Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lyric infection of its host

H. Ozawa, H. Tanaka, Yuki Ichinose, T. Shiraishi, T. Yamada

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solanacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter, and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E. coli NM522 cells harboring the plasmid with the bacteriolyric gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.

Original languageEnglish
Pages (from-to)95-101
Number of pages7
JournalMGG Molecular & General Genetics
Volume265
Issue number1
Publication statusPublished - 2001
Externally publishedYes

Fingerprint

Ralstonia solanacearum
Bacteriophages
Parasites
Infection
Genes
Proteins
Tobacco
DNA
Plasmids
Molecular Weight
Amino Acids
Oligonucleotide Probes
Escherichia coli Proteins
Genetically Modified Plants
Amino Acid Sequence
Suspensions
Japan
Escherichia coli

Keywords

  • Bacteriolytic gene
  • Bacteriophage
  • Ralstonia solanacearum

ASJC Scopus subject areas

  • Genetics

Cite this

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lyric infection of its host. / Ozawa, H.; Tanaka, H.; Ichinose, Yuki; Shiraishi, T.; Yamada, T.

In: MGG Molecular & General Genetics, Vol. 265, No. 1, 2001, p. 95-101.

Research output: Contribution to journalArticle

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abstract = "To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solanacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter, and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E. coli NM522 cells harboring the plasmid with the bacteriolyric gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.",
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