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PubMed1995

Gamma-tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts.

We report that in Drosophila, gamma-tubulin is required for the structure as well as the function of microtubule organizing centres (MTOCs). This conclusion is based on the identification and phenotypic characterization of a mutant allele of the gamma-tubulin gene located at region 23C of the polytene chromosome map. This mutation, which we have called gamma-tub23CPl, is caused by the insertion of a P-element within the 5' untranslated leader of the gamma-tubulin transcript. Northern and Western analysis show that gamma-tub23CPl is either a null or a very severe hypomorph as no gamma-tubulin mRNA or protein can be detected in mutant individuals. Visualization of DNA, MTOCs and microtubules by confocal laser scanning microscopy of cells from individuals homozygous for gamma-tub23CPl reveals a series of phenotypic abnormalities. Some of these are similar to those observed after disruption of gamma-tubulin function in other organisms, including mitotic arrest and a dramatic decrease in the number of microtubules, but, in addition, we have observed that mutation in this gene also results in highly abnormal MTOCs which show a variety of shapes and sizes which we never observed in wild type cells. These results show that gamma-tubulin is required for both structural and functional roles in the MTOCs.

Animals↗
PubMed2001

Comprehensive identification of conditionally essential genes in mycobacteria.

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

Base Sequence↗
PubMed1995

Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.

The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.

Bacteriophage mu↗
PubMed2001

Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance.

The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance.

Amino Acid Sequence↗
PubMed1989

Mutagenesis of the Tra1 core region of RK2 by using Tn5: identification of plasmid-specific transfer genes.

The conjugation system of the IncP alpha plasmid RK2/RP4 is encoded by transfer regions designated Tra1, Tra2, and Tra3. The Tra1 core region, cloned on plasmid pDG4 delta 22, consists of the origin of transfer (oriT) and 2.6 kilobases of flanking DNA providing IncP alpha plasmid-specific functions that allow pDG4 delta 22 to be mobilized by the heterologous IncP beta plasmid R751. Tn5 insertions in pDG4 delta 22 define a minimal 2.2-kilobase region required for plasmid-specific transfer of oriT. The Tra1 core contains the traJ and traK genes as well as an 18-kilodalton open reading frame downstream of traJ. The traJ and traK genes were shown to be required for transfer by complementation of inserts within these genes. Genetic evidence for the role of the 18-kilodalton open reading frame in transfer was obtained, although this protein has not been detected in cell lysates. These studies indicate that at least three transfer proteins are involved in plasmid-specific interactions at oriT.

Bacterial Proteins↗
PubMed2004

Critical variations of conjugational DNA transfer into secondary metabolite multiproducing Sorangium cellulosum strains So ce12 and So ce56: development of a mariner-based transposon mutagenesis system.

Myxobacteria increasingly gain attention as a source of bioactive natural products. The genus Sorangium produces almost half of the secondary metabolites isolated from these microorganisms. Nevertheless, genetic systems for Sorangium strains are poorly developed, which makes the identification of the genes directing natural product biosynthesis difficult. Using biparental and triparental mating, we have developed methodologies for DNA transfer from Escherichia coli via conjugation for the genome sequencing model strain So ce56 and the secondary metabolite multiproducing strain So ce12. The conjugation protocol developed for strain So ce56 is not applicable to other Sorangium strains. Crucial points for the conjugation are the ratio of E. coli and Sorangium cellulosum cells, the choice of liquid or solid medium, the time used for the conjugation process and antibiotic selection in liquid medium prior to the plating of cells. A mariner-based transposon containing a hygromycin resistance gene was generated and used as the selectable marker for S. cellulosum. The transposon randomly integrates into the chromosome of both strains. As a proof of principle, S. cellulosum So ce12 transposon mutants were screened using an overlay assay to target the chivosazole biosynthetic gene cluster.

Conjugation, Genetic↗
PubMed2005

Functional comparison of the transposition core machineries of phage Mu and Haemophilus influenzae Mu-like prophage Hin-Mu reveals interchangeable components.

Bacteriophage Mu uses DNA transposition for propagation and is a model for transposition studies in general. Recent identification of Mu-like prophages within bacterial genomes offers new material for evolutionary and comparative functional studies. One such prophage, Hin-Mu of Haemophilus influenzae Rd, was studied for its transpositional properties. The components of its transposition core machinery, the encoded transposase (MuA(Hin)) and the transposase binding sites, were evaluated for functional properties by sequence comparisons and DNase I footprinting. Transpositional activity of Hin-Mu was examined by in vitro assays directly assessing the assembly and catalytic function of the transposition core machinery. The Hin-Mu components readily assembled catalytically competent protein-DNA complexes, transpososomes. Thus, Hin-Mu encodes a functional transposase and contains critical transposase binding sites. Despite marked sequence differences, components of the Hin-Mu and Mu transposition core machineries are partially interchangeable, reflecting both conservation and flexibility in the functionally important regions within the transpososome structure.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of the linear IS3 molecules generated by staggered breaks.

