Plasmid pSE21 of Streptomyces erythraeus strains

Crossing of S. erythraeus 4, a laboratory strain (NRRL 2338) containing a family of plasmids with S. erythraeus 1, a plasmid-free strain resulted in formation of strain 6. A multi-copy plasmid pSE21 11.5 kb in length was isolated from S. erythraeus 6. A detailed restriction map of plasmid pSE21 was constructed. Its cloning to E. coli YM83 on vector pUC19 showed that plasmid pSE21 was not stable in E. coli. It was found that the status of plasmid pSE21 changed in relation to the physiological state of S. erythraeus 6. Southern hybridization of the plasmid pSE21 DNA with the total DNA of the cultures of S. erythraeus 1 maintained for various periods at 4 degrees C demonstrated that plasmid pSE21 was present in S. erythraeus 1 in an autonomous state in 0.1 to 0.2 copies per genome. The number of the plasmid pSE21 copies could be decreased. The chromosomal DNA of S. erythraeus 1 contained the DNA sequences highly homologous to those of plasmid pSE21. It was assumed that during the crossing of S. erythraeus 4 with S. erythraeus 1 the genetic element from the donor strain was transferred to the recipient strain which in some way changed the plasmid pSE21 status and imparted the multicopy pattern to it. Further investigation of the plasmid pSE21 properties and construction of a vector for S. erythraeus on the plasmid basis are under way.     

赤腹松鼠一新亚种

在查对中国南部赤腹松鼠标本(401号)的基础上,发现分布于云南东北部昭通地区的赤腹松鼠与赤腹松鼠其他亚种有明显的区别.毛色特征:背部棕黄色,背中央区域稍带黑色;腹部至前胸栗红色;喉、颏部棕灰色;尾毛背腹无明显差异,尾毛末端棕黄、次末端黑,尾末端具棕黄色(稍黄白)区域;前后足背棕褐色,稍带黑色.进一步对头骨可量性状数据进行分析(差异系数),结果表明:分布于该地区的赤腹松鼠分别与赤腹松鼠其他13个亚种两两之间至少有一项差异系数大于1.28,系一新亚种Callosciurus erythraeus zhaotongensis subsp.nov..     

Population dynamics of Dremomys pernyi and Callosciurus erythraeus in protective and non-protective pine forests at different ages

Four pine forests (6-10,11-15,16-20,and 31-40 year-old)located in the Cangshan Mountain and Erhai Lake National Reserve and 7 pine forests (1-5,6-10,11-15,16-20,21-30,31-40,and more than 50 year-old)located in the non-protective area near the national reserve were selected.Three replications of each forest was set and a total of 33 sites were investigated.At each site,we quantified 6 habitat variables (species richness,abundance,and percentage of grasses and shrubs coverage respectively at the bottom layer of forests)within randomly determined 5 m×5 m areas.One hundred cages were set in five lines at each site to trap small mammals,whose species and numbers were recorded.Dominance of Dremomys pernyi and Callosciurus erythraeus in small mammal communities,time niche breadth,and time niche overlap between the two small mammals were calculated,respectively.Step-wise regression was used to analyze the relationship between small mammals and habitat factors.Our results indicated that D.pernyi occurred earlier than C.erythraeus in protective pine forests.D.pernyi was captured in 6-10 year-old forest initially,and C.erythraeus was captured in 16-20 year-old forest initially.D.pernyi and C.erythraeus were captured in the 31-40 and 21-30 year-oldforests initially in the non-protective area,respectively.Populations of D.pernyi and C.erythraeus in the 31-40 year-old protective forests were 3 and 3.75 times of those in the sameaged non-protective forests,respectively.Shrubs significantly influenced the populations of the two small mammals.The population of D.pernyi was positively correlated with the density of shrubs;the population of C.allosciurus erythraeus was positively correlated with the coverage of shrubs,and negatively correlated with the coverage of grasses.D.remomys pernyi and C.allosciurus erythraeus were important for pine forests to scatter pine seeds.Human activities in the nonprotective pine forests decreased the vegetation heterogeneity at the bottom layer of pine forests,postponed the occurrence of D.pernyi and C.erythraeus,and decreased the populations of the two small mammals.     

