Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

4-Thiouridine (s⁴U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s⁴U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s⁴U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s⁴U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s⁴U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s⁴U. In the absence of an RLD, MMP1354 uses Cys²⁶⁵ and Cys²⁶⁸ located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S²⁻ is a physiologically relevant sulfur donor for s⁴U formation catalyzed by MMP1354 (K_m for Na₂S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s⁴U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.     

Identification of Nucleotide Residues Essential for RNase P Activity from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg²⁺ ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Identification of an Internal RNA Element Essential for Replication and Translational Enhancement of Tobacco Necrosis Virus A C

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-A^C coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-A^C RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-A^C, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

Cysteine Is Not the Sulfur Source for Iron-Sulfur Cluster and Methionine Biosynthesis in the Methanogenic Archaeon Methanococcus maripaludis

Three multiprotein systems are known for iron-sulfur (Fe-S) cluster biogenesis in prokaryotes and eukaryotes as follows: the NIF (nitrogen fixation), the ISC (iron-sulfur cluster), and the SUF (mobilization of sulfur) systems. In all three, cysteine is the physiological sulfur source, and the sulfur is transferred from cysteine desulfurase through a persulfidic intermediate to a scaffold protein. However, the biochemical nature of the sulfur source for Fe-S cluster assembly in archaea is unknown, and many archaea lack homologs of cysteine desulfurases. Methanococcus maripaludis is a methanogenic archaeon that contains a high amount of protein-bound Fe-S clusters (45 nmol/mg protein). Cysteine in this archaeon is synthesized primarily via the tRNA-dependent SepRS/SepCysS pathway. When a ΔsepS mutant (a cysteine auxotroph) was grown with ³⁴S-labeled sulfide and unlabeled cysteine, <8% of the cysteine, >92% of the methionine, and >87% of the sulfur in the Fe-S clusters in proteins were labeled, suggesting that the sulfur in methionine and Fe-S clusters was derived predominantly from exogenous sulfide instead of cysteine. Therefore, this investigation challenges the concept that cysteine is always the sulfur source for Fe-S cluster biosynthesis in vivo and suggests that Fe-S clusters are derived from sulfide in those organisms, which live in sulfide-rich habitats.     

Identification of Metabolic Pathways Essential for Fitness of Salmonella Typhimurium In Vivo

Bacterial infections remain a threat to human and animal health worldwide, and there is an urgent need to find novel targets for intervention. In the current study we used a computer model of the metabolic network of Salmonella enterica serovar Typhimurium and identified pairs of reactions (cut sets) predicted to be required for growth in vivo. We termed such cut sets synthetic auxotrophic pairs. We tested whether these would reveal possible combined targets for new antibiotics by analyzing the performance of selected single and double mutants in systemic mouse infections. One hundred and two cut sets were identified. Sixty-three of these included only pathways encoded by fully annotated genes, and from this sub-set we selected five cut sets involved in amino acid or polyamine biosynthesis. One cut set (asnA/asnB) demonstrated redundancy in vitro and in vivo and showed that asparagine is essential for S. Typhimurium during infection. trpB/trpA as well as single mutants were attenuated for growth in vitro, while only the double mutant was a cut set in vivo, underlining previous observations that tryptophan is essential for successful outcome of infection. speB/speF,speC was not affected in vitro but was attenuated during infection showing that polyamines are essential for virulence apparently in a growth independent manner. The serA/glyA cut-set was found to be growth attenuated as predicted by the model. However, not only the double mutant, but also the glyA mutant, were found to be attenuated for virulence. This adds glycine production or conversion of glycine to THF to the list of essential reactions during infection. One pair (thrC/kbl) showed true redundancy in vitro but not in vivo demonstrating that threonine is available to the bacterium during infection. These data add to the existing knowledge of available nutrients in the intra-host environment, and have identified possible new targets for antibiotics.     

Activation of Methanogenesis by Cadmium in the Marine Archaeon Methanosarcina acetivorans

Methanosarcina acetivorans was cultured in the presence of CdCl₂ to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd²⁺ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.     

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.     

Identification of a Novel Palmitylation Site Essential for Membrane Association and Isomerohydrolase Activity of RPE65

RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.Both rod and cone visual pigments in vertebrates require 11-cis-retinal as the chromophore. Isomerization of 11-cis-retinal to all-trans-retinal by a photon triggers the phototransduction cascade and initiates vision (1, 2). Recycling of 11-cis-retinal through the retinoid visual cycle is an essential process for the regeneration of visual pigments and for normal vision (3, 4). The key step in the visual cycle is to isomerize all-trans-retinyl ester to 11-cis-retinol in retinal pigment epithelium (RPE)² (5, 6). This isomerization process is known to be catalyzed by an isomerohydrolase in the RPE. Several recent lines of evidence suggest that RPE65 is the isomerohydrolase in the visual cycle (7-9).RPE65 is a microsomal protein, abundantly expressed in the RPE (10-12). RPE65 knock-out (Rpe65^-/-) mice showed a lack of 11-cis-retinoids, overaccumulation of all-trans-retinyl ester, impaired visual function, and early degeneration of cone photoreceptors (7-9). RPE65 is an iron(II)-dependent enzyme, in which an iron is coordinated by four conserved histidine (His) residues (His-180, -241, -313, and -527) based on molecular modeling using a crystal structure of apocarotenoid monooxygenase as a template (8, 13-15). RPE65 lacks any predicted transmembrane helix and is associated with the microsomal membrane (11). Previous studies have shown that membrane association of RPE65 is essential for its isomerohydrolase activity (7). However, the structural basis for its membrane association has not been revealed. An earlier study showed that three Cys residues (Cys-231, 329 and 330) in RPE65 were palmitylated, which were suggested to be responsible for its membrane association (16). However, triple mutations of all the three Cys residues did not completely dissociate RPE65 from the membrane (17, 18). Moreover, the triple Cys mutant remains palmitylated (17). These results suggested that either the site of palmitylation responsible for the membrane association of RPE65 had not yet been identified or other mechanisms, such as hydrophobic interactions, anchor the protein to cellular membranes (17, 19).In this study, we used the combination of mass spectrometric analysis and site-directed mutagenesis to identify the palmitylated site in RPE65. Moreover, we determined the role of this site in the membrane association and enzymatic activity of RPE65.     

