The use of Pseudomonas sp. M plasmid pM3 for the transposon mutagenesis of Erwinia

The transposon containing derivatives pMTF9 (Tn9), pMTF10 (Tn10) and pMTF59 (Tn5, Tn9) of the Pseudomonas sp. M conjugative plasmid pM3 demonstrating temperature-dependent instability in Erwinia cells incubated at 37 degrees C have been isolated. The obtained plasmids have been shown to be usable for transposon-mediated mutability in the bacterial cells of Erwinia generum incubated at 37 degrees C.     

Vectors for transposon mutagenesis of non-enteric bacteria

Summary We have constructed a series of transposon delivery vectors derived from pRK2013. Since pRK2013 has a broad host range transfer system and a ColE1 replicon, it can be transferred to, but not replicated in, many nonenteric gram-negative bacteria. Thus pRK2013 provides an effective mechanism for the transient introduction of a transposon. Delivery vectors containing Tn7 (tmp str), Tn10 (tet), Tn10 HH104 (tet), or Tn5-132 (tet) have been constructed. When transposition in Caulobacter crescentus was examined, both Tn7 and Tn5-132 were found to transpose efficiently. In contrast, although the antibiotic resistances of Tn10 and Tn501 (mer) were expressed in C. crescentus, no transposition was observed with either transposon. However, transposition of Tn10 from the Tn10 vectors did occur in Acinetobacter calcoaceticus, and transposition of Tn501 from pMD100 has been demonstrated in Rhizobium japonicum (Bullerjahn and Benzinger 1984). Thus, transposon-host interactions play an important role in the determination of whether a particular transposon can transpose in a given host. Futhermore, the results with C. crescentus indicate that there must be different requirements for host interactions for Tn10 and Tn501 than for Tn5 and Tn7.     

Recent advances in large-scale transposon mutagenesis

Transposons were identified as mobile genetic elements over fifty years ago and subsequently became powerful tools for molecular-genetic studies. Recently, transposon-mutagenesis strategies have been developed to identify essential and pathogenicity-related genes in pathogenic microorganisms. Also, a number of in vitro transposition systems have been used to facilitate genome sequence analysis. Finally, transposon mutagenesis of yeast and complex eukaryotes has provided valuable functional genomic information to complement genome-sequencing projects.     

A series of Tn5 variants with various drug-resistance markers and suicide vector for transposon mutagenesis

A series of variants of transposon Tn5 were constructed by replacement of the 2.7-kb central segment which encodes kanamycin resistance with various other resistance-coding genes: tetracycline, chloramphenicol, gentamicin, trimethoprim, streptomycin or ampicillin. A thermosensitive replication mutant of the broad-host-range transmissible plasmid R388 was also constructed for use as a suicide vector for the delivery of transposable elements.     

Steady-state transposon mutagenesis in inbred maize

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.     

Characteristics of Pseudomonas solanacearum

Summary: A collection of 185 isolates of Pseudomonas solanacearum has been classified into 4 biotypes according to their capacity to oxidize 3 disaccharides (lactose, maltose and cellobiose) and 3 hexose alcohols (mannitol, sorbitol and dulcitol). Biotype 2 which oxidized the disaccharides but not the hexose alcohols appears to have a restricted host range; it was obtained solely from two plant hosts, potato and tomato, whereas biotype 1, which oxidized neither group of carbohydrates, and biotype 3 which oxidized both, were obtained from a diversity of plant hosts. A bacteriophage isolated from material infected with biotype 2 showed a high degree of specificity for isolates of biotype 2. The present known distribution of the 4 biotypes is given and their relationship to host range is discussed. A neotype culture for Ps. solanacearum is proposed.     

Lipopolysaccharide of Pseudomonas solanacearum

A novel monoterpene, 2(E)-(4-methyl-3-pentenyl)butenedial (α-acaridial (1) for the trivial name), was isolated from the secretion of the acarid mite Tyrophagus perniciosus. The structure was clarified in the light of spectral data, and the geometry of the double bond in the butenedial moiety was assigned based on the γ-deshielding effect on a methylene and on an aldehyde group. Coupling the Grignard reagent (CH₃)₂C =CHCH₂CH₂MgBr to THP-OCH₂(C = O)CH₂CH₂O-THP, and dehydration and deprotection of the OH groups gave α-(E)- and α-(Z)-acaridiol, which were fully assigned byMS and NMR. Matching the spectral data of the synthetic alcohols with those of the alcohol derived from the natural product, or of natural acaridial with those of synthetic α-(E)- and α-(Z)-acaridial, corroborated beyond all doubt the structure of this new monoterpene dial.     

