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With the ability to colonize in the guts of a broad range of mosquito larvae, Bacillus cereus Cx5 has a potential to be utilized as a new host cell for the production of mosquito-larvicidal toxins aimed for mosquito control. However, the presence of one single (entFM) and two triple (hblCDA and nheABC) enterotoxin genes were previously confirmed in strain Cx5, raising concerns in its immediate use in the environment. Cx5 cells indeed showed recognizable levels of haemolytic and Vero cell cytotoxic activities. In this study, the single enterotoxin gene in B. cereus Cx5 (entCx5) has been inactivated in order to study the relationship between the presence of this gene and the cytotoxic and haemolytic activities found in the strain. We constructed a gene disruption plasmid, pΔTentCx5TV2, harbouring a truncated entCx5 gene in the temperature-sensitive shuttle vector, pUCTV2. After introducing the plasmid into B. cereus Cx5, we found that the plasmid was integrated via single crossover into the chromosome as expected at the entCx5 locus, disrupting the gene. Analysis of one mutant strain revealed that Vero cell cytotoxicity and haemolytic activity of the mutant were dramatically decreased compared to that of the wild type strain. This indicates an involvement of the entCx5 gene in haemolytic and Vero cell cytotoxic activities. The results also imply that there is a high possibility to generate an effective, and safe, host cell based on B. cereus Cx5 for the production of mosquito-larvicidal toxin. [ABSTRACT FROM AUTHOR]

Tetrahydrofolate is a cofactor involved in C1 metabolism including biosynthesis pathways for adenine and serine. In the classical tetrahydrofolate biosynthesis pathway, the steps removing three phosphate groups from the precursor 7,8-dihydroneopterin triphosphate (DHNTP) remain unclear in many bacteria. DHNTP pyrophosphohydrolase hydrolyzes pyrophosphate from DHNTP and produces 7,8-dihydroneopterin monophosphate. Although two structurally distinct DHNTP pyrophosphohydrolases have been identified in the intestinal bacteria Lactococcus lactis and Escherichia coli, the distribution of their homologs is limited. Here, we aimed to identify a third DHNTP pyrophosphohydrolase gene in the intestinal lactic acid bacterium Limosilactobacillus reuteri. In a gene operon including genes involved in dihydrofolate biosynthesis, we focused on the lreu_1276 gene, annotated as Ham1 family protein or XTP/dITP diphosphohydrolase, as a candidate encoding DHNTP pyrophosphohydrolase. The Lreu_1276 recombinant protein was prepared using E. coli and purified. Biochemical analyses of the reaction product revealed that the Lreu_1276 protein displays significant pyrophosphohydrolase activity toward DHNTP. The optimal reaction temperature and pH were 35°C and around 7, respectively. Substrate specificity was relatively strict among 17 tested compounds. Although previously characterized DHNTP pyrophosphohydrolases prefer Mg2+, the Lreu_1276 protein exhibited maximum activity in the presence of Mn2+, with a specific activity of 28.2 ± 2.0 µmol min-1 mg-1 in the presence of 1 mM Mn2+. The three DHNTP pyrophosphohydrolases do not share structural similarity to one another, and the distribution of their homologs does not overlap, implying that the Lreu_1276 protein represents a third structurally novel DHNTP pyrophosphohydrolase in bacteria. [ABSTRACT FROM AUTHOR]

We recently discovered a biosynthetic system using a novel amino group carrier protein called LysW for lysine biosynthesis via α-aminoadipate (AAA), and revealed that this system is also utilized in the biosynthesis of arginine by Sulfolobus. In the present study, we focused on the biosynthesis of lysine and ornithine in the hyperthermophilic archaeon Thermococcus kodakarensis, and showed that their biosynthesis is accomplished by a single set of metabolic enzymes. We also determined the crystal structure of the LysX family protein from T. kodakarensis, which catalyzes the conjugation of LysW with either AAA or glutamate, in a complex with LysW-γ-AAA. This crystal structure is the first example to show how LysX recognizes AAA as a substrate and provides a structural basis for the bifunctionality of the LysX family protein from T. kodakarensis. Based on comparisons with other LysX family proteins, we propose a mechanism for substrate recognition and its relationship with molecular evolution among LysX family proteins, which have different substrate specificities. [ABSTRACT FROM AUTHOR]

Regulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. maritima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated Tm-KPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase (Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 µM CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated by feedback inhibition targeting PanK, although Tm-PanK is a type III enzyme. [ABSTRACT FROM AUTHOR]

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment. [ABSTRACT FROM AUTHOR]

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway. [ABSTRACT FROM AUTHOR]

NAD+ is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD+ is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD+ concentrations and possibly remove and/or reuse undesirable degradation products of NAD+. Here we confirmed that at 85°C, thermal degradation of NAD+ results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD+ in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD+ breakdown. [ABSTRACT FROM AUTHOR]

