Transposon Tn5 mutagenesis of genes for the photosynthetic apparatus in Rhodopseudomonas capsulata

Summary Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.Abbreviations Bchl Bacteriochlorophyll - Cm chloramphenicol - Km kanamycin - Tc tetracycline - Ap ampicillin - Gm gentamicin - Spc spectinomycin     

Heat Shock Tolerant Mutants of Rhizobium meliloti Forming Ineffective Nodules

Six heat shock tolerant mutants of Rhizobium meliloti Rmd201 were isolated through transposon Tn5 mutagenesis. The symbiotic assays of these mutants with alfalfa plants, showed four of these mutants to be affected in nitrogenase effectivity also. These four mutants could be classified into two separate complementation groups hssA and hssC through R-prime mediated merodiploid constructions. The hssC mutant Rmd1040 also showed poor interaction with phages indicating surface alterations. The results indicated possible involvement of these loci in symbiosis as well as heat shock response.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Sequence Analysis of a Novel Insertion Site of Transposon IS10

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5′-CTGAGAGATCCCCTCATAATTT-3′ and 5′-AAATCATTAGGGGATTCATCAG-3′, which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5′-TGCTTGGTT-3′ instead of the previously reported 5′-NGCTNAGCN-3′. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5α and that there were two copies in the DH5α genome.     

recA-dependent genetic switch generated by transposon Tn10

We describe a new type of regulatory switch generated in bacteriophage lambda by transposon Tn10. By this switch, phage genes alternate reversibly between expressed and non-expressed states as the direct consequence of a reversible DNA rearrangement. The switch itself has arisen via Tn10-promoted recombination. The subsequent “flip-flop” in gene expression occurs by general recombination between two IS10 elements serving as “portable regions of homology”.     

A specific tetracycline-induced, low-molecular-weight RNA encoded by the inverted repeat of Tn10 (IS10)

The tetracycline (Tc)-resistance transposon Tn10 has previously been shown to encode a small (~4 S) RNA species in Escherichia coli minicells. When Tn10-containing mini-cells were labeled in the presence of Tc a new RNA species was detected that hybridized specifically to DNA sequences from the outer 400 bp of each inverted repeat sequence (IS10).     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

Genetic evidence for absence of transposition functions from the internal part of Tn981 a relative of Tn9

Summary Inverse transposition of the DNA of pBR322 was found to be mediated by the small transposon Tn981 a relative of Tn9 flanked by direct repeats of IS1. Since the resulting structure IS1:: pBR322::IS1 (Tn983) is transposed in a second step in the absence of Tn981, it is concluded that all the functions necessary for transposition of IS1 flanked transposons are coded for by IS1 itself or the E. coli chromosome, respectively.     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 10⁴-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium.     

Structure and stability of transposon 5-mediated cointegrates

We have determined the structure of a set of independently derived, Tn5-mediated cointegrates and examined the stability of several examples. A variety of cointegrate structures was found, including those mediated by the entire compound transposon, and those mediated by a single flanking IS50 element, which was always IS50-R, and never IS50-L. IS50-R but not IS50-L is reported to code for a protein(s) required for transposition. This finding confirms that IS50-L is relatively inactive and suggests that the active transposition protein(s) acts largely in cis on IS50-R. Another class of cointegrate was created by inverse transposition of Tn5 (using the inside ends of the flanking elements). In addition, we found an unexpectedly large set of cointegrates, in which the joint between the two plasmids was not adjacent to the transposon. All cointegrates analysed were found to be stable. This suggests that Tn5, unlike the transposon Tn3, does not transpose via an obligate cointegrate intermediate. This finding is compared to previous results with Tn5 and Tn9, and is discussed in terms of current models of transposition.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Insertional inactivation studies of the csmA and csmC genes of the green sulfur bacterium Chlorobium vibrioforme 8327: the chlorosome protein CsmA is required for viability but CsmC is dispensable

Targeted mutagenesis was used to investigate the roles of the CsmA and CsmC proteins of the chlorosomes of the green bacteria Chlorobium tepidum and Chlorobium vibrioforme 8327. Under the photoautotrophic growth conditions employed, CsmA is required for the viability of the cells but CsmC is dispensable. The absence of CsmC caused a small red shift in the near-infrared absorption maximum of bacteriochlorophyll d in whole cells and chlorosomes, but chlorosomes were assembled in and could be isolated from the csmC mutant. The doubling time of the csmC mutant was approximately twice that of the wild-type strain. Fluorescence emission measurements suggested that energy transfer from the bulk bacteriochlorophyll d to another pigment, perhaps bacteriochlorophyll a, emitting at 800–804 nm, was less efficient in the csmC mutant cells than in wild-type cells. These studies establish that transformation and homologous recombination can be employed in targeted mutagenesis of Chlorobium sp. and further demonstrate that chlorosome proteins play important roles in the structure and function of these light-harvesting organelles.     

Transposon-mediated insertion of R factor into bacterial chromosome

Summary Insertion of transposon Tn1 into the E. coli JC411 chromosome results in a sharp increase of plasmid RP4 integration frequency. This effect is absent in JC1553 recA cells. The RP4 integration with the chromosome is probably accomplished via recA-dependent recombination between transposon Tn1 inserted into the chromosome and the same transposon in the RP4 plasmid.     

Tn10 mediated integration of the plasmid R100.1 into the bacterial chromosome: Inverse transposition

Summary Upon integration into the bacterial chromosome the drug resistance plasmid R100.1 often loses its tetracycline resistance character. We have analyzed an Hfr strain formed by such an integration and an R-prime plasmid derived from it. We find that integration took place within the Tn10 transposon, that the two IS10 sequences were retained, but that at least 80% of the transposon segment located between them, and carrying the tetracycline resistance genes, had been lost. We suggest that integration of R100.1 was mediated by an inverse transposition using the IS10 sequences.     

Transposition mutagenesis in Streptomyces fradiae: identification of a neutral site for the stable insertion of DNA by transposon exchange

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.