Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria

Coq, Dominique Le; Steinmetz, Michel O.; Berkelman, Tom; Kado, Clarence I.

doi:10.1128/jb.164.2.918-921.1985

Cited by 446 publications

(144 citation statements)

References 22 publications

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“…Puri-fied transconjugants were grown i n low-salt-Bertani medium overnight and 105-10q cells were spread on low-salt Luria-Bertani medium plates containing IS % sucrose. The presence of an intact sacB gene encoding the periplasmic enzyme levansucrase kills gram-negative bacteria due to the production of levan in the periplasm (Gay et al, 1985). Thus, positive selection for sucrose resistance allows the collection of mutants with an allelic exchange.…”

Section: Strains and Plasmidsmentioning

confidence: 99%

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

Dernedde

Eitinger

Patenge

et al. 1996

European Journal of Biochemistry

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In Alcaligenes eutrophus HI6 the hyp gene complex consists of six open reading frames hypAl, B l , F I , C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NADreducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypHl and hypFl were originally considered to form a single open reading frame designated hypB (Dernedde, J., Eitinger, M. & Friedrich, B. (1 993) Arch. Microhiol. 159, 545-5531. Re-examination of the relevant sequence identified hypBl and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, BI and F l deletion mutants (cl 11) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. lmmunoblot analysis showed the presence of the H,-activating SH subunit (HoxH) at levels comparable to those observed in the wildtype strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. "Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class I1 mutants bearing deletions in hypAl, B l and F I showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.Keywords: Alculigenes eutrophus; hyp genes ; in-frame deletions; hydrogenase processing; nickel incorporation.Alculigenes eutrophus HI 6 , a facultative chemolithoautotrophic bacterium, is able to grow on molecular hydrogen as the sole energy source. Oxidation of hydrogen is mediated by two NiFe-containing hydrogenases: a heteroditneric membranebound enzyme (MBH;Schink and Schlegel, 1979) and a cytoplasmic heterotetrameric protein (SH; Schneider and Schlegel, 1976). MBH is considered to donate electrons to the respiratory chain via a membrane-bound b-type cytochroine as has been shown for Wolinella succinogenes hydrogenase (Dross et al., 1992). The FMN-containjng SH transfers electrons to NAD as the physiological acceptor. The genes (hox) encoding the two hydrogenases of A. eutrophus are located on a transmissible 450-kb megaplasmid. They are organised in two separate operons Corresporrdenw to

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Section: Strains and Plasmidsmentioning

confidence: 99%

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

Dernedde

Eitinger

Patenge

et al. 1996

European Journal of Biochemistry

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“…and analysing their plasmid profiles. Rhizobial cells cured of the labelled plasmid and thus rendered capable of growing on sucrose (Gay et al 1985) can be identified and recovered by growing on sucrose-containing medium and confirmed by their loss of resistance to Km and by plasmid profile analysis.…”

Section: Introductionmentioning

confidence: 97%

Sequential heat-curing of Tn5-Mob-sac labelled plasmids from Rhizobium to obtain derivatives with various combinations of plasmids and no plasmid

Moënne‐Loccoz

Baldani

Weaver

1995

Lett Appl Microbiol

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All four plasmids from Rhizobium leguminosarum bv. trifolii W14‐2 were sequentially labelled with Tn5‐Mob‐sac, which codes for resistance to kanamycin and sensitivity to sucrose, and cured by exposing rhizobia to supra‐optimal temperatures. This is the first report where a plasmid‐less derivative of Rhizobium was obtained. No relationship was found between plasmid size and elimination. The efficiency of Hynes' selection system decreased when used to cure plasmids from rhizobial derivatives already lacking one plasmid or more. The authors' results suggest that previous reports of failure in curing Tn5‐Mob‐sac labelled plasmids may only be due to an insufficient number of colonies being screened.

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“…It seems likely that while the pUC oriV is functional in PstQ, its transcription signals [especially the RNAII promoter (Polaczek & Ciesla, 1984)] are not well recognised in Pseudomonas. This situation sets up an unusual type of IS trap based on activation of plasmid replication, rather than on activation of silent resistance genes (Schneider et al, 2000), or inactivation of toxic genes (Gay et al, 1985;Simon et al, 1991). The repliconactivation IS trap discovered here may be useful in other contexts for detection of MGE activity and discovery of MGEs in other bacteria.…”

Section: Discussionmentioning

confidence: 99%

Insertion sequence ISPst4 activates pUC plasmid replication inPseudomonas stutzeri

Coleman

Richardson-Harris

Wilson

et al. 2014

FEMS Microbiol Lett

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Insertion sequences (IS) are important drivers of bacterial evolution. Here, we report a previously undescribed IS element (ISPst4) in Pseudomonas stutzeri, and its unusual interaction with plasmids introduced into this species. Transformation of the pUC19 derivative plasmid pUS23 into P. stutzeri yielded ampicillin-resistant transformants in P. stutzeri, but these grew very poorly. Plasmids recovered from the transformants frequently contained insertions of the IS elements ISPst4 and ISPst5. Hybridisation analysis showed that these two IS elements were common in P. stutzeri strains, but were not found in other pseudomonads. Insertions of ISPst4 in pUS23 were found predominantly between bla and oriV, and plasmids with this type of insertion were capable of robust replication in P. stutzeri, unlike pUS23. A promoter-containing region was localised to a 74 bp NcoI-SacI fragment within ISPst4, and we postulate that this promoter drives expression of the pUC oriV in P. stutzeri. This is the first report of IS transposition directly leading to an expansion of the effective host range of a plasmid, adding a new dimension to our understanding of the relationship between plasmids and IS elements.

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Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria

Cited by 446 publications

References 22 publications

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

Sequential heat-curing of Tn5-Mob-sac labelled plasmids from Rhizobium to obtain derivatives with various combinations of plasmids and no plasmid

Insertion sequence ISPst4 activates pUC plasmid replication inPseudomonas stutzeri

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