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PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗

PubMed1989

Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa.

Pleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp-5 and xcp-54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD... xcp-5/xcp-54/met-9011/oru-314/trpF/leu-10. Restriction mapping and transposon (Tn1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp-5 gene has been subcloned on a 4kb EcoRI fragment.

Bacterial Proteins↗

PubMed1998

Tam3 in Antirrhinum majus is exceptional transposon in resistant to alteration by abortive gap repair: identification of nested transposons.

Most transposon families consist of heterogeneous copies with varying sizes. In contrast, the Tam3 copies in Antirrhinum majus are known to have exceptionally conserved structures of uniform size. Gap repair has been reported to be involved in the structural alteration of copies from several transposon families. In this study, we have asked whether or not gap repair has affected Tam3 copies. Five Tam3 copies carrying aberrant sequences were selected from 40 independent Tam3 clones and their sequences were analyzed. Two of the five copies contain insertions in the Tam3 sequence. These two insertions, designated Tam356 and Tam661, are typical transposon-like sequences, which have terminal inverted repeats and cause target site duplication. These nested transposons were obviously associated with transpositional events, and did not originate from the gap-repair process. The remaining three copies had lost large parts of the Tam3 sequence. We could not find any relationship between the deletions of Tam3 sequence in the three copies and gap repair. PCR analysis of a Tam3 excision site in the nivea(recurrence:Tam3) mutant also showed that most of the repair events after the Tam3 excision involved end-joining. In addition to the results obtained here, among the other clones isolated, we could not find any of the internally deleted copies that comprise a major part of other transposon families. All of these data suggest that some feature of the Tam3 structure suppresses the structural alterations that are otherwise generated during the gap repair process.

Alleles↗

PubMed2000

Rice transposable elements: a survey of 73,000 sequence-tagged-connectors.

As part of an international effort to sequence the rice genome, the Clemson University Genomics Institute is developing a sequence-tagged-connector (STC) framework. This framework includes the generation of deep-coverage BAC libraries from O. sativa ssp. japonica c.v. Nipponbare and the sequencing of both ends of the genomic DNA insert of the BAC clones. Here, we report a survey of the transposable elements (TE) in >73,000 STCs. A total of 6848 STCs were found homologous to regions of known TE sequences (E<10(-5)) by FASTX search of STCs against a set of 1358 TE protein sequences obtained from GenBank. Of these TE-containing STCs (TE-STCs), 88% (6027) are related to retroelements and the remaining are transposase homologs. Nearly all DNA transposons known previously in plants were present in the STCs, including maize Ac/Ds, En/Spm, Mutator, and mariner-like elements. In addition, 2746 STCs were found to contain regions homologous to known miniature inverted-repeat transposable elements (MITEs). The distribution of these MITEs in regions near genes was confirmed by EST comparisons to MITE-containing STCs, and our results showed that the association of MITEs with known EST transcripts varies by MITE type. Unlike the biased distribution of retroelements in maize, we found no evidence for the presence of gene islands when we correlated TE-STCs with a physical map of the CUGI BAC library. These analyses of TEs in nearly 50 Mb of rice genomic DNA provide an interesting and informative preview of the rice genome.

Arabidopsis↗

PubMed1988

Molecular cloning of genes that specify virulence in Pseudomonas solanacearum.

The suicide plasmid pSUP2021 was used to introduce Tn5 into the Pseudomonas solanacearum wild-type strain K60. We isolated eight avirulent mutants after screening 6,000 kanamycin-resistant transconjugants by inoculating eggplant (Solanum melongena L. cv. Black Beauty) and tobacco (Nicotiana tabacum L. cv. Bottom Special) seedlings. The Tn5-containing EcoRI fragments from the eight mutants were unique, suggesting that numerous genes specify virulence in this species. These EcoRI fragments were cloned into pBR322 or pUC12, and one of the clones, pKD810, was transformed into K60. All of the kanamycin-resistant, ampicillin-sensitive transformants were avirulent. Three randomly selected avirulent transformants were shown to carry the Tn5-containing fragment in place of the wild-type fragment and to exhibit the same hybridization pattern as the original KD810 mutant did. With pKD810 as a probe, we identified cosmids carrying the wild-type virulence genes by using a genomic library of K60 prepared in pLAFR3. Two of the homologous cosmids, pL810A and pL810C, when introduced into KD810 by transformation, restored virulence and normal growth of this mutant in tobacco. Altogether, these data indicate that the gene(s) interrupted by Tn5 insertion in KD810 is essential for the virulence of P. solanacearum. Further characterization of this gene is now being completed by subcloning, transposon mutagenesis, and complementation analysis.

