Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.
AUTOR(ES)
Singer, J T
RESUMO
The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=209901Documentos Relacionados
- Complete Sequence of Virulence Plasmid pJM1 from the Marine Fish Pathogen Vibrio anguillarum Strain 775
- Iron uptake system medicated by Vibrio anguillarum plasmid pJM1.
- Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1).
- Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1.
- Polypeptides p40, pOM2, and pAngR are required for iron uptake and for virulence of the marine fish pathogen Vibrio anguillarum 775.