Purification and activation of a recombinant histidine-tagged pro-transglutaminase after soluble expression in Escherichia coli and partial characterization of the active enzyme

Pro-transglutaminase from Streptomyces mobaraensis was expressed in Escherichia coli as a fusion protein carrying a C-terminal histidine tag (pro-MTG-His₆). The recombinant organism was cultivated in 15 L bioreactor scale and pro-MTG-His₆ was purified by immobilized metal affinity chromatography. Activation of the inactive pro-enzyme using trypsin resulted in an unexpected degradation of the transglutaminase and a concomitant loss of activity. Therefore, a set of commercially available proteases was investigated for their activation potential without destroying the target enzyme. Besides trypsin, chymotrypsin and proteinase K were found to activate but hydrolyze the (pro-MTG-His₆). Cathepsin B, dispase I, and thrombin were shown to specifically hydrolyze pro-MTG-His₆ without deactivation. TAMEP, the endogeneous protease from S. mobaraensis was purified for comparison and also found to activate the recombinant histidine-tagged transglutaminase without degradation. The TAMEP activated MTG-His₆ was purified and characterized. The specific activity (23 U/mg) of the recombinant histidine-tagged transglutaminase, the temperature optimum (50 °C), and the temperature stability (t_1/2 at 60 °C = 1.7 min) were comparable to the wild-type enzyme. A C-terminal peptide tag did neither affect the activity nor the stability but facilitated the purification. The purification of the histidine-tagged protein is possible before or after activation.     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Application of lat gene disruption to increase the clavulanic acid production of Streptomyces clavuligerus

A 1.7 kb fragment of lat was obtained from Streptomyces clavuligerus NRRL 3585, and recombinant plasmid pKC1139-lat, which was used to disrupt the lat gene was constructed. pKC1139-lat was introduced into S. clavuligerus by bi-parental conjugation from Escherichia coli ET12567 to S. clavuligerus. The apramcin-resistant transformants were obtained and through homogeneous single-crossover between recombinant plasmid pKC1139-lat and the S. clavuligerus chromosome lat disrupted mutant strains were obtained. The genome of S. clavuligerus NRRL 3585 and the lat disrupted mutants were analyzed by PCR technique, the bioactivity of cephamycin C in the two kinds of strains were also tested. Both results proved that lat was disrupted by the insertion of pKC1139 in the lat disrupted mutants. And the production of clavulanic acid of these two kinds of strains were analyzed by HPLC with different incubation time interval (96 and 120 h), and the yield in the lat mutants was approximately 2.6 fold higher at their highest production point.     

青海地区解磷微生物的筛选及对小油菜生长的影响

为了筛选出能适应青海省冷凉气候环境的解磷菌株,以磷酸钙、卵磷脂、植酸为单一磷源,对胶冻样芽孢杆菌、巨大芽孢杆菌、蜡状芽孢杆菌、紫变异链霉菌、肉桂褐链霉菌、黄团孢链霉菌进行固体平板培养基初筛和液体培养基复筛,通过综合比较固体平板培养基中解磷圈的大小和液体培养基可溶性磷含量,初步筛选出解磷效果较好的3株解磷菌,分别为紫变异链霉菌、肉桂褐链霉菌和胶冻样芽孢杆菌。将这3株解磷菌制成液体菌剂,在9月冷凉气候下采用两种不同肥力土壤进行小油菜盆栽试验。结果表明:与对照相比,施加紫变异链霉菌剂后,高肥力耕地土的小油菜收获时株高、鲜重、根长、根重分别增加了35.5%、191.0%、26.2%、282.7%,植株磷吸收量、根际土壤全磷、速效磷分别增加了968.9%、5.1%、2.1%;低肥力自然土的小油菜收获时株高和鲜重分别增加了45.8%和61.3%,根长和根重分别减少了2.6%和4.4%,植株磷吸收量、根际土壤全磷、速效磷分别增加了91.5%、29.1%和213.7%。其他两种解磷菌剂效果不如紫变异链霉菌明显,表明紫变异链霉菌为适合青海地区冷凉气候环境的解磷菌株。     