Cloning, Molecular↗

PubMed1995

Genes required for cellulose synthesis in Agrobacterium tumefaciens.

A region of the chromosome of Agrobacterium tumefaciens 11 kb long containing two operons required for cellulose synthesis and a part of a gene homologous to the fixR gene of Bradyrhizobium japonicum has been sequenced. One of the cellulose synthesis operons contained a gene (celA) homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum. The same operon also contained a gene (celC) homologous to endoglucanase genes from A. xylinum, Cellulomonas uda, and Erwinia chrysanthemi. The middle gene of this operon (celB) and both the genes of the other operon required for cellulose synthesis (celDE) showed no significant homology to genes contained in the databases. Transposon insertions showed that at least the last gene of each of these operons (celC and celE) was required for cellulose synthesis in A. tumefaciens.

Agrobacterium tumefaciens↗

PubMed2002

Progress in maize gene discovery: a project update.

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu). Current achievements of the MGDP (NSF 98-72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor. In addition, ZmDB contains links to other plant and genetics websites.

Alleles↗

PubMed1992

Transposon Tn5-259 mutagenesis of Pseudomonas cepacia to isolate mutants deficient in antifungal activity.

Transposon Tn5-259 was inserted into the chromosome of Pseudomonas cepacia by mating with an Escherichia coli strain harboring a self-mobilizable, temperature-sensitive plasmid, pME12. Data from Southern blots and auxotroph analyses indicated that a single copy of the transposon was inserted in several places into the chromosome of P. cepacia. Among 1500 Tn5-259 transconjugants, only one mutant was found to be defective in the production of an antifungal compound, pyrrolnitrin. In addition, this mutant lost its ability to antagonize fungal phytopathogens. Using flanking DNA of the mutated gene as a probe, we have isolated four overlapping cosmid clones from a genomic library of P. cepacia. However, we were unable to complement the mutant because of difficulty in mobilizing the cosmids from E. coli to P. cepacia.

Antifungal Agents↗

PubMed1985

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti.

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.

DNA Transposable Elements↗

PubMed2001

Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence.

PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini-Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini-Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini-Tn10 transposons mapped in a five-gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini-Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a DeltaplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum-sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in DeltaoppB, Deltaspo0A and DeltaoppB Deltaspo0A mutants indicates that Opp is required for plcR expression via a Spo0A-independent mechanism.

Animals↗

PubMed1996

Identification of sigma S-regulated genes in Salmonella typhimurium: complementary regulatory interactions between sigma S and cyclic AMP receptor protein.

sigma S (RpoS)-regulated lacZ transcriptional fusions in Salmonella typhimurium were identified from a MudJ transposon library by placing the rpoS gene under the control of the araBAD promoter and detecting lacZ expression in the presence or absence of arabinose supplementation. Western blot (immunoblot) analysis of bacteria carrying PBAD::rpoS demonstrated arabinose-dependent rpoS expression during all phases of growth. sigma S-dependent gene expression of individual gene fusions was confirmed by P22-mediated transduction of the MudJ insertions into wild-type or rpoS backgrounds. Analysis of six insertions revealed the known sigma S-regulated gene otsA, as well as five novel loci. Each of these genes is maximally expressed in stationary phase, and all but one show evidence of cyclic AMP receptor protein-dependent repression during logarithmic growth which is relieved in stationary phase. For these genes, as well as for the sigma S-regulated spvB plasmid virulence gene, a combination of rpoS overexpression and crp inactivation can result in high-level expression during logarithmic growth. The approach used to identify sigma S-regulated genes in this study provides a general method for the identification of genes controlled by trans-acting regulatory factors.

Amino Acid Sequence↗

PubMed1997

Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis.

A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene. This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants. Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.

Alleles↗

PubMed1996

Lack of expression of the global regulator OxyR in Haemophilus influenzae has a profound effect on growth phenotype.

