Archaeon Methanosarcina Acetivorans Research Articles

Characterization of LIC11207, a novel leptospiral protein that is recognized by human convalescent sera and prevents apoptosis of polymorphonuclear leukocytes

We report the study of a predicted outer-membrane leptospiral protein encoded by the gene lic11207. This protein is conserved in several pathogenic leptospiral strains but is absent in the saprophyte Leptospira biflexa. This putative outer-membrane protein has a domain of unknown function (DUF) 1565 found in several phylogenetically diverse bacteria and in the archaeon Methanosarcina acetivorans. The gene was cloned and expressed in Escherichia coli BL21 (SI) strain using the expression vector pDEST17. The 34 kDa recombinant protein was tagged with N-terminal hexahistidine and purified by metal-charged chromatography. The purified protein was used to assess: reactivity with human convalescent sera; in vivo expression; ability to activate endothelial cells (EC); and ability to modulate the apoptosis of polymorphonuclear cells (PMNs). The LIC11207 coding sequence was identified in vivo in the hamster renal tubules during experimental infection with Leptospira interrogans. The rLIC11207 showed significant antigenicity against human convalescent sera when compared with sera from healthy donors. The recombinant protein did not alter the surface expression of E-selectin or intercellular adhesion molecule 1 (ICAM-1) in EC and failed to induce the release of von Willebrand factor (vWF). Interestingly, rLIC11207 delayed apoptosis of PMNs suggesting a possible role of this protein during the infection.

A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans

BackgroundCorrect annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability.ResultsThe target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed.ConclusionsIn all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.

Reverse-Chaperoning Activity of an AAA+ Protein

Speed and processivity of replicative DNA polymerases can be enhanced via coupling to a sliding clamp. Due to the closed ring shape of the clamp, a clamp loader protein, belonging to the AAA+ class of ATPases, needs to open the ring-shaped clamp before loading it to DNA. Here, we developed real-time fluorescence assays to study the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans. Unexpectedly, we discovered that RFC can assemble a PCNA ring from monomers in solution. A motion-based DNA polymerization assay showed that the PCNA assembled by RFC is functional. This PCNA assembly activity required the ATP-bound conformation of RFC. Our work demonstrates a reverse-chaperoning activity for an AAA+ protein that can act as a template for the assembly of another protein complex.

Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans

Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs(Ma), utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP(i) were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP(i). These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs(Ma) structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys(256), Cys(298), Gly(351), Trp(259), Trp(237), and Trp(254), were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.

Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

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 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta 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.

Dynamics of An Archael DNA Polymerase Revealed By Single Molecule Fret

In the archaeon Methanosarcina acetivorans, DNA replication is done by DNA polymerase BI. The processivity of this enzyme is greatly enhanced by a conserved cofactor known as PCNA, which plays a crucial role in orchestrating many replication-related processes. To understand the dynamics of these proteins, we have used single molecule FRET to examine the behavior of PolBI labeled with FRET donor on various DNA structures labeled with FRET acceptor and the effect of PCNA on the dynamics of PolBI. The binding of PolBI to DNA was observed in the low nanomolar concentration range as expected. Interestingly, this polymerase is highly mobile on the DNA structures with two nonadjacent primer strands that are complementary to two different regions in the template strand, 20 nucleotides apart, as evidenced by the frequent transitions between two long-lived FRET states exhibited in single molecule trajectories. To explore the nature of this spontaneous motion, we considered several possible mechanisms including translocations along single- or double-stranded DNA, polymerase binding orientation flipping, and polymerase active site switching. We observed that changes made downstream of the primer/template (P/T) junction have a significant impact on the dynamics of PolBI, indicating the translocation along the single-stranded DNA as the mechanism for the spontaneous motion revealed by FRET. In addition, we have found PCNA not only improves the binding affinity of its cognate polymerase, but suppresses the frequent movement of PolBI from the P/T junction. In summary, many of the dynamics discussed here are reported for the first time and will provide a new perspective for understanding the orchestration of replication-related processes in archaea.

The Stepwise Evolution of Early Life Driven by Energy Conservation

Two main theories have emerged for the origin and early evolution of life based on heterotrophic versus chemoautotrophic metabolisms. With the exception of a role for CO, the theories have little common ground. Here we propose an alternative theory for the early evolution of the cell which combines principal features of the widely disparate theories. The theory is based on the extant pathway for conversion of CO to methane and acetate, largely deduced from the genomic analysis of the archaeon Methanosarcina acetivorans. In contrast to current paradigms, we propose that an energy-conservation pathway was the major force which powered and directed the early evolution of the cell. We envision the proposed primitive energy-conservation pathway to have developed sometime after a period of chemical evolution but prior to the establishment of diverse protein-based anaerobic metabolisms. We further propose that energy conservation played the predominant role in the later evolution of anaerobic metabolisms which explains the origin and evolution of extant methanogenic pathways.