Mariner-based transposon mutagenesis of Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that grows directly within the cytoplasm of its host cell, unbounded by a vacuolar membrane. The obligate intracytoplasmic nature of rickettsial growth places severe restrictions on the genetic analysis of this distinctive human pathogen. In order to expand the repertoire of genetic tools available for the study of this pathogen, we have employed the versatile mariner-based, Himar1 transposon system to generate insertional mutants of R. prowazekii. A transposon containing the R. prowazekii arr-2 rifampin resistance gene and a gene coding for a green fluorescent protein (GFP(UV)) was constructed and placed on a plasmid expressing the Himar1 transposase. Electroporation of this plasmid into R. prowazekii resulted in numerous transpositions into the rickettsial genome. Transposon insertion sites were identified by rescue cloning, followed by DNA sequencing. Random transpositions integrating at TA sites in both gene coding and intergenic regions were identified. Individual rickettsial clones were isolated by the limiting-dilution technique. Using both fixed and live-cell techniques, R. prowazekii transformants expressing GFP(UV) were easily visible by fluorescence microscopy. Thus, a mariner-based system provides an additional mechanism for generating rickettsial mutants that can be screened using GFP(UV) fluorescence.     

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367. Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.     

Large-scale transposon mutagenesis of Mycoplasma pulmonis

To obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn 4001T was determined for 1700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared with those reported for Mycoplasma genitalium and Bacillus subtilis . Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn 4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA , uvrA , uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N -acetylglucosamine was identified.     

Site-selected transposon mutagenesis at the hcf106 locus in maize.

The High chlorophyll fluorescence106 (Hcf106) gene in maize is required for chloroplast membrane biogenesis, and the hcf106-mum1 allele is caused by the insertion of a Robertson's Mutator Mu1 element into the promoter of the gene. Seedlings homozygous for hcf106-mum1 are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcf106 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf106-mum1 were screened by PCR for insertions and deletions associated with Robertson's Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcf106-mum1 allele and the other was in the wild-type allele, but all three new alleles were found to have break-points at the same nucleotide in the first intron. Unlike the hcf-106-mum1 progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of mutator activity. However, the second insertion had a weak, viable phenotype under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 in Kenya.

Genetic diversity among isolates of the bacterial plant pathogen Burkholderia solanacearum (synonym Pseudomonas solanacearum) race 3 biovar II of Kenya was determined by PCR with repetitive sequences (ERIC and BOX repetitive primer sets) and pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE). The study comprised 46 isolates collected during 1992 from the major potato-growing regions of Kenya (45 were identified as race 3 biovar II, and 1 belonged to race 3 biovar N2) and 39 reference isolates from 19 other countries. RC-PFGE identified 10 distinct profile types among the Kenyan race 3 biovar II isolates (29 of the isolates exhibited identical profiles) and a further 27 distinct profile types among the reference isolates. ERIC and BOX primer sets were unable to differentiate race 3 biovar II isolates within the Kenyan population but differentiated a further two distinct profile types among the reference isolates. The race 3 biovar N2 isolate had a highly distinct RC-PFGE and repetitive sequence PCR profile. Statistical analysis of the data identified biogeographic trends consistent with conclusions drawn from previous studies on the origin and worldwide dissemination of race 3 biovar II isolates; however, genomic fingerprinting by RC-PFGE revealed a level of genetic diversity previously unrealized.     

New transposon delivery plasmids for insertional mutagenesis in Bacillus anthracis

Two new transposon delivery vector systems utilizing Mariner and mini-Tn10 transposons have been developed for in vivo insertional mutagenesis in Bacillus anthracis and other compatible Gram-positive species. The utility of both systems was directly demonstrated through the mutagenesis of a widely used B. anthracis strain.     

Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum.

Several Pseudomonas solanacearum strains which produced no detectable extracellular polysaccharide (EPS) in planta had been reported to remain highly virulent when tested at high inoculum concentrations (P. Xu, M. Iwata, S. Leong, and L. Sequeira, J. Bacteriol. 172:3946-3951, 1990; P. Xu, S. Leong, and L. Sequeira, J. Bacteriol. 170:617-622, 1988). Two of these mutants, KD700 and KD710, have now been molecularly and genetically mapped to the EPSI gene cluster described by Denny and Baek (Mol. Plant-Microbe Interact. 4:198-206, 1991). When a range of inoculum concentrations was used, these two mutants and all other EPS-defective mutants tested were found to be reduced in virulence to eggplants and tobacco relative to the wild-type strain. Thus, EPS consistently is required for the wild-type level of virulence in P. solanacearum.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

The lipopolysaccharides of Pseudomonas solanacearum i Pseudomonas cichorii

Structure analysis by the methods of methylation, 1H- and 13C-n. m. r. spectroscopy has shown that O-specific polysaccharides of typical strains of Pseudomonas solanacearum (biovar I) and P. cichorii are identical by their structure and constructed of branched pentasaccharide repeating links which include three residues of rhamnose (one of them is in the branching node), one residue of beta-xylose (it occupies terminal position) and one reside of N-acetyl-beta-glucosamine. The other strain of P. solanacearum of biovar I and two strains belonging to biovars III and IV also produce structure-similar O-specific polysaccharides, constructed of linear tetrasaccharide repeating links which include three residues of alpha-L-rhamnose and one residue of N-acetyl-alpha-D-glucosamine.     

The Flagella of Pseudomonas solanacearum

Summary: Electron microscopic examination of 5 strains of Pseudomonas solanacearum , including the proposed neotype, NCPPB 325, showed that the cells of these organisms possess polar flagella.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.