Study on thermostability of phospholipase D from Streptomyces sp

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.     

Streptomyces manipurensis sp. nov., a novel actinomycete isolated from a limestone deposit site in Manipur, India

A novel actinobacterium, designated MBRL 201(T), was isolated from a sample collected from a limestone quarry at Hundung, Manipur, India. The strain was characterized using polyphasic taxonomy. Comparison of the 16S rRNA gene sequence of strain MBRL 201(T) and other Streptomyces species showed sequence similarities ranging from 93.0 to 99.6 % and strain MBRL 201(T) showed closest similarities to Streptomyces virginiae NBRC 12827(T) (99.6 %) and Streptomyces cinnamonensis NBRC 15873(T) (99.6 %). The DNA relatedness between MBRL 201(T) and the type strains of S. virginiae NBRC 12827(T) and S. cinnamonensis NBRC 15873(T) were 44.5 and 35.6 % respectively. Strain MBRL 201(T) contained LL: -diaminopimelic acid (A(2)pm) as the diagnostic diamino acid, with glucose as the main sugar, while small amounts of galactose, glucose, mannose, rhamnose, ribose and xylose were also present in cell-wall hydrolysates. The major fatty acids identified were anteiso-C(15:0) (38.9 %), iso-C(15:0) (19.9 %) and anteiso-C(17:1) (14.7 %). The predominant menaquinones detected were MK-9(H(6)) and MK-9(H(8)), while the polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositolmannosides, with other unknown phospholipids and lipids. The G+C content of the genomic DNA was 72.9 %. The phenotypic and genotypic data showed that strain MBRL 201(T) merits recognition as a representative of a novel species of the genus Streptomyces. It is proposed that the isolate should be classified in the genus Streptomyces as a novel species, Streptomyces manipurensis sp. nov. The type strain is MBRL 201(T) (=DSM 42029(T)?=?JCM 17351(T)).     

嗜麦芽寡养单胞菌D2株2套Ⅱ型分泌系统

嗜麦芽寡养单胞菌D2株经前期研究发现有2套Ⅱ型分泌系统（T2SS）,根据嗜麦芽寡养单胞菌K279a、R551—3、JV3、D457的T2SS基因簇序列,以及D2株的部分测序结果设计引物,采用基因移步法逐一扩增2套T2SS基因序列,产物连接至T载体,经酶切鉴定后测序、拼接。发现有22个完整的开放多码框架（ORF）,比对分析后发现T2SSl的基因簇与同种属细菌相应基因簇序列同源性均在80％以上,对应氨基酸序列同源性均能达到97％以上,而T2SS2基因簇与K279a、R551—3对应基因序列和氨基酸序列的同源性均不及T2SS1高。2套T2SS的GspE、F、I基因同源性在50％以上,其他对应基因同源性在35％～68％之间不等,氨基酸序列同源性则为15％～61％。2套他ss与铜绿假单胞菌PA01的同源性高于不同科的小肠结肠炎耶尔森菌。T2SS的序列测定及同源性分析为进一步研究细菌蛋白分泌机制奠定基础。     

海洋链霉菌Streptomyces sp.B-17卤化酶基因的克隆及生物信息学分析

海洋放线菌是生理活性物质重要的产生菌,而海洋放线菌产生的卤化酶可催化生理活性物质的卤化,极大提高了其抑菌和抗癌活性。从大连海域分离出1株链霉菌Streptomyces sp.B-17,从其基因组DNA中扩增一524 bp的基因片段,经与pMD-19T载体连接,转化大肠埃希菌JM109感受态细胞,重组质粒经鉴定证明正向插入到pMD-19T载体。生物信息学分析表明该序列与Actinocorallia herbida DSM 44252的2个依赖FADH2卤化酶基因的同源性为88%,拟编码氨基酸与色氨酸卤化酶(CP001848)的相似性也达到74%,证明所克隆的基因片段为卤化酶基因片段,为后续的基因功能分析及表达奠定了基础。     

