The Evolution of RecD Outside of the RecBCD Complex

The common understanding of the function of RecD, as derived predominantly from studies in Escherichia coli, is that RecD is one of three enzymes in the RecBCD double-stranded break repair DNA recombination complex. However, comparative genomics has revealed that many organisms possess a recD gene even though the other members of the complex, recB and recC, are not present. Further, bioinformatic analyses have shown that there is substantial sequence dissimilarity between recD genes associated with recB and recC (recD1), and those that are not associated with recBC (recD2). Deinococcus radiodurans, known for its extraordinary DNA repair capability, is one such organism that does not possess either recB or recC, and yet does possess a recD gene. The recD of D. radiodurans was deleted and this mutant was shown to have a capacity to repair double-stranded DNA breaks equivalent to wild-type. The phylogenetic history of recD was studied using a dataset of 120 recD genes from 91 fully sequenced species. The analysis focused upon the role of gene duplication and functional genomic context in the evolution of recD2, which appears to have undergone numerous independent events resulting in duplicate recD2 genes. The role of RecD as part of the RecBCD complex appears to have a divergence from an earlier ancestral RecD function still preserved in many species including D. radiodurans.     

Extracellular Production of Yeast Protease

The groEL gene of the alkaliphilic Bacillus sp. strain C-125 was cloned in Escherichia coli and sequenced. The groEL gene encoded a polypeptide of 544 amino acids and was preceded by the incomplete groES gene, lacking its 5′-end. The sequence of the derived amino acids was 87.5% identical to that of B. subtilis, 85.4% identical to that of B. stearothemophilus, and 60.9% identical to that of E. coli. The GroEL protein was expressed in E. coli. Purified GroEL protected yeast a-glucosidase from irreversible aggregation at a high temperature and the addition of Mg-ATP was essential for reactivation of the a-glucosidase. The addition of E. coli GroES increased recovery of the enzyme activity, indicating that C-125 GroEL could function in coordination with E. coli GroES.     

Structural Analysis of an Extracellular Polysaccharide of Porodisculus pendulus

The aroL gene, encoding shikimate kinase of Brevibacterium lactofermentum, a coryneform glutamic acid-producing bacterium, was cloned. Recombinant plasmids containing the aroL gene caused elevated levels of shikimate kinase synthesis in B. lactofermentum. It was found that in addition to the aroL gene, the aroB and aroE genes, encoding dehydroquinate synthase and shikimate dehydrogenase, respectively, also existed on these recombinant plasmids, in complementation tests with various Escherichia coli and B. lactofermentum aromatic amino acid auxotrophs. The aroL, aroB and aroE genes of B. lactofermentum are located closely on the cloned DNA fragment, in that order. It was shown that at least these three aro genes form a cluster on the chromosome of B. lactofermentum.     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

Recombination-deficient mutants of Salmonella typhimurium are avirulent and sensitive to the oxidative burst of macrophages

Mutations in the genes recA and recBC were constructed in the virulent Salmonella typhimurium strain 14028s. Both the recA and recBC mutants were attenuated in mice. The mutants were also sensitive to killing by macrophages in vitro. The recombination mutants were no longer macrophage sensitive in a variant line of J774 macrophage-like cells that fail to generate superoxide. This suggests that repair of DNA damage by Salmonella is necessary for full virulence in vivo and that the oxidative burst of phagocytes is one source of such DNA damage.     

Dependence of the Composition of the Avermectin Complex of Streptomyces avermitilis on the Glucose Content in the Medium

The effects of the glucose concentration in the medium and O-methyl-L-threonine resistance on the ratio of components of the avermectin complex produced by Streptomyces avermitilishave been studied. Glucose deficiency increases the ratio of components A and ain the complex, while decreasing that of components 1. A mutation that renders the microorganisms resistant to O-methyl-L-threonine (an analogue of isoleucine) increases the ratio of components a in the complex, while decreasing that of components 1. The distribution of aand b in fractions 1and 2 remains constant: the values of the ratio a/b in the fractions amount, respectively, to 1 : 1 and 2 : 1. The relation of the variations in the composition of the avermectin complex to changes in the carbohydrate metabolism of the producer stain, underlain by availability of the source of carbon, is discussed.     

