation of multi-layered biofilms of Methanosphaera stadtmanae and Methanobrevibacter smithii was observed as well. Stable biofilm formation was further confirmed by scanning electron microscopy analysis. Methanosarcina mazei and Methanobrevibacter smithii also formed multi-layered biofilms in uncoated plastic μ-dishesTM, which were very similar in morphology and reached a height of up to 40 μm. In contrast, biofilms formed by Methanosphaera stadtmanae reached only a height of 2 μm. Staining with the two lectins ConA and IB4 indicated that all three strains produced relatively low amounts of extracellular polysaccharides most likely containing glucose, mannose, and galactose. Taken together, this study provides the first evidence that methanoarchaea can develop and form biofilms on different substrates and thus, will contribute to our knowledge on the appearance and physiological role of Methanobrevibacter smithii and Methanosphaera stadtmanae in the human intestine.
doi:10.3389/fmicb.2014.00353
PMCID: PMC4086402  PMID: 25071757
biofilms; methanoarchaea; human gut; microbiota
2.  The Intestinal Archaea Methanosphaera stadtmanae and Methanobrevibacter smithii Activate Human Dendritic Cells 
PLoS ONE  2014;9(6):e99411.
The methanoarchaea Methanosphaera stadtmanae and Methanobrevibacter smithii are known to be part of the indigenous human gut microbiota. Although the immunomodulatory effects of bacterial gut commensals have been studied extensively in the last decade, the impact of methanoarchaea in human's health and disease was rarely examined. Consequently, we studied and report here on the effects of M. stadtmanae and M. smithii on human immune cells. Whereas exposure to M. stadtmanae leads to substantial release of proinflammatory cytokines in monocyte-derived dendritic cells (moDCs), only weak activation was detected after incubation with M. smithii. Phagocytosis of M. stadtmanae by moDCs was demonstrated by confocal microscopy as well as transmission electronic microscopy (TEM) and shown to be crucial for cellular activation by using specific inhibitors. Both strains, albeit to different extents, initiate a maturation program in moDCs as revealed by up-regulation of the cell-surface receptors CD86 and CD197 suggesting additional activation of adaptive immune responses. Furthermore, M. stadtmanae and M. smithii were capable to alter the gene expression of antimicrobial peptides in moDCs to different extents.
Taken together, our findings strongly argue that the archaeal gut inhabitants M. stadtmanae and M. smithii are specifically recognized by the human innate immune system. Moreover, both strains are capable of inducing an inflammatory cytokine response to different extents arguing that they might have diverse immunomodulatory functions. In conclusion, we propose that the impact of intestinal methanoarchaea on pathological conditions involving the gut microbiota has been underestimated until now.
doi:10.1371/journal.pone.0099411
PMCID: PMC4051749  PMID: 24915454
3.  Two CRISPR-Cas systems inMethanosarcina mazeistrain Gö1 display common processing features despite belonging to different types I and III 
RNA Biology  2013;10(5):779-791.
The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5′ repeat tags. The 3′-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3′-tag). The analysis further revealed a 5′-hydroxy and 3′-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.
doi:10.4161/rna.23928
PMCID: PMC3737336  PMID: 23619576
methanoarchaea; CRISPR-Cas system; immunity of prokaryotes; regulatory RNA; phages; Methanosarcina mazei
4.  Effects of β-Lactam Antibiotics and Fluoroquinolones on Human Gut Microbiota in Relation to Clostridium difficile Associated Diarrhea 
PLoS ONE  2014;9(2):e89417.
Clostridium difficile infections are an emerging health problem in the modern hospital environment. Severe alterations of the gut microbiome with loss of resistance to colonization against C. difficile are thought to be the major trigger, but there is no clear concept of how C. difficile infection evolves and which microbiological factors are involved. We sequenced 16S rRNA amplicons generated from DNA and RNA/cDNA of fecal samples from three groups of individuals by FLX technology: (i) healthy controls (no antibiotic therapy); (ii) individuals receiving antibiotic therapy (Ampicillin/Sulbactam, cephalosporins, and fluoroquinolones with subsequent development of C. difficile infection or (iii) individuals receiving antibiotic therapy without C. difficile infection. We compared the effects of the three different antibiotic classes on the intestinal microbiome and the effects of alterations of the gut microbiome on C. difficile infection at the DNA (total microbiota) and rRNA (potentially active) levels. A comparison of antibiotic classes showed significant differences at DNA level, but not at RNA level. Among individuals that developed or did not develop a C. difficile infection under antibiotics we found no significant differences. We identified single species that were up- or down regulated in individuals receiving antibiotics who developed the infection compared to non-infected individuals. We found no significant differences in the global composition of the transcriptionally active gut microbiome associated with C. difficile infections. We suggest that up- and down regulation of specific bacterial species may be involved in colonization resistance against C. difficile providing a potential therapeutic approach through specific manipulation of the intestinal microbiome.
doi:10.1371/journal.pone.0089417
PMCID: PMC3938479  PMID: 24586762
5.  Composition of Microbial Oral Biofilms during Maturation in Young Healthy Adults 
PLoS ONE  2014;9(2):e87449.
