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1.  Identification of the Major Expressed S-Layer and Cell Surface-Layer-Related Proteins in the Model Methanogenic Archaea: Methanosarcina barkeri Fusaro and Methanosarcina acetivorans C2A 
Archaea  2012;2012:873589.
Many archaeal cell envelopes contain a protein coat or sheath composed of one or more surface exposed proteins. These surface layer (S-layer) proteins contribute structural integrity and protect the lipid membrane from environmental challenges. To explore the species diversity of these layers in the Methanosarcinaceae, the major S-layer protein in Methanosarcina barkeri strain Fusaro was identified using proteomics. The Mbar_A1758 gene product was present in multiple forms with apparent sizes of 130, 120, and 100 kDa, consistent with post-translational modifications including signal peptide excision and protein glycosylation. A protein with features related to the surface layer proteins found in Methanosarcina acetivorans C2A and Methanosarcina mazei Goel was identified in the M. barkeri genome. These data reveal a distinct conserved protein signature with features and implied cell surface architecture in the Methanosarcinaceae that is absent in other archaea. Paralogous gene expression patterns in two Methanosarcina species revealed abundant expression of a single S-layer paralog in each strain. Respective promoter elements were identified and shown to be conserved in mRNA coding and upstream untranslated regions. Prior M. acetivorans genome annotations assigned S-layer or surface layer associated roles of eighty genes: however, of 68 examined none was significantly expressed relative to the experimentally determined S-layer gene.
doi:10.1155/2012/873589
PMCID: PMC3361143  PMID: 22666082
2.  Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells 
Environmental microbiology  2011;13(9):2587-2599.
Summary
Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.
doi:10.1111/j.1462-2920.2011.02531.x
PMCID: PMC3700383  PMID: 21854518
3.  EcoCyc: fusing model organism databases with systems biology 
Nucleic Acids Research  2012;41(Database issue):D605-D612.
EcoCyc (http://EcoCyc.org) is a model organism database built on the genome sequence of Escherichia coli K-12 MG1655. Expert manual curation of the functions of individual E. coli gene products in EcoCyc has been based on information found in the experimental literature for E. coli K-12-derived strains. Updates to EcoCyc content continue to improve the comprehensive picture of E. coli biology. The utility of EcoCyc is enhanced by new tools available on the EcoCyc web site, and the development of EcoCyc as a teaching tool is increasing the impact of the knowledge collected in EcoCyc.
doi:10.1093/nar/gks1027
PMCID: PMC3531154  PMID: 23143106
4.  Complete genome sequence of Syntrophobacter fumaroxidans strain (MPOBT) 
Standards in Genomic Sciences  2012;7(1):91-106.
Syntrophobacter fumaroxidans strain MPOBT is the best-studied species of the genus Syntrophobacter. The species is of interest because of its anaerobic syntrophic lifestyle, its involvement in the conversion of propionate to acetate, H2 and CO2 during the overall degradation of organic matter, and its release of products that serve as substrates for other microorganisms. The strain is able to ferment fumarate in pure culture to CO2 and succinate, and is also able to grow as a sulfate reducer with propionate as an electron donor. This is the first complete genome sequence of a member of the genus Syntrophobacter and a member genus in the family Syntrophobacteraceae. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,990,251 bp long genome with its 4,098 protein-coding and 81 RNA genes is a part of the Microbial Genome Program (MGP) and the Genomes to Life (GTL) Program project.
doi:10.4056/sigs.2996379
PMCID: PMC3570798  PMID: 23450070
Anaerobic; Gram-negative; syntrophy; sulfate reducer; mesophile; propionate conversion; host-defense systems; Syntrophobacteraceae; Syntrophobacter fumaroxidans; Methanospirillum hungatei
5.  Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans  
Crystal structures of ModA from M. acetivorans in the apo and ligand-bound conformations confirm domain rotation upon ligand binding.