转CiCHS基因拟南芥的黄酮代谢及抗氧化能力分析

该研究克隆了中间锦鸡儿的查尔酮合成酶基因(CiCHS)并转入野生型拟南芥和tt4突变体,用qRT-PCR检测了转基因拟南芥中内源AtCHS基因的表达量,用分光光度法分析了转基因拟南芥的总黄酮、丙二醛含量及DPPH自由基清除能力,用HPLC法检测了转基因拟南芥的柚皮苷含量。结果显示:(1)转基因拟南芥中,内源AtCHS基因的表达量约为野生型的十分之一,总黄酮含量明显高于野生型;HPLC测得转基因株系中柚皮苷含量高于野生型;紫外照射处理前后转基因拟南芥中丙二醛积累量明显少于野生型。(2)转基因株系提取物对DPPH自由基清除能力显著高于野生型。(3)CiCHS基因互补拟南芥tt4突变体,转基因株系的种皮呈现浅棕色。研究表明,中间锦鸡儿CiCHS基因异源表达后生成了柚皮苷,使转基因植物的抗氧化性增强,部分恢复了tt4突变体的种皮颜色。     

Comparison of lantibiotic gene clusters and encoded proteins

Lantibiotics form a group of modified peptides with unique structures, containing post-translationally modified amino acids such as dehydrated and lanthionine residues. In the gram-positive bacteria that secrete these lantibiotics, the gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized. The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the lantibiotic gene clusters shows that they contain conserved genes that probably encode similar functions.The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all lantibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but they do contain a much larger lanM gene that is the lanC gene homologue. Most lantibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding and ABC transporter likely to be involved in the export of (precursors of) the lantibiotics. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lanK, have been located that code for a two-component regulatory system.Finally, non-homologous genes are found in some lantibiotic gene clusters. The nisl and spal genes encode lipoproteins that are involved in immunity, the pepI gene encodes a membrane-located immunity protein, and epiD encodes an enzyme involved in a post-translational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster.A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanM, LanP, LanR and LanT gene products that may be essential for structure and function. This database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.     

Enhancement of root hydraulic conductivity by methyl jasmonate and the role of calcium and abscisic acid in this process

The role of jasmonic acid in the induction of stomatal closure is well known. However, its role in regulating root hydraulic conductivity (L) has not yet been explored. The objectives of the present research were to evaluate how JA regulates L and how calcium and abscisic acid (ABA) could be involved in such regulation. We found that exogenous methyl jasmonate (MeJA) increased L of Phaseolus vulgaris, Solanum lycopersicum and Arabidopsis thaliana roots. Tomato plants defective in JA biosynthesis had lower values of L than wild‐type plants, and that L was restored by addition of MeJA. The increase of L by MeJA was accompanied by an increase of the phosphorylation state of the aquaporin PIP2. We observed that MeJA addition increased the concentration of cytosolic calcium and that calcium channel blockers inhibited the rise of L caused by MeJA. Treatment with fluoridone, an inhibitor of ABA biosynthesis, partially inhibited the increase of L caused by MeJA, and tomato plants defective in ABA biosynthesis increased their L after application of MeJA. It is concluded that JA enhances L and that this enhancement is linked to calcium and ABA dependent and independent signalling pathways.     

八仙山不同类型松栎林群落主要特征分析

为探究八仙山保护区不同类型森林群落的更新潜力、多样性程度以及稳定性水平,阐明三者间的关系,该文以保护区内油松林、蒙古栎林和油松-栓皮栎混交林(松栎混交林) 3种不同类型天然次生林为对象,调查建群种径级结构和更新潜力,计算不同层次群落多样性,测定M. Godron稳定性,并采用主成分法建立评价模型。结果表明:(1)油松种群径级结构近似正态分布,处于成熟期,但幼苗较少;栓皮栎、蒙古栎以及阔叶杂木的幼苗、幼树较多,更新潜力较大。(2)松栎混交林的乔木层多样性较高,而灌木层多样性最低,油松林的草本层多样性最低;松栎混交林群落总体物种丰富度最低而均匀度最高。(3) M. Godron稳定性表明,蒙古栎林距离稳定点较近,而油松林偏离较远。(4) PCA双序图表明,M. Godron稳定性与种群更新潜力、Alatalo均匀度呈较强正相关,综合排序依次为松栎混交林、蒙古栎林和油松林。综上结果表明,建群种更新潜力和物种均匀度对群落稳定性影响较大,林地管理应注重对幼苗幼树的保护。     

