Transposon mutagenesis of coryneform bacteria

Vertès, Alain A.; Asai, Yoko; Inui, Masayuki; Kobayashi, Miki; Kurusu, Yasurou; Yukawa, Hideaki

doi:10.1007/bf00302251

Cited by 40 publications

(30 citation statements)

References 20 publications

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“…However, both ends of the 8-nt target duplication are rich in GC with an AT-rich central region, for example, TCATTG CCC (endogenous copy), GCCAAAAC (mutant A1), and AC GAAAGT (mutant A27). For the transposons derived from IS1096 and IS31831 (18,37), the same pattern was reported for the insertion sites. This may often lead to insertions within promoter regions in these high-GC gram-positive bacteria; for example, this may have occurred in mutant A27.…”

Section: Discussionsupporting

confidence: 63%

“…The transposition rates of about 10 3 transpositions/g of DNA used in electroporation of Arthrobacter are in the same range as those obtained with transposons constructed from IS1096 (3) and IS31831 (37). Since transposition rates strictly depend on the efficiency of transformation, transposon mutagenesis requires optimization of the conditions for electroporation.…”

Section: Discussionmentioning

confidence: 74%

“…For IS31831 and ISPs1, the formation of excised transposon circles was reported (2,38). Integration of the delivery plasmid at low frequency, which was also observed for IS31831 (37), could arise either by recombination or cointegrate formation.…”

Section: Discussionmentioning

confidence: 86%

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Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

Gartemann

Eichenlaub

2001

J Bacteriol

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Insertion elements (IS elements) are small mobile non-selfreplicating DNA regions specifying only the gene(s) required for their transposition (16). According to the phylogenetic relationship of the transposases and some features involved in the transposition (e.g., the inverted repeats which constitute the ends of most elements, as well as the recognition sites of the transposase and the target duplication caused by most elements), they can be grouped in different families (16). At least for some IS elements, a target-sequence-dependent site preference has been demonstrated, while members of other families apparently transpose in a random fashion. They are widespread in bacteria and contribute to the plasticity of bacterial genomes due to their transposition ability and to their role as target sites for homologous recombination, which can give rise to deletions, inversions, or more complex rearrangements (20). Some composite transposons derived from IS elements, in addition to the genes required for transposition, harbor genes encoding resistances to antibiotics or degradative pathways.Arthrobacter, a very common soil bacterium, and related genera like Micrococcus form one of the major branches of the actinomycetes, the Micrococcaceae (30). Some Arthrobacter strains are known to degrade aromatic compounds as well as chlorinated aromatic compounds such as 4-chlorobenzoate (4-CBA). Hydrolytic dehalogenation of 4-CBA by Arthrobacter sp. strain SU DSM20407 requires three genes organized in an operon which maps on plasmid pASU1 (25). The same set of genes is also present in Arthrobacter sp. strain TM1 (17). In the course of cloning and sequencing of these genes (unpublished data), we identified an IS element upstream of the dehalogenase operon in Arthrobacter sp. strain TM1. So far, only one other IS element in Arthrobacter and the Micrococcaceae, IS1473 from Arthrobacter nicotinovorans (which belongs to the IS3 family), has been described (19). A growing number of genes specifying degradation of aromatic compounds are known to be located on or associated with transposable elements (for recent reviews see references 31 and 41), with the best known examples being the very large catabolic transposons Tn4651 and Tn4653 on the TOL plasmids of pseudomonads. Thus, it seems possible that the genes for the hydrolytic dehalogenation of 4-CBA are located on a transposon.In order to demonstrate that IS1409 is a functional transposable element, a series of transposons were constructed based on a slightly modified IS1409 and carrying antibiotic resistance genes. These transposons are the first which can be used for transposon mutagenesis in Arthrobacter. MATERIALS AND METHODSBacterial strains, plasmids, culture media, and antibiotics. Bacterial strains and plasmids used in this study are listed in Table 1. 4-CBA-degrading strains were grown at 28°C in a minimal medium as described by Seiler (27) at pH 8.0, supplemented with 1 g of 4-CBA/liter as the only carbon and energy source and 0.2 ml of trace element solution/liter (9). Al...

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Section: Discussionsupporting

confidence: 63%

Section: Discussionmentioning

confidence: 74%

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Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

Gartemann

Eichenlaub

2001

J Bacteriol

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“…IS31831 exhibits cointegrate formation and resolution (47) and IS1096 was shown to contain a putative resolvase (12). It was suggested that IS1411 might transpose via circle formation (23), as has been shown to occur during the transfer of conjugative plasmids (37).…”

Section: Discussionmentioning

confidence: 99%

ISAfe1, an ISL3 Family Insertion Sequence fromAcidithiobacillus ferrooxidansATCC 19859