The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apo­protein conformation at 1.95 and 1.69 Å resolution and in the molybdate-bound conformation at 2.25 and 2.45 Å resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed α/β domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the ‘Venus flytrap’ model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.
doi:10.1107/S1744309109055158
PMCID: PMC2833028  PMID: 20208152
ModA; molybdate; Methanosarcina acetivorans; periplasmic binding proteins
6.  Syntrophy in Anaerobic Global Carbon Cycles 
Current opinion in biotechnology  2009;20(6):623-632.
Summary of recent advances
Syntrophy is an essential intermediary step in the anaerobic conversion of organic matter to methane where metabolically distinct microorganisms are tightly linked by the need to maintain the exchanged metabolites at very low concentrations. The need for syntrophy is thermodynamically constrained, and is probably a prime reason why it is difficult to culture microbes as these approaches disrupt consortia. Reconstruction of artificial syntrophic consortia has allowed uncultured syntrophic metabolizers and methanogens to be optimally grown and studied biochemically. The pathways for syntrophic acetate, propionate and longer chain fatty acid metabolism are mostly understood, but key steps involved in benzoate breakdown and cyclohexane carboxylate formation are unclear. Syntrophic metabolism requires reverse electron transfer, close physical contact, and metabolic synchronization of the syntrophic partners. Genomic analyses reveal that multiple mechanisms exist for reverse electron transfer. Surprisingly, the flagellum functions were implicated in ensuring close physical proximity and synchronization of the syntrophic partners.
doi:10.1016/j.copbio.2009.10.001
PMCID: PMC2790021  PMID: 19897353
syntrophy; methanogenesis; reversed electron transfer; fatty acids; benzoate; hydrocarbons; Syntrophus; Syntrophomonas; Pelotomaculum; Syntrophobacter
7.  EcoCyc: a comprehensive database of Escherichia coli biology 
Nucleic Acids Research  2010;39(Database issue):D583-D590.
EcoCyc (http://EcoCyc.org) is a comprehensive model organism database for Escherichia coli K-12 MG1655. From the scientific literature, EcoCyc captures the functions of individual E. coli gene products; their regulation at the transcriptional, post-transcriptional and protein level; and their organization into operons, complexes and pathways. EcoCyc users can search and browse the information in multiple ways. Recent improvements to the EcoCyc Web interface include combined gene/protein pages and a Regulation Summary Diagram displaying a graphical overview of all known regulatory inputs to gene expression and protein activity. The graphical representation of signal transduction pathways has been updated, and the cellular and regulatory overviews were enhanced with new functionality. A specialized undergraduate teaching resource using EcoCyc is being developed.
doi:10.1093/nar/gkq1143
PMCID: PMC3013716  PMID: 21097882
8.  Reconstruction of the Archaeal Isoprenoid Ether Lipid Biosynthesis Pathway in Escherichia coli Through Digeranylgeranylglyceryl Phosphate 
Metabolic engineering  2009;11(3):184-191.
The membrane lipids of archaea are characterized by unique isoprenoid biochemistry, which typically is based on two core lipid structures, sn-2,3-diphytanylglycerol diether (archaeol) and sn-2,3-dibiphytanyldiglycerol tetraether (caldarchaeol). The biosynthetic pathway for the tetraether lipid entails unprecedented head-to-head coupling of isoprenoid intermediates by an unknown mechanism involving unidentified enzymes. To investigate the isoprenoid ether lipid biosynthesis pathway of the hyperthermophilic archaeon, Archaeoglobus fulgidus, its lipid synthesis machinery was reconstructed in an engineered E. coli strain in an effort to demonstrate, for the first time, efficient isoprenoid ether lipid biosynthesis for the production of the intermediate, digeranylgeranylglyceryl phosphate (DGGGP). The biosynthesis of DGGGP was verified using a LC/MS/MS technique and was accomplished by cloning and expressing the native E. coli gene for IPP isomerase (idi), along with the A. fulgidus genes for G1P dehydrogenase (egsA) and GGPP synthase (gps), under the control of the lac promoter. The A. fulgidus genes for GGGP synthase (GGGPS) and DGGGP synthase (DGGGPS), under the control of the araBAD promoter, were then introduced and expressed to enable DGGGP biosynthesis in vivo. This investigation established roles for four A. fulgidus genes in the isoprenoid ether lipid pathway for DGGGP biosynthesis and provides a platform useful for identification of subsequent, currently unknown, steps in tetraether lipid biosynthesis proceeding from DGGGP, which is the presumed substrate for the head-to-head coupling reaction yielding unsaturated caldarchaeol.