Biochemical and Mutational Analyses of a Unique Clamp Loader Complex in the Archaeon Methanosarcina acetivorans

Clamp loaders orchestrate the switch from distributive to processive DNA synthesis. Their importance in cellular processes is underscored by their conservation across all forms of life. Here, we describe a new form of clamp loader from the archaeon Methanosarcina acetivorans. Unlike previously described archaeal clamp loaders, which are composed of one small subunit and one large subunit, the M. acetivorans clamp loader comprises two similar small subunits (M. acetivorans replication factor C small subunit (MacRFCS)) and one large subunit (MacRFCL). The relatedness of the archaeal and eukaryotic clamp loaders (which are made up of four similar small subunits and one large subunit) suggests that the M. acetivorans clamp loader may be an intermediate form in the archaeal/eukaryotic sister lineages. The clamp loader complex reconstituted from the three subunits MacRFCS1, MacRFCS2, and MacRFCL stimulated DNA synthesis by a cognate DNA polymerase in the presence of its sliding clamp. We used site-directed mutagenesis in the Walker A and SRC motifs to examine the contribution of each subunit to the function of the M. acetivorans clamp loader. Although mutations in MacRFCL and MacRFCS2 did not impair clamp loading activity, any mutant clamp loader harboring a mutation in MacRFCS1 was devoid of the clamp loading property. Mac-RFCS1 is therefore critical to the clamp loading activity of the M. acetivorans clamp loader. It is our anticipation that the discovery of this unique replication factor C homolog will lead to critical insights into the evolution of more complex clamp loaders from simpler ones as more complex organisms evolved in the archaeal/eukaryotic sister lineages.

Novel Chaperonins in a Prokaryote

Group II chaperonins belong to the Hsp60 family occurring in archaea and eukaryotes. The archaeal chaperonins build the thermosome, which is similar to the eukaryotic CCT (chaperonin-containing TCP-1). Eukaryotes have eight subunits, and up until now, it was thought that archaea had between one and three subunits, depending on the species. We now report two novel subunits, termed Hsp60-4 and Hsp60-5, in the archaeon Methanosarcina acetivorans, which also has Hsp60-1, Hsp60-2, and Hsp60-3 with orthologs in Methanosarcinae. Hsp60-4 and Hsp60-5 occur only in M. acetivorans, which makes this organism unique in that it has the highest number of chaperonin subunits ever described for an archaeon. Evolutionary analysis suggests that either Hsp60-4 or Hsp60-5 paralogs have arisen by gene duplication with vastly increased accepted substitution rates or that they represent ancestral types found only in this species.

Functional Analysis of Multiple Single-stranded DNA-binding Proteins from Methanosarcina acetivorans and Their Effects on DNA Synthesis by DNA Polymerase BI

Single-stranded DNA-binding proteins and their functional homologs, replication protein A, are essential components of cellular DNA replication, repair and recombination. We describe here the isolation and characterization of multiple replication protein A homologs, RPA1, RPA2, and RPA3, from the archaeon Methanosarcina acetivorans. RPA1 comprises four single-stranded DNA-binding domains, while RPA2 and RPA3 are each composed of two such domains and a zinc finger domain. Gel filtration analysis suggested that RPA1 exists as homotetramers and homodimers in solution, while RPA2 and RPA3 form only homodimers. Unlike the multiple RPA proteins found in other Archaea and eukaryotes, each of the M. acetivorans RPAs can act as a distinct single-stranded DNA-binding protein. Fluorescence resonance energy transfer and fluorescence polarization anisotropy studies revealed that the M. acetivorans RPAs bind to as few as 10 single-stranded DNA bases. However, more stable binding is achieved with single-stranded DNA of 18-23 bases, and for such substrates the estimated Kd was 3.82 +/- 0.28 nM, 173.6 +/- 105.17 nM, and 5.92 +/- 0.23 nM, for RPA1, RPA2, and RPA3, respectively. The architectures of the M. acetivorans RPAs are different from those of hitherto reported homologs. Thus, these proteins may represent novel forms of replication protein A. Most importantly, our results show that the three RPAs and their combinations highly stimulate the primer extension capacity of M. acetivorans DNA polymerase BI. Although bacterial SSB and eukaryotic RPA have been shown to stimulate DNA synthesis by their cognate DNA polymerases, our findings provide the first in vitro biochemical evidence for the conservation of this property in an archaeon.