An Mr 29000 protein is essential for mini-F maintenance in E. coli

Plasmids consisting of mini-F inserted into multicopy vectors were constructed. Derivatives of these hybrid replicons were isolated which contained the transposon Tn5. The polypeptides encoded by these plasmids were identified by Escherichia coli minicell analysis. We show that a previously unidentified polypeptide of 29000 Mr is encoded by the mini-F gene E between 45.1 and 46.2 F kb on the mini-F plasmid map, and that this coding sequence (E gene) is transcribed rightward. Hybrid plasmids carrying Tn5 inserted into the E gene are unable to replicate in a polA- strain. Hence the E protein is essential for mini-F replication. Mutations in the A and B genes of mini-F affect E gene expression, and the results suggest that E protein synthesis is stimulated by A protein.     

The purB gene controls rhizosphere colonization by Pantoea agglomerans

Aims: To isolate the rhizosphere competence‐defective transposon Tn5 mutant of Pantoea agglomerans NBRISRM (SRM) and to identify the gene causing defect in its root colonization ability. Methods and Results: From over 5000 clones containing Tn5, one mutant P. agglomerans NBRISRMT (SRMT) showing 6 log units less colonization when compared with SRM, after 30 days in sand‐nonsterilized soil assay system was selected for further work to determine the effects of the mutation on rhizosphere competence. Southern hybridization analysis of restricted genomic DNA of SRMT demonstrated that the mutant had a single Tn5 insert. SRM increased in titre to about 2 × 10⁸ CFU g^?1 root, compared with the indigenous bacterial population of heterotrophs of about 5 × 10⁷ CFU g^?1 root. In contrast, 30 days later, the titre value of SRMT was almost undetectable at 1 × 10² CFU g^?1 root, demonstrating its inability to survive and colonize the rhizosphere. Sequencing of the flanking region of the Tn5 mutant revealed that Tn5 disrupted the purB gene. Conclusions: A defect in the colonization phenotype of the SRMT was attributed to the disruption in adenylosuccinate lyase (EC 4.3.2.2) which is encoded by the pur B gene and is required for rhizosphere colonization in P. agglomerans. Significantly less exopolysaccharide and biofilm was formed by SRMT when compared to SRM, because of the disruption of the purB gene. Significance and Impact of the Study: This work provides the first evidence for a functional role of purB gene in rhizosphere competence and root colonization by any rhizobacteria.     

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.     

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.     

Further characterization of the binding properties of two monoclonal antibodies recognizing human Tn red blood cells

The terminal alpha anomeric Ga1NAc residue is an essential sugar for the Tn glycotope, human blood group A determinant, and Forssman antigen. In a previous study [King M.J., Parson S.F., Wu A,M., Jones N., Transfusion 31: 142-149, 1991] we defined two monoclonal antibodies (MoAbs, BRIC66 and BRIC111) reacting with human Tn red blood cells. However, more advanced studies of these two MoAbs were hampered by the lack of availability of Gal/GalNAc related glycotopes. In order to use these antibodies as powerful probes to elucidate structural changes during life processes, we have characterized in detail the combining sites of these two MoAbs using enzyme-linked immunosorbent (ELISA) and inhibition assays with an extended glycan/ligand collection. From the results, it has been established that BRIC66 demonstrated multiple specificities and its reactivity towards glycotopes was defined as: Ga1NAc alpha1-->Ser/Thr (Tn) > or = Ga1NAc alpha1-->3(LFuc alpha1-->2)Gal (Ah) > Ga1NAcalpha1-->3Galbeta1-->4Glc (AL) > Ga1NAalpha1-->3Gal (A) GalNAc alpha1-->3GalNAc > Gal or Glc. Another MoAb, BRIC111, mainly bound Tn-glycophorin. The best ligand for this MoAb was Tn-containing glycopeptides (M.W. < 3.0 x 10(3) Da) from asialo ovine salivary mucin (OSM), which was approximately 70 and 58 times more active than Ga1NAc and monomeric Ga1NAc alpha1-->Ser/Thr (Tn), respectively, suggesting that the active glycotopes present in glycophorin for BRIC111 binding also exist in OSM. The N-acetyl group at carbon-2 and configuration at carbon-2 and carbon-4 of the alpha anomeric Ga1NAc are required for the binding of either MoAb. Identification of these binding properties should aid in the selection of these MoAbs and the conditions required for biological studies and clinical applications.     

Selection of Tn5::lacZ mutants isogenic to wild type Azospirillum brasilense strains capable of growing at sub-optimal temperature

Azospirillum brasilense strains, CDJA and A40, capable of growing at sub-optimal temperature were tagged with stable chromogenic marker Tn5-lacZ. Mutants were screened for plant growth promoting activities at 20, 22, 25, 30 and 37 °C. Mutants MC48 and MA3 were found to fix nitrogen upto 85% and produced indole acetic acid (IAA) and siderophore in isogenic manner to their respective wild type strains, CDJA and A40, at sub-optimal temperatures. Co-inoculation of mutants with their respective parent (1:1 ratio) to the wheat revealed that colonization potential of the mutants was affected greatly. Tn5-lacZ tagged mutants MC48 and MA3 were found isogenic to their respective wild type Azospirillum strain, with regards to plant growth promoting activities and root colonization ability. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dual modes of CRISPR-associated transposon homing

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AChE在Tn~+人白血病Jurkat细胞中的表达状况研究

通过研究Tn+人白血病Jurkat细胞中ACh E的表达状况,可以分析Jurkat细胞ACh E表达与Tn抗原表达水平的关系,从而为ACh E用于肿瘤的预后判断或临床治疗提供实验依据。本论文利用ELISA、色度法试验在蛋白水平分析了ACh E的表达状况,利用RT-PCR以及Real-time PCR试验在m RNA水平分析了ACh E的表达状况。结果显示:Jurkat细胞中ACh E的含量及活性明显低于Tn-的白血病细胞K562,且二者又均低于正常白细胞。由此认为,人白血病细胞中ACh E的含量及活性低于正常细胞,ACh E的表达与Tn抗原的表达水平呈负相关,为肿瘤的临床判断提供了有力的依据。     

Photorhabdus luminescens LN2转座突变菌株的研究

发光杆菌属Photorhabdus细菌与异小杆属Heterorhabditis昆虫病原线虫的共生关系是这类生物杀虫剂产业化生产和田间应用的基础。本文采用Tn5转座方法构建了共生细菌P. luminescens LN2突变体库;从中筛选出一个对其共生线虫H. indica LN2的生长繁殖有显著促进作用的突变菌株（LN2-M2716）;测定了该突变菌株的菌落特征、对大蜡螟Galleria mellonella及非特异共生线虫H. bacteriophora H06的毒性、对线虫产量的影响。结果显示,LN2-M2716菌株在菌落形态、色素分泌、过氧化氢酶反应、荧光、食物信息作用以及对大蜡螟毒力等方面与野生型菌株差异不明显;但对非特异共生线虫H. bacteriophora H06的毒性及对特异共生线虫H. indica LN2生长繁殖的促进作用方面均明显高于野生型菌株。论文结果为构建支持线虫高产的菌株提供了关键技术。