doi:10.1016/j.ymben.2009.01.008
PMCID: PMC2726270  PMID: 19558961
Archaeoglobus fulgidus; isoprenoid; ether lipid; DGGGP
9.  S-layer Surface-Accessible and Concanavalin A Binding Proteins of Methanosarcina acetivorans and Methanosarcina mazei 
Journal of proteome research  2009;8(4):1972-1982.
The outermost cell envelope structure of many archaea and bacteria contains a proteinaceous lattice termed the surface layer or S-layer. It is typically composed of only one or two abundant, often post-translationally modified proteins that self-assemble to form the highly organized arrays. Surprisingly, over a hundred proteins were annotated to be S-layer components in the archaeal species Methanosarcina acetivorans C2A and Methanosarcina mazei Gö1, reflecting limitations of current predictions. An in vivo biotinylation methodology was devised to affinity tag surface-exposed proteins while overcoming unique challenges in working with these fragile organisms. Cells were adapted to growth under N2 fixing conditions, thus minimizing free amines reactive to the NHS-label, and high pH media compatible with the acylation chemistry was used. A 3-phase separation procedure was employed to isolate intact, labeled cells from lysed-cell derived proteins. Streptavidin affinity enrichment followed by stringent wash conditions removed non-specifically bound proteins. This methodology revealed S-layer proteins in M. acetivorans C2A and M. mazei Gö1 to be MA0829 and MM1976, respectively. Each was demonstrated to exist as multiple glycosylated forms using SDS-PAGE coupled with glycoprotein-specific staining, and by interaction with the lectin, Concanavalin A. A number of additional surface-exposed proteins and glycoproteins were identified and included all three subunits of the thermosome: the latter suggests that the chaperonin complex is both surface- and cytoplasmically-localized. This approach provides an alternative strategy to study surface proteins in the archaea.
doi:10.1021/pr800923e
PMCID: PMC2666069  PMID: 19228054
S-layer; surface proteins; Methanosarcina acetivorans; Methanosarcina mazei; biotinylation; mass spectrometry; glycoproteins; Concanavalin A
10.  Carbon-dependent control of electron transfer and central carbon pathway genes for methane biosynthesis in the Archaean, Methanosarcina acetivorans strain C2A 
BMC Microbiology  2010;10:62.
Background
The archaeon, Methanosarcina acetivorans strain C2A forms methane, a potent greenhouse gas, from a variety of one-carbon substrates and acetate. Whereas the biochemical pathways leading to methane formation are well understood, little is known about the expression of the many of the genes that encode proteins needed for carbon flow, electron transfer and/or energy conservation. Quantitative transcript analysis was performed on twenty gene clusters encompassing over one hundred genes in M. acetivorans that encode enzymes/proteins with known or potential roles in substrate conversion to methane.
Results
The expression of many seemingly "redundant" genes/gene clusters establish substrate dependent control of approximately seventy genes for methane production by the pathways for methanol and acetate utilization. These include genes for soluble-type and membrane-type heterodisulfide reductases (hdr), hydrogenases including genes for a vht-type F420 non-reducing hydrogenase, molybdenum-type (fmd) as well as tungsten-type (fwd) formylmethanofuran dehydrogenases, genes for rnf and mrp-type electron transfer complexes, for acetate uptake, plus multiple genes for aha- and atp-type ATP synthesis complexes. Analysis of promoters for seven gene clusters reveal UTR leaders of 51-137 nucleotides in length, raising the possibility of both transcriptional and translational levels of control.