Resistoflavine, cytotoxic compound from a marine actinomycete, Streptomyces chibaensis AUBN1/7

In our systematic screening programme for marine actinomycetes, a bioactive Streptomycete was isolated from marine sediment samples of Bay of Bengal, India. The taxonomic studies indicated that the isolate belongs to Streptomyces chibaensis and it was designated as S. chibaensis AUBN₁/7. The isolate yielded a cytotoxic compound. It was obtained by solvent extraction followed by the chromatographic purification. Based on the spectral data of the pure compound, it was identified as quinone-related antibiotic, resistoflavine (1). It showed a potent cytotoxic activity against cell lines viz. HMO2 (Gastric adenocarcinoma) and HePG2 (Hepatic carcinoma) in vitro and also exhibited weak antibacterial activities against Gram-positive and Gram-negative bacteria.     

The preparation of low-molecular-weight chitosan using chitinolytic complex from Streptomyces kurssanovii

Streptomyces kurssanovii are Gram-positive mycelial bacteria ubiquitous in soil. They have a saprophytic way of life and produce many extracellular enzymes with polymer-degrading properties, for example, chitinase (EC 3.2.1.14) and N-acetyl-β- -glucosaminidase (EC3.2.1.30). Biochemical aspects of chitosan degradation were presented. Low-molecular-weight (LMW) chitosans with molecular weight 4–8 kDa were prepared from commercial crab chitosan by means of chitinolytic a complex from S. kurssanovii. The optimum conditions of process in solution (temperature, pH, enzyme-substrate ratio) have been determined. Yields of LMW chitosan were 70–80%.     

Involvement of an Inducible Cytochrome P-450 in Precocene II Oxidation by Streptomyces Griseus

Streptomyces griseus oxidizes the insecticide precocene II to its cis- and trans-dihydrodiols and 3-chromenol after growth on an enriched medium containing soybean flour. Oxidation of precocene II is dependent on the level of cytochrome P-450 in this organism. Extracts of cells grown on media lacking soybean flour were devoid of cytochrome P-450 and could not oxidize precocene II. In an in vitro reconstituted system containing NADPH, spinach ferredoxin reductase, spinach ferredoxin and ammonium sulfate fractions enriched in cytochrome P-450, precocene II was oxidized to its dihydrodiols. An aerial mycelium-negative variant of S. griseus (AMY mutant), that was unable to elicit cytochrome P-450 when grown on soybean flour-enriched medium, failed to oxidize precocene II.     

Morphological relationships among populations in the Sorex caecutiens/shinto group (Eulipotyphla, Soricidae) in East Asia, with a description of a new subspecies from Cheju Island, Korea

We investigated the morphological relationships among eight populations of Sorex caecutiens/shinto group in East Asia using 11 cranial and dental characters and four external characters. Univariate and multivariate analyses of these characters failed to distinguish S. caecutiens and S. shinto. Morphological characters were, in fact, continuous between populations. Sorex shinto from Honshu was similar to S. caecutiens from the Korean peninsula and Primorye in skull dimensions and to S. caecutiens from Hokkaido-Sakhalin in external dimensions. Sorex caecutiens from Cheju Island is morphologically similar to S. shinto from Sado and Shikoku islands. These three insular populations were characterized by having large body sizes. Sorex caecutiens from Cheju was the largest of the S. caecutiens/shinto group in East Asia. This shrew from Cheju was classified definitively as S. caecutiens on DNA data, but has a unique morphology among S. caecutiens populations in East Asia. We therefore regard this Sorex shrew on Cheju Island as a new subspecies of S. caecutiens and designate it S. c. hallamontanus Abe and Oh.     