Cladistics of Sunaristes,a genus of harpacticoid copepods associated with hermit crabs

Four species of Sunaristes (Canuellidae) are known to live in association with hermit crabs and exhibit a degree of host specificity. Although hermit crabs are common in many parts of the world, Sunaristes is notably absent from waters of the New World. The phylogeny of Sunaristes is here examined through a cladistic analysis. The reconstructed phologeny indicates that S. inaequalis and S. japonicus form a sister group, and its closest relative is S. tranteri. The sister taxon of this 3-species clade is S. paquri. It is assumed that Sunaristes evolved in the Paleocene on the north shore of the Tethys seaway. The constructed cladogram forms the basis for the analysis of vicariant events that led to the speciation and development of the present pattern of distribution of Sunaristes.     

菘蓝IiEMF基因的克隆与功能分析

该研究以菘蓝(Isatis indigotica Fort.)转录组数据为基础,克隆得到菘蓝EMF基因的cDNA全长,命名为IiEMF。(1)序列分析表明,IiEMF基因开放阅读框长度为1896 bp,编码631个氨基酸。进化树分析表明,菘蓝IiEMF蛋白与甘蓝(Brassica oleracea)EMF蛋白亲缘关系最为接近。(2)实时定量PCR结果显示,IiEMF在菘蓝不同器官中均有表达,且在叶中表达量最高,果实中表达量最低;IiEMF基因在菘蓝抽薹开花过程中叶内的表达量呈先升后降的趋势,并于初花期表达量达到最高后逐渐降低回落;在花/果期IiEMF基因表达量较花蕾中明显降低。(3)成功构建了超表达载体pCAMBIA1300-EMF,经农杆菌介导侵染拟南芥,PCR鉴定表明,有7株为超表达转IiEMF基因植株。(4)表型观察发现,在长日照和短日照条件下,与野生型相比2个转IiEMF基因拟南芥株系的开花时间都明显较早(提前6~10 d),且转IiEMF基因株系的莲座叶数比野生型多10片以上,叶片也比野生型大而肥厚。(5)qRT-PCR检测结果显示,在拟南芥营养生长过程中,过表达IiEMF显著抑制了拟南芥AtAP1、AtCO和AtLFY的表达,而促进了AtFLC的表达;当拟南芥开花时,转基因株系中的AtAP1和AtFLC表达量均高于野生型,AtCO和AtLFY的表达量显著低于野生型。研究表明,过量表达IiEMF基因能够促使拟南芥提前开花,且IiEMF可能是通过影响多种开花途径来共同调节促进拟南芥的早花。     

Genetic and phenotypic characterization of cop1 mutants of Arabidopsis thaliana

Ten Arabidopsis lines that carry recessive mutations in the cop1 (constitutively photomorphogenic) locus have been isolated. These lines define at least four different alleles. All of the mutant lines produce dark-grown seedlings that mimic wild-type seedlings grown in the light. The phenotype of the dark-grown mutant seedlings includes: short hypocotyls, open and enlarged cotyledons, accumulation of anthocyanin, cell-type differentiation and chloroplast-like plastid differentiation in cotyledons. Moreover, in more prolonged dark-growth periods the mutants exhibit true leaf development that parallels that in light-grown siblings. The four mutant alleles represent two types of mutations: three alleles (cop 1-1, cop 1-2, and cop 1-3) have severely affected phenotypes whereas one allele (cop 1-4) has a less severe phenotype. Compared to the severe alleles, the cop 1-4 mutant has slightly longer hypocotyls in dark-grown seedlings and does not accumulate abnormal levels of anthocyanin. The cop1–1/cop1-4 hybrid seedlings are intermediate in many physiological properties under both dark- and light-growth conditions, relative to the two parents. These results may suggest that the extent of residual cop1 gene activity in the mutants dictates the degree to which the aberrant plant phenotype is expressed. Analysis of plants carrying both cop1 and hy, a mutation that results in a deficiency of active phyto-chrome, suggests that the cop1 gene product acts downstream of phytochrome. The differentiation of chloroplasts in the roots of light-grown cop1 plants but not in wild-type plants suggests that the wild-type cop1 gene product also normally plays a role in suppressing chloroplast development in the roots of light-grown plants. To aid the eventual molecular cloning of the cop1 locus, its chromosomal location has been mapped and a molecular marker that is located about 1 centimorgan away from the cop1 locus obtained.     