Conclusions
The above findings establish the differential and coordinated expression of two major gene families in M. acetivorans in response to carbon/energy supply. Furthermore, the quantitative mRNA measurements demonstrate the dynamic range for modulating transcript abundance. Since many of these gene clusters in M. acetivorans are also present in other Methanosarcina species including M. mazei, and in M. barkeri, these findings provide a basis for predicting related control in these environmentally significant methanogens.
doi:10.1186/1471-2180-10-62
PMCID: PMC2838876  PMID: 20178638
11.  Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol* 
Journal of proteome research  2007;6(2):759-771.
Summary
Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate.
doi:10.1021/pr060383l
PMCID: PMC2577390  PMID: 17269732
Quantitative proteomics; metabolic labeling; microarray; methanogenesis; acetate; methanol
12.  EcoCyc: A comprehensive view of Escherichia coli biology 
Nucleic Acids Research  2008;37(Database issue):D464-D470.
EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc.
doi:10.1093/nar/gkn751
PMCID: PMC2686493  PMID: 18974181
13.  Heat Shock Response of Archaeoglobus fulgidus†  
Journal of Bacteriology  2005;187(17):6046-6057.
The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.
doi:10.1128/JB.187.17.6046-6057.2005
PMCID: PMC1196131  PMID: 16109946
14.  Coordinate Regulation of the Escherichia coli Formate Dehydrogenase fdnGHI and fdhF Genes in Response to Nitrate, Nitrite, and Formate: Roles for NarL and NarP 
Journal of Bacteriology  2003;185(17):5076-5085.
Escherichia coli possesses three distinct formate dehydrogenase enzymes encoded by the fdnGHI, fdhF, and fdoGHI operons. To examine how two of the formate dehyrogenase operons (fdnGHI and fdhF) are expressed anaerobically in the presence of low, intermediate, and high levels of nitrate, nitrite, and formate, chemostat culture techniques were employed with fdnG-lacZ and fdhF-lacZ reporter fusions. Complementary patterns of gene expression were seen. Optimal fdhF-lacZ expression occurred only at low to intermediate levels of nitrate, while high nitrate levels caused up to 10-fold inhibition of gene expression. In contrast, fdnG-lacZ expression was induced 25-fold in the presence of intermediate to high nitrate concentrations. Consistent with prior reports, NarL was able to induce fdnG-lacZ expression. However, NarP could not induce expression; rather, it functioned as an antagonist of fdnG-lacZ expression under low-nitrate conditions (i.e., it was a negative regulator). Nitrite, a reported signal for the Nar sensory system, was unable to stimulate or suppress expression of either formate dehydrogenase operon via NarL and NarP. The different gene expression profiles of the alternative formate dehydrogenase operons suggest that the two enzymes have complementary physiological roles under environmental conditions when nitrate and formate levels are changing. Revised regulatory schemes for NarL- and NarP-dependent nitrate control are presented for each operon.
doi:10.1128/JB.185.17.5076-5085.2003
PMCID: PMC180993  PMID: 12923080
15.  Oxygen and nitrate-dependent regulation of dmsABC operon expression in Escherichia coli: sites for Fnr and NarL protein interactions 
BMC Microbiology  2002;2:13.
Background
Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.
Results
The regions of dmsA regulatory DNA required for Fnr and NarL interactions in response to anaerobiosis and nitrate, respectively, were examined. Mutations within the Fnr site that deviated from the wild type sequence, TTGATaccgAACAA, or that removed an entire half-site, either impaired or abolished the anaerobic activation of dmsA-lacZ expression. The region for phosphorylated NarL (NarL-phosphate) binding at the dmsA promoter was identified by DNase I and hydroxyl radical footprinting methods. A large 97 bp region that overlaps the Fnr and RNA polymerase recognition sites was protected by NarL-phosphate but not by the non-phosphorylated form of NarL. Hydroxyl radical footprinting analysis confirmed the NarL-phosphate DNase I protections of both dmsA strands and revealed 8–9 protected sites of 3–5 bp occurring at ten bp intervals that are offset by 3 bp in the 3' direction.