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-Alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobntyrate (GABA). The recombinant TK18 protein displayed not only GAD activity but also ADC activity using pyridoxal 5'-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Gin, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4'-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK18 functions as an ADC in T. kodakarensis, providing the β-Alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK 1814 homologs are distributed in a wide range of archaea and may be responsible for β-Alanine biosynthesis in these organisms. [ABSTRACT FROM AUTHOR]

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralo-gous genes encoding putative a subunits of nucleoside diphosphate (NDP)-formingacyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two a subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α2β 2 structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS IIITk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICSTk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICSTk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales. [ABSTRACT FROM AUTHOR]

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined.Wethus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward D-allose, D-lyxose, D-tagatose, D-talose, D-xylose, and D-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate. [ABSTRACT FROM AUTHOR]

Serine kinase catalyzes the phosphorylation of free serine (Ser) to produce O-phosphoserine (Sep). An ADP-dependent Ser kinase in the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-SerK) is involved in cysteine (Cys) biosynthesis and most likely Ser assimilation. An ATP-dependent Ser kinase in the mesophilic bacterium Staphylococcus aureus is involved in siderophore biosynthesis. Although proteins displaying various degrees of similarity with Tk-SerK are distributed in a wide range of organisms, it is unclear if they are actually Ser kinases. Here, we examined proteins from Desulfurococcales species in Crenarchaeota that display moderate similarity with Tk-SerK from Euryarchaeota (42 to 45% identical). Tk-serK homologs from Staphylothermus marinus (Smar_0555), Desulfurococcus amylolyticus (DKAM_0858), and Desulfurococcus mucosus (Desmu_0904) were expressed in Escherichia coli. All three partially purified recombinant proteins exhibited Ser kinase activity utilizing ATP rather than ADP as a phosphate donor. Purified Smar_0555 protein displayed activity for l-Ser but not other compounds, including d-Ser, l-threonine, and l-homoserine. The enzyme utilized ATP, UTP, GTP, CTP, and the inorganic polyphosphates triphosphate and tetraphosphate as phosphate donors. Kinetic analysis indicated that the Smar_0555 protein preferred nucleoside 5'-triphosphates over triphosphate as a phosphate donor. Transcript levels and Ser kinase activity in S. marinus cells grown with or without serine suggested that the Smar_0555 gene is constitutively expressed. The genes encoding Ser kinases examined here form an operon with genes most likely responsible for the conversion between Sep and 3-phosphoglycerate of central sugar metabolism, suggesting that the ATP-dependent Ser kinases from Desulfurococcales play a role in the assimilation of Ser. [ABSTRACT FROM AUTHOR]

Members of Thermococcales harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis. The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (Pcsg). The purified protein did not display aminotransferase activity but exhibited racemase activity. An examination of most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal medium supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo. When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase but displayed cell yield similar to that observed in medium with l-Leu. In contrast, the ΔTK1211 strain displayed growth in medium with l-Leu but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensis. [ABSTRACT FROM AUTHOR]

tRNA m²G10/m²2G10 methyltransferase (archaeal Trm11) methylates the 2-amino group in guanosine at position 10 in tRNA and forms N²,N²-dimethylguanosine (m²2G10) via N²-methylguanosine (m²G10). We determined the complete sequence of tRNATrp, one of the substrate tRNAs for archaeal Trm11 from Thermococcus kodakarensis, a hyperthermophilic archaeon. Liquid chromatography/mass spectrometry following enzymatic digestion of tRNATrp identified 15 types of modified nucleoside at 21 positions. Several modifications were found at novel positions in tRNA, including 2′-O-methylcytidine at position 6, 2-thiocytidine at position 17, 2′-O-methyluridine at position 20, 5,2′-O-dimethylcytidine at position 32, and 2′-O-methylguanosine at position 42. Furthermore, methylwyosine was found at position 37 in this tRNATrp, although 1-methylguanosine is generally found at this location in tRNATrp from other archaea. We constructed trm11 (Δtrm11) and some gene disruptant strains and compared their tRNATrp with that of the wild-type strain, which confirmed the absence of m²2G10 and other corresponding modifications, respectively. The lack of 2-methylguanosine (m²G) at position 67 in the trm11 trm14 double disruptant strain suggested that this methylation is mediated by Trm14, which was previously identified as an m²G6 methyltransferase. The Δtrm11 strain grew poorly at 95°C, indicating that archaeal Trm11 is required for T. kodakarensis survival at high temperatures. The m²2G10 modification might have effects on stabilization of tRNA and/or correct folding of tRNA at the high temperatures. Collectively, these results provide new clues to the function of modifications and the substrate specificities of modification enzymes in archaeal tRNA, enabling us to propose a strategy for tRNA stabilization of this archaeon at high temperatures. [ABSTRACT FROM AUTHOR]

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM-1 · s-1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota. [ABSTRACT FROM AUTHOR]