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PubMed1994

Hypertransposing derivatives of the streptomycete insertion sequence IS493.

Transposons derived from the Streptomyces lividans insertion sequence IS493 are useful for the genetic analysis and manipulation of a number of Streptomyces spp. Tn5099-10, an IS493 derivative that contains a spontaneous deletion terminating in the left inverted repeat (IR-L), transposed at a 1000-fold higher frequency in Streptomyces griseofuscus, and at a tenfold higher frequency in Streptomyces fradiae, than the IS493 derivatives, Tn5096 and Tn5099. The IR-L from Tn5099-10 was used to construct a cassette which hypertransposes from plasmids containing the transposon genes, ORFA and ORFB, outside of the inverted repeats. The target sequences of two Tn5099-10 insertions conformed to the consensus target sequence of the other IS493 derivatives, gNCaNTgNNy (where lower-case letters indicate that other nt have been observed at this position and N is any nt). Transposition mutant libraries of S. griseofuscus and S. fradiae can be easily prepared in broth culture by using the hypertransposing elements and a temperature-sensitive delivery plasmid.

Base Sequence↗

PubMed1991

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.

Bacteriophage lambda↗

PubMed1985

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.

Cloning, Molecular↗

PubMed1995

Cloning and characterization of the genes encoding the hemolysin of Haemophilus ducreyi.

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi. In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35,000-1, was further characterized. Sequences flanking the Tn916 element in strain 35,000-1 were employed to identify clones from a lambda DASHII library of H. ducreyi strain 35,000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35,000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB, were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens.

Amino Acid Sequence↗

PubMed2004

Assaying gene function by growth competition experiment.

High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

Animals↗

PubMed1991

Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor sigma B of Bacillus subtilis.

We describe a general, in vivo method for identifying Bacillus subtilis genes controlled by specific, nonessential regulatory factors. We establish the use of this approach by identifying, isolating, and characterizing a gene dependent on sigma B, an alternate transcription factor which is found early in stationary phase but which is not essential for sporulation. The method relies on two features: (i) a plate transformation technique to introduce a null mutation into the regulatory gene of interest and (ii) random transcriptional fusions to a reporter gene to monitor gene expression in the presence and absence of a functional regulatory product. We applied this genetic approach to isolate genes comprising the sigma B regulon. We screened a random Tn917lacZ library for fusions that required an intact sigma B structural gene (sigB) for greatest expression, converting the library strains from wild-type sigB+ to sigB delta::cat directly on plates selective for chloramphenicol resistance. We isolated one such fusion, csbA::Tn917lacZ (csb for controlled by sigma B), which mapped between hisA and degSU on the B. subtilis chromosome. We cloned the region surrounding the insertion, identified the csbA reading frame containing the transposon, and found that this frame encoded a predicted 76-residue product which was extremely hydrophobic and highly basic. Primer extension and promoter activity experiments identified a sigma B-dependent promoter 83 bp upstream of the csbA coding sequence. A weaker, tandem, sigma A-like promoter was likewise identified 28 bp upstream of csbA. The csbA fusion was maximally expressed during early stationary phase in cells grown in Luria broth containing 5% glucose and 0.2% glutamine. This timing of expression and medium dependence were very similar to those for ctc, the only other recognized gene dependent on sigma B.

Amino Acid Sequence↗

PubMed1998

A 3.3-kb plasmid of enterohemorrhagic Escherichia coli O157:H7 is closely related to the core region of the Salmonella typhimurium antibiotic resistance plasmid NTP16.

A restriction enzyme map was constructed of a 3.3-kb plasmid derived from enterohemorrhagic Escherichia coli O157:H7 strain 4821 using the enzymes SmaI, HpaI, FokI, HaeIII, StyI, RsaI, Bg/II, ClaI, and EcoRV. The molecular size of p4821 was 3307 bp, determined by nucleotide sequencing. Homology searches in the EMBL database library revealed that the nucleotide sequence of P4821 is similar (> 98%) to the core region of the antibiotic resistance plasmid NTP16 of Salmonella typhimurium strains. Nucleotide sequence analysis showed that p4821 contains all the information necessary for its replication, stability, and mobilization. However, the lack of tra genes indicates that the plasmid is nonconjugative. A possible transposon insertion site was found in p4821 but two such sites were found at the boundaries between the core region and the antibiotic resistance encoding transposons in plasmid NTP16. Using a hybridization assay, we determined that of 50 E. coli O157:H7 strains isolated in 1996 in Würzburg, Germany from patients with diarrhea and hemolytic-uremic syndrome, 4 strains (8%) contained plasmid p4821-specific sequences.

Diarrhea↗

PubMed1998

TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

Amino Acid Sequence↗

PubMed2003

Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection.

Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1,440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function.

Animals↗

PubMed1990

The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing.

The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease activity and others with increased protease activity. These results suggested that mspA expression might be regulated. Msp was shown to be produced at a much higher level in L. pneumophila grown in rich compared to semidefined media. A Tn9 insertion which abolishes Msp expression was introduced into the L. pneumophila genome. This mspA::Tn9 L. pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain. This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.

Bacterial Proteins↗

PubMed2003

A transposon encoding the complete 2,4-dichlorophenoxyacetic acid degradation pathway in the alkalitolerant strain Delftia acidovorans P4a.

