Draft Genome Sequence of Turicella otitidis ATCC 51513, Isolated from Middle Ear Fluid from a Child with Otitis Media

Turicella otitidis is an unusual corynebacterium with a controversial role in otitis media in children. Metabolic capabilities deduced from the draft genome indicate its adaptation to habitats on the human skin and in the intestine. The lack of candidate virulence factors implies that T. otitidis has a low pathogenic potential.     

Partial Purification and Characterization of a Thermostable Actinomycete beta-Amylase

A thermostable amylase, possibly a beta-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60 degrees C and pH 7 and by retention of 70% activity at 70 degrees C (30 min). It was stimulated by Mn and Fe but strongly inhibited by Hg. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Preliminary Characterization of Bacteriophages Infecting the Thermophilic Actinomycete Thermomonospora

Bacteriophages lysing strains of Thermomonospora alba and T. fusca were isolated, following specific enrichment, from vegetable composts. Four Thermomonospora phages were distinguished by plaque morphology and host range. Electron microscopy of phage particles, termperature inactivation profiles, and electrophoretic analyses of major virion proteins and genomic DNA were used in the comparison and initial characterization of these phages. The four phages studied possessed polyhedral heads and long tails; genomes were linear double-stranded DNA molecules, 35 to 45 kilobases in length, which probably contain cohesive ends. Transfection of Thermomonospora protoplasts with purified genomic DNA from one of the phages was demonstrated.     

Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.     

Isolation and Characterization of Actinomycete Antagonists of a Fungal Root Pathogen

By use of selective media, 267 actinomycete strains were isolated from four rhizosphere-associated and four non-rhizosphere-associated British soils. Organic media with low nutrient concentrations were found to be best for isolating diverse actinomycetes while avoiding contamination and overgrowth of isolation media by eubacteria and fungi. While all isolates grew well at pHs 6.5 to 8.0, a few were unable to grow at pH 6.0 and a significant number failed to grow at pH 5.5. Eighty-two selected isolates were screened for in vitro antagonism towards Pythium ultimum by use of a Difco cornmeal agar assay procedure. Five isolates were very strong antagonists of the fungus, four were strong antagonists, and ten others were weakly antagonistic. The remaining isolates showed no antagonism by this assay. Additional studies showed that several of the P. ultimum antagonists also strongly inhibited growth of other root-pathogenic fungi. Twelve isolates showing antifungal activity in the in vitro assay were also tested for their effects on the germination and short-term growth of lettuce plants in glasshouse pot studies in the absence of pathogens. None of the actinomycetes prevented seed germination, although half of the isolates retarded seed germination and outgrowth of the plants by 1 to 3 days. During 18-day growth experiments, biomass yields of some actinomycete-inoculated plants were reduced in comparison with untreated control plants, although all plants appeared healthy and well rooted. None of the actinomycetes significantly enhanced plant growth over these short-term experiments. For some, but not all, actinomycetes, some correlations between delayed seed germination and reduced 18-day plant biomass yields were seen. For others, plant biomass yields were not reduced despite an actinomycete-associated delay in seed germination and plant outgrowth. Preliminary glasshouse experiments indicated that some of the actinomycetes protect germinating lettuce seeds against damping-off caused by P. ultimum.     

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.     

深海放线菌08A4的鉴定及其抗真菌活性产物研究

从南海深海分离得到1株放线菌08A4,其发酵产物具有抗植物病原真菌活性,分离纯化得到3个化合物,通过1H-NMR初步鉴定为抗霉素类物质。结合形态学鉴定方法与16S rDNA序列分析方法,鉴定该菌株为微白黄链霉菌(Streptomyces albidoflavus)。     

Characterization of a large subunit catalase truncated by proteolytic cleavage

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Formation of a transfer product from stevioside by the cultures of Actinomycete.

An Actinomycete, strain K-128, which was isolated from soil, formed a transfer product when the strain was cultured in a medium containing both stevioside and curdlan. The transfer product in culture broth was separated by DIAION HP-20 column and TOYOPEARL HW-40F column chromatographies. From structural studies, the transfer product was identified as C13-O-beta-6(2)-beta-glucosylsophorosyl-C19-O-beta-glucopyranosyl steviol. This product was the first to be obtained by a culture of an Actinomycete.     

The isolation and restriction mapping of a miniplasmid from the Actinomycete Nocardia corallina

Abstract A variety of plasmids has been identified as covalently closed circular and linear DNA in certain Actinomycetes, such as Streptomyces . This paper describes the first isolation and characterisation of a plasmid from the genus Nocardia . The plasmid pKU100 isolated from Nocardia corallina is a cccDNA molecule, 2.7 kb in length. This plasmid has been mapped with a wide variety of restriction enzymes and contains a number of unique restriction sites making it suitable for development as a cloning vector.     