Kinetic characterization of diphenolase activity from Streptomyces antibioticus tyrosinase in the presence and absence of cyclodextrins

Streptomyces antibioticus tyrosinase was kinetically characterized after purification by PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation using tert-butylcathechol (TBC) and dopamine. The enzyme showed an optimal pH at 6.5 and a K_M of 1.2 mM and 8.4 mM, respectively. The effect of several modulators was studied on this Gram-positive bacterium tyrosinase. In addition, previously undescribed characterization of apparent inhibition and activation of a bacterial tyrosinase using different kinds of cyclodextrins was carried out. When a hydrophobic substrate of S. antibioticus tyrosinase, in this case, tert-butylcatechol was used, a marked substrate sequestrant effect was observed in the presence of hydroxypropyl-β-cyclodextrins (OH-β-CDs) and gamma cyclodextrins (γ-CDs). This sequestrant effect was due to the complexation of TBC into the CD cavity. Moreover, the effect of some hydrophobic inhibitors in the presence of OH-β-CDs and γ-CDs was studied using dopamine, a hydrophilic substrate of S. antibioticus tyrosinase. Increasing concentrations of CDs in the presence of inhibitors like hexestrol or hinokitiol, were able to reactivate the inhibited enzyme to reach the non-inhibited level, as a result of the complexation of these inhibitory compounds in the hydrophobic core of the CDs. This dual effect of CDs as apparent inhibitor and activator has never before described being observed in bacteria.     

The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri

The aminoglycoside antibiotic paromomycin that is highly toxic to the green alga Volvox carteri is efficiently inactivated by aminoglycoside 3′-phosphotransferase from Streptomyces rimosus. Therefore, we made constructs in which the bacterial aphH gene encoding this enzyme was combined with Volvox cis-regulatory elements in an attempt to develop a new dominant selectable marker – paromomycin resistance (Pm^R) – for use in Volvox nuclear transformation. The construct that provided the most efficient transformation was one in which aphH was placed between a chimeric promoter that was generated by fusing the Volvox hsp70 and rbcS3 promoters and the 3′ UTR of the Volvox rbcS3 gene. When this plasmid was used in combination with a high-impact biolistic device, the frequency of stable Pm^R transformants ranged about 15 per 10⁶ target cells. Due to rapid and sharp selection, Pm^R transformants were readily isolated after six days, which is half the time required for previously used markers. Co-transformation of an unselected marker ranged about 30%. The chimeric aphH gene was stably integrated into the Volvox genome, frequently as tandem multiple copies, and was expressed at a level that made selection of Pm^R transformants simple and unambiguous. This makes the engineered bacterial aphH gene an efficient dominant selection marker for the transformation and co-transformation of a broad range of V. carteri strains without the recurring need for using auxotrophic recipient strains.     

Compatibility of Steinernema feltiae (Nematoda: Steinernematidae) with Pesticides and Plant Growth Regulators Used in Glasshouse Plant Production

Compatibility of the entomopathogenic nematode, Steinernema feltiae Filipjev with 17 different pesticides and three plant growth regulators, recommended for drench application in glasshouses, was evaluated. The products tested were two biofungicides, Streptomyces griseoviridis (Mycostop) and Trichoderma harzianum (Rootshield), nine chemical fungicides, iprodione (Chipco 26GT), thiophanate-methyl (Fungo Flo 4.5F), azoxystrobin (Heritage), fludioxonil (Medallion), flutolanil (Prostar), mefenoxam (SudDue Maxx 21.3ME), PCNB (Terraclor 400F), triflumizole (Terraguard), and etridiazole (Terrazole), two bioinsecticides, spinosad (Conserve SC) and Bacillus thuringiensis subsp. israelensis (Gnatrol), three chemical insecticides diflubenzuron (Adept IGR), acephate (Orthene), and fenoxycarb (Precision 25WP), one herbicide, clethodim (Envoy) and three plant growth regulators, ancymidol (A-Rest), paclobutrazol (Bonzi), and uniconazole-P (Sumagic). Infective juvenile nematodes were exposed to each product at the highest recommended concentration in 24-well plates at 22°C. Observations on the viability and infectivity of the nematodes were made at 4, 24, and 72 h after exposure. We found that S. feltiae is compatible with the majority of the tested formulations with no loss in viability and infectivity up to 24 h of exposure. The viability of S. feltiae was more than 80% in all the products even after 72 h of exposure. Three pesticide formulations, Prostar (1%), Gnatrol (9%), and Terrazole (10%) decreased the viability of S. feltiae significantly within 24 h compared to the controls. However, during the 24 to 72 hr incubation period, eight pesticides affected the viability of S. feltiae with Gnatrol and Terrazole causing the highest decrease (17% and 15%, respectively). Only Terrazole decreased the infectivity of S. feltiae to Galleria mellonella larvae compared to the control when tested after 24 h exposure. At 72 hr, Orthene and Terrazole caused significant decrease in the infectivity of S. feltiae (10% and 15%, respectively) and Gnatrol caused a significant increase in the infectivity (11%) compared to the control. Our results suggest that S. feltiae can be tank-mixed and applied in combination with all the tested formulations, except Terrazole.     

