Ferritin from the obligate anaerobe Porphyromonas gingivalis: purification, gene cloning and mutant studies

Department of Microbiology, Faculty of Dentistry 1 and Department of Bacteriology, Faculty of Medicine 2 , Kyushu University, Fukuoka 812-8582, Japan Author for correspondence: Koji Nakayama. Tel: +81 92 642 6332. Fax: +81 92 642 6263. e-mail: knak{at}dent.kyushu-u.ac.jp Porphyromonas gingivalis is an obligate anaerobe that utilizes haem, transferrin and haemoglobin efficiently as sources of iron for growth, and has the ability to store haem on its cell surface, resulting in black pigmentation of colonies on blood agar plates. However, little is known about intracellular iron storage in this organism. Ferritin is one of the intracellular iron-storage proteins and may also contribute to the protection of organisms against oxidative stresses generated by intracellular free iron. A ferritin-like protein was purified from P. gingivalis and the encoding gene ( ftn ) was cloned from chromosomal DNA using information on its amino-terminal amino acid sequence. Comparison of the amino acid sequence deduced from the nucleotide sequence of ftn with those of known ferritins and bacterioferritins identified the protein as a ferritin and positioned it between proteins from the Proteobacteria and Thermotogales . The P. gingivalis ferritin was found to contain non-haem iron, thus confirming its identity. Construction and characterization of a P. gingivalis ferritin-deficient mutant revealed that the ferritin was particularly important for the bacterium to survive under iron-depleted conditions (both haemin and transferrin starvation), indicating that intracellular iron is stored in ferritin regardless of the iron source and that the iron stored in ferritin is utilized under iron-restricted conditions. However, the ferritin appeared not to contribute to protection against oxidative stresses caused by peroxides and atmospheric oxygen. Keywords: Porphyromonas gingivalis , ferritin, iron storage, oxidative stress Abbreviations: Em, erythromycin; TMBZ, 3,3',5,5'-tetramethylbenzidine The GenBank accession number for the sequence reported in this paper is AB016086 .