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

Identification of the mcrC gene product in Methanococcus vannielii

Abstract The polypeptide encoded by the mcr C gene has been identified in Methanococcus vannielii by immunoblotting using rabbit antibodies raised against the product of a lac Z- mcr C gene fusion synthesized and purified from Escherichia coli . The mcr C gene product (gp mcr C) was located in both supernatant and pellet fractions after centrifugation of Mc. vannielii cell extracts for 2 h at 100 000 × g . When anaerobic reducing conditions were maintained during purification, gp mcr C co-sedimented through sucrose gradients to the same position as molecules of the methyl coenzyme M reductase holoenzyme (approx. 300 kDa). This co-sedimentation was lost under aerobic, nonreducing conditions.     

Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k_cat/K_m) measured at 15°C for the peptidyl substrates, N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM⁻¹ s⁻¹, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicus FK506-binding protein). The MTFK gene (462 bp) was cloned from an M. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.     

Essential Role for an M17 Leucine Aminopeptidase in Encystation of Acanthamoeba castellanii

Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.     

Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. ΔleuB and ΔtrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.     

Domain Interplay Mediated by an Essential Disulfide Linkage Is Critical for the Activity and Secretion of the Metastasis-promoting Enzyme Autotaxin

Autotaxin or NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2) is a secreted lysophospholipase-D that promotes metastasis and tumor growth by its ability to generate lysophosphatidic acid. Considerable evidence suggests that inhibitors of NPP2 can be used as a novel therapy for the treatment of cancer. Although most attention is currently directed toward the development of inhibitors of the catalytic site, we have explored whether NPP2 can also be targeted through its non-catalytic nuclease-like domain. We demonstrate here that the catalytic and nuclease-like domains are covalently linked by an essential disulfide bridge between Cys⁴¹³ and Cys⁸⁰⁵. Within the nuclease-like domain, residues 829–850 are involved in the secretion of NPP2, and Lys⁸⁵² is required for the expression of catalytic activity. These data show that the nuclease-like domain is crucial for catalysis by NPP2 and is a possible target to generate inhibitors.NPP2 ³ or autotaxin is a secreted lysophospholipase-D that acts in a paracrine or autocrine manner. The major substrate of NPP2 is lysophosphatidylcholine, which is converted into choline and lysophosphatidic acid. The latter promotes signaling through specific G-protein-coupled receptors that stimulate cell proliferation, differentiation, and motility (1). NPP2 functions in processes as diverse as the homing of lymphocytes, blood vessel formation, and wound healing but also promotes tumorigenesis (2–6). The metastasis-enhancing properties of NPP2 have been attributed to its ability to promote the invasive properties of cancer cells and to stimulate angiogenesis. Importantly, NPP2 is highly expressed by various cancers including breast carcinoma (7), Hodgkin lymphoma (8), and glioblastoma multiforme (9), and this correlates with an increased metastasis and angiogenesis. Therefore, NPP2 is considered to be an attractive target for a novel anticancer therapy, in particular because it acts extracellularly, and interfering drugs thus do not need to be cell-permeable (10).NPP2 is one of the seven mammalian members of the NPP-type family of ectophosphodiesterases, belonging to the superfamily of phospho-/sulfo-coordinating metalloenzymes (11). These enzymes all have a structurally related catalytic domain and the same catalytic mechanism but show a different substrate specificity. For example, NPP1 only recognizes nucleotide substrates, whereas NPP2 preferentially hydrolyzes lysophospholipids. Interestingly, NPP1–3 share a C-terminal non-catalytic domain, commonly referred to as the nuclease-like domain (NLD) (12). The latter is related to DNA/RNA-nonspecific endonucleases but lacks key residues that are necessary for activity and is therefore believed to have a structural or regulatory role. It is not known whether the NLD acts as a positive or negative regulator of NPP activity, but swapping experiments of the NLD between NPP1 and NPP2 suggested that it harbors isoform-specific determinants for catalysis (13).To examine the therapeutic potential of NPP2, in particular for the treatment of cancer, a number of small molecule inhibitors have been developed (14, 15). However, these inhibitors are all directed against the catalytic site and may therefore also interfere with other phospho-/sulfo-coordinating metalloenzymes. We report here that the NLD is essential for the expression of catalytic activity and can be used as an alternative target to inhibit NPP2.     

Identification of the Methanococcus voltae S-layer structural gene.

We have established that the gene which we had previously identified as encoding the Methanococcus voltae P-type ATPase is, in fact, the structural gene for the M. voltae S-layer protein. This conclusion is based on a comparison of the N-terminal sequence of S-layer protein prepared by two independent methods with that derived from the nucleotide sequence of the cloned gene. This conclusion was further supported by immunocytochemical localization of the antigen directed against the antibodies used in the cloning experiments.