Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Sep;66(9):3778-83.
doi: 10.1128/aem.66.9.3778-3783.2000.

A Serine Protease-Encoding Gene (aprII) of Alteromonas Sp. Strain O-7 Is Regulated by the Iron Uptake Regulator (Fur) Protein

Affiliations
Free PMC article

A Serine Protease-Encoding Gene (aprII) of Alteromonas Sp. Strain O-7 Is Regulated by the Iron Uptake Regulator (Fur) Protein

H Tsujibo et al. Appl Environ Microbiol. .
Free PMC article

Abstract

The ferric uptake regulator (Fur) box-like sequence was located upstream of the serine protease-encoding gene (aprII) from a marine bacterium, Alteromonas sp. strain O-7. To clarify whether the production of AprII (the gene product of aprII) is regulated by the environmental iron concentrations, this strain was cultured under iron-depleted or iron-rich conditions and the level of AprII in the culture supernatant was analyzed by Western blotting. The production of AprII was significantly repressed under iron-rich conditions. Northern hybridization analysis demonstrated that AprII biosynthesis was regulated by iron through the control of transcription. These results indicate that aprII is a new member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the gene encoding Fur was cloned and sequenced. The deduced amino acid sequence of the cloned Fur showed high sequence similarity with that from gram-negative bacteria.

Figures

FIG. 1
FIG. 1
(A) Restriction map of pAP661; (B) domain structure of AprII. (A) Arrows indicate the ORF and the direction of transcription. aprII, serine protease-encoding gene; aprR, putative transcriptional regulator-encoding gene. (B) The arrows indicate the repeat amino acid sequence. Abbreviations: SP, signal peptide; AprII-N, N-terminal proregion; AprII-M, mature AprII; AprII-C, C-terminal proregion.
FIG. 2
FIG. 2
Western blot analysis of AprII. (A) Coomassie blue staining. Alteromonas sp. strain O-7 was cultured in iron-depleted (−) or iron-rich (+) medium. Each of the culture supernatants (900 μl) was added to 100 μl of 20% trichloroacetic acid and centrifuged. The pellets were dissolved with 10 μl of SDS-PAGE sample buffer and subjected to SDS–12% PAGE. Lane M, prestained molecular weight marker. (B) Western blot analysis of AprII. Samples were subjected to SDS-PAGE followed by immunodetection with 1:25,000 dilutions of AprII antibodies.
FIG. 3
FIG. 3
Northern blot analysis of the aprII transcript. Alteromonas sp. strain O-7 was cultured in iron-depleted (−) or iron-rich (+) medium. Lane M, RNA marker. (A) Formaldehyde-agarose gel electrophoresis of the total RNA from the strain; (B) Northern blot analysis.
FIG. 4
FIG. 4
Determination of the transcription start site of aprII. (A) Primer extension and nucleotide sequencing were performed with the same FITC-labeled primer. The nucleotide sequence around the transcription start site is shown in lanes A, C, G, and T. The transcriptional start site is shown by an arrow (lane P). (B) Nucleotide sequence of the 5′ upstream regions of aprII. The deduced amino acid sequence of AprII is shown below the nucleotide sequence. The Fur box-like sequence is boxed. The transcriptional start site is indicated with +1. The position of the FITC-labeled primer is shown below the nucleotide sequence by an arrow. The putative −35 and −10 regions are marked with solid and broken lines.
FIG. 5
FIG. 5
Transcriptional regulation of aprII in E. coli. E. coli JM109(pAP661) and E. coli H1780(pAP661) were cultured in iron-depleted (−) or iron-rich (+) LB medium. Lane M, RNA marker. (A) Formaldehyde-agarose gel electrophoresis of the total RNA; (B) Northern blot analysis of the aprII transcript.
FIG. 6
FIG. 6
Nucleotide sequence of fur and the 3′ region of fldA. The deduced amino acid sequences of Fur and FldA are shown below the nucleotide sequence. The inverted repeat sequence is indicated by convergent dashed arrows. The PCR primers furF, furR, furC, and furN are shown below the nucleotide sequence by arrows. The putative Shine-Dalgarno sequence is marked with a broken line.

Similar articles

See all similar articles

Cited by 3 articles

MeSH terms

LinkOut - more resources

Feedback