Structural analysis of loci involved in pSAM2 site-specific integration in Streptomyces

pSAM2 is an 11-kb plasmid integrated in the Streptomyces ambofaciens ATCC23877 and ATCC15154 genomes and found additionally as a free replicon in an uv derivative. After transfer into S. ambofaciens DSM40697 (devoid of pSAM2) or into Streptomyces lividans, specific integration of pSAM2 occurred very efficiently. A 58-bp sequence (att) present in both pSAM2 (attP) and S. ambofaciens strain DSM40697 (attB) attachment regions is found at the boundaries (attL and attR) of integrated pSAM2 in S. ambofaciens strain ATCC23877. The S. lividans chromosomal integration zone contained an imperfectly conserved att sequence (attB), and the integration event of pSAM2 was located within a 49-bp sequence of attB. Only one primary functional attB sequence was present in the S. lividans or S. ambofaciens DSM40697 total DNA. The integration zone of S. lividans hybridized with the integration zone of S. ambofaciens DSM40697. The two integration zones were homologous only to the right side of the att sequence. The conserved region contained an open reading frame (ORF A) with a stop codon located 99 bp from the attB sequence in both strains. S. ambofaciens DSM40697 contained DNA sequences related to pSAM2 on the left side of the att site. The att sequence was included in a region conserved in Streptomyces antibioticus, Streptomyces actuosus, Streptomyces bikiniensis, Streptomyces coelicolor, Streptomyces glaucescens, and Streptomyces parvulus. Site-specific integration of a pSAM2 derivative was characterized in another unrelated strain, Streptomyces griseofuscus. This strain contained an imperfectly conserved 58-bp attB sequence, and the integration event took place within a 45-bp sequence of attB. Site-specific integration of pSAM2 in three nonrelated Streptomyces strains suggests the wide host range of pSAM2 integration in Streptomyces.     

Draft Genome Sequence of Lactobacillus rossiae DSM 15814T

The draft genome sequence of Lactobacillus rossiae DSM 15814(T) (CS1, ATCC BAA-88) was determined by a whole-genome shotgun approach. Reads were assembled to a 2.9-Mb draft version. RAST genome annotation evidenced 2,723 predicted coding sequences. Many carbohydrate, amino acid, and amino acid derivative subsystem features were found.     

Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes Tü57.

A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.     

A new xylanase from Streptomyces megasporus DSM 41476 with high yield of xylobiose

A new xylanase gene, xynBM4, was cloned from Streptomyces megasporus DSM 41476 and expressed in Pichia pastoris. The full-length gene consists of 1,443 bp and encodes 480 amino acids including a putative 49-residue signal peptide. The deduced amino acid sequence of xynBM4 shows the highest identity of 66.3% to the xylanase Xys1L from Streptomyces halstedii JM8. The purified recombinant XYNBM4 had a high specific activity of 350.7 U mg^-1 towards soluble wheat arabinoxylan, exhibited optimal activity at pH 6.0 and 57°C, showed broad pH adaptability (>75% of the maximum activity at pH 2.5–9.0), was resistant to neutral proteases and most chemicals, and produced simple products. The hydrolysis products of birchwood xylan and corncob xylan were predominantly xylobiose (76.9 and 90.8%, respectively) and no xylose. These characteristics suggest that XYNBM4 has potential in various applications, especially in the food industry.     

一株氯氰菊酯降解菌16SrDNA，gyrB和GyrB的系统发育分析

从农药厂污水处理池中分离得到一株氯氰菊酯降解菌,在30℃,pH7．0的条件下,无机盐培养基中100mg／L的氯氰菊酯,经过7．5天,能够降解大约52．3％,外加碳源能够明显提高其降解性能。生理生化实验结合16SrDNA,册诏和GyrB的系统发育分析,将其归为Gordonia菌属。在16SrDNA水平上,其与G．amicalis DSM44461和G．hydrophobica DSM44015^T的相似值最高,为98．1％;而在gyrB和GyrB水平上,其与G.hydrophobica JCM10086的相似值最高,分别为86．8％和91,1％。通过对所构建的系统发育树进行评估,表明16SrDNA序列适用于将分离菌株鉴定到属的水平上,而gyrB和GyrB更适用于在属内种的水平上进行系统发育的分析。     