Holmes¹,

Zhao²,

Levicán³

et al. 2001

J Bacteriol

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Acidithiobacillus ferrooxidans, formerly Thiobacillus ferrooxidans (24), is a gram-negative bacterium that has been shown to be active in the solubilization of copper and in the processing of refractory gold ores in bioleaching operations (reviewed in references 21 and 36). It is also a major contributor to acid mine drainage in copper and coal mines and in certain natural environments. It is a chemolithotroph, deriving energy and electrons from the oxidation of ferrous iron and/or sulfur and various reduced sulfur compounds at pH 2 to 4, using oxygen as the ultimate electron acceptor (22). It fixes CO 2 by the Calvin-Bassham scheme. It can also anaerobically oxidize hydrogen at pH 5.5 (15). Recently, the almost complete genome sequence of A. ferrooxidans was used to detect and inventory the genes involved in amino acid metabolism (40).A mutant of A. ferrooxidans ATCC 19859 has been isolated that is able to switch reversibly, and with high frequency, between a wild-type state, in which it can oxidize both ferrous iron and sulfur compounds, and a mutant state, in which it has lost the capacity to oxidize iron (39). This phenomenon resembles other states of instability associated with the transposition of insertion sequences that have been described in other organisms and led us to investigate whether phenotypic switching might similarly be explained in A. ferrooxidans.Evidence was recently presented (5) that implicated a member of the so-called family 1 repetitive elements (50) in phenotypic switching. This repetitive element was tentatively identified as an insertion sequence and termed IST1 (renamed ISAfe1 here). Phenotypic switching was shown to be correlated with the high frequency insertion and excision of ISAfe1 into, and out of, the resB gene (5). ResB encodes a cytochrome c-type maturation protein (reviewed in reference 45), and a model was proposed in which insertion of ISAfe1 into resB eliminated the capacity of ResB to satisfactorily mature a c-type cytochrome and that this resulted, in turn, in the loss of the ability to oxidize iron but not sulfur (5).In order to describe and explain the phenomenon of phenotypic switching, we carried out a partial molecular characterization of ISAfe1. We further demonstrate that ISAfe1 can promote plasmid integration and resolution in E. coli, opening up the future possibility of exploiting E. coli to test experimentally certain characteristics of this insertion sequence. MATERIALS AND METHODSBacterial strains and media. Strains and plasmids used in this study are listed in Table 1. A. ferrooxidans ATCC 19859 was grown on Mackintosh medium or in modified 9K-ferrous iron medium (50). E. coli was grown in Luria-Bertani (LB) medium (30).Construction of plasmids. Construction of pTf85. A member of family 1 repeated DNA from A. ferrooxidans ATCC 19859 was cloned into pBR322, and the resulting plasmid was designated pTf11 (50). An internal SphI fragment of pTf1 1 was subcloned into pBR322 and termed pTf1 1-sph. pTf1 1-sph was subsequently used as a probe in a Southern blot...

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“…Information is emerging about the existence of such mobile elements (insertion sequence [IS] elements or transposons) in corynebacteria. Vertès et al (41) described the presence in Corynebacterium glutamicum of the element IS31831 and used it to construct two artificial transposons (Tn31831 and miniTn31831) which were functional in a Brevibacterium flavum strain (39). A new IS element (IS1206) from C. glutamicum has been described by Bonamy et al (5), and a different one (IS13869) has been isolated from Brevibacterium lactofermentum (7).…”

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confidence: 99%

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation

et al. 1996

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Conjugative transfer of mobilizable derivatives of the Escherichia coli narrow-host-range plasmids pBR322, pBR325, pACYC177, and pACYC184 from E. coli to species of the gram-positive genera Corynebacterium and Brevibacterium resulted in the integration of the plasmids into the genomes of the recipient bacteria. Transconjugants appeared at low frequencies and reproducibly with a delay of 2 to 3 days compared with matings with replicative vectors. Southern analysis of corynebacterial transconjugants and nucleotide sequences from insertion sites revealed that integration occurs at different locations and that different parts of the vector are involved in the process. Integration is not dependent on indigenous insertion sequence elements but results from recombination between very short homologous DNA segments (8 to 12 bp) present in the vector and in the host DNA. In the majority of the cases (90%), integration led to cointegrate formation, and in some cases, deletions or rearrangements occurred during the recombination event. Insertions were found to be quite stable even in the absence of selective pressure.Coryneform bacteria are gram-positive, nonsporulating soil microorganisms widely distributed in nature. Species of the genera Corynebacterium and Brevibacterium are of special interest for the industrial production of amino acids, nucleotides, and other compounds (22). In the last 10 years, efficient vector and transfer systems have been developed and a variety of genes involved in amino acid biosynthesis have been cloned (21,43). Conjugal transfer of plasmids from Escherichia coli to coryneform bacteria (31) is the most efficient method to introduce heterologous DNA into Corynebacterium and Brevibacterium strains, reaching transfer frequencies of up to 10 Ϫ2 per final donor colony, with 10 7 to 10 8 transconjugants per mating assay. Restriction systems in coryneform bacteria impairing intergeneric conjugation have been shown to be stress sensitive and can be inactivated by short stress treatments (30,32). Gene disruption and gene replacement techniques have been applied to selectively inactivate genes in corynebacteria by the use of transformation techniques (20,40) or by conjugation (33, 34) with mobilizable E. coli plasmids carrying internal fragments of the genes. In addition, because of their high frequency of conjugal transfer, corynebacteria are suitable candidates in which to search for and apply mobile elements for mutagenesis. Information is emerging about the existence of such mobile elements (insertion sequence [IS] elements or transposons) in corynebacteria. Vertès et al. (41) described the presence in Corynebacterium glutamicum of the element IS31831 and used it to construct two artificial transposons (Tn31831 and miniTn31831) which were functional in a Brevibacterium flavum strain (39). A new IS element (IS1206) from C. glutamicum has been described by Bonamy et al. (5), and a different one (IS13869) has been isolated from Brevibacterium lactofermentum (7). In addition, an extensive survey on th...

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Transposon mutagenesis of coryneform bacteria

Cited by 40 publications

References 20 publications

Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

Isolation and Characterization of IS 1409 , an Insertion Element of 4-Chlorobenzoate-Degrading Arthrobacter sp. Strain TM1, and Development of a System for Transposon Mutagenesis

ISAfe1, an ISL3 Family Insertion Sequence fromAcidithiobacillus ferrooxidansATCC 19859

Integration of narrow-host-range vectors from Escherichia coli into the genomes of amino acid-producing corynebacteria after intergeneric conjugation

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