Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.

The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.     

Cloning and analysis of the xylAB operon and characterization of xylose isomerase from Thermoanaerobacter ethanolicus

Three genes, xylA-like, xylA and xylB, were cloned and sequenced from the chromosome of Thermoanaerobacter ethanolicus JW200. xylA and xylB share an operon and encode xylose isomerase and xylulokinase, respectively. The xylA-like gene locates upstream of xylAB operon and encodes a hypothetical protein that lacks xylose isomerase activity. The xylose isomerase was expressed in Escherichia coli and purified by heat treatment and an ion-exchange chromatography. The enzyme had highest activity at 85°C and pH 7.0, and a half-life for 1 h at 85°C. The K (m) and V (max) values for xylose were 11 mM and 25 U/mg, respectively. The high level of expression, easy purification, and thermostability of the XylA from T. ethanolicus JW200 suggests industrial usefulness.     

Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter

Thermoanaerobacter brockii, T. ethanolicus, T. thermohydrosulfuricus, T. finnii, and Thermoanaerobacter strain SEBR 5268 (an isolate from an oil-producing well) were studied for their ability to oxidize proteinaceous compounds that included gelatin, peptides, and casamino acids. All bacteria tested used peptides and amino acids, but only slightly. However, in the presence of thiosulfate all the Thermoanaerobacter species showed a substantial improvement in growth and/or the production of acetate, isovalerate, isobutyrate, and sulfide. Propionate was a minor product of peptide or amino acid oxidation. The reduction of thiosulfate during growth on peptides by members of the Thermoanaerobacter species is a trait that closely resembles that of archaeal hyperthermophiles during growth on peptides and amino acids with elemental sulfur as electron acceptor.     

嗜热乙醇杆菌中醛/醇脱氢酶的双启动子分析

克隆了嗜热乙醇杆菌(Thermoanaerobacter ethanolicus)中乙醇代谢的关键酶之一醛/醇脱氢酶(alcohol/acetaldehydedehy drogenase,AdhE)基因的上游假定启动子序列,并进行了结构分析。结果表明,adhE的上游序列是启动子,能启动报告基因在大肠杆菌中持续表达。首次发现adhE的启动子序列中存在两个独立的启动子(P172和P37)和核糖体结合位点(SD172和SD37),分别都具有完整功能,但其活性均低于完整的启动子序列。由此推测嗜热乙醇杆菌中adhE的表达受这两个启动子协同调控。     

A heat stable paraoxonase (O,O-diethyl O-p-nitrophenyl phosphate O-p-nitrophenyl hydrolase) from Russell's viper venom.

Fractionation of Russell's viper venom revealed separate phosphohydrolase activities directed against p-nitrophenyl phosphate, bis(p-nitrophenyl) phosphate, p-nitrophenylthymidylic acid, and O,O-diethyl p-nitrophenyl phosphate (paraoxon). On gel fractionation, the first two activities are eluted ahead of the latter. They could be resolved further by phosphocellulose cation exchange chromatography. The hydrolytic activities directed against p-nitrophenylthymidylic acid hydrolyzing component is heat labile, while the paraoxon hydrolyzing component manifests an unusually high degree of heat stability. Gel filtration yields 9600 for the molecular weight of the "paraoxonase". This enzyme, as all known enzymes of this type, requires the presence of a divalent cation. Maximum activity is obtained in the presence of Ca2+. In the presence of Sr2+ the reaction rate is 50% of that of Ca2+; other divalent cations show lower activities. The presence of the enzyme is species specific. Of four species tested, only Russell's viper venom showed significant paraoxonase activity. Enzyme activity is intact following incubation with iodoacetate of p-chloromercuribenzoate. Activity is partially preserved even in the presence of 8 M urea.     

Non-diauxic fermentation of acid hydrolysed pine by a strain of Clostridium thermohydrosulfuricum isolated from a New Zealand hot spring

Abstract Fermentation of dilute acid hydrolysed pine could be achieved by all of 18 thermophilic glycolytic anaerobic bacteria tested, while only seven isolates representing the species Clostridium thermohydrosulfuricum, Thermoanaerobium brockii and Thermoanaerobacter ethanolicus were able to ferment undiluted hydrolysate. A strain of C. thermohydrosulfuricum , designated Rt8.B1, was notably better at fermenting the hydrolysate and was studied further. Studies showed that glucose and xylose present in the hydrolysate were utilized simultaneously, a phenomenon which also occurred in a medium containing glucose and xylose as the added carbohydrates.     

The aldehyde/alcohol dehydrogenase (AdhE) in relation to the ethanol formation in Thermoanaerobacter ethanolicus JW200

A bifunctional aldehyde/alcohol dehydrogenase gene (adhE) from Thermoanaerobacter ethanolicus JW200 was identified and cloned. To unambiguously characterize the activity of AdhE, the recombinant protein was purified. The purified AdhE exhibited high enzymatic activity attributed to aldehyde dehydrogenase (11.0+/-0.3U/mg) and low alcohol dehydrogenase activity (2.6+/-0.2U/mg). Analysis of adhE homologous expression in T. ethanolicus showed that AdhE affected ethanol production.     

Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.     

Mutation of cysteine-295 to alanine in secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus affects the enantioselectivity and substrate specificity of ketone reductions.

The mutation of Cys-295 to alanine in Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) was performed to give C295A SADH, on the basis of molecular modeling studies utilizing the X-ray crystal structure coordinates of the highly homologous T. brockii secondary alcohol dehydrogenase (1YKF.PDB). This mutant SADH has activity for 2-propanol comparable to wild-type SADH. However, the C295A mutation was found to cause a significant shift of enantioselectivity toward the (S)-configuration in the reduction of some ethynylketones to the corresponding chiral propargyl alcohols. This result confirms our prediction that Cys-295 is part of a small alkyl group binding pocket whose size determines the binding orientation of ketone substrates, and, hence, the stereochemical configuration of the product alcohol. Furthermore, C295A SADH has much higher activity towards t-butyl and some alpha-branched ketones than does wild-type SADH. The C295A mutation does not affect the thioester reductase activity of SADH. The broader substrate specificity and altered stereoselectivity for C295A SADH make it a potentially useful tool for asymmetric reductions.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Oligonucleotide probes for the detection of Thermoanaerobacter

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection and identification of representatives of the genus Thermoanaerobacter. To increase the specificity level of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5'-GCTTCCGCDYCCCACACCTA-3') detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5'-TTAACTACGGCACGRAATGCTTC-3') was specific for the first group of the species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5'-CACTAMYGGGGTTTACAACC-3') targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5'-TC-CTCCATCAGGATGCCCTA-3') was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).     

Molecular cloning, nucleotide sequencing, and expression of genes encoding alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii and the mesophile Clostridium beijerinckii

Proteins play a pivotal role in thermophily. Comparing the molecular properties of homologous proteins from thermophilic and mesophilic bacteria is important for understanding the mechanisms of microbial adaptation to extreme environments. The thermophile Thermoanaerobacter (Thermoanaerobium) brockii and the mesophile Clostridium beijerinckii contain an NADP(H)-linked, zinc-containing secondary alcohol dehydrogenase (TBADH and CBADH) showing a similarly broad substrate range. The structural genes encoding the TBADH and the CBADH were cloned, sequenced, and highly expressed in Escherichia coli. The coding sequences of the TB adh and the CB adh genes are, respectively, 1056 and 1053 nucleotides long. The TB adh gene encoded an amino acid sequence identical to that of the purified TBADH. Alignment of the deduced amino acid sequences of the TB and CB adh genes showed a 76% identity and a 86% similarity, and the two genes had a similar preference for codons with A or T in the third position. Multiple sequence alignment of ADHs from different sources revealed that two (Cys-46 and His-67) of the three ligands for the catalytic Zn atom of the horse-liver ADH are preserved in TBADH and CBADH. Both the TBADH and CBADH were homotetramers. The substrate specificities and thermostabilities of the TBADH and CBADH expressed inE. coli were identical to those of the enzymes isolated from T. brockii and C. beijerinckii, respectively. A comparison of the amino acid composition of the two ADHs suggests that the presence of eight additional proline residues in TBADH than in CBADH and the exchange of hydrophilic and large hydrophobic residues in CBADH for the small hydrophobic amino acids Pro, Ala, and Val in TBADH might contribute to the higher thermostability of the T. brockii enzyme.     

Thermostability of alkaline phosphatase of Meithermus ruber bacteria: cloning of the gene, expression in Escherichia coli cells and biochemical characterization of the recombinant protein

The Meiothermus ruber alkaline phosphatase gene was cloned, expressed in Escherichia coli cells, and sequenced. The enzyme precursor, including the putative signal peptide, was shown to consist of 503 residues (deduced molecular mass 54,229 Da). The recombinant enzyme showed the maximal activity at 60-65 degrees C and pH 11.0 and had K(m) = 0.055 mM as estimated with p-nitrophenyl phosphate (pNPP). The enzyme proved to be moderately thermostable, retaining 50% activity after 6 h incubation at 60 degrees C and being completely inactivated in 2 h at 80 degrees C. In substrate specificity assays, the highest enzymic activity was observed with pNPP and dATP. Vanadate, inorganic phosphate, and SDS inhibited M. ruber alkaline phosphatase, while thiol-reducing agents had virtually no effect. The enzymic activity strongly depended on exogenous Mg2+ and declined in the presence of EDTA.     

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.     

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.     

Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs.

Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.     

Analysis of T4 bacteriophage deletion mutants that lack td and frd genes.

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.