Purification and N-terminal amino-acid sequences of bacterial malate dehydrogenases from six actinomycetales strains and from Phenylobacterium immobile, strain E

Malate dehydrogenases from Streptosporangium roseum (DSM 43021), Planomonospora venezuelensis (DSM 43178), Microtetraspora glauca (ATCC 23057), Actinoplanes missouriensis (DSM 43046), Streptomyces atratus (ATCC 14046), Kibdelosporangium aridum (ATCC 39323), and from Phenylobacterium immobile, strain E (DSM 1986) were purified to homogeneity. The N-terminal amino-acid sequences were determined and compared with known prokaryotic and eukaryotic sequence data. The partial sequences from Actinomycetales enzymes include a string of amino acids which is also present in the N-terminal region of malate dehydrogenases from Thermus flavus and from mammalian cytoplasm.     

Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases.

The gene (chiD) encoding the precursor of chitinase D was found to be located immediately upstream of the chiA gene, encoding chitinase A1, which is a key enzyme in the chitinase system of Bacillus circulans WL-12. Sequencing analysis revealed that the deduced polypeptide encoded by the chiD gene was 488 amino acids long and the distance between the coding regions of the chiA and chiD genes was 103 bp. Remarkable similarity was observed between the N-terminal one-third of chitinase D and the C-terminal one-third of chitinase A1. The N-terminal 47-amino-acid segment (named ND) of chitinase D showed a 61.7% amino acid match with the C-terminal segment (CA) of chitinase A1. The following 95-amino-acid segment (R-D) of chitinase D showed 62.8 and 60.6% amino acid matches, respectively, to the previously reported type III-like repeating units R-1 and R-2 in chitinase A1, which were shown to be homologous to the fibronectin type III sequence. A 73-amino-acid segment (residues 247 to 319) located in the putative activity domain of chitinase D was found to show considerable sequence similarity not only to other bacterial chitinases and class III higher-plant chitinases but also to Streptomyces plicatus endo-beta-N-acetylglucosaminidase H and the Kluyveromyces lactis killer toxin alpha subunit. The evolutionary and functional meanings of these similarities are discussed.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

New beta -lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 and its roles on the morphological differentiation

A new beta-lactamase inhibitory protein (BLIP-I) from Streptomyces exfoliatus SMF19 was purified and characterized. The molecular mass of BLIP-I was estimated to be 17.5 kDa by gel filtration fast protein liquid chromatography. The N-terminal sequence was NH(2)-Asn-Ser-Gly-Phe-Ser-Ala-Glu-Lys-Tyr-Glu-Gln-Ile-Gln-Phe-Gly. BLIP-I inhibited Bacto(R) Penase (Difco), and plasmid encoded TEM-1 beta-lactamase, whereas it did not inhibit Enterobacter cloacae beta-lactamases. The K(i) value of BLIP-I against TEM-1 beta-lactamase was determined to be 0.047 nm. The gene (bliA) encoding BLIP-I protein was identified by screening a genomic library using an oligonucleotide probe with a sequence based on the N-terminal sequence of BLIP-I. Analysis of the nucleotide sequence revealed that the gene was 558 base pairs in length and encoded a mature protein of 157 amino acid residues preceded by a 29-amino acid signal sequence. Pairwise comparison of the deduced amino acid sequence showed 38% identity with BLIP of Streptomyces clavuligerus. Furthermore, the 49th amino acid residue of BLIP-I was identical to Asp-49 of BLIP that was characterized to be an important residue for the inhibitory activity of BLIP. A modified BLIP-I in which Asp-49 was replaced by alanine (D49A) was obtained by site-directed mutagenesis. The inhibitory activities of recombinant (r) BLIP-I and its D49A mutant derivative, expressed in Escherichia coli, were compared. The K(i) value of rBLIP-I against TEM-1 beta-lactamase was similar to that of wild-type BLIP-I, but the D49A mutation increased the K(i) of rBLIP-I inhibition approximately 200-fold. A disruption mutant of the bliA gene in S. exfoliatus SMF19 was obtained by replacing the wild-type bliA gene with a copy inactivated by inserting a hygromycin resistance gene. The disruption mutant showed a bald phenotype, indicating that the bliA gene plays a role in morphological differentiation.     

