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Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706(T).

Iiyama K, Mon H, Mori K, Mitsudome T, Lee JM, Kusakabe T, Tashiro K, Asano S, Yasunaga-Aoki C - Meta Gene (2015)

Bottom Line: A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae.Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16.A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Japan.

ABSTRACT
A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706(T) were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706(T) and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

No MeSH data available.


Related in: MedlinePlus

Pull-down assay to test interaction between recombinant Orf8 and Orf16 proteins. Total proteins were detected by CBB staining (upper panel). FLAG-tagged Orf8 was detected with a FLAG antibody.
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f0025: Pull-down assay to test interaction between recombinant Orf8 and Orf16 proteins. Total proteins were detected by CBB staining (upper panel). FLAG-tagged Orf8 was detected with a FLAG antibody.

Mentions: When FLAG-tagged Orf8 alone was applied to the pull-down assay as a control, it was detected in the flow-through fraction but not in the eluent (Fig. 5). Co-incubation with His-tagged Orf16, however, brought the FLAG-tagged Orf8 into the flow-through and elution fractions. Western blot analysis indicated the FLAG-tagged Orf8 interacted with His-tagged Orf16, suggesting heteromultimer formation.


Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706(T).

Iiyama K, Mon H, Mori K, Mitsudome T, Lee JM, Kusakabe T, Tashiro K, Asano S, Yasunaga-Aoki C - Meta Gene (2015)

Pull-down assay to test interaction between recombinant Orf8 and Orf16 proteins. Total proteins were detected by CBB staining (upper panel). FLAG-tagged Orf8 was detected with a FLAG antibody.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372654&req=5

f0025: Pull-down assay to test interaction between recombinant Orf8 and Orf16 proteins. Total proteins were detected by CBB staining (upper panel). FLAG-tagged Orf8 was detected with a FLAG antibody.
Mentions: When FLAG-tagged Orf8 alone was applied to the pull-down assay as a control, it was detected in the flow-through fraction but not in the eluent (Fig. 5). Co-incubation with His-tagged Orf16, however, brought the FLAG-tagged Orf8 into the flow-through and elution fractions. Western blot analysis indicated the FLAG-tagged Orf8 interacted with His-tagged Orf16, suggesting heteromultimer formation.

Bottom Line: A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae.Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16.A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Insect Pathology and Microbial Control, Institute of Biological Control, Faculty of Agriculture, Graduate School, Kyushu University, Japan.

ABSTRACT
A scaffold obtained from whole-genome shotgun sequencing of Paenibacillus popilliae ATCC 14706(T) shares partial homology with plasmids found in other strains of P. popilliae. PCR and sequencing for gap enclosure indicated that the scaffold originated from a 15,929-bp circular DNA. The restriction patterns of a plasmid isolated from P. popilliae ATCC 14706(T) were identical to those expected from the sequence; thus, this circular DNA was identified as a plasmid of ATCC 14706(T) and designated pPOP15.9. The plasmid encodes 17 putative open reading frames. Orfs 1, 5, 7, 8, and 9 are homologous to Orfs 11, 12, 15, 16, and 17, respectively. Orf1 and Orf11 are annotated as replication initiation proteins. Orf8 and Orf16 are homologs of KfrA, a plasmid-stabilizing protein in Gram-negative bacteria. Recombinant Orf8 and Orf16 proteins were assessed for the properties of KfrA. Indeed, they formed multimers and bound to inverted repeat sequences in upstream regions of both orf8 and orf16. A phylogenetic tree based on amino acid sequences of Orf8, Orf16 and Kfr proteins did not correlate with species lineage.

No MeSH data available.


Related in: MedlinePlus