Novel Protein Mg2046 Regulates Magnetosome Synthesis in Magnetospirillum gryphiswaldense MSR-1 by Modulating a Proper Redox Status

Abstract

Magnetotactic bacteria (MTB) are a large, polyphyletic group of aquatic microorganisms capable of absorbing large amounts of iron and synthesizing intercellular nano-scaled nanoparticles termed magnetosomes. In our previous transcriptomic studies, we discovered that a novel gene (MGMSRv2_2046, termed as mg2046) in Magnetospirillum gryphiswaldense strain MSR-1 was significantly up-regulated during the period of magnetosome synthesis. In the present study, we constructed a MSR-1 mutant strain with deletion of mg2046 (termed Δmg2046) in order to evaluate the role of this gene in cell physiological status and magnetosome formation process. In comparison with wild-type MSR-1, Δmg2046 showed similar cell growth, but much lower cell magnetic response, smaller number and size of magnetosomes, and reduced iron absorption ability. mg2046 deletion evidently disrupted iron uptake, and redox equilibrium, and strongly inhibited transcription of dissimilatory denitrification pathway genes. Our experimental findings, taken together with results of gene homology analysis, indicate that Mg2046 acts as a positive regulator in MSR-1 under microaerobic conditions, responding to hypoxia signals and participating in regulation of oxygen metabolism, in part as a co-regulator of dissimilatory denitrification pathway with oxygen sensor MgFnr (MGMSRv2_2946, termed as Mg2946). Mg2046 is clearly involved in coupled regulation of cellular oxygen, iron and nitrogen metabolism under micro-aerobic or anaerobic conditions. Our findings help explain how MSR-1 cells initiate dissimilatory denitrification pathway and overcome energy deficiency under microaerobic conditions, and have broader implications regarding bacterial survival and energy metabolism strategies under hypoxia.

Keywords: Magnetospirillum gryphiswaldense; Mg2046; dissimilatory denitrification pathway; magnetosome; redox status; regulator.

FIGURE 1

Construction and verification of mg2046 null strain. (A) Schematic representation of mg2046 and flanking genes in MSR-1 genome. Arrows: genes. Brackets: interval gaps between genes. (B) Schematic representation of homologous double-crossover. pUXsuc_mg2046: suicide plasmid. Following double-crossover, mg2046 gene was replaced by gentamicin gene in MSR-1 genome. (C) Detection of mam genes in mutant and complementary strain by PCR. #, Mutant genome; +, complementary strain genome; –, ddH₂O used as template. Mutant and complementary strains were successfully constructed without auto-deletion of mam/mms genes.

FIGURE 2

Cell growth and magnetic response of WT and Δmg2046. (A) Cell growth (OD₅₆₅). (B) Magnetic response (Cmag). Growth was comparable for the two strains, but magnetic response was much lower for Δmg2046.

FIGURE 3

Characteristics of magnetosomes synthesized by WT and Δmg2046. (A) TEM images, with progressive magnification from left to right. Bars: 1 μm, 500 nm, 200 nm. (B) High-resolution TEM image of nanoparticles. Both strains showed typical ferroferric oxide lattice structure. Bar: 10 nm. (C,D) Box-plot charts showing statistical analysis of magnetosome diameter and number. ^∗∗∗∗p < 0.05. (E) FORCs diagram of WT. (F) FORCs diagram of Δmg2046. (G) Relative saturation magnetization based on comparison of hysteresis loops of WT (blue) and Δmg2046 (red). Max movement of WT is defined as ±1. Magnetosomes synthesized by Δmg2046 were fewer, smaller, and more weakly magnetic.

FIGURE 4

Iron utilization by WT and Δmg2046. (A) Iron absorption rate. (B) Intracellular iron content. Both parameters were lower for Δmg2046 than for WT.

FIGURE 5

Relative transcription levels of key pathway genes in WT and Δmg2046. (A) First genes of mam/mms operons (see text). (B) Important mam/mms genes. (C) Iron metabolism related genes. (D) Terminal oxidase genes. Transcription levels of genes in WT 8 h sample were used as reference (defined as 1) for expression of fold changes in other samples. (E) Fold changes between samples. Yellow: fold change >2. Pink: fold change <0.5. Δmg2046 showed disrupted transcription of mms, terminal oxidase, iron uptake, and storage related genes.

FIGURE 6

Transcription levels of nap, nir, nor, nos genes, and enzyme activity of nitrate reductase, in WT and Δmg2046. (A) Relative transcription levels of first genes (nap, nir, nor, nos) of four operons of dissimilatory denitrification pathway (see text) in WT 8 h, WT 16 h, Δmg2046 8 h, and Δmg2046 16 h. (B) Nitrate reductase activity at times 6, 9, 12, 15, and 18 h.