Conclusion
These findings suggest that multiple molecules of phosphorylated NarL bind along one face of the DNA and may interfere with Fnr and/or RNA polymerase interactions at the dmsA regulatory region. The interplay of these transcription factors insures a hierarchical expression of the dmsABC genes when respiration of the preferred electron acceptors, oxygen and nitrate, is not possible.
doi:10.1186/1471-2180-2-13
PMCID: PMC116602  PMID: 12079504
16.  The Molybdate-Responsive Escherichia coli ModE Transcriptional Regulator Coordinates Periplasmic Nitrate Reductase (napFDAGHBC) Operon Expression with Nitrate and Molybdate Availability 
Journal of Bacteriology  2002;184(12):3253-3259.
Expression of the Escherichia coli napFDAGHBC operon (also known as aeg46.5), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a napF-lacZ operon fusion, we demonstrate that napF gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the napF promoter with an apparent Kd of about 35 nM at a position centered at −133.5 relative to the start of napF transcription. Although the ModE binding site sequence is similar to other E. coli ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a modE phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered napF expression independent of modE. These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent napF expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of napF gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.
doi:10.1128/JB.184.12.3253-3259.2002
PMCID: PMC135098  PMID: 12029041
17.  The nrfA and nirB Nitrite Reductase Operons in Escherichia coli Are Expressed Differently in Response to Nitrate than to Nitrite 
Journal of Bacteriology  2000;182(20):5813-5822.
Escherichia coli possesses two distinct nitrite reductase enzymes encoded by the nrfA and nirB operons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using nrfA-lacZ and nirB-lacZ reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimal nrfA-lacZ expression occurred only at low to intermediate levels of nitrate and where nirB-lacZ expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed nrfA gene expression. While either NarL or NarP was able to induce nrfA-lacZ expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by nirBDC under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress nirB expression. Nitrite was also an inducer of nirB and nrfA gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since nrfA operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the nirB nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.
PMCID: PMC94705  PMID: 11004182
18.  Glycine Betaine Transport in the Obligate Halophilic Archaeon Methanohalophilus portucalensis 
Journal of Bacteriology  2000;182(17):5020-5024.
Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed Kt of 23 μM and a Vmax of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [14C]betaine uptake. The proton-conducting uncoupler 2,4-dinitrophenol and the ATPase inhibitor N,N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 μM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, α-glutamate, β-glutamine, and Nɛ-acetyl-β-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.
PMCID: PMC111389  PMID: 10940053
19.  The napF and narG Nitrate Reductase Operons in Escherichia coli Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite 
Journal of Bacteriology  1999;181(17):5303-5308.
Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The napF operon apparently encodes a “low-substrate-induced” reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a “high-substrate-induced” operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered.
PMCID: PMC94036  PMID: 10464201
20.  Signal-Dependent Phosphorylation of the Membrane-Bound NarX Two-Component Sensor-Transmitter Protein of Escherichia coli: Nitrate Elicits a Superior Anion Ligand Response Compared to Nitrite 
Journal of Bacteriology  1999;181(17):5309-5316.
