MamX encoded by the mamXY operon is involved in control of magnetosome maturation in Magnetospirillum gryphiswaldense MSR-1

Abstract

Background: Magnetotactic bacteria produce membrane-enveloped magnetite crystals (magnetosomes) whose formation is controlled primarily by a gene island termed the magnetosome island (MAI). Characterization of single gene and operon function in MAI has elucidated in part the genetic basis of magnetosome formation. The mamX gene, located in the mamXY operon, is highly conserved in the MAI of all Magnetospirillum strains studied to date. Little is known regarding the function of mamX in the process of biomineralization.

Results: A mamX deletion mutant (∆mamX) and its complemented strain (CmamX) by conjugation in M. gryphiswaldense strain MSR-1 were constructed. There were no striking differences in cell growth among ∆mamX, CmamX, and wild-type strain (WT). ∆mamX displayed a much weaker magnetic response than WT. Transmission electron microscopy revealed the presence of irregular, superparamagnetic magnetite particles in ∆mamX, in contrast to regular, single-domain particles in WT and CmamX. The phenotype of ∆mamX was similar to that of an ftsZ-like deleted mutant and mamXY operon deleted mutant reported previously. Quantitative real-time RT-PCR (qPCR) results indicated that the deletion of mamX had differential effects on the transcription levels of the other three genes in the operon.

Conclusions: The MamX protein plays an important role in controlling magnetosome size, maturation, and crystal form. The four MamXY proteins appear to have redundant functions involved in magnetosome formation. Our findings provide new insights into the coordinated function of MAI genes and operons in magnetosome formation.

Figure 1

Comparison of cell growth and magnetic response (C_mag) in WT, mutant (∆mamX), and complemented strains (CmamX). All experiments were performed in triplicate. A: There were no striking differences among the growth curves of the three strains. B: The C_mag value of ∆mamX was consistently zero. The C_mag value of WT increased from 0.17 at 0 hr to a maximum of 0.89 at 10 hr and then gradually decreased. The C_mag value of CmamX increased from 0.14 at 0 hr to 0.45 at 10 hr.

Figure 2

Intracellular iron contents during culture of WT, ∆mamX, and CmamX. The intracellular iron content was much lower for ∆mamX (0.20%) than for WT and CmamX (both 0.47%). **, The difference between WT and ∆mamX was statistically significant (P < 0.01, by t test).

Figure 3

HR-TEM observation of different cells. HR-TEM of WT (A, B, C), ∆mamX(D, E, F), and CmamX(G, H, I). A, D, G: cell phenotype and magnetosome location. B, E, H: magnetosome chain organization. C, F, I: crystal lattice structure. Arrows: standard Fe₃O₄ crystal lattice. Scale bars: A, D, G: 200 nm; B, E, H: 100 nm; C, F, I: 10 nm.

Figure 4

Measurements of magnetism in deferent cells. (A):WT, (B): ΔmamX and (C): CmamX. Left: room-temperature hysteresis loops. Right: FORCs diagrams.

Figure 5

Absolute qPCR results for transcription levels of the four genes (mamY , mamX, mamZ, ftsZ-like) in the mamXY operon in WT. Each of the genes had a high transcription level from 12 to 18 hr, corresponding to the log phase of growth. The transcription level of mamZ was much higher than those of the other three genes at all four sampling times. *, 1/3 of original transcription level of mamZ in the figure was showed for better display of the other gene transcriptions.

Figure 6

Transcription levels of four genes in WT, ΔmamX, and CmamX strains. All experiments were performed in triplicate. A: The content of MamY was similar in ∆mamX and WT at 6 and 12 hr but was twice as high in ∆mamX as in WT at 20 hr. B: Deletion of mamX had no striking effect on mamZ transcription. The transcriptional disparity between mamZ and the other three genes was large in WT but much smaller in ∆mamX. C: The level of ftsZ-like showed dramatic changes during cell growth in ∆mamX. The level was twice as high as in WT at 6 hr, decreased 6-fold by 12 hr, increased >4-fold by 18 hr, and then gradually declined until 24 hr. For the highest transcription of all four genes appeared at 18h in WT (see Figure 5), the Student t-test was used to analyze the differences between transcription levels of WT and ∆mamX at this time point. *, the difference was statistically significant (P < 0.05, by t test). **, the difference was statistically extremely significant (P < 0.01, by t test).