Streptomyces blattellae,a novel actinomycete isolated from the in vivo of a Blattella germanica

During a screening for novel and useful actinobacteria in desert animal, a new actinomycete was isolated and designated strain TRM63209^T. The strain was isolated from in vivo of a Blattella germanica in Tarim University in Alar City, Xinjiang, north-west China. The strain was found to exhibit an inhibitory effect on biofilm formation by Candida albicans ATCC 18,804. The strain was observed to form abundant aerial mycelium, occasionally twisted and which differentiated into spiral spore chains. Spores of TRM63209^T were observed to be oval-shaped, with a smooth surface. Strain TRM63209^T was found to grow optimally at 28 °C, pH 8 and in the presence of 1% (w/v) NaCl. The whole-cell sugars of strain TRM63209^T were rhamnose ribose, xylose, mannose, galactose and glucose, and the principal polarlipids were found to be diphosphatidylglycerol, phos-phatidylethanolamine, phosphatidylcholine, phosphatidylinositol mannoside, phosphatidylinositol and an unknown phospholipid(L). The diagnostic cell wall amino acid was identified as LL-diaminopimelic acid. The predominant menaquinone was found to be MK-9(H6) (14.64%), MK-9(H2) (19.65%), MK-9(H8) (22.34%), MK-10(H2) (25.37%). The major cellular fatty acids were identified as iso-C16:0, 16:0, anteiso-C15:0, anteiso-C17:0, iso-C15:0 and Sum in Feature 3. Analysis of the 16S rRNA sequence showed that strain TRM63209^T exhibits high sequence similarity to Streptomyces bungoensis strain DSM 41781^T 98.20%. A multi-locus sequence analysis of five house-keeping genes (atpD, gyrB, rpoB, recA and trpB) and phylogenomic analysis also illustrated that strain TRM63209^T should be assigned to the genus Streptomyces. The DNA G?+?C content of the strain was determined to be 70.2 mol%. Average nucleotide identity (ANI) between strain TRM63209^T and S. bungoensis DSM 41781^T, Streptomyces phyllanthi PA1-07^T, Streptomyces longwoodensis DSM 41677^T and Streptomyces caeruleatus NRRL B-24802^T were 82.76%, 82.54%, 82.65%, 84.02%, respectively. Digtal DNA-DNA (dDDH) hybridization were 26.30%, 25.10%, 26.20%, 29.50%, respectively. Therefore, it is concluded that strain TRM63209^T represents a novel species of the genus Streptomyces, for which the name Streptomyces blattelae is proposed. The type strain is TRM63209^T (CCTCC AA 2018093^T?=?LMG 31,403?=?TRM63209^T).

     

<Emphasis Type="Italic">Streptomyces roseoalbus</Emphasis> sp. nov., an actinomycete isolated from soil in Yunnan,China

A strain YIM 33098T (= CCTCC AA001027T = DSM 41831T) was isolated from a forest soil sample collected from Nanning in Guangxi Province, China, in the course of screening for producers of new drug lead compounds. This strain was identified by using a polyphasic approach. The results showed that it should be assigned to the genus Streptomyces. An almost complete 16S rRNA gene sequence of the strain was determined and compared with those of representative Streptomyces species. Strain YIM 33098T was clustered in the same subclade with Streptomyces tendae ATCC19812T and Streptomyces eurythermus ATCC14975T. Similarities of strain YIM 33098T with the two strains were 97.35% and 97.42%, respectively. Based on the phenotypic and genotypic evidence, it is therefore proposed that strain YIM 33098T should be classified in the genus Streptomyces as a new species under the name of Streptomyces nanningensis sp. nov.     

