IS1491fromPseudomonas alcaligenesNCIB 9867: Characterization and Distribution amongPseudomonasSpecies

A new insertion sequence, IS1491,has been cloned and sequenced. The 2489-bp IS1491was isolated from aPseudomonas alcaligenesNCIB 9867 (strain P25X) 4.8-kbPstI chromosomal fragment. IS1491is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2. Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21family of insertion sequences, which include two IS elements previously isolated fromP. alcaligenesP25X, IS1474,and IS1475(Yeo, C. C., and Poh, C. L. (1997).FEMS Microbiol. Lett.149,257–263). Transposition assays showed that IS1491transposed at a frequency of approximately 1.4 × 10⁻⁶. Transposition of IS1491into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected. IS1491-like sequences were detected in total DNA ofPseudomonas putidaNCIB 9869 (strain P35X),Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans,andComomonas testosteroniby hybridization with IS1491DNA.     

Isolation and Characterization of Group II Introns from Pseudomonas alcaligenes and Pseudomonas putida

Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P. putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96%. The genome of P. alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP). Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators. These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition. One of the group II introns found in P. alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.     

Sequence of the 50-kb Conjugative Multiresistance Plasmid pRE25 from Enterococcus faecalis RE25

The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.     

肠道病毒ECHO25河南分离株基因特征分析

首次对ECHO25病毒进行分子生物学分析,阐明ECHO25(Entric Cytopathic Human Orphanviruses Type25)病毒河南分离株的分子生物学特征及其与世界其它分离株的基因关系。逆转录-聚合酶链反应(RT-PCR)扩增出VP1蛋白编码基因并进行序列测定,将所测4株ECHO25病毒的VP1序列与GenBank上已发表的ECH-O25病毒VP1区进行同源性比较及遗传进化分析发现:河南省4株ECHO25与标准株JV-4核苷酸同源性为79.2%～80.1%,氨基酸同源性为89.0%～92.4%;河南省4株ECHO25核苷酸同源性为93.0%～99.0%,氨基酸同源性为92.4%～97.5%;HN-01分离株与HN-26分离株高度同源,其核苷酸同源性达99.0%;河南省4株ECHO25同属B1基因亚型。     

Characterization of the Cryptic Plasmids of thePseudomonas alcaligenesType Strain

The species type strain ofPseudomonas alcaligenescontains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively. Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related. pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C. This suggested that pECB3 is especially rich in these bases. Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1. A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kbPseudomonas-specific plasmid isolated fromPseudomonas syringaepv.savastonoi.The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced. Introduction of the putative replicon into theEscherichia coliplasmid pUC19 created a recombinant vector able to replicate in bothE. coliandPseudomonasspp. Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1—a finding supported by sequencing studies. Attempts at plasmid curing were unsuccessful. A phenotypic comparison with a non-plasmid-harboring strain ofP. alcaligenes,based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth. Transformation of the non-harboring strain with pECB1–3, followed by selection on a minimal medium containing benzoate, gave no colonies. The advantages gained by possession of pECB1–3, if any, are at present unknown.     

Molecular Characterization of the Lactococcus lactis LlaKR2I Restriction-Modification System and Effect of an IS982 Element Positioned between the Restriction and Modification Genes

The nucleotide sequence of the plasmid-encoded LlaKR2I restriction-modification (R-M) system of Lactococcus lactis subsp. lactis biovar diacetylactis KR2 was determined. This R-M system comprises divergently transcribed endonuclease (llaKR2IR) and methyltransferase (llaKR2IM) genes; located in the intergenic region is a copy of the insertion element IS982, whose putative transposase gene is codirectionally transcribed with llaKR2IM. The deduced sequence of the LlaKR2I endonuclease shared homology with the type II endonuclease Sau3AI and with the MutH mismatch repair protein, both of which recognize and cleave the sequence 5′ GATC 3′. In addition, M·LlaKR2I displayed homology with the 5-methylcytosine methyltransferase family of proteins, exhibiting greatest identity with M·Sau3AI. Both of these proteins shared notable homology throughout their putative target recognition domains. Furthermore, subclones of the native parental lactococcal plasmid pKR223, which encode M·LlaKR2I, all remained undigested after treatment with Sau3AI despite the presence of multiple 5′ GATC 3′ sites. The combination of these data suggested that the specificity of the LlaKR2I R-M system was likely to be 5′ GATC 3′, with the cytosine residue being modified to 5-methylcytosine. The IS982 element located within the LlaKR2I R-M system contained at its extremities two 16-bp perfect inverted repeats flanked by two 7-bp direct repeats. A perfect extended promoter consensus, which represented the likely original promoter of the llaKR2IR gene, was shown to overlap the direct repeat sequence on the other side of IS982. Specific deletion of IS982 and one of these direct repeats via a PCR strategy indicated that the LlaKR2I R-M determinants do not rely on elements within IS982 for expression and that the efficiency of bacteriophage restriction was not impaired.     

