The terminal oxidase cbb3 functions in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense

Abstract

The biomineralization of magnetosomes in Magnetospirillum gryphiswaldense and other magnetotactic bacteria occurs only under suboxic conditions. However, the mechanism of oxygen regulation and redox control of biosynthesis of the mixed-valence iron oxide magnetite [FeII(FeIII)2O4] is still unclear. Here, we set out to investigate the role of aerobic respiration in both energy metabolism and magnetite biomineralization of M. gryphiswaldense. Although three operons encoding putative terminal cbb3-type, aa3-type, and bd-type oxidases were identified in the genome assembly of M. gryphiswaldense, genetic and biochemical analyses revealed that only cbb3 and bd are required for oxygen respiration, whereas aa3 had no physiological significance under the tested conditions. While the loss of bd had no effects on growth and magnetosome synthesis, inactivation of cbb3 caused pleiotropic effects under microaerobic conditions in the presence of nitrate. In addition to their incapability of simultaneous nitrate and oxygen reduction, cbb3-deficient cells had complex magnetosome phenotypes and aberrant morphologies, probably by disturbing the redox balance required for proper growth and magnetite biomineralization. Altogether, besides being the primary terminal oxidase for aerobic respiration, cbb3 oxidase may serve as an oxygen sensor and have a further role in poising proper redox conditions required for magnetite biomineralization.

FIG 1

(A) Molecular organization of putative terminal oxidase in the genome assembly of MSR-1. Dashed lines indicate the extent of deletions in mutant strains. (B and C) Transcription of putative terminal oxidase operons with gusA as a reporter in WT (B) and ΔMgfnr mutant (C) cells. Cultures were grown aerobically (21% O₂) in nitrate and ammonium medium or microaerobically (2% O₂) in nitrate and ammonium medium. Expression was measured by β-glucuronidase activities.

FIG 2

(A to C) Growth (OD₅₆₅) and magnetic response (C_mag) of MSR-1 WT, Δcbb₃, and Δaa₃ Δcbb₃ strains under different conditions. (A) Aerobic conditions in nitrate medium; (B) aerobic conditions in ammonium medium; (C) microaerobic conditions in ammonium medium. (D) TEM images of microaerobically grown WT (i), Δcbb₃ mutant (ii), and Δaa₃ Δcbb₃ mutant (iii) strains in ammonium medium. Bars, 500 nm (whole cells) and 100 nm (magnetosomes). (E) Measurements of crystal sizes for MSR-1 WT, Δcbb₃, and Δaa₃ Δcbb₃ strains at different time points in microaerobic ammonium medium. Results from representative experiments were determined in triplicate, and values are given as means and standard deviations.

FIG 3

(A) Growth (OD₅₆₅) and magnetic response (C_mag) of MSR-1 WT, Δcbb₃, and Δaa₃ Δcbb₃ strains in microaerobic nitrate medium. (B) Time courses of nitrate and nitrite utilization during microaerobic growth in nitrate medium. (C) Measurements of crystal sizes for MSR-1 WT, Δcbb₃, and Δaa₃ Δcbb₃ strains at different time points in microaerobic nitrate medium. The number of crystals measured for each strain (n) is shown. (D) Magnetosome morphotypes in Δcbb₃ and Δaa₃ Δcbb₃ mutants and proportion of each morphotype at different time points. The number of cells measured for each strain is presented.

FIG 4

(A) Nadi assay of the WT and various mutant strains. This method is commonly used to specifically detect cytochrome c oxidase activity (26), which is based on the rapid formation of indophenol blue from colorless α-naphthol catalyzed by cytochrome c oxidases with N,N-dimethyl-p-phenylenediamine monohydrochloride as an exogenous electron donor. Five microliters of cultures grown anaerobically for 24 h, which were adjusted to about 10⁷ CFU/ml, were dropped onto an agar plate in the presence of nitrate. Strains were incubated at 30°C for 4 days under anaerobic conditions and photographed after a 5-min Nadi reaction. (B) Nadi assay of anaerobically grown complementation strains. Plasmid pLYJ138 contains a WT cbb₃ allele, while pLYJ139 and pLYJ140 harbor WT aa₃ and bd alleles, respectively. (C) TEM images of Δcbb₃ and Δaa₃ Δcbb₃ strains complemented with plasmid pLY138, harboring the WT cbb₃ allele, grown in microaerobic nitrate medium. Bars, 500 nm (whole cells) and 100 nm (magnetosomes).

FIG 5

Biomineralization phenotypes of Δcbb₃ and Δaa₃ Δcbb₃ mutants incubated for 9 h in microaerobic nitrate medium. (A and C) TEM images of whole cells of the Δcbb₃ (A) and Δaa₃ Δcbb₃ (C) mutants. Bar, 500 nm. (B and D) Close-up views of magnetosome crystals shown in panels A and C, respectively. Bar, 100 nm. Irregularly shaped particles are indicated by arrows.

FIG 6

Morphologies found in different cells of the Δcbb₃ and Δaa₃ Δcbb₃ mutants in microaerobic nitrate medium. (A) WT-like spiral-shaped mutant cells; (B) thicker spiral cells; (C and D) thicker and smaller vibrioid cells. Bar, 500 nm.

FIG 7

Cellular NAD⁺/NADH ratio measurements of WT, Δcbb₃, and Δaa₃ Δcbb₃ strains under different conditions. NAD⁺ and NADH were extracted from cells grown in liquid medium, measured, normalized by the luminescence signal, and plotted. Means ± standard deviations are shown (n = 3).