Insertion sequences IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB. The OrfAB transframe protein that is IS3 transposase is produced by -1 translational frameshifting between orfA and orfB. Efficient production of the IS3 transposase in the cells harboring the IS3-carrying plasmid has been shown to generate miniplasmids as well as characteristic minicircles, called IS3 circles, consisting of the entire IS3 sequence and one of the 3-base pair sequences flanking IS3 in the parental plasmid. Here, we show that the IS3 transposase also generates the linear molecules of IS3 with 3-nucleotide overhangs at the 5'-ends. The nucleotide sequences of the overhangs are the same as those flanking IS3 in the parental plasmid, suggesting that the linear IS3 molecules are generated from the parental plasmid DNA by staggered double strand breaks at the end regions of IS3. The linear IS3 molecules are likely to be the early intermediates in the transposition reaction, which proceeds in a non-replicative manner.

Base Sequence↗
PubMed1989

Identification, molecular cloning and sequence analysis of a gene cluster encoding the class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli.

To investigate a possible chromosomal clustering of glycolytic enzyme genes, the complete nucleotide sequence of the 8029 bp insert of Escherichia coli DNA in the ColE1 plasmid pLC33-5 of the Clarke and Carbon collection (Clark and Carbon, 1976) was determined. Genes (pgk, fda) encoding the phosphoglycerate kinase and Class II fructose 1,6-bisphosphate aldolase, respectively, of E. coli were identified. The phosphoglycerate kinase was found to be highly homologous in primary structure to the same enzyme from eukaryotic organisms. A further large open reading frame, designated gapB, was also identified, which on the basis of sequence homology, appears to encode another glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. This putative gene differs significantly from that (designated gapA) already identified as coding for this enzyme in E. coli and which maps elsewhere on the chromosome. The products, if any, of several other open reading frames remain to be identified.

Amino Acid Sequence↗
PubMed1987

Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa.

The phospholipase C (PLC) gene of Pseudomonas aeruginosa encodes a heat-labile secreted hemolysin which is part of a Pi-regulated operon. The structural gene for PLC, plcS, was mutated in vitro by insertion of a tetracycline resistance gene cartridge. Gene replacement techniques were used to introduce the mutated plcS gene into the P. aeruginosa chromosome in place of the wild-type gene. The precise replacement of wild-type sequences by mutant sequences was confirmed by Southern hybridization. The mutant strain, designated PLC S, is nonhemolytic and lacks a 78-kilodalton protein corresponding to the size of the wild-type PLC. However, there is an additional phospholipase activity present in PLC S capable of hydrolyzing p-nitrophenylphosphorylcholine, a synthetic PLC substrate, and phosphatidylcholine. This enzymatic activity is not a result of a truncated product produced from the mutated plcS gene. The phospholipase activity of PLC S was identified as a nonhemolytic PLC.

Chromatography, Thin Layer↗
PubMed2003

Identification and distribution of insertion sequences of Paracoccus solventivorans.

Three novel insertion sequences (ISs) (ISPso1, ISPso2, and ISPso3) of the soil bacterium Paracoccus solventivorans DSM 11592 were identified by transposition into entrapment vector pMEC1. ISPso1 (1,400 bp) carries one large open reading frame (ORF) encoding a putative basic protein (with a DDE motif conserved among transposases [Tnps] of elements belonging to the IS256 family) with the highest levels of similarity with the hypothetical Tnps of Rhodospirillum rubrum and Sphingopyxis macrogoltabida. ISPso2 (832 bp) appeared to be closely related to ISPpa2 of Paracoccus pantotrophus DSM 11072 and IS1248 of Paracoccus denitrificans PdX22, both of which belong to the IS427 group (IS5 family). These elements contain two overlapping ORFs and a putative frameshift motif (AAAAG) responsible for production of a putative transframe Tnp. ISPso3 (1,286 bp) contains a single ORF, whose putative product showed homology with Tnps of ISs classified as members of a distinct subgroup of the IS5 group of the IS5 family. The highest levels of similarity were observed with ISSsp126 of Sphingomonas sp. and IS1169 of Bacteroides fragilis. Analysis of the distribution of ISs of P. solventivorans revealed that ISPso2-like elements are the most widely spread of the elements in nine species of the genus PARACOCCUS: ISPso1 and ISPso3 are present in only a few paracoccal strains, which suggests that they were acquired by lateral transfer. Phylogenetic analysis of Tnps of the novel ISs and their closest relatives showed their evolutionary relationships and possible directions of lateral transfer between various bacterial hosts.

Amino Acid Sequence↗
PubMed2001

A SNP resource for human chromosome 22: extracting dense clusters of SNPs from the genomic sequence.