Genetic mobility of plasmids of Streptomyces erythraeus

Grossing of S. erythraeus 4 with S. erythraeus 1 resulted in transfer of genetic elements from strain 4 to strain 1 as evidence by the 20 and 18 kb fragments in the experiments on DNA-DNA hybridization. The presence of the genetic elements in strain 1 was the cause of plasmid pSE 21 mobility. In strain 6, a derivative of S. erythraeus 1 plasmid pSE 21 was accompanied by other extrachromosomal DNAs characterized by high instability. During storage of the strain at a temperature of 4 degrees C for more than 1 or 2 months the number of the plasmid pSE 21 copies decreased. When the strain was stored for longer periods (6 months or more) the plasmid DNA was not detectable even with the DNA-DNA hybridization procedure. The results of hybridization of a fraction of the extrachromosomal DNA of S. erythraeus 6, the Bam HIB fragment of plasmid pSE 21 with the total DNA of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus and hybridization of DNA of plasmid pSE 21 with the total DNA of S. erythraeus 6 and 1 showed that (1) strains 1, 5 and BTCC 2 had the same hybridization patterns, (2) the other extrachromosomal DNAs present in the fraction were homologous with the Bam HIA fragment of plasmid pSE 21, (3) chromosomes of strains 1, 4, 5, 6 and BTCC 2 of S. erythraeus also contained DNA homologous to the plasmid Bam HIA fragment. It was suggested that plasmid pSE 21 could be used as a basis for constructing the integrative vector for S. erythraeus.     

珀氏长吻松鼠和赤腹松鼠在保护区与非保护区各年龄松林内的种群动态

2004年6~7月,在云南省大理白族自治州苍山和洱海国家自然保护区选取4种年龄段(6~10、11~15、16~20、31~40年)的松林和保护区周围的非保护区选取7种年龄段(1~5、6~10、11~15、16~20、21~30、31~40、50年以上)的松林,每种松林设3个重复,共33个样地,在样地内随机选取3个5m×5m的样方,调查并记录样方内草本植物和灌木的种类、数量、覆盖度。在每个样地按5条样线布笼100个捕捉小兽,每天检查捕获的种类和数量。计算珀氏长吻松鼠和赤腹松鼠在小兽群落中物种优势度、时间生态位宽度、两种小兽的时间生态位重叠度;用逐步回归分析两种松鼠与松林栖境因子的关系。上述结果表明,在保护区珀氏长吻松鼠出现的时间早于(6~10年的松林开始捕获到)赤腹松鼠(16~20年的松林内开始捕获到);在非保护区,分别在31~40年和21~30年的松林内才捕到珀氏长吻松鼠和赤腹松鼠。保护区31~40年的松林内珀氏长吻松鼠和赤腹松鼠种群数量分别是同年龄段非保护区松林的3倍和3·75倍。松林底层的灌木对两种小兽的种群数量有重要影响。珀氏长吻松鼠种群数量与灌木密度呈正相关;赤腹松鼠种群数量与灌木覆盖度呈正相关,而与草本植物覆盖度呈负相关。非保护区树底植被的异质性降低,延迟了两种松鼠在松林里建立种群的时间。     

Cloning of clusters of erythromycin biosynthesis genes of Streptomyces erythraeus (Saccharopolyspora erythraea)

The erythromycin resistance gene (ermE) and part of erythromycin biosynthesis genes located in the same cluster with the ermE gene were cloned from S. erythraeus 3 subjected to improvement with respect to erythromycin production. For isolating the erythromycin biosynthesis genes, the plasmid vector pUC18 and the phage vector lambda EMBL3 were used. The ermE gene DNA was used as a labeled probe for analysis of the recombinant plasmids and phages. The recombinant phages lambda ermE1 and ermE4 containing fragments of the chromosomal DNA collinear to the genome DNA of S. erythraeus 3 were analyzed. The size of the cloned fragment of the chromosomal DNA of S. erythraeus 3 was about 20 kb. Subcloning with the vector pUS18 resulted in isolation of plasmids pSU235-pSU244 containing BamHI fragments of chromosomal DNA from S. erythraeus 3. The restriction map of the chromosomal region of S. erythraeus 3 containing the ermE gene was constructed. The cloned genes of erythromycin biosynthesis are useful in the study of their structure and functions, construction of integrative vectors, improvement of cultures producing macrolide antibiotics and isolation of genes responsible for biosynthesis of other polyketide antibiotics.     

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.     