In the present study we aimed to analyze the bacterial community structure of oral biofilms at different maturation stages in young healthy adults. Oral biofilms established on membrane filters were collected from 32 human subjects after 5 different maturation intervals (1, 3, 5, 9 and 14 days) and the respective phylogenetic diversity was analyzed by 16S rDNA amplicon sequencing. Our analyses revealed highly diverse entire colonization profiles, spread into 8 phyla/candidate divisions and in 15 different bacterial classes. A large inter-individual difference in the subjects’ microbiota was observed, comprising 35% of the total variance, but lacking conspicuous general temporal trends in both alpha and beta diversity. We further obtained strong evidence that subjects can be categorized into three clusters based on three differently occurring and mutually exclusive species clusters.
doi:10.1371/journal.pone.0087449
PMCID: PMC3913613  PMID: 24503584
6.  A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii* 
The Journal of Biological Chemistry  2014;289(10):7164-7177.
Background: The Cas6 protein is required for generating crRNAs in CRISPR-Cas I and III systems.
Results: The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax.
Conclusion: A Cascade-like complex is required in the type I-B system for a stable crRNA population.
Significance: The CRISPR-Cas system I-B has a similar Cascade complex like types I-A and I-E.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.
doi:10.1074/jbc.M113.508184
PMCID: PMC3945376  PMID: 24459147
Archaea; Microbiology; Molecular Biology; Molecular Genetics; Protein Complexes; CRISPR/Cas; Cas6; Haloferax volcanii; crRNA; Type I-B
7.  Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy 
PLoS ONE  2013;8(8):e68661.
In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km2, which contained ∼2.2×104 tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ∼440 km3 the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ∼30% of the photoautotrophic carbon fixation.
Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ waters.
doi:10.1371/journal.pone.0068661
PMCID: PMC3749208  PMID: 23990875
8.  An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains 
Nucleic Acids Research  2012;40(21):10964-10979.
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.
doi:10.1093/nar/gks847
PMCID: PMC3510493  PMID: 22965121
9.  Small RNAs for defence and regulation in archaea 
Extremophiles  2012;16(5):685-696.
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.
doi:10.1007/s00792-012-0469-5
PMCID: PMC3432209  PMID: 22763819
sRNA; Lsm; Hfq; Archaea; CRISPR; crRNA
10.  Connection between Multimetal(loid) Methylation in Methanoarchaea and Central Intermediates of Methanogenesis▿† 
Applied and Environmental Microbiology  2011;77(24):8669-8675.
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.
doi:10.1128/AEM.06406-11
PMCID: PMC3233109  PMID: 22003009
11.  Count-Rate Performance of the Discovery STE PET Scanner Using Partial Collimation 
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.
doi:10.1109/NSSMIC.2006.354416
PMCID: PMC3210021  PMID: 22072860
12.  Simulations of the Effect of Collimation on Count Rates of an LSO PET 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.
doi:10.1109/NSSMIC.2006.356517
PMCID: PMC3210022  PMID: 22072861
13.  Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes 
Archaea  2011;2011:439608.
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.
doi:10.1155/2011/439608
PMCID: PMC3177094  PMID: 21941461
14.  Insights into Membrane Association of Klebsiella pneumoniae NifL under Nitrogen-Fixing Conditions from Mutational Analysis▿  
Journal of Bacteriology  2010;193(3):695-705.
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.
doi:10.1128/JB.00775-10
PMCID: PMC3021237  PMID: 21057007
15.  Modeling block detectors in SimSET 
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.
PMCID: PMC2999359  PMID: 21151857
16.  Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments 
PLoS ONE  2010;5(11):e13846.
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.
doi:10.1371/journal.pone.0013846
PMCID: PMC2974642  PMID: 21079771
17.  Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems▿ †  
Applied and Environmental Microbiology  2009;75(12):4035-4045.
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.
doi:10.1128/AEM.00515-09
PMCID: PMC2698369  PMID: 19376903
18.  Estimating Live-Time for New PET Scanner Configurations 
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.
doi:10.1109/NSSMIC.2007.4436736
PMCID: PMC2637558  PMID: 19209237
19.  Image Reconstruction for a Partially Collimated Whole Body PET Scanner 
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.
doi:10.1109/TNS.2008.921938
PMCID: PMC2605072  PMID: 19096731
Collimation; fully 3-D PET; normalization; scatter estimation; septa; 2.5D
20.  Optimization of Noise Equivalent Count Rate Performance for a Partially Collimated PET Scanner by Varying the Number of Septa 
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.
doi:10.1109/TMI.2007.895485
PMCID: PMC2592607  PMID: 17649907
Biomedical nuclear imaging; collimation; partial collimation; positron emission tomography; sensitivity; simulation
21.  Effects of Nitrogen and Carbon Sources on Transcription of Soluble Methyltransferases in Methanosarcina mazei Strain Gö1† 
Journal of Bacteriology  2005;187(17):6147-6154.
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.
doi:10.1128/JB.187.17.6147-6154.2005
PMCID: PMC1196137  PMID: 16109956
22.  Functional organization of a single nif cluster in the mesophilic archaeon Methanosarcina mazei strain Gö1 
Archaea  2002;1(2):143-150.
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.
PMCID: PMC2685556  PMID: 15803652
GlnB-like proteins; nif genes; nitrogen fixation; nitrogen regulation
24.  Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1 
Journal of Bacteriology  2002;184(4):1028-1040.
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.
doi:10.1128/jb.184.4.1028-1040.2002
PMCID: PMC134814  PMID: 11807063
25.  Fnr Is Required for NifL-Dependent Oxygen Control of nif Gene Expression in Klebsiella pneumoniae 
Journal of Bacteriology  2001;183(4):1385-1393.
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.
doi:10.1128/JB.183.4.1385-1393.2001
PMCID: PMC95013  PMID: 11157952

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