Cloning And Functional Analysis Of Genes Related To The Magnetosome Biosynthesis In Magnetospirillum Gryphiswaldense MSR-1

The genetic analysis of the mechanism of magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1 has been hampered by the lack of an appropriate genetic manipulation system. Here reported on the establishment of a genetic manipulation system that can be used more readily than that of the others for Magnetospirillum. This system included method of solid medium plate enveloped with Parafilm to form colony, conjugational gene transfer in a selective medium, screening the nonmagnetic mutants by magnet adsorption technique. Two magnetosome deletion mutants were constructed by conjugative transposon mutagensis and the application of this genetic system. The two magnetosome deletion mutants were named as NM4 and NM21 respectively. A Southern blotting analysis using Clal fragment of mini-Tn5 lacZl as probe revealed that each of two magnetosome deleted mutants contained a single mini-Tn5 lacZl fusion and that the location of each insertion was distinct. It indicated that the magnetosome deleted phenotype was caused by Tn5 insertion.The sequences flanking Tn5 in magnetosome deleted mutant NM4 was cloned by Anchored PCR ,and was used as tag to extend the 5'and 3'terminal sequences of the target DNA fragment by Anchored PCR constinuously. A 5045bp fragment which contains six putative open reading frames (ORF) was obtained. The Tn5 inserted in the ORF4. It was demonstrated that this fragment was related to magnetosome biosynthesis by experiment of functional complement. The ORF4 encodes a protein which is homologous to the ATPase from Magnetospirillum magnetotacticum MS-1 and contains a REC domain which has a CheY-homologous receiver domain. This domain receives the signal from the sensor partner in bacterial two-component systems. These results indicated that the protein of the ORF4 works as ATPase function involved in the synthesis of magnetosome or takes part in the signal transduction relate to the promotion of magnetosome biosynthesis.Sequences flanking Tn5 in NM21 was cloned by Anchored PCR. A 3073bp fragment which contains three putative open reading frames (ORF) and shows strong homology with the Magnetospirillum magnetotacticum MS-1 was obtained , The Tn5 inserted in the ORF1. There are 29 bp interval between ORF1 and ORF2, and 14 bp overlaped between ORF2 and ORF3. Hydrophobicity and transmembrane domain analysis show that the protein encoded by ORF1 contains five Hydrophobic domains and four transmembrane domains. It maybe a transmembrane protein. It was demonstrated that this fragment was related to magnetosome biosynthesis by experiment of functional complement. The protein encoded by ORF1 show strong homology with cation efflux family protein from Geobacter sulfurreducens PCA [Identities = 65/272 (23%)], and contains MMT1-like putative conserved domains KOG1485 ,MMT1 contains conserved domains KOG1485 is mitochondrial Fe2+ transporter (cation diffusion facilitator superfamily).So it can be considered that the protein encoded by ORF1 is Fe2+ transporter involved in magnetosome biosynthesis in Magnetospirillum gryphiswaldense MSR-1.