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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

Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.
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f0020: Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.

Mentions: Broad bands of 180 kDa were detected when His-tagged proteins were incubated at pH 6.5 or higher, indicating multimer formation (Fig. 4A). A previous report of RK2-encoded KfrA cross-linking yielded a clear dimer band and a faint tetramer band that was difficult to reproduce (Jagura-Burdzy and Thomas, 1992). We did not observe the dimer band in this study. Although the detected bands may suggest tetramer formation, low resolution in the high molecular mass range and the observed broad band could not rule out other levels of multimerization; thus, we adopted the term “multimer” in this manuscript.


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)

Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0020: Effect of pH on homomultimer formation of His-tagged Orf8 and Orf16 proteins. The recombinant proteins were incubated at various pH values and cross-linked with glutaraldehyde. The samples (250 ng/lane) were electrophoresed and CBB stained (A). The recombinant proteins were incubated at pH 2.5 and pH 7.5. After ultrafiltration (100-kDa cutoff), permeate (P) and retentate (R) fractions were electrophoresed and CBB stained (B). To ascertain pH stability, the recombinant proteins were incubated at various pH values, diluted, and shifted to pH 7–8. After cross-linking, the samples (25 ng/lane) were electrophoresed and silver stained (C). In all panels, large closed and open triangles indicate multimers and monomers, respectively.
Mentions: Broad bands of 180 kDa were detected when His-tagged proteins were incubated at pH 6.5 or higher, indicating multimer formation (Fig. 4A). A previous report of RK2-encoded KfrA cross-linking yielded a clear dimer band and a faint tetramer band that was difficult to reproduce (Jagura-Burdzy and Thomas, 1992). We did not observe the dimer band in this study. Although the detected bands may suggest tetramer formation, low resolution in the high molecular mass range and the observed broad band could not rule out other levels of multimerization; thus, we adopted the term “multimer” in this manuscript.

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