Gene detection of staphylococcal enterotoxins in production strain of staphylococcin injection and superantigenic activity of rSEK and rSEQ

In China, staphylococcin injection has been commonly used in the combined treatment of cancer to enhance the systemic immune response and reduce the toxicities associated with chemotherapy and radiation therapy. It is claimed that the main active component in the injection is staphylococcal enterotoxin C2 (SEC2). To determine whether other serological types of staphylococcal enterotoxins (SEs) could also be present in the injection products, in this study, the distribution of se genes (from sea to see, from seg to seu) in the one and only production strain of Staphylococcus aureus from one manufacturing company was analyzed by PCR method. In addition, sek and seq genes were cloned from the strain and the corresponding recombinant proteins, rSEK and rSEQ, were expressed in Escherichia coli and purified by affinity chromatography and anion-exchange chromatography. The superantigenic properties of the two recombinant proteins were then measured by MTT method. The PCR results showed that seven se genes are harbored by the production strain. However, sec2 gene was not detected. The results of MTT assay showed that rSEK and rSEQ could elicit strong stimulatory effects on proliferation and cytotoxicity of murine splenocytes in vitro. Overall, the results in this study indicated that one or a plurality of the seven SEs may be present in the related products, and that the two recombinant SEs are promising candidates as immunomodulatory agents for cancer therapy.     

Delineation and phylogeny of Centaurea sect. Acrocentron based on DNA sequences: a restoration of the genus Crocodylium and indirect evidence of introgression

 The Mediterranean group Acrocentron of the genus Centaurea is defined mainly on the basis of pollen type, but also by achene characters and involucral bracts morphology. We have revised the delineation of the group by cladistically comparing the sequences of the ITS spacers of the nuclear ribosomal DNA. Our results confirm that the Acrocentron group is a natural one, with a different delimitation from the commonly accepted. The ITS phylogeny supports that Centaurea sect. Chamaecyanus and sect. Stephanochilus belong to the Acrocentron group and suggests that sect. Chamaecyanus should be merged in sect. Acrocentron as a subsection. Contrary, sect. Aegialophila and sect. Crocodylium form a natural group that cannot be placed in the Acrocentron group and should be considered a different genus. The inclusion of Centaurea crocodylium in Aegialophila makes that the prioritary name for the generic level is Crocodylium; thus, two new nomenclatural combinations are proposed: Crocodylium creticum and Crocodylium pumilum. The groups suggested by the ITS sequences are correlated to the main geographical centers of speciation of Acrocentron. However, support for internal nodes of the tree is extremely poor. The low support within the tree and the absence of correlation between karyology and molecular phylogeny suggest that hybridization has played an important role in the diversification of Acrocentron. Received June 6, 2001; accepted April 2, 2002 Published online: November 7, 2002 Address of the authors: Mònica Font, Teresa Garnatje, Núria Garcia-Jacas, and Alfonso Susanna, Botanic Institute of Barcelona (CSIC-Ajuntament de Barcelona), Av.Muntanyans s.n., E-08038 Barcelona, Spain.     

