The periplasmic nitrate reductase nap is required for anaerobic growth and involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense

Abstract

The magnetosomes of many magnetotactic bacteria consist of membrane-enveloped magnetite crystals, whose synthesis is favored by a low redox potential. However, the cellular redox processes governing the biomineralization of the mixed-valence iron oxide have remained unknown. Here, we show that in the alphaproteobacterium Magnetospirillum gryphiswaldense, magnetite biomineralization is linked to dissimilatory nitrate reduction. A complete denitrification pathway, including gene functions for nitrate (nap), nitrite (nir), nitric oxide (nor), and nitrous oxide reduction (nos), was identified. Transcriptional gusA fusions as reporters revealed that except for nap, the highest expression of the denitrification genes coincided with conditions permitting maximum magnetite synthesis. Whereas microaerobic denitrification overlapped with oxygen respiration, nitrate was the only electron acceptor supporting growth in the entire absence of oxygen, and only the deletion of nap genes, encoding a periplasmic nitrate reductase, and not deletion of nor or nos genes, abolished anaerobic growth and also delayed aerobic growth in both nitrate and ammonium media. While loss of nosZ or norCB had no or relatively weak effects on magnetosome synthesis, deletion of nap severely impaired magnetite biomineralization and resulted in fewer, smaller, and irregular crystals during denitrification and also microaerobic respiration, probably by disturbing the proper redox balance required for magnetite synthesis. In contrast to the case for the wild type, biomineralization in Δnap cells was independent of the oxidation state of carbon substrates. Altogether, our data demonstrate that in addition to its essential role in anaerobic respiration, the periplasmic nitrate reductase Nap has a further key function by participating in redox reactions required for magnetite biomineralization.

Fig 1

Effect of oxygen and nitrogen sources on magnetosome formation. (A) TEM micrographs of whole cells of the WT under the indicated conditions. Scale bars, 500 nm. (B) Close-up views of the magnetosome crystals shown in panel A. Scale bars, 100 nm.

Fig 2

Molecular organization of identified denitrification genes in MSR-1. Dashed lines indicate the extent of deletions in mutant strains.

Fig 3

Growth (OD₅₆₅) and magnetic response (C_mag) of WT MSR-1 and the Δnap, ΔnorCB, and ΔnosZ mutants under different conditions. Under aerobic conditions, the C_mag values were always zero and not shown. (A) Aerobic, ammonium medium; (B) aerobic, nitrate medium; (C) microaerobic, ammonium medium; (D) microaerobic, nitrate medium; (E) anaerobic, nitrate medium. Results from representative experiments were measured in triplicate, and values are given as means and standard deviations.

Fig 4

(A) TEM micrographs of Δnap, ΔnorCB, and ΔnosZ whole cells under the indicated conditions. Scale bars, 500 nm. (B) Close-up views of the magnetosome crystals shown in panel A. Scale bars, 100 nm. (C and D) TEM micrographs of anaerobically grown ΔnorCB (C) and Δnap (D) cells complemented with plasmids pLYJ75 and pLYJ80, respectively, harboring their WT alleles. Scale bars, 500 nm. (E) TEM micrographs of WT cells grown anaerobically on pyruvate and acetate. Scale bars, 500 nm. (F) Close-up views of the magnetosome crystals shown in panel E. Scale bars, 100 nm.

Fig 5

Growth (OD₅₆₅) and magnetic response (C_mag) of WT MSR-1 and the Δnap mutant under microaerobic conditions with 500 μM nitrite added to ammonium medium.