Molecular Characterization of the Klebicin B Plasmid of Klebsiella pneumoniae

The nucleotide sequence of a bacteriocin-encoding plasmid isolated from Klebsiella pneumoniae (pKlebB-K17/80) has been determined. The encoded klebicin B protein is similar in sequence to the DNase pyocins and colicins, suggesting that klebicin B functions as a nonspecific endonuclease. The klebicin gene cluster, as well as the plasmid backbone, is a chimera, with regions similar to those of pore-former colicins, nuclease pyocins and colicins as well as noncolicinogenic plasmids. Similarities between pKlebB plasmid maintenance functions and those of the colicin E1 plasmid suggest that pKlebB is a member of the ColE1 plasmid replication family.     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

A Circular Plasmid from the YeastTorulaspora delbrueckii

A new member of the 2-μm family of plasmids, named pTD1, was found in the yeastTorulaspora delbrueckii,a widespread yeast associated with food. Nucleotide sequences revealed the presence of a pair of inverted repeats and three open reading frames, one of which is a homologue of the FLP recombinase gene of 2-μm plasmid. An ARS region was identified, by replication inSaccharomyces cerevisiaeandT. delbrueckii,near one of the inverted repeats. By the use of pTD1 derivatives and auxotrophic mutant hosts, an efficient host–vector system was established forT. delbrueckii.So far, the 2-μm family of plasmids is restricted to four closely related genera (Q6 group):Saccharomyces, Zygosaccharomyces, Kluyveromyces,andTorulaspora.After a survey of 2500 strains belonging to about 500 species (80 genera) of yeast, no circular plasmids were found in other genera.     

Physical Characterization of Plasmid pMQV10 from a Steroid Biotransforming Strain of Micrococcus

The presence of a single plasmid 8.5 kb in size has been demonstrated in a cholesterol biotransforming strain of Micrococcus. A detailed physical map of the plasmid has been constructed using various restriction enzymes. Streptomycin resistance has been localized on a 1.8-kb fragment of pMQV10.     

拐枣七内生细菌溶磷相关基因的鉴定

为了鉴定拐枣七内生细菌Pseudomonas sp.GZJR-8的pqqE和GDH基因在溶磷方面的功能,该研究通过同源重组技术分别获得pqqE和GDH基因的缺失突变体ΔpqqE和ΔGDH,采用电转法获得它们的互补菌株ΔpqqE(pqqE)和ΔGDH(GDH);难溶性无机磷培养基(PKO)定性检测结果显示,与野生型菌株(WT)相比,ΔpqqE和ΔGDH不能产生溶磷圈,ΔpqqE(pqqE)和ΔGDH(GDH)能够产生溶磷圈;抗坏血酸-钼蓝显色定磷法定量检测结果显示,WT、ΔpqqE、ΔpqqE(pqqE)、ΔGDH、ΔGDH(GDH)产生的有效磷总量分别为1 939.000 mg/L、1 279.000mg/L、1 999.000mg/L、439.000mg/L、2 314.000mg/L,与WT产生的有效磷总量相比,ΔpqqE降低1.52倍,ΔpqqE(pqqE)增加1.03倍,ΔGDH降低4.42倍,ΔGDH(GDH)增加1.19倍;培养液离心后上清的pH测定结果显示,WT、ΔpqqE、ΔpqqE(pqqE)、ΔGDH、ΔGDH(GDH)的pH分别为4.08、4.34、4.03、4.71、4.00,与WT的pH相比,ΔpqqE上升1.06倍,ΔpqqE(pqqE)降低0.27倍,ΔGDH上升1.15倍,ΔGDH(GDH)降低1.02倍。研究表明:pqqE和GDH基因具有溶磷能力,其中ΔpqqE和ΔGDH较WT的溶磷能力下降,但并未完全丧失溶磷能力,而ΔpqqE(pqqE)和ΔGDH(GDH)可以恢复到WT的溶磷能力,拐枣七内生细菌Pseudomonas sp.GZJR-8是通过产生酸性物质来溶磷,在农业生产方面具有潜在应用价值。     

Characterization of Insecticidal Genes of Bacillus thuringiensis Strains Isolated from Arid Environments