The recent publication of the complete sequence of human chromosome 22 provides a platform from which to investigate genomic sequence variation. We report the identification and characterization of 12,267 potential variants (SNPs and other small insertions/deletions) of human chromosome 22, discovered in the overlaps of 460 clones used for the chromosome sequencing. We found, on average, 1 potential variant every 1.07 kb and approximately 18% of the potential variants involve insertions/deletions. The SNPs have been positioned both relative to each other, and to genes, predicted genes, repeat sequences, other genetic markers, and the 2730 SNPs previously identified on the chromosome. A subset of the SNPs were verified experimentally using either PCR-RFLP or genomic Invader assays. These experiments confirmed 92% of the potential variants in a panel of 92 individuals. [Details of the SNPs and RFLP assays can be found at http://www.sanger.ac.uk and in dbSNP.]

Base Composition↗
PubMed1994

Identification of mutations in DNA polymerase beta mRNAs from patients with Werner syndrome.

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by prematurely aged appearance. Genetic linkage analysis has placed the relevant gene in subchromosomal band 8p12. DNA polymerase beta gene has been mapped to chromosome 8p12-11 and thought to be involved in DNA repair and possibly in recombination. Somatic cells from WS patients exhibit chromosomal instability, a markedly reduced replicative life span and slow growth. The functions of DNA polymerase beta gene and its position prompted us to examine this gene in WS patients. We have found the novel DNA polymerase beta cDNA species in blood samples from WS patients, which contain 107 bp insertions or 87 bp deletions in the catalytic domain of DNA polymerase beta. These mutations change the structure of DNA polymerase beta and thus the capacity of the DNA repair system would be impaired, which may account for the high mutation rate observed in WS.

Amino Acid Sequence↗
PubMed1999

Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis.

Herpesviruses are important pathogens in animals and humans. The large DNA genomes of several herpesviruses have been sequenced, but the function of the majority of putative genes is elusive. Determining which genes are essential for their replication is important for identifying potential chemotherapy targets, designing herpesvirus vectors, and generating attenuated vaccines. For this purpose, we recently reported that herpesvirus genomes can be maintained as infectious bacterial artificial chromosomes (BAC) in Escherichia coli. Here we describe a one-step procedure for random-insertion mutagenesis of a herpesvirus BAC using a Tn1721-based transposon system. Transposon insertion sites were determined by direct sequencing, and infectious virus was recovered by transfecting cultured cells with the mutant genomes. Lethal mutations were rescued by cotransfecting cells containing noninfectious genomes with the corresponding wild-type subgenomic fragments. We also constructed revertant genomes by allelic exchange in bacteria. These methods, which are generally applicable to any cloned herpesvirus genome, will facilitate analysis of gene function for this virus family.

Base Sequence↗
PubMed1994

Identification and characterization of genes involved in excision of the Lactococcus lactis conjugative transposon Tn5276.

The 70-kb transposon Tn5276, originally detected in Lactococcus lactis NIZO R5 and carrying the genes for nisin production and sucrose fermentation, can be conjugally transferred to other L. lactis strains. Sequence analysis and complementation studies showed that the right end of Tn5276 contains two genes, designated xis and int, which are involved in excision. The 379-amino-acid int gene product shows high (up to 50%) similarity with various integrases, including that of the Tn916-related conjugative transposons. The xis gene product, like almost all known excisionase (Xis) proteins, is a small (68-residue), basic protein. Expression of both the Tn5276 int and xis genes is required for efficient excision of the ends of Tn5276 in Escherichia coli that appeared to be circularized in the excision process. Mutational analysis of the xis and int genes showed that excision efficiency is dependent on the integrity of the int gene but that an intact xis gene is also required for efficient excision.

Amino Acid Sequence↗
PubMed2004

[Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene].

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. The inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kdul-kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.

Base Sequence↗
PubMed1998

Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti.

Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be up-regulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB- mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.

Base Sequence↗
PubMed2004

Identification of Burkholderia cenocepacia genes required for bacterial survival in vivo.

Burkholderia cenocepacia (formerly Burkholderia cepacia complex genomovar III) causes chronic lung infections in patients with cystic fibrosis. In this work, we used a modified signature-tagged mutagenesis (STM) strategy for the isolation of B. cenocepacia mutants that cannot survive in vivo. Thirty-seven specialized plasposons, each carrying a unique oligonucleotide tag signature, were constructed and used to examine the survival of 2,627 B. cenocepacia transposon mutants, arranged in pools of 37 unique mutants, after a 10-day lung infection in rats by using the agar bead model. The recovered mutants were screened by real-time PCR, resulting in the identification of 260 mutants which presumably did not survive within the lungs. These mutants were repooled into smaller pools, and the infections were repeated. After a second screen, we isolated 102 mutants unable to survive in the rat model. The location of the transposon in each of these mutants was mapped within the B. cenocepacia chromosomes. We identified mutations in genes involved in cellular metabolism, global regulation, DNA replication and repair, and those encoding bacterial surface structures, including transmembrane proteins and cell surface polysaccharides. Also, we found 18 genes of unknown function, which are conserved in other bacteria. A subset of 12 representative mutants that were individually examined using the rat model in competition with the wild-type strain displayed reduced survival, confirming the predictive value of our STM screen. This study provides a blueprint to investigate at the molecular level the basis for survival and persistence of B. cenocepacia within the airways.

Animals↗