The bacterial strain Delftia acidovorans P4a, isolated from an extreme environment (heavily contaminated with organochlorines, highly alkaline conditions in an aqueous environment), was found to mineralize 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid under alkaline conditions. Screening a genomic DNA library of the alkalitolerant strain for 2,4-D genes revealed the presence of the two 2,4-D gene clusters tfdCDEF and tfdC(II)E(II)BKA, tfdR genes being located in the vicinity of each tfd gene cluster. The results showed that the putative genes of the complete 2,4-D degradation pathway are organized in a single genomic unit. Sequence similarities to homologous gene clusters indicate that the individual tfd elements of strain P4a do not share a common origin, but were brought together by recombination events. The entire region is flanked by insertion elements of the IS1071 and IS1380 families, forming a transposon-like structure of about 30 kb, of which 28.4 kb were analysed. This element was shown to be located on the bacterial chromosome. The present study provides the first reported case of a chromosomally located catabolic transposon which carries the genes for the complete 2,4-D degradation pathway.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1992

Adhesive properties conferred by the plasminogen activator of Yersinia pestis.

A genomic library of Yersinia pestis EV76c created in a cosmid vector was screened for clones capable of binding type IV collagen. An unexpectedly high number of such clones was observed. One recombinant plasmid was selected for further study, and the locus controlling collagen binding was mapped by subcloning, transposon mutagenesis and exonuclease digestion. The outer-membrane protein profiles of transposon insertion mutants were correlated with phenotype to implicate a 36 kDa polypeptide in type IV collagen binding. Fine substructure restriction mapping and limited DNA sequence analysis showed the cloned locus to be identical to the locus (pla) for the plasminogen activator, previously characterized genetically and biochemically. The pla locus is resident on a 9.5 kb plasmid in wild-type Y. pestis strains. Curing of this plasmid resulted in negligible reduction in collagen-binding capacity, implying the existence of a chromosomally located determinant for collagen binding. The affinity of the plasminogen activator for collagen was relatively weak. When the cloned pla locus was introduced into E. coli, it conferred upon the cell the ability to bind to cells from a number of cell lines. Binding to glycolipids separated by thin-layer chromatography demonstrated that the receptor was a member of the globo-series of glycolipids. Since it has been reported that mutation of pla dramatically reduces virulence, we propose that this hitherto undescribed function of the gene product could contribute to the biological activities necessary for full virulence.

Bacterial Adhesion↗

PubMed1997

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.

Amino Acid Sequence↗

PubMed2005

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus.

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.

Base Sequence↗

PubMed1995

Epitope mapping of recombinant antigens by transposon mutagenesis.

We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.

Antibodies, Monoclonal↗

PubMed2006

PLOTREP: a web tool for defragmentation and visual analysis of dispersed genomic repeats.

Identification of dispersed or interspersed repeats, most of which are derived from transposons, retrotransposons or retrovirus-like elements, is an important step in genome annotation. Software tools that compare genomic sequences with precompiled repeat reference libraries using sensitive similarity-based methods provide reliable means of finding the positions of fragments homologous to known repeats. However, their output is often incomplete and fragmented owing to the mutations (nucleotide substitutions, deletions or insertions) that can result in considerable divergence from the reference sequence. Merging these fragments to identify the whole region that represents an ancient copy of a mobile element is challenging, particularly if the element is large and suffered multiple deletions or insertions. Here we report PLOTREP, a tool designed to post-process results obtained by sequence similarity search and merge fragments belonging to the same copy of a repeat. The software allows rapid visual inspection of the results using a dot-plot like graphical output. The web implementation of PLOTREP is available at http://bioinformatics.abc.hu/PLOTREP/.

Computer Graphics↗

PubMed1989

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.

Alleles↗

PubMedDate not supplied

Identification of a family of avirulence genes from Xanthomonas oryzae pv. oryzae.

Races of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, interact with cultivars of rice in a gene-for-gene specific manner. Multiple DNA fragments of various sizes from all strains of X. o. pv. oryzae hybridized with avrBs3, an avirulence gene from Xanthomonas campestris pv. vesicatoria, in Southern blots; this suggests the presence of several homologs and possibly a gene family. A genomic library of a race 2 strain of X. o. pv. oryzae, which is avirulent on rice cultivars carrying resistance genes xa-5, Xa-7, and Xa-10, was constructed. Six library clones, which hybridized to avrBs3, altered the interaction phenotype with rice cultivars carrying either xa-5, Xa-7, or Xa-10 when present in a virulent race 6 strain. Two avirulence genes, avrXa7 and avrXa10, which correspond to resistance genes Xa-7 and Xa-10, respectively, were identified and partially characterized from the hybridizing clones. On the basis of transposon insertion mutagenesis, sequence homology, restriction mapping, and the presence of a repeated sequence, both genes are homologs of avirulence genes from dicot xanthomonad pathogens. Two BamHI fragments that are homologous to avrBs3 and correspond to avrXa7 and avrXa10 contain a different number of copies of a 102-bp direct repeat. The DNA sequence of avrXa10 is nearly identical to avrBs3. We suggest that avrXa7 and avrXa10 are members of an avirulence gene family from xanthomonads that control the elicitation of resistance in mono- and dicotyledonous plants.

Amino Acid Sequence↗