This study aimed at characterizing the insecticidal genes of eight Bacillus thuringiensis isolates that were recovered from the local environment of western Saudi Arabia. The screening for the presence of lepidopteran-specific cry1A family and vip3A genes, dipteran-specific cry4 family and coleopteran-specific cry3A, vip1A and vip2A genes, was carried out by PCR. All eight isolates produced PCR products that confirmed the presence of cry1Aa, cry1Ab, cry1Ac, cry4A, cry4B genes, but not cry3A, vip1A and vip2A genes. However, three isolates only were found to carry vip3A genes as revealed by PCR. The observation of cry1 and cry4 genes suggests that these eight isolates may have dual activity against Lepidoptera and Diptera species, while three isolates possessed vip3 genes in addition to cry1 and cry4 which suggests that these three isolates have toxic crystals and vegetative proteins. The results of this study are interesting in the sense that they may help developing new strategies for controlling insects of economic and medical importance in Saudi Arabia, using B. thuringiensis strains that naturally exist in the local environment instead of the current control strategies that are based solely on chemical insecticides.     

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.     

Characterization and Sequence Analysis of the Replicator Region of the Novel Plasmid pALC1 from Paracoccus alcaliphilus

The replicator region of a low-copy-number plasmid, pALC1, of Paracoccus alcaliphilus JCM 7364 was cloned in a form of the minireplicon pALC100 (3.6 kb). The host range of the minireplicon embraces several species of genus Paracoccus, as well as Agrobacterium tumefaciens, Rhizobium leguminosarum, and Rhodobacter sphaeroides (all belonging to alpha-Proteobacteria), but not Escherichia coli. The complete nucleotide sequence of the replicator region (2276 bp) revealed the presence of one complete open reading frame coding for the 28.4-kDa protein (RepA) with similarity to replication proteins of plasmid pSW500 of Erwinia stewartii and pVS1 of Pseudomonas fluorescens. The iteron-like region was identified upstream of the repA gene and consisted of two clusters of repeated sequences (17 bp long) separated by a putative DnaA box. Analysis of the predicted amino acid sequence of two adjacent incomplete ORFs suggests the localization of repA between genes involved in conjugation (traG) and partitioning (parA) within the pALC1 genome.     

盐角草和盐芥3个超氧化物歧化酶基因的克隆和功能分析

为了探究盐生植物的耐盐机制,比较不同盐生植物超氧化物歧化酶(SOD)基因耐盐性的大小,采用RACE技术克隆了盐角草锰和铜/锌两种SOD的cDNA全长。序列分析表明,盐角草MnSOD基因(SeMSD,GenBank登录号为JQ061158)含有699bp的开放阅读框,编码一个长233个氨基酸的多肽,其预测相对分子量为25.7kD。Cu/ZnSOD基因(SeCSD,GenBank登录号为JQ061160)开放阅读框长为684bp,并编码228个氨基酸的多肽,相对分子量为23.3kD。根据已获得的这两个cDNA序列和GenBank公布的盐芥MnSOD基因序列(ThMSD,EF140719),构建了3个原核表达载体pET30a-SeMSD、pET30a-SeCSD和pET30a-ThMSD,并将它们转化大肠杆菌BL21,成功表达出了目的蛋白。通过优化IPTG的诱导浓度,在6.5%和7.5%盐度下对这3个SOD基因的耐盐能力验证结果显示,相比对照菌BL21(pET30a),转化了pET30a-SeMSD和pET30a-ThMSD载体的重组菌具有更高的耐盐性,而转化pET30a-SeCSD载体的重组菌没有表现出耐盐性的提高。     

变铅青链霉菌内源线性质粒接合转移必需功能区的鉴定

通常细菌间环型质粒在接合转移过程中,单链质粒DNA在质粒内部“oriT”接合转移起始位点发生缺刻.随后,打开的单链质粒DNA通过细胞膜的Ⅳ型分泌系统转移到受体菌中.但是,链霉菌中的接合型线型质粒带有游离3′端,5′端与末端蛋白结合,因而不能以细胞-细胞间方式转移单链缺刻DNA.报道了变铅青链霉菌线型质粒SLP2衍生的环型质粒,与SLP2一样可以高频高效接合转移,并鉴定了接合转移功能区.质粒有效的接合转移功能区包含6个共转录的基因,分别编码一个Tra样的DNA转移酶、胞壁水解酶、2个膜蛋白(可以与ATP结合蛋白相互作用)和一个功能未知的蛋白质.从SalⅠR-/M-向SalⅠR/M宿主转移的质粒频率下降表明,线型和环型的质粒都是以双链的形式转移的.上述研究结果表明SLP2衍生的线型质粒和环型质粒以相似的与细胞膜/胞壁功能相关的机理进行接合转移.