Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

Summary A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.     

Structural interpretation of the two-site binding of troponin on the muscle thin filament

Conditions are described for the quantitative removal of amino acid residues 274 to 284 from rabbit muscle α-tropomyosin with carboxypeptidase A. The product, non-polymerizable tropomyosin, has a much reduced affinity for the tropomyosinbinding fragment CB1 (residues 1 to 151) of troponin-T. Iodination of α-tropomyosin and non-polymerizable tropomyosin by ¹²⁵I and lactoperoxidase was carried out in the presence and absence of CB1. Following tryptic digestion and peptide mapping, the radioactivities of the labeled tyrosine peptides were compared. In the presence of CB1, tyrosine residues 261 and 267 were iodinated only to the extent of 30 to 40% as compared with the same tyrosine residues in the absence of CB1, All other tyrosine residues (60, 162, 214 and 221) were iodinated to a similar level in the absence or presence of CB1. With non-polymerizable tropomyosin, the presence of CB1 had a much reduced effect on the level of labeling of the tyrosine residues. We conclude that the highly helical region of troponin-T (residues 71 to 151) binds close to or at the COOH-terminal end of the tropomyosin molecule. Taken together with other considerations and recent observations, the results can be interpreted in terms of the two-site model for troponin attachment to the thin filament. A calcium-insensitive site would involve interaction of the highly helical CB2 region of troponin-T (residues 71 to 151) and the COOH-terminal region of tropomyosin (residues 258 to 284) and perhaps the NH₂-terminal overlap region (residues 1 to 9). A calcium-sensitive site would involve the interaction of troponin-T in the neighborhood of cysteine 190 of tropomyosin in F-actin-tropomyosin assemblies both directly and indirectly through the association of its COOH and NH₂-terminal regions with the troponin-I and C components.     

Absence of direct repeats flanking transposons resulting from intramolecular transposition

Tn2660 is an ampicillin-resistance-conferring transposon with a high degree of homology for the transposon Tn3. The nucleotide sequences flanking the termini of Tn2660 have been determined on plasmids inferred to have resulted from both inter- and intramolecular transposition of Tn2660. In all cases, transposition of Tn2660, as of Tn3, creates 5-bp flanking direct repeats, except following intramolecular transposition resulting from trans ligation. In this case, in R6K replicons, the nucleotide sequence between the two Tn2660 elements is stably inverted from the normal orientation, and 5-bp direct repeats do not flank each transposon, but instead flank opposite ends of the two transposon copies.     

玉米联合固氮菌Kosakonia radicincitans GXGL-4A转座突变体系的构建

【背景】长期以来,农业生产上过度施用化学氮肥造成了农田生态环境的破坏,引起土壤板结、次生盐渍化和重金属污染。此外,由于田间大量的氮素流失和淋溶导致水体富营养化和地下水污染,使得农产品硝酸盐含量超标,最终可能通过食物链危及人类的健康。因此,通过合理地开发和利用生物固氮菌,从而减少化学氮肥施用量,对于保护生态环境,促进农业的可持续生产具有十分重要的意义。【目的】对从玉米根部分离得到的联合固氮菌Kosakonia radicincitans GXGL-4A进行Tn5转座突变,从而创制大量的突变体,经筛选后应用于对固氮及其调控的分子机制和氮代谢调控网络解析等研究。【方法】以固氮菌K.radicincitans GXGL-4A为研究对象,通过PCR法克隆得到GXGL-4A菌株的亚硝酸还原酶基因nirBD。通过构建重组质粒pMOD-egfp-tet,并利用电击转化法将转座复合体导入GXGL-4A野生株,进行Tn5转座突变,从而获得大量突变菌株。【结果】筛选到4株亚硝酸盐还原酶活性显著降低的突变株,并克隆了突变株M36突变位点的侧翼序列。【结论】对玉米联合固氮菌K.radicincitans GXGL-4A进行Tn5转座突变是可行的,初步建立了该菌株稳定有效的插入突变技术体系。     

Analysis of transposable elements inserted in the genomes of bacteriophages Mu and P1.