Identification of an iridovirus in Acetes erythraeus (Sergestidae) and the development of in situ hybridization and PCR method for its detection

An iridovirus (tentatively named SIV, sergestid iridovirus) that causes high mortality in the sergestid shrimp, Acetes erythraeus, was found in Madagascar in 2004. Severely affected shrimp exhibit a blue-green opalescence. Histological examination revealed massive cytoplasmic inclusions in the cuticular epithelial cells, connective tissues, ovary and testes. The electron microscopic examination showed paracrystalline arrays of virions at a size of 140nm, suggesting infection with an iridovirus. A pair of PCR primers were selected from the conserved region of the major capsid protein (MCP)-coding sequence among insect iridoviruses and used to amplify a 1.0kb fragment from the infected A. erythraeus. This fragment was cloned, sequenced and found to be highly similar (upto 80% similarity in translated amino acids with an E value of 1e-124) to the MCP of invertebrate iridoviruses. This clone was then labeled with digoxigenin-11-dUTP and hybridized to tissue sections of infected A. erythraeus, which reacted positively to the probe. The reacting tissues included epithelial cells, connective tissues, and the germinal cells; the same cells as those with inclusions. A PCR method was also developed from the MCP coding sequence for detecting SIV.     

Restriction mapping and close relationship of the DNA of Streptomyces erythraeus phages 121 and SE-5

The biological properties and genome structure of two actinophages, 121 and SE-5, infecting Streptomyces erythraeus were characterized. They had the same host range (limited to S. erythraeus) and similar DNA G + C contents (around 60 mol %). Restriction maps of their genomes also showed many similarities. The close relationship between the two phages was confirmed by DNA hybridization experiments: large parts of their genomes were homologous, except for a segment in the middle of the map, where no hybridization was detected.     

Ectoparasites of the Pallas squirrel, Callosciurus erythraeus, introduced to Japan

The squirrel Callosciurus erythraeus (Pallas) (Rodentia: Sciuridae) was intentionally introduced to Japan in 1935 and has become established throughout much of the country. Although they live mainly in forests, Pallas squirrels come into gardens and are frequently fed by people or kept as pets, so their ectoparasites could be of potential medical as well as veterinary importance. During 2001-2003 we conducted the first ectoparasite survey of Pallas squirrels in Japan. From 105 C. erythraeus captured in Kamakura District of Kanagawa Prefecture on Honshu Island, three types of ectoparasite were found: 52 specimens of the sucking louse Neohaematopinus callosciuri Johnson (Anoplura: Haematopinidae), 26 fleas Ceratophyllus (Monopsyllus) anisus Rothschild (Siphonaptera: Ceratophyllidae) and four nymphs of the tick Haemaphysalis flava Neumann (Acari: Ixodidae) on 22, 13 and one squirrels, respectively. Evidently in Japan C. erythraeus carries relatively few ectoparasite species; this may be a contributory factor to their invasive success. Further investigations are needed to assess risks of zoonotic transmission of plague or murine typhus by C. anisus, of louse-borne typhus by N. callosciuri and of tularaemia and especially Japanese spotted fever (Rickettsia japonica) by H. flava.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Recovery of gold from thiourea solutions using microorganisms

The recovery of gold from gold-thiourea solutions using various types of waste biomass was investigated. All organisms tested, namely, Saccharomyces cerevisiae, Spirulina platensis and Streptomyces erythraeus removed gold rapidly from gold–thiourea solutions. The process of gold accumulation was pH-dependent for Saccharomyces ceresvisiae and Streptomyces erythraeus and independent of pH in the case of Spirulina platensis. Of all strains of microorganisms examined, Spirulina platensis had the highest affinity and capacity for gold even at low pH values. Thus, all three microorganisms tested for their ability to recover gold from gold–thiourea solutions can therefore be used in biotechnological applications, especially Spirulina platensis which has the highest binding capacity for gold at low pH values. © Rapid Science 1998     

Streptomyces erythraeus trypsin inactivates α1-antitrypsin

Streptomyces erythraeus trypsin (SET) is a serine protease that is secreted extracellularly by S. erythraeus. We investigated the inhibitory effect of α(1)-antitrypsin on the catalytic activity of SET. Intriguingly, we found that SET is not inhibited by α(1)-antitrypsin. Our investigations into the molecular mechanism underlying this observation revealed that SET hydrolyzes the Met-Ser bond in the reaction center loop of α(1)-antitrypsin. However, SET somehow avoids entrapment by α(1)-antitrypsin. We also confirmed that α(1)-antitrypsin loses its inhibitory activity after incubation with SET. Thus, our study demonstrates that SET is not only resistant to α(1)-antitrypsin but also inactivates α(1)-antitrypsin.     