抗肿瘤活性海洋放线菌的筛选及菌株HGF26的初步鉴定

对采自连云港海域的海泥样品进行放线菌选择性分离,用其发酵液进行抗肿瘤活性筛选,并对活性较好的菌株HGF26进行了初步鉴定。从海泥样品中共分离得到放线菌78株,以人肝癌细胞HepG2为靶标,活性筛选得到细胞毒活性达60％以上的阳性菌株3株,以其他5株肿瘤细胞为靶标的复筛表明,菌株HGF26的发酵液对多种肿瘤细胞具有显著的细胞毒活性,其中对胃癌细胞BGC823的细胞毒活性为79％,活性产物具有较好的酸碱和热稳定性。通过对菌株的培养特征、形态特征、生理生化特征和细胞壁成分分析,将菌株HGF26初步鉴定为微白黄链霉菌的海洋变种。     

产低温纤维素酶放线菌的筛选、鉴定及酶学性质初步研究

从青藏高原采集的牦牛粪中,经过CMC平板分离得到一株产低温纤维素酶能力较强的放菌Tibet-YD5227-2,经16S rDNA序列比对分析将其初步鉴定为链霉菌属(Streptomyces sp.),目前尚无链霉菌产低温纤维素酶的研究报道。Tibet-YD5227-2液体摇瓶培养产生低温CMC酶活力高达145U/mL,最适产酶温度25℃,最适产酶pH值为8.0。酶学性质初步研究显示,Tibet-YD5227-2产生的CMC酶反应温度以35℃左右为适,反应的pH值以8.0左右为适,该菌株所产的纤维素酶在中碱性条件具有较强的稳定性;K+、Fe2+、Mg2+对酶反应有促进作用,Hg2+、Cu2+对酶反应有抑制作用。     

一株嗜碱放线菌发酵液 中的抗肿瘤活性成分(英文)

从嗜碱放线菌YIM 80149的发酵液中分离得到4个抗肿瘤活性成分.根据波谱数据,鉴定为4',7-dihydroxyisoflavone(1)、4',5,7-trihydroxyflavone(2)、3-(4-hydroxy-3-methoxyphenyl)-2(E)-propenoic acid(3)、2-methoxy-1,4-naphthoquinone(4).     

Isolation and Characterization of Novel Marine-Derived Actinomycete Taxa Rich in Bioactive Metabolites

A unique selective enrichment procedure has resulted in the isolation and identification of two new genera of marine-derived actinobacteria. Approximately 90% of the microorganisms cultured by using the presented method were from the prospective new genera, a result indicative of its high selectivity. In this study, 102 actinomycetes were isolated from subtidal marine sediments collected from the Bismarck Sea and the Solomon Sea off the coast of Papua New Guinea. A combination of physiological parameters, chemotaxonomic characteristics, distinguishing 16S rRNA gene sequences, and phylogenetic analysis based on 16S rRNA genes provided strong evidence for the two new genera (represented by strains of the PNG1 clade and strain UMM518) within the family Micromonosporaceae. Biological activity testing of fermentation products from the new marine-derived actinomycetes revealed that several had activities against multidrug-resistant gram-positive pathogens, malignant cells, and vaccinia virus replication.     

Characterization of mannooligosaccharide caps in mycobacterial lipoarabinomannan by capillary electrophoresis/electrospray mass spectrometry

A new analytical approach based on capillary electrophoresis-electrospray mass spectrometry (CE/ESI-MS) has provided new insight into the characterization of mannooligosaccharide caps from lipoarabinomannans (LAMs), which are key molecules in the immunopathogenesis of tuberculosis. This analytical approach requires oligosaccharide labeling with the fluorophore 1-aminopyrene-3,6,8-trisulfonate (APTS) by reductive amination at the reducing termini. Optimization of the separation and ionization conditions, such as the choice of capillary electrophoresis (CE) electrolyte buffers, is presented and discussed. Anionic separation of the mono and oligosaccharide APTS derivatives was finally achieved with aqueous triethylammonium formate buffer. It was found that in contrast to the triethylammonium phosphate buffer, the triethylammonium formate buffer was appropriate for CE/ESI-MS coupling analysis of APTS-carbohydrate derivatives. In this case, negative ESI-mass spectra of APTS-carbohydrate adducts showed mainly (M-2H)2-pseudomolecular ions and some sequence fragment ions allowing their non-ambiguous structural characterization at the picomolar level. This analytical approach was successfully applied to more complex mixtures of carbohydrates released by mild acid hydrolysis of the lipoarabinomannans from Mycobacterium bovis BCG. The APTS-mannooligosaccharide cap adducts were separated by CE and their structural characterization achieved by CE/ESI-MS analyses. Mannooligosaccharide caps were routinely analyzed by capillary electrophoresis-laser induced fluorescence (CE-LIF) from 50 fmol of lipoarabinomannans with mannosyl capping (ManLAMs) but sensitivity was about 50 times lower using ESI-MS detection.     

Characterization of an unusual tropoelastin with truncated C-terminus in the frog

Tropoelastin is the monomeric form of elastin, a major polymeric protein of the extracellular elastic matrix of vertebrate tissues with properties of extensibility and elastic recoil. Mammalian and avian species contain a single gene for tropoelastin. A tropoelastin gene has also previously been identified in amphibians. In contrast, two tropoelastin genes with different tissue expression patterns have been described in teleosts. While general characteristics of tropoelastins, such as alternating arrangements of hydrophobic and crosslinking domains, are conserved across a wide phylogenetic range, sequences of these domains are highly variable, particularly when amphibian and teleost tropoelastins are included. For this reason exon-to-exon correspondence is not clear, and overall alignment of tropoelastin sequences across all species is not possible. An exception to this is the C-terminal exon, whose coding sequence has been very well-conserved across all species described to date. In mammalians this C-terminal domain has been shown to be important for interactions with cells and other matrix-associated proteins involved in matrix assembly. Here we identify and characterize a second tropoelastin gene in the frog with several unusual characteristics, the most striking of which is truncation of the C-terminal domain, deleting normally conserved sequence motifs. We demonstrate that, in spite of the absence of these motifs, both frog tropoelastin genes are expressed and incorporated into the elastic matrix, although with differential tissue localizations.