The resolvase protein from the transposon Tn21

Summary The tac promoter was inserted into Tn21 upstream of the tnpR gene and the resultant plasmid was used to generate substantial amounts of resolvase. This protein was purified to homogeneity. The protein was characterized by amino acid sequence studies (which showed that an open-reading frame previously identified by DNA sequencing had been correctly assigned to the tnpR gene) and by molecular weight measurements (which demonstrated that the only active for of the protein in solution was dimeric). Pure Tn21 resolvase catalysed site-specific recombinations between directly repeated res sites from Tn21 or Tn1721 but not from Tn3 nor on inverted res sites from Tn21.     

Evidence for a common mechanism for the insertion of the Tn10 transposon and for the generation of Tn10-stimulated deletions

Summary Mutations in and near the Salmonella typhimurium histidine transport operon were generated by insertion of the translocatable tetracycline-resistance element Tn10. Deletion mutants affecting histidine transport genes were subsequently isolated in several of the Tn10-containing strains. Tn10 insertions in hisJ occurred preferentially at one site, designated site A. This same site was also the preferential endpoint of deletions originating from Tn10 insertions at two neighboring sites. Thus, Tn10 insertion and Tn10-stimulated deletion formation appear to involve a common DNA-recogition step.     

Coupling sequences flanking Tn916 do not determine the affinity of binding of integrase to the transposon ends and adjacent bacterial DNA

Coupling sequences are the 6 bp flanking the conjugative transposon Tn916 and are thought to play a role in determining the frequency of conjugative transposition. The affinity of binding of a chimeric protein, which consisted of maltose binding protein fused to the carboxy-terminal DNA binding domain of Tn916 integrase (Int), to different double-stranded oligonucleotide substrates containing coupling sequences associated with high- and low-frequency conjugative transposition was measured using a competition binding assay. The relative affinity of the chimeric protein was unaffected by the nature of the coupling sequences tested. The same results were obtained when the coupling sequences were placed in a different surrounding sequence context. It therefore appears that the effects of different coupling sequences on the frequency of conjugative transposition are not due simply to differences in Int binding.     

The small envelope protein E is not essential for murine coronavirus replication

The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (Delta E) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The Delta E mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the Delta E mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.     

The bleomycin resistance gene of transposon Tn5 is an excellent marker for transformation of corynebacteria

Summary Corynebacteria are highly sensitive to the glycopeptide antibiotic bleomycin. The bleomycin resistance gene of transposon Tn5 is expressed very efficiently in Brevibacterium lactofermentum. This gene constitutes an excellent marker for selection of transformants of corynebacteria. The bleomycin resistance gene is expressed from the same promoter as the neomycin resistance gene, which is already used as marker in many vectors of corynebacteria. The promoter of the neo-ble cluster is expressed in a variety of Gram-negative and Gram-positive microorganisms and eucaryotic organisms.Offprint requests to: J. F. Martín     

Xis protein binding to the left arm stimulates excision of conjugative transposon Tn916

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.     

Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ

The 6.3 kb Clostridium perfringens transposon Tn 4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RS_A site, and an RS_A-like sequence has been identified upstream of the tnpZ gene. In Escherichia coli , in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RS_A site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens . Site-directed mutagenesis of two bases within the RS_A site resulted in a significant reduction in mobilization frequency, demonstrating that the RS_A site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn 4451 is the first mobilizable but non-self-transmissible transposon to be identified from a Gram-positive bacterium.     

Ribosomal protein S1 is not essential for the trans-translation machinery

In eubacteria, ribosome stalling during protein synthesis is rescued by a tmRNA-derived trans-translation system. Because ribosomal protein S1 specifically binds to tmRNA with high affinity, it is considered to be involved in the trans-translation system. However, the role of S1 in trans-translation is still unclear. To study the function of S1 in the trans-translation system, we constructed an S1-free cell-free translation system. We found that trans-translation proceeded even in the absence of S1. Addition of S1 into the S1-free system did not affect trans-translation efficiency. These results suggest that S1 does not play a role in the trans-translation machinery.     

Extracellular matrix-associated protein Sc1 is not essential for mouse development

Sc1 is an extracellular matrix-associated protein whose function is unknown. During early embryonic development, Sc1 is widely expressed, and from embryonic day 12 (E12), Sc1 is expressed primarily in the developing nervous system. This switch in Sc1 expression at E12 suggests an importance for nervous-system development. To gain insight into Sc1 function, we used gene targeting to inactivate mouse Sc1. The Sc1-null mice showed no obvious deficits in any organs. These mice were born at the expected ratios, were fertile, and had no obvious histological abnormalities, and their long-term survival did not differ from littermate controls. Therefore, the function of Sc1 during development is not critical or, in its absence, is subserved by another protein.     