The Nar two-component regulatory system, consisting of the dual sensor-transmitters NarX and NarQ and the dual response regulators NarL and NarP, controls the expression of various anaerobic respiratory pathway genes and fermentation pathway genes. Although both NarX and NarQ are known to detect the two environmental signals nitrate and nitrite, little is known regarding the sensitivity and selectivity of ligand for detection or activation of the sensor-transmitters. In this study, we have developed a sensitive anion-specific in vitro assay for NarX autophosphorylation by using Escherichia coli membranes highly enriched in the full-length NarX protein. In this ATP- and magnesium-dependent reaction, nitrate elicited a greater signal output (i.e., NarX autophosphorylation) than did nitrite. Nitrate stimulation occurred at concentrations as low as 5 μM, and the half-maximal level of NarX autophosphorylation occurred at approximately 35 μM nitrate. In contrast, nitrite-dependent stimulation was detected only at 500 μM, while 3.5 mM nitrite was needed to achieve half-maximal NarX autophosphorylation. Maximal nitrate- and nitrite-stimulated levels of NarX phosphorylation were five and two times, respectively, over the basal level of NarX autophosphorylation. The presence of Triton X-100 eliminated the nitrate-stimulated kinase activity and lowered the basal level of activity, suggesting that the membrane environment plays a crucial role in nitrate detection and/or regulation of kinase activity. These results provide in vitro evidence for the differential detection of dual signaling ligands by the NarX sensor-transmitter protein, which modulates the cytoplasmic NarX autokinase activity and phosphotransfer to NarL, the cognate response regulator.
PMCID: PMC94037  PMID: 10464202
21.  Functional Dissection of the Molybdate-Responsive Transcription Regulator, ModE, from Escherichia coli 
Journal of Bacteriology  1998;180(17):4638-4643.
The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of λ CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.
PMCID: PMC107478  PMID: 9721306
22.  Disaggregation of Methanosarcina spp. and Growth as Single Cells at Elevated Osmolarity 
Applied and Environmental Microbiology  1993;59(11):3832-3839.
The effect of medium osmolarity on the morphology and growth of Methanosarcina barkeri, Methanosarcina thermophila, Methanosarcina mazei, Methanosarcina vacuolata, and Methanosarcina acetivorans was examined. Each strain was adapted for growth in NaCl concentrations ranging from 0.05 to 1.0 M. Methanosarcina spp. isolated from both marine and nonmarine sources exhibited similar growth characteristics at all NaCl concentrations tested, demonstrating that these species are capable of adapting to a similar range of medium osmolarities. Concomitant with the adaptation in 0.4 to 1.0 M NaCl, all strains disaggregated and grew as single cells rather than in the characteristic multicellular aggregates. Aggregated cells had a methanochondroitin outer layer, while disaggregated single cells lacked the outer layer but retained the protein S-layer adjacent to the cell membrane. Synthesis of glucuronic acid, a major component of methanochondroitin, was reduced 20-fold in the single-cell form of M. barkeri when compared with synthesis in aggregated cells. Strains with the methanochondroitin outer cell layer exhibited enhanced stability at low (<0.2 M NaCl) osmolarity and grew at higher temperatures. Disaggregated cells could be converted back to aggregated cells by gradually readapting cultures to lower NaCl (<0.2 M) and Mg2+ (<0.005 M) concentrations. Disaggregated Methanosarcina spp. could also be colonized and replica plated with greater than 95% recovery rates on solidified agar basal medium that contained 0.4 to 0.6 M NaCl and either trimethylamine, methanol, or acetate as the substrate. The ability to disaggregate and grow Methanosarcina spp. as viable, detergent-sensitive, single cells on agar medium makes these species amenable to mutant selection and screening for genetic studies and enables cells to be gently lysed for the isolation of intact genetic material.
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PMCID: PMC182538  PMID: 16349092
23.  Composition, Variation, and Dynamics of Major Osmotic Solutes in Methanohalophilus Strain FDF1 
Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, β-glutamine, and Nε-acetyl-β-lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as α-glucosylglycerate from the 13C and 1H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. 13CH3OH-pulse/12CH3OH-chase experiments were used to determine half-lifes for these organic solute pools in the cells. l-α-Glutamate showed a rapid loss of heavy isotope, indicating that l-α-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both β-glutamine, which acts as an osmolyte, and α-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and Nε-acetyl-β-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.
PMCID: PMC195800  PMID: 16348748

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