A novel role for Crp in controlling magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1

Abstract

Magnetotactic bacteria (MTB) are specialized microorganisms that synthesize intracellular magnetite particles called magnetosomes. Although many studies have focused on the mechanism of magnetosome synthesis, it remains unclear how these structures are formed. Recent reports have suggested that magnetosome formation is energy dependent. To investigate the relationship between magnetosome formation and energy metabolism, a global regulator, named Crp, which mainly controls energy and carbon metabolism in most microorganisms, was genetically disrupted in Magnetospirillum gryphiswaldense MSR-1. Compared with the wild-type or complemented strains, the growth, ferromagnetism and intracellular iron content of crp-deficient mutant cells were dramatically decreased. Transmission electron microscopy (TEM) showed that magnetosome synthesis was strongly impaired by the disruption of crp. Further gene expression profile analysis showed that the disruption of crp not only influenced genes related to energy and carbon metabolism, but a series of crucial magnetosome island (MAI) genes were also down regulated. These results indicate that Crp is essential for magnetosome formation in MSR-1. This is the first time to demonstrate that Crp plays an important role in controlling magnetosome biomineralization and provides reliable expression profile data that elucidate the mechanism of Crp regulation of magnetosome formation in MSR-1.

Figure 1. Multiple alignments of Crp/Fnr family transcriptional regulators from MSR-1 (MGR_1896 and MGR_2553), E. coli (U068_c0718) and C. crescentus (Caul_2975).

Conserved amino acid residues were highlighted by navy blue, proposed cyclic nucleotide-binding domain was encircled by blue and helix-turn-helix DNA-binding domain was encircled by red.

Figure 2. Growth, magnetism, and iron content analysis of various strains.

(A) Growth measurements of the WT, crp-M and crp-C strains under the same cultivation conditions; (B) Cmag measurements of the WT, crp-M and crp-C strains under the same cultivation conditions; (C) Residual iron levels in the medium and intracellular iron concentrations detected in cultures with the WT, crp-M and crp-C strains. All experiments were independently repeated three times to ensure their reproducibility.

Figure 3

Transmission electron microscopy (TEM) images of the WT (A), crp-M (B) and crp-C (C) strains, magnetosomes or magnetosome chains were denoted by arrows.

Figure 4

GO classification of genes from the MSR-1 expression profile results (A). The results are summarized in three broad categories: biological process (B), cellular component (C) and molecular function (D).

Figure 5

GO classifications of genes differentially expressed between WT and crp-M strains under three categories: biological process (A), cellular component (B) and molecular function (C).

Figure 6. Genes selected for validation by qRT-PCR, samples taken at 24 and 36 h were used for this assay, relative expression level of WT for each gene was set as 1 and not shown in the data.

(A) expression levels of select genes related to energy or carbon metabolism as determined by qRT-PCR; (B) expression level of certain MAI genes that are crucial for magnetosome synthesis as determined by qRT-PCR; (C) The expression level of select energy or carbon metabolism genes as determined by RNA-seq; (D) The expression levels of select MAI genes as determined by RNA-seq.