A pBR322-based library of chromosomal DNA from the nontypeable Haemophilus influenzae TN106 was screened for the expression of transferrin-binding activity in Escherichia coli. A recombinant clone expressing transferrin-binding activity contained a 3.7-kb fragment of nontypeable H. influenzae DNA. Nucleotide sequence analysis of this insert revealed the presence of two complete open reading frames encoding proteins of approximately 26 and 34 kDa. Mini-Tn10kan transposon mutagenesis at different sites within the open reading frame encoding the 34-kDa protein resulted in the abolition of transferrin-binding activity in the recombinant E. coli clone. The deduced amino acid sequence of the 34-kDa protein had 70% identity with the OxyR protein of E. coli; this latter macromolecule is a member of the LysR family of transcriptional activators. When a mutated H. influenzae oxyR gene was introduced into the chromosome of the wild-type H. influenzae strain by allelic exchange, the resulting oxyR mutant still exhibited wild-type levels of transferrin-binding activity but was unable to grow on media containing the heme precursor protoporphyrin IX (PPIX) in place of heme. This mutant also exhibited reduced growth around disks impregnated with heme sources. Supplementation of the PPIX-based growth media with catalase or sodium pyruvate resulted in normal growth of the H. influenzae oxyR mutant. Provision of the wild-type H. influenzae oxyR gene in trans also permitted the growth of this mutant on a PPIX-based medium. Exogenously supplied catalase restored the growth of this mutant with heme sources to nearly wild-type levels. These results indicate that expression of a wild-type OxyR protein by H. influenzae is essential to allow this organism to protect itself against oxidative stresses in vitro.

Amino Acid Sequence↗

PubMed1998

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons.

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.

Base Sequence↗

PubMed2003

Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium.

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.

Amino Acid Sequence↗

PubMed1996

Cloning of the fliI gene from Rhodobacter sphaeroides WS8 by analysis of a transposon mutant with impaired motility.

A transposon mutant of Rhodobacter sphaeroides WS8 was isolated that showed reduced swarming on soft agar plates. Liquid cultures of this mutant (M18) showed a low percentage of motile swimming cells in mid-exponential phase and a low level of extracellular flagellin protein by Western blotting. M18 was complemented by a clone from a library of R. sphaeroides WS8 DNA, and restriction mapping of the site of TnphoA insertion in the mutant, coupled with DNA sequencing, showed that it had a defect in the fliI gene. To determine if a partly functional fliI gene was giving the low-motility phenotype of M18, a drug resistance omega cartridge was inserted into the gene to give a complete null mutant. This null strain also produced a low percentage of motile cells. Possible reasons for this apparent fliI-independent flagellar formation are discussed.

Amino Acid Sequence↗

PubMed1996

Insertional inactivation of an intrageneric coaggregation-relevant adhesin locus from Streptococcus gordonii DL1 (Challis).

Transposon Tn916 was used to insertionally inactivate a coaggregation-relevant locus of Streptococcus gordonii DL1 (Challis). One mutant (F11) was isolated that lost the ability to coaggregate with the streptococcal partners of DL1 but retained the ability to coaggregate with partners belonging to other genera. A probe specific for the region flanking the Tn916 insertion was used to isolate a locus-specific fragment from a chromosomal lambda library. Southern analysis of the resulting phagemids revealed that a 0.5-kb EcoRI fragment hybridized with the F11 probe. Cloning of the 0.5-kb EcoRI fragment into the E. coli-streptococcal insertion vector p(omega) yielded pCW4, which was used to insertionally inactivate the putative coaggregation-relevant gene in DL1. Insertion mutants showed altered coaggregation with streptococci but retained wild-type coaggregation properties with other genera of bacteria. Comparison of immunoblots of cell surface proteins showed a 100-kDa protein in DL1 which was not detected in the Tn916 and pCW4 insertion mutants. These results indicate that the 0.5-kb EcoRI fragment is part of an adhesin-relevant locus that is involved in the production of a 100-kDa protein at the cell surface.

Adhesins, Bacterial↗

PubMed2002

The IrrE protein of Deinococcus radiodurans R1 is a novel regulator of recA expression.

IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation in an uncharacterized locus designated irrE. Five overlapping cosmids capable of restoring ionizing radiation resistance to IRS24 were isolated from a genomic library. The ends of each cloned insert were sequenced, and these sequences were used to localize irrE to a 970-bp region on chromosome I of D. radiodurans R1. The irrE gene corresponds to coding sequence DR0167 in the R1 genome. The irrE gene encodes a 35,000-Da protein that has no similarity to any previously characterized peptide. The irrE locus of R1 was also inactivated by transposon mutagenesis, and this strain was sensitive to ionizing radiation, UV light, and mitomycin C. Preliminary findings indicate that IrrE is a novel regulatory protein that stimulates transcription of the recA gene following exposure to ionizing radiation.

Bacterial Proteins↗