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the T1rA and T1rD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related (‘erm-type’) genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.     

Rapid and reliable identification of Staphylococcus equorum by a species-specific PCR assay targeting the sodA gene

Rapid and reliable identification of Staphylococcus (S.) equorum was achieved by species-specific PCR assays. A set of primers targeting the manganese-dependent superoxide dismutase (sodA) gene of S.equorum was designed. Species-specificity of the primer set was evaluated by using a total of 112 strains (including 27 reference strains of the DSM collection), representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria, and different strains of Macrococcus caseolyticus. By using primers SdAEqF and SdAEqR the expected PCR fragment was obtained only when DNA from S. equorum strains was used as template. The rapidity (about 4 h from DNA isolation to results) and reliability of the PCR procedures established suggests that the method may be profitably applied for specific detection and identification of S. equorum strains.     

A new approach to directed gene evolution by recombined extension on truncated templates (RETT)

We describe a new approach to in vitro DNA recombination technique termed recombined extension on truncated templates (RETT). RETT generates random recombinant gene library by template-switching of unidirectionally growing polynucleotides from primers in the presence of unidirectional single-stranded DNA fragments used as templates. RETT was applied to the recombination of two homologous chitinase genes from S. marcescens ATCC 21074 and S. liquefaciens GM1403. When the shuffled genes were examined by restriction mapping and sequence analysis, it was found that chimeric genes were produced at a high frequency (more than 70%) between two chitinase genes with 83% of sequence identity. The number of crossovers within each chimeric gene ranged from one to four, and the recombination points were randomly distributed along entire DNA sequence. We also applied RETT to directed evolution of a chitinase variant for enhancing thermostability. Chimeric chitinases that were more thermostable than the parental enzyme were successfully obtained by RETT-based recombination.     

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode

Sparganum proliferum is characterized by continuous branching and budding, the resulting progeny invading all tissues of the human body, causing fatal sparganosis. Its life cycle, definitive hosts and the route of infection to humans have not yet been disclosed. Because its morphology is similar to Spirometra erinacei, the phylogeny of S. proliferum has been thought to be identical to or closely related to S. erinacei. However, the taxonomy of S. proliferum has not been established up to present due to the lack of definitive observations. In order to clarify the phylogenetic relationship between S. proliferum and S. erinacei, nucleotide sequences of mitochondrial NADH dehydrogenase subunit 3 gene (ND3) and four mitochondrial tRNA coding genes of S. proliferum and other pseudophyllidean cestodes were analyzed. The sequences of S. proliferum showed high similarity to those of S. erinacei, although they were clearly different from each other, indicating that the phylogeny of S. proliferum and S. erinacei is distinct. This is the first report showing the phylogenetic relationship among S. proliferum and other pseudophyllidean cestodes at the DNA sequence level.     

Antioxidative defense and proline/phytochelatin accumulation in a newly discovered Cd-hyperaccumulator, Solanum nigrum L.