Arginine-55 in the beta-arm is essential for the activity of DNA-binding protein HU from Bacillus stearothermophilus

DNA-binding protein HU (BstHU) from Bacillus stearothermophilus is a homodimeric protein which binds to DNA in a sequence-nonspecific manner. In order to identify the Arg residues essential for DNA binding, four Arg residues (Arg-53, Arg-55, Arg-58, and Arg-61) within the beta-arm structure were replaced either by Gln, Lys, or Glu residues, and the resulting mutants were characterized with respect to their DNA-binding activity by a filter-binding analysis and surface plasmon resonance analysis. The results indicate that three Arg residues (Arg-55, Arg-58, and Arg-61) play a crucial role in DNA binding as positively charged recognition groups in the order of Arg-55 > Arg-58 > Arg-61 and that these are required to decrease the dissociation rate constant for BstHU-DNA interaction. In contrast, the Arg-53 residue was found to make no contribution to the binding activity of BstHU.     

A tetracycline efflux gene on Bacteroides transposon Tn4400 does not contribute to tetracycline resistance.

Previously, we demonstrated that the Bacteroides transposon Tn4351, which confers tetracycline resistance only on aerobically grown Escherichia coli, carries a gene that codes for a tetracycline-inactivating enzyme (B. S. Speer and A. A. Salyers, J. Bacteriol. 170:1423-1429, 1988). However, Park et al. (B. H. Park, M. Hendricks, M. H. Malamy, F. P. Tally, and S. B. Levy, Antimicrob. Agents Chemother. 31:1739-1743, 1987) showed that E. coli carrying a closely related transposon, Tn4400, exhibits energy-dependent efflux of tetracycline as well as tetracycline-inactivating activity (B. H. Park and S. B. Levy, Antimicrob. Agents Chemother. 32:1797-1800, 1988). This result raised the question of whether efflux or inactivation or a combination of the two was necessary for resistance conferred by both transposons. We showed that cells carrying Tn4351 did not exhibit the clear-cut efflux activity seen with cells carrying Tn4400 but rather exhibited a tetracycline accumulation profile which could be explained solely on the basis of inactivation of tetracycline in the cytoplasm and rapid diffusion of altered tetracycline out of the cell. Additionally, we were able to clone the efflux and tetracycline-modifying genes of Tn4400 separately. The region carrying the efflux gene spanned one of the two regions in which Tn4400 differs from Tn4351. A clone containing the corresponding region of Tn4351 did not exhibit efflux. Thus, it appears that Tn4351 does not have the efflux gene and that efflux makes no contribution to the resistance conferred by Tn4351. The MIC for cells carrying the subclone from Tn4400 that contained only the gene for tetracycline inactivation was the same that for cells carrying both the inactivation and efflux genes. Cells carrying only the gene for tetracycline efflux were tetracycline sensitive. This was true even when the efflux gene was on a high-copy-number plasmid which increased the level of efflux to that associated with the Tcr gene on pBR328. These results indicate that efflux activity does not contribute significantly to the tetracycline resistance conferred by Tn4400.     

The Aspergillus niger annexin, anxC3.1 is constitutively expressed and is not essential for protein secretion

An annexin, anxC3.1, was isolated and characterised from the industrially important filamentous fungus Aspergillus niger. anxC3.1 is a single copy gene encoding a 506 amino acid predicted protein which contains four annexin repeats. Disruption of the anxC3.1 gene did not lead to any visible changes in phenotype, nor in the levels of secreted protein, nor specifically in glucoamylase production, suggesting no major role in secretion. anxC3.1 expression was found to be unaltered under a variety of conditions such as increased secretion, altered nitrogen source, heat shock, and decreased Ca2+ levels, indicating that anxC3.1 is constitutively expressed. This is the first reported functional characterisation of a fungal annexin.     

Mind bomb 1 is essential for generating functional Notch ligands to activate Notch

The Delta-Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism essential for cell fate specification. Mind bomb 1 (Mib1) has been identified as a ubiquitin ligase that promotes the endocytosis of Delta. We now report that mice lacking Mib1 die prior to embryonic day 11.5, with pan-Notch defects in somitogenesis, neurogenesis, vasculogenesis and cardiogenesis. The Mib1-/- embryos exhibit reduced expression of Notch target genes Hes5, Hey1, Hey2 and Heyl, with the loss of N1icd generation. Interestingly, in the Mib1-/- mutants, Dll1 accumulated in the plasma membrane, while it was localized in the cytoplasm near the nucleus in the wild types, indicating that Mib1 is essential for the endocytosis of Notch ligand. In accordance with the pan-Notch defects in Mib1-/- embryos, Mib1 interacts with and regulates all of the Notch ligands, jagged 1 and jagged 2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1 is an essential regulator, but not a potentiator, for generating functional Notch ligands to activate Notch signaling.     

Apicoplast lipoic acid protein ligase B is not essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.     

The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants.

Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence. In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used. An A. tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed. Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers. As expected, deletion of the virD operon led to an avirulent phenotype. The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans. No requirement for virD3 in tumor formation was observed in these assays.