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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

DNA binding capacity of Orf8 and Orf16 by electrophoretic mobility shift assays (EMSAs). Preparation of DNA substrate is graphically shown in panel A. US8 and US16 indicate upstream regions of orf8 and orf16 genes. T indicates TasI site, and the numbers beside TasI map mean length of fragment in bp. 205US8 and 198US16 indicate 205-bp upstream region of orf8 and 195-bp upstream region of orf16, respectively. Sequences of 205US8 and 198US16 are boxed. In the sequences, underlined letters and arrows indicate TasI sites and inverted repeat sequences, respectively. FITC-UnivF and UnivR show primers for FITC labeling. US8, US16 (panel B), TasI digested US8, US16 (panel C) and FITC-labeled EV and IR1V–IR4V (panels D and E) were used as DNA substrates. Panel E shows the EMSA result for non-labeled IR1–IR4 competitors. The ethidium bromide and FITC signals were detected in panels A, B and C, D, respectively.
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f0030: DNA binding capacity of Orf8 and Orf16 by electrophoretic mobility shift assays (EMSAs). Preparation of DNA substrate is graphically shown in panel A. US8 and US16 indicate upstream regions of orf8 and orf16 genes. T indicates TasI site, and the numbers beside TasI map mean length of fragment in bp. 205US8 and 198US16 indicate 205-bp upstream region of orf8 and 195-bp upstream region of orf16, respectively. Sequences of 205US8 and 198US16 are boxed. In the sequences, underlined letters and arrows indicate TasI sites and inverted repeat sequences, respectively. FITC-UnivF and UnivR show primers for FITC labeling. US8, US16 (panel B), TasI digested US8, US16 (panel C) and FITC-labeled EV and IR1V–IR4V (panels D and E) were used as DNA substrates. Panel E shows the EMSA result for non-labeled IR1–IR4 competitors. The ethidium bromide and FITC signals were detected in panels A, B and C, D, respectively.

Mentions: Electrophoresis mobility shift assays (EMSAs) were performed to investigate the DNA-binding capacities of Orf8 and Orf16. DNA substrates were prepared as described below (Fig. 6A). A 547-bp fragment containing a 476-bp upstream segment of orf8 (US8) was amplified from the plasmid with primers orf8(-476)f and orf8-16(71)r and Phusion DNA polymerase (New England Biolabs Japan Inc., Tokyo, Japan). Similarly, a 641-bp fragment containing a 570-bp upstream segment of orf16 (US16) was amplified with primers orf16(-570)f and orf8-16(71)r. The purified PCR products (US8 and US16) and their TasI digests were used as substrates for EMSAs. For further EMSA, synthetic double stranded DNA, IR1, IR2, IR3 and IR4 were created with appropriate oligonucleotides, IR1f–IR4r (Table 1). These dsDNA, IR1, IR2, IR3 and IR4 were ligated into SacI–Asp718 sites of pBBR1MCS2 to construct pIR1, pIR2, pIR3 and pIR4, respectively. PCR was carried out using universal primers, FITC-UnivF and UnivR to label the insert DNA of the plasmids with FITC. The PCR products were purified to remove unreacted primers. Since the PCR products contained vector sequences, the labeled DNA fragments were termed IR1V, IR2V, IR3V, and IR4V to avoid confusion with IR1–IR4.


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)

DNA binding capacity of Orf8 and Orf16 by electrophoretic mobility shift assays (EMSAs). Preparation of DNA substrate is graphically shown in panel A. US8 and US16 indicate upstream regions of orf8 and orf16 genes. T indicates TasI site, and the numbers beside TasI map mean length of fragment in bp. 205US8 and 198US16 indicate 205-bp upstream region of orf8 and 195-bp upstream region of orf16, respectively. Sequences of 205US8 and 198US16 are boxed. In the sequences, underlined letters and arrows indicate TasI sites and inverted repeat sequences, respectively. FITC-UnivF and UnivR show primers for FITC labeling. US8, US16 (panel B), TasI digested US8, US16 (panel C) and FITC-labeled EV and IR1V–IR4V (panels D and E) were used as DNA substrates. Panel E shows the EMSA result for non-labeled IR1–IR4 competitors. The ethidium bromide and FITC signals were detected in panels A, B and C, D, respectively.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0030: DNA binding capacity of Orf8 and Orf16 by electrophoretic mobility shift assays (EMSAs). Preparation of DNA substrate is graphically shown in panel A. US8 and US16 indicate upstream regions of orf8 and orf16 genes. T indicates TasI site, and the numbers beside TasI map mean length of fragment in bp. 205US8 and 198US16 indicate 205-bp upstream region of orf8 and 195-bp upstream region of orf16, respectively. Sequences of 205US8 and 198US16 are boxed. In the sequences, underlined letters and arrows indicate TasI sites and inverted repeat sequences, respectively. FITC-UnivF and UnivR show primers for FITC labeling. US8, US16 (panel B), TasI digested US8, US16 (panel C) and FITC-labeled EV and IR1V–IR4V (panels D and E) were used as DNA substrates. Panel E shows the EMSA result for non-labeled IR1–IR4 competitors. The ethidium bromide and FITC signals were detected in panels A, B and C, D, respectively.
Mentions: Electrophoresis mobility shift assays (EMSAs) were performed to investigate the DNA-binding capacities of Orf8 and Orf16. DNA substrates were prepared as described below (Fig. 6A). A 547-bp fragment containing a 476-bp upstream segment of orf8 (US8) was amplified from the plasmid with primers orf8(-476)f and orf8-16(71)r and Phusion DNA polymerase (New England Biolabs Japan Inc., Tokyo, Japan). Similarly, a 641-bp fragment containing a 570-bp upstream segment of orf16 (US16) was amplified with primers orf16(-570)f and orf8-16(71)r. The purified PCR products (US8 and US16) and their TasI digests were used as substrates for EMSAs. For further EMSA, synthetic double stranded DNA, IR1, IR2, IR3 and IR4 were created with appropriate oligonucleotides, IR1f–IR4r (Table 1). These dsDNA, IR1, IR2, IR3 and IR4 were ligated into SacI–Asp718 sites of pBBR1MCS2 to construct pIR1, pIR2, pIR3 and pIR4, respectively. PCR was carried out using universal primers, FITC-UnivF and UnivR to label the insert DNA of the plasmids with FITC. The PCR products were purified to remove unreacted primers. Since the PCR products contained vector sequences, the labeled DNA fragments were termed IR1V, IR2V, IR3V, and IR4V to avoid confusion with IR1–IR4.

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