肉苁蓉寄主梭梭根际土壤微生物种类及群落结构特征

为探索梭梭根际土壤微生物结构特征及其与肉苁蓉寄生的关系,应用磷脂脂肪酸(PLFA)法分析了5—8月份梭梭生长季节的根际土壤微生物种类及群落结构特征,采用湿筛倾注-蔗糖离心法对其根际土壤AM真菌进行了初步分离和鉴定,并分析了肉苁蓉寄生与梭梭根际微生物及环境因子间的相关性。结果表明,5—7月3个月份的梭梭根际土壤微生物磷脂脂肪酸种类及含量均显著高于8月份,总磷脂脂肪酸和AM真菌磷脂脂肪酸以6月份含量最高。梭梭根际土壤共鉴定出AM真菌4属35种,它们分别为球囊霉属(Glomus)22种、无梗囊霉属(Acaulospora)7种、多孢囊霉属(Diversispora)3种和巨孢囊霉属(Gigaspora)3种。其中以黑球囊霉(Glomus melanosporum)和双网无梗囊霉(Acaulospora bireticulata)为优势种群,并且发现了与寄生有关的巨孢囊霉属AM真菌。6月份和8月份的AM真菌孢子数量最多,而5月份的AM真菌孢子数量最低。6月份梭梭根际土壤提取液得到的肉苁蓉种子萌发率(65.94%)和田间接种寄生率(59.19%)均为最高值,而5月份土壤提取液测试得到的肉苁蓉种子萌发率最低。因此,推测梭梭根际AM真菌可能参与了肉苁蓉的寄生过程。相关分析表明梭梭根际土壤微生物种类和数量主要与土壤温湿度和土壤理化性质相关性较大,其中可能与寄生有关的真菌数量与土壤温度呈显著正相关;肉苁蓉寄生率与土壤温度及土壤养分呈显著负相关。研究为解析梭梭根际土壤微生物在肉苁蓉寄生过程中的作用以及指导肉苁蓉人工种植提供参考。     

中国桑科植物属的空间分布及多样性

该研究以FRPS《中国植物志》全文电子版网站、中国在线植物志(eFlora)网站和国家标本资源共享平台(NSII)网站收录的全部中国桑科植物数据为基础,以部分省的植物志以及正式发表的论文为补充,查找每一个桑科植物的具体分布地点(精确到县一级),并采用地理信息系统技术,以县为空间数据的基本单元,以桑科12属的植物为研究对象,制作属的空间分布图,计算空间相似性系数,分析桑科植物各属的空间多样性及其差异。结果表明:(1)中国桑科植物中桑属的分布最广,橙桑属的分布最狭窄。(2)橙桑属与其他属的空间分布相似性系数均较低(0~0.0444),其中橙桑属与见血封喉属和牛筋藤属的相似性系数均为0,表明橙桑属与其他属的分布几乎没有重叠区;榕属与构属和柘属的空间分布相似性系数分别为0.7394和0.6795,表明这3属的空间分布有较多的重叠区;见血封喉属的分布范围较广,从热带到亚热带地区均有。(3)中国桑科植物属的多样化中心(保护区域)集中在热带和亚热带地区,其中波罗蜜属和葎草属的多样化中心均在云南,鹊肾树属的多样化中心在海南,柘属的多样化中心从热带、南亚热带扩大至中亚热带地区;榕属在中国有98个种,多样化中心分布在甘肃东南部、贵州东北部、云南南部、广西西南部、台湾南部和海南西部;桑属(11个种)的多样化中心分布在重庆南部、湖北南部、湖南西北部、贵州中南部、云南东部和广西西部。研究认为,中国桑科植物属的多样化中心各有特点,基于县的空间分布及多样性研究结果能够具体确定中国桑科植物属的最小保护区域;且该研究结果支持贵州地区是桑属植物的分化中心和过渡中心。     

Characterization of the pleiotropic phenotype of an ompR strain of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogen that forms a symbiotic association with the nematode, Steinernema carpocapsae. Xenorhabdus is carried into the insect host by the nematode, is released into the hemolymph and participates in killing the insect. The bacteria grow to high concentrations supporting the development of the nematode in the hemolymph. OmpR is a global regulatory protein involved in the regulation of porin genes, motility, acid tolerance and virulence in several enteric bacteria. To study the role of ompR in the lifecyle of Xenorhabdus, an ompR -minus strain was constructed. The ompR strain produced markedly reduced levels of the porin protein, OpnP and was both hypermotile and exhibited a hyperhemolysis phenotype. Inactivation of flhDC, the master regulator for flagella synthesis, eliminated hemolysin production in the ompR strain, suggesting that ompR regulates hemolysin production via flhDC. The ompR mutant strain was virulent towards insect hosts. However, when nematodes were grown on a mixture of the wild-type and the ompR strain, only the wild-type strain was recovered indicating that ompR is required for competitive symbiotic interaction with the nematode. The role of ompR in the symbiosis between the bacterium and the nematode is under investigation. This revised version was published online in August 2006 with corrections to the Cover Date.