Genetic system in<i>Pseudomonas alcaligenes</i>NCIB 9867

Poh, Chit Laa; Tham, Jill Maelan

doi:10.1111/j.1574-6968.1993.tb05972.x

“…Pseudomonas alcaligenes NCIB 9867 (P25X) is able to degrade 2,5‐xylenol, 3,5‐xylenol and m ‐cresol via the gentisate pathway [1]. The native plasmid of P25X, pRA2 (33 kbp), has a narrow‐host‐range and is self‐transmissible but attempts at curing it have proven unsuccessful [2, 3]. The plasmid of P. alcaligenes C‐0 [4] and more recently those of P. alcaligenes type strain [5] have also been reported to be incurable.…”

Section: Introductionmentioning

confidence: 99%

Sequence analysis of plasmid pRA2 fromPseudomonas alcaligenesNCIB 9867 (P25X) reveals a novel replication region

Kwong

¹

,

Yeo

²

,

Chuah

³

et al. 1998

FEMS Microbiology Letters

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The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1.

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“…Previously, a genetic system in P. alcaligenes NCIB 9867 was identified with both auxotrophic and catabolic chromosomal markers (29). Transfer of chromosomal DNA was established to occur in a bidirectional manner at frequencies ranging from 10 Ϫ5 for auxotrophic markers to 10 Ϫ7 for catabolic markers.…”

Section: ϫ9mentioning

confidence: 99%

Characterization of the Endogenous Plasmid from Pseudomonas alcaligenes NCIB 9867: DNA Sequence and Mechanism of Transfer

Kwong

¹

,

Yeo

²

,

Suwanto

³

et al. 2000

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The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G؉C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized.An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the ⍀Str r /Spc r gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 ؋ 10 ؊4 per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.The adaptive abilities of Pseudomonas species that allow them to grow in the polluted environment have made them ideal biocatalysts in bioremediation studies. Genetic diversity greatly enhances the adaptive abilities of bacteria, and this diversity can be accelerated by the horizontal exchange of genetic information, processes in which plasmids play a prominent role. Horizontal transfer of plasmids can occur by conjugation, transformation, and transduction. Both conjugation and transformation are active bacterial processes that require genetic information that is provided by either plasmids or their bacterial hosts.Investigations into the processes of conjugal transfer among gram-negative bacteria have shown that conjugative plasmids require a cis-acting component known as the origin of transfer (oriT) and numerous genes involved in DNA processing and mating pair formation (52). In IncP and related plasmids, the process of conjugal transfer is initiated by the binding of TraJ to oriT (53), followed by strand-specific nicking by TraI (27). A third DNA-binding protein, TraK, was also found to be important to the formation of this protein-DNA complex (55), and when it is assembled, it is often referred to as the relaxosome. These plasmid-encoded proteins seem to have developed a specific activity for their own oriT sequences and ar...

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“…Pseudomonas alcaligenes NCIB 9867 (P25X) is able to degrade 2,5-xylenol, 3,5-xylenol and m-cresol via the gentisate pathway [1]. The native plasmid of P25X, pRA2 (33 kbp), has a narrow-host-range and is self-transmissible but attempts at curing it have proven unsuccessful [2,3]. The plasmid of P. alcaligenes C-0 [4] and more recently those of P. alcaligenes type strain [5] have also been reported to be incurable.…”

Section: Introductionmentioning

confidence: 99%

Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region

Kwong¹

1998

FEMS Microbiology Letters

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The replication region of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (strain P25X) was localized within a 5.9-kbp DNA fragment and its sequence was determined. An interesting feature of the sequence is the presence of a 1.3-kbp region containing seven, highly conserved, direct repeats of 72 bp in length. The pRA2 replication region has two open reading frames (ORFs). ORF1 appeared to be essential for replication and had the potential to encode a novel 30-kDa protein with a predicted helix-turn-helix motif located at the C-terminal end. ORF2 was not essential for replication and may encode for a 37-kDa protein which shares 41% and 27% amino acid sequence identity to the KfrA proteins from plasmids RK2 and R751, respectively. The essential region of replication was narrowed down to 2819 nucleotides and included four of the seven 72-bp direct repeats, a potential DnaA-binding site and ORF1. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

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Genetic system inPseudomonas alcaligenesNCIB 9867

Cited by 3 publications

References 13 publications

Sequence analysis of plasmid pRA2 fromPseudomonas alcaligenesNCIB 9867 (P25X) reveals a novel replication region

Sequence analysis of plasmid pRA2 fromPseudomonas alcaligenesNCIB 9867 (P25X) reveals a novel replication region

Characterization of the Endogenous Plasmid from Pseudomonas alcaligenes NCIB 9867: DNA Sequence and Mechanism of Transfer

Sequence analysis of plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 (P25X) reveals a novel replication region

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