Excision and integration of a self-transmissible replicon of Streptomyces ambofaciens

When Streptomyces ambofaciens OSF was crossed with the plasmid-free Streptomyces lividans TK24, almost all S. lividans exconjugants contained the free 11.1-kb plasmid pOS1. Southern hybridizations showed that pOS1 was derived from the integrated copy of previously recognized plasmid pSAM2 present in strain OSF. A shorter derivative of pOS1 was constructed carrying the tsr gene in a non-essential region, and this pOS7 plasmid was used in transformation experiments with protoplasts of S. ambofaciens ATCC23877 (containing pSAM2 only as an integrated sequence) and S. ambofaciens DSM40697 (devoid of pSAM2-related forms). In both cases, some clones carrying pOS7 in an integrated state were found. Integration into strain ATCC23877 was into the pre-existing integrated copy of pSAM2. In contrast, plasmid pOS7 integrated through specific plasmidic and chromosomal sites into strain DSM40697. Thus it is probable that pSAM2 integrates by interaction between preferred regions of the plasmid and host genomes.     

Identification ofStreptomyces violaceoruber Tü22 genes involved in the biosynthesis of granaticin

A 50 kb region of DNA fromStreptomyces violaceoruber Tü22, containing genes encoding proteins involved in the biosynthesis of granaticin, was isolated. The DNA sequence of a 7.3 kb fragment from this region, located approximately 10 kb from the genes that encode the polyketide synthetase responsible for formation of the benzoisochromane quinone skeleton, revealed five open reading frames (ORF1-ORF5). The deduced amino acid sequence of GraE, encoded by ORF2, shows 60.8% identity (75.2% similarity) to a dTDP-glucose dehydratase (StrE) fromStreptomyces griseus. Cultures ofEscherichia coli containing plasmids with ORF2, on a 2.1 kbBamHI fragment, were able to catalyze the formation of dTDP-4-keto-6-deoxy-d-glucose from dTDP-glucose at 5 times the rate of control cultures, confirming that ORF2 encodes a dTDP-glucose dehydratase. The amino acid sequence encoded by ORF3 (GraD) is 51.4% identical (69.9% similar) to that of StrD, a dTDP-glucose synthase fromStreptomyces griseus. The amino acid sequence encoded by ORF4 shares similarities with proteins that confer resistance to tetracycline and methylenomycin, and is suggested to be involved in transporting granaticin out of the cells by an active efflux mechanism.     

<Emphasis Type="Italic">Acinetobacter soli</Emphasis> sp. nov., isolated from forest soil

A non-motile and rod shaped bacterium, designated strain B1(T), was isolated from forest soil at Mt. Baekwoon, Republic of Korea. Cells were Gram-negative, catalase-positive, and oxidase-negative. The major fatty acids were 9-octadecenoic acid (C(18:1) omega9c; 42%) and hexadecanoic acid (C(16:0); 25.9%) and summed feature 3 (comprising iso-C(15:0) 2-OH and/or C(16:1) omega7c; 10.0%). The DNA G+C content was 44.1 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain B1(T) formed a lineage within the genus Acinetobacter and was closely related to A. baylyi DSM 14961(T) (98.6% sequence similarity), followed by A. baumannii DSM 30007(T) (97.4%), A. calcoaceticus DSM 30006(T) (97.0%) and 3 genomic species (96.8 approximately 7.6%). Phenotypic characteristics, gyrB gene sequence analysis and DNA-DNA relatedness data distinguished strain B1(T) from type strains of A. baylyi, A. baumannii, and A. calcoaceticus. On the basis of the evidence presented in this study, strain B1(T) represents a novel species of the genus Acinetobacter, for which the name Acinetobacter soli sp. nov. is proposed. The type strain is B1(T) (= KCTC 22184(T)= JCM 15062(T)).