We have examined the genomes of the temperate bacteriophages Mu and P1 and some of their insertion mutants for hybridization with the prokaryotic transposable elements IS1 and IS2. We used the DNA blotting-hybridization technique in which denatured DNA fragments are transferred to nitrocellulose paper directly from agarose gels and hybridized to 32P-labeled probe DNA. The 800 base pair insertion in an X mutant of Mu was found to hybridize with IS1. The chloramphenicol resistance transposon, Tn9, in Mu X cam mutants was found to be located at or close to the sites of IS1 insertion in X mutants; Tn9 also hybridized with IS1. The restriction endonuclease BalI cleaved IS1 once; it cleaved Tn9 in all Mu X cam mutants twice to release a fragment of about 1700 base pairs. These results support the conclusion that Tn9 contains one copy of IS1 at each end. In the P1cam isolate, from which Tn9 was transposed to Mu, BalI made a third cut in Tn9 giving rise to fragments of about 850 base pairs. The data further suggested that Tn9 is present in tandem copies in the P1cam isolate we examined. P1 itself was found to harbor IS1. The two P1 strains tested had a common fragment containing IS1; one strain had an additional copy of IS1. The IS1 element common to the P1 strains was shown to be the site of the Tn9 insertion in the P1cam isolate examined. No hybridization between IS2 and any of the Mu and P1 strains could be detected.     

黏蛋白型O-聚糖：结构、功能及与肿瘤的相关性

黏蛋白是细胞表面的或分泌的、具有高度O-糖基化修饰的糖蛋白。黏蛋白型O-聚糖是由多肽：N-乙酰氨基半乳糖转移酶（ppGalNAc-T）家族催化起始合成的,在肿瘤中常常伴随着黏蛋白型O-聚糖结构和数量上的改变,形成肿瘤特异聚糖结构（cancer-associated glycans）,如肿瘤Tn和T抗原等。肿瘤特异聚糖使肿瘤细胞的抗原性和黏附能力发生改变,促进肿瘤细胞的恶性增生与转移。而这些肿瘤特异聚糖结构,也为肿瘤的诊断与抗肿瘤药物或疫苗开发提供了理论基础。     

Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET

To obtain information on Ca(2+)-induced tropomyosin (Tm) movement in Ca(2+)-regulated muscle thin filaments, frequency-domain fluorescence energy transfer data were collected between 5-(2-iodoacetyl-amino-ethyl-amino)naphthalene-1-sulfonic acid at Cys-190 of Tm and phalloidin-tetramethylrhodamine B isothiocyanate bound to F-actin. Two models were used to fit the experimental data: an atomic coordinate (AC) model coupled with a search algorithm that varies the position and orientation of Tm on F-actin, and a double Gaussian distance distribution (DD) model. The AC model showed that little or no change in transfer efficiency is to be expected between different sites on F-actin and Tm if Ca(2+) causes azimuthal movement of Tm of the magnitude suggested by structural data (C. Xu, R. Craig, L. Tobacman, R. Horowitz, and W. Lehman. 1999. Biophys. J. 77:985-992). However, Ca(2+) produced a small but significant change in our phase/modulation versus frequency data, showing that changes in lifetime decay can be detected even when a change of the steady-state transfer efficiency is very small. A change in Tm azimuthal position of 17 on the actin filament obtained with the AC model indicates that solution data are in reasonable agreement with EM image reconstruction data. In addition, the data indicate that Tm also appears to rotate about its axis, resulting in a rolling motion over the F-actin surface. The DD model showed that the distance from one of the two chains of Tm to F-actin was mainly affected, further verifying that Ca(2+) causes Tm to roll over the F-actin surface. The width of the distance distributions indicated that the position of Tm in absence and in presence of Ca(2+) is well defined with appreciable local flexibility.