Changes in the activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and the contents of malondialdehyde (MDA), chlorophyll, free proline and phytochelatins (PCs) in Solanum nigrum, the newly discovered Cd-hyperaccumulator were examined and compared with a non-hyperaccumulator Solanum melongena. It was indicated that leaf SOD and POD activity of S. nigrum was significantly higher than that of S. melongena. The Cd treatments significantly increased root SOD activity, leaf POD activity, and CAT activity and free proline content in the leaves and roots of S. nigrum. On the contrary, the Cd treatments decreased SOD activity, and did not change CAT activity in the leaves and roots of S. melongena. Moreover, there were no significant differences in free proline levels in the roots of S. melongena. These results validated that S. nigrum had a greater capacity than S. melongena to adapt to oxidative stress caused by Cd and free proline accumulation might be responsible for the tolerance of S. nigrum to Cd. Treated with 10 μg Cd g⁻¹, growth of S. nigrum and its contents of chlorophyll and MDA were basically unaffected. In contrast, there were a decrease in the growth and chlorophyll content, and an increase in MDA in the roots of S. melongena. Although lipid peroxidation was promoted in both the hyperaccumulator and non-hyperaccumulator by high Cd stress, the greater increase took place in the tissues of S. melongena. The PCs level in roots of S. nigrum was significantly lower than that of S. melongena. On the contrary, the content of leaf PCs was much higher in S. nigrum than that in S. melongena. These results further suggested that antioxidative defense in the Cd-hyperaccumulator might play an important role in Cd tolerance, and PCs synthesis is not the primary reason for Cd tolerance although PCs in S. nigrum increased significantly by Cd.     

Host specificity testing and suitability of a European biotype of the braconid parasitoid Microctonus aethiopoides as a biological control agent against Sitona lepidus (Coleoptera: Curculionidae) in New Zealand

The European biotype of the parasitoid Microctonus aethiopoides Loan (Hymenoptera: Braconidae) is being considered for release against Sitona lepidus Gyllenhal (Coleoptera: Curculionidae) in New Zealand. Host specificity was evaluated in the laboratory using both endemic and introduced weed biological control curculionid species, with 12 no-choice and three choice experiments carried out comparing the S. lepidus and test weevils. Two further no-choice tests used the Moroccan M. aethiopoides biotype to compare attack rate between European and Moroccan M. aethiopoides, the latter released in 1982 to control the lucerne pest S. discoideus. Across all experiments, total parasitism of S. lepidus was 69% compared with 15% for the test weevils. European M. aethiopoides was able to develop in the native weevils Irenimus aequalis, Nicaeana cervina, Catoptes cuspidatus, Protolobus porculus and Steriphus variabilis with parasitism rates of 13, 28, 2, 7 and 8%, respectively. These levels were significantly less than those in the corresponding S. lepidus control. Total parasitism of I. aequalis and C. cuspidatus increased significantly in the presence of S. lepidus than recorded under no-choice conditions. The presence of European M. aethiopoides caused minor, if any, test weevil mortality prior to the onset of prepupal emergence and there was no significant reproductive suppression in parasitoid-exposed test weevils. Parasitism of the introduced weed control agent R. conicus by European M. aethiopoides was significantly lower (1.1%) compared to the Moroccan biotype (47.5%). Based on these and other experiments, should the European M. aethiopoides be released as a biological control agent of S. lepidus, its ecological impacts are likely to be less severe than those already exhibited by the Moroccan M. aethiopoides.     

ITS2 ribosomal RNA indicates Schistosoma hippopotami is a distinct species

The internal transcribed spacer region of the ribosomal RNA, ITS2, was sequenced from a singlé specimen of S. hippopotami collected from a pulmonary artery of the hippopotamus, Hippopotamus amphibius in South Africa. The nucleotide sequence was aligned with those of S. mansoni, S. rodhaini, S. haematobium, S. intercalatum, S. curassoni. S bovis and S. japonicum. Both maximum parsimony and genetic distance analyses were performed on these data sets. Using S. japonicum as outgroup to the African schistosomes, a single most-parsimonious tree was obtained of length 64 steps with a consistency index of 1. S. hippopotami was the sister-group to the remaining African species. This species has lateral-spined eggs and its basal position in the tree suggests that this condition is primitive and that terminal-spined eggs developed secondarily. Molecular data clearly show that S. hippopotami cannot be considered synonymous with S. mansoni. Assuming the hippopotamus is the normal host of S. hippopotami, phylogenetic analysis is consistent with an ancient association between schistosomes and ungulates.