The use of a chromosome integration vector to map erythromycin resistance and production genes in Saccharopolyspora erythraea (Streptomyces erythraeus)

The thiostrepton-resistance-conferring plasmid pIJ702 was integrated into the ermE region of the chromosome of erythromycin (Er)-producing bacterium Saccharopolyspora erythraea (Streptomyces erythraeus) by single, reciprocal (Campbell) recombination between DNA cloned in the vector and homologous nucleotide sequences in the chromosome. Genetic mapping experiments by conjugational transfer were used to establish that the ErR gene, ermE, was located close to the Er-production loci eryA34 and eryB25.     

Site of action of a ribosomal RNA methylase responsible for resistance to erythromycin and other antibiotics

The enzyme which confers resistance to erythromycin in the producing organism Streptomyces erythraeus dimethylates a single adenine residue in Bacillus stearothermophilus 23 S rRNA. This corresponds to residue Ade 2058 in Escherichia coli 23 S RNA. The methylase responsible for resistance to macrolides, lincomycin, and streptogramin B-related antibiotics in Staphylococcus aureus also acts at this site.     

Development of integrative vectors for Actinomycetes--producers of antibiotics]

The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus.     

赤腹松鼠采食选择的季节性变化

2008年9月至2010年8月,对广西宜州龙江河畔赤腹松鼠采食选择的季节性变化进行分析.赤腹松鼠对榕树的采食量最大,约占总采食量的36.53%,食物采食选择的种类存在显著差异(P<0.05),各季节采食的种类无显著变化(P>0.05);取食偏好分析、食物生态位宽度和重叠度结果表明,夏季的生态位宽度最大,为4.1545,秋季的生态位宽度最小,为3.5001,夏季和秋季的生态位重叠度最大,为0.9882,夏季和冬季的生态位重叠度最小,为0.9343.     

Expression of the macrolide-lincosamide-streptogramin-B-resistance methylase gene, ermE, from Streptomyces erythraeus in Escherichia coli results in N6-monomethylation and N6,N6-dimethylation of ribosomal RNA

The ermE gene was cloned from Streptomyces erythraeus into Escherichia coli on a series of plasmids. When transcribed from the lac promoter, ermE conferred high-level resistance to erythromycin and other macrolide-lincosamide-streptogramin-B (MLS) antibiotics. A methylase activity capable of N6-mono- and N6,N6-dimethylation of adenine residues in E. coli rRNA was detected in extracts of MLS-resistant cells. In addition, rRNA extracted from MLS-resistant E. coli contained N6-mono- and N6,N6-dimethylated adenine residues.     

常春油麻藤有气味花蜜及其生态功能

一些被子植物能够分泌有气味的花蜜,但这一自然现象很少被关注.作为嗅觉信号线索,有气味的花蜜可能是将访花者和气味信号结合在一起的特征,它与传粉者及盗蜜者的关系值得探索.本研究以常春油麻藤(Mucuna sempervirens)为对象,研究了其开花动态及泊氏长吻松鼠(Dremomys pernyi)和赤腹松鼠(Callosciurus erythraeus)的访花行为,采用顶空固相微萃取和气相色谱-质谱法收集并分析了花蜜的挥发物成分,探讨了花蜜对中华蜜蜂(Apis cerana cerana)的吸引作用及对酸臭蚁(Tapinoma sp.)的毒杀作用.结果表明:常春油麻藤花蜜释放的挥发物以脂肪族化合物为主(87.2％),其中酮类占56.1％,无含硫挥发性成分,这和该属其他蝙蝠传粉种类花蜜释放含硫化合物的结果不一致.此外,常春油麻藤的花蜜对酸臭蚁有慢性毒杀作用,而对中华蜜蜂则有吸引作用.未发现蝙蝠访花,但观察到泊氏长吻松鼠和赤腹松鼠可能为常春油麻藤传粉.因此,常春油麻藤可能不属于蝙蝠传粉的种类.希望本研究能为该属亚洲类群的传粉机制提供数据,并为其他植物类群花蜜成分及功能研究提供新的视角.     

Methylation of 23S rRNA caused by tlrA (ermSF), a tylosin resistance determinant from Streptomyces fradiae.

Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus).