Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

Comparative analysis of magnetosome gene clusters in magnetotactic bacteria provides further evidence for horizontal gene transfer

The organization of magnetosome genes was analysed in all available complete or partial genomic sequences of magnetotactic bacteria (MTB), including the magnetosome island (MAI) of the magnetotactic marine vibrio strain MV‐1 determined in this study. The MAI was found to differ in gene content and organization between Magnetospirillum species and strains MV‐1 or MC‐1. Although a similar organization of magnetosome genes was found in all MTB, distinct variations in gene order and sequence similarity were uncovered that may account for the observed diversity of biomineralization, cell biology and magnetotaxis found in various MTB. While several magnetosome genes were present in all MTB, others were confined to Magnetospirillum species, indicating that the minimal set of genes required for magnetosome biomineralization might be smaller than previously suggested. A number of novel candidate genes were implicated in magnetosome formation by gene cluster comparison. Based on phylogenetic and compositional evidence we present a model for the evolution of magnetotaxis within the Alphaproteobacteria, which suggests the independent horizontal transfer of magnetosome genes from an unknown ancestor of magnetospirilla into strains MC‐1 and MV‐1.     

Magnetospirillum magneticum AMB-1 peroxiredoxins contribute to the aerotolerance and genetic stability of the genomic magnetosome island

The magnetotactic bacterium Magnetospirillum magneticum AMB-1 can grow at variable oxygen concentrations, although the intracellular magnetic structures, magnetosomes, are only synthesized under microaerobic or anaerobic conditions. Three members of the peroxiredoxin family were identified in M. magneticum AMB-1. All purified recombinant proteins displayed thiol-dependent peroxidase activities. Allelic replacement mutagenesis revealed that, although the absence of the three peroxidase genes had no effect on either the growth or the formation of magnetosome under anaerobic conditions, the growth of mutants was compromised in an aerobic culture. Moreover, an accelerated loss in the genomic 'magnetosome island' (MAI) was observed in the null mutants cultured in the presence of oxygen. Taken together, these data suggest that the thiol-peroxidases identified act as key antioxidants in magnetotactic bacteria and, as a result, contribute to maintaining their capacity to synthesize magnetosome by shielding the genetic stability of the genomic MAI in adaptation to constant physiological change and stress.     

Compromised DNA Damage Repair Promotes Genetic Instability of the Genomic Magnetosome Island in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) are capable of synthesizing nano-sized, intracellular membrane-bound magnetosomes. To learn more about the genetic factors involved in magnetosome formation, transposon mutagenesis was carried out by conjugation using a hyperactive mariner transposon to obtain nonmagnetic mutants of Magnetospirillum magneticum AMB-1. A mutant with defect in uvrA gene encoding the DNA binding subunit of the UvrABC complex responsible for the process of nucleotide excision repair, was obtained. Growth, magnetosome formation and maintenance of magnetosome island (MAI) were further analyzed in the absence of UvrA. Interruption of uvrA led to decreased capacity to form magnetosome when cultured in the presence of oxygen. The deficiency in UvrA also resulted in an accelerated loss of the MAI under aerobic conditions indicating that the nucleotide excision repair system guards against the instability of the MAI. The incapacity of MTB to efficiently initiate recombination mediated by RecA rescued the instability of MAI observed in uvrA mutant. Elevated recombination activity resulting from the accumulation of unrepaired mutations may thus account for the instability of MAI in the absence of UvrA.     

Frequent mutations within the genomic magnetosome island of Magnetospirillum gryphiswaldense are mediated by RecA

Genes for magnetosome formation in magnetotactic bacteria are clustered in large genomic magnetosome islands (MAI). Spontaneous deletions and rearrangements were frequently observed within these regions upon metabolic stress. This instability was speculated to be due to RecA-dependent homologous recombination between the numerous sequence repeats present within the MAI. Here we show that a RecA-deficient strain of Magnetospirillum gryphiswaldense (IK-1) no longer exhibits genetic instability of magnetosome formation. Strain IK-1 displayed higher sensitivity to oxygen and UV irradiation. Furthermore, the lack of RecA abolished allelic exchange in the mutant. Cells of strain IK-1 displayed a slightly altered (i.e., more elongated) morphology, whereas the absence of RecA did not affect the ability to synthesize wild-type-like magnetosomes. Our data provide evidence that the observed genetic instability of magnetosome formation in the wild type is due predominantly to RecA-mediated recombination. In addition, increased genetic stability could make strain IK-1 a useful tool for the expression of genes and further genetic engineering, as well as for biotechnological production of bacterial magnetosomes.     

Monophyletic origin of magnetotaxis and the first magnetosomes

Horizontal gene transfer (HGT), the transfer of genetic material other than by descent, is thought to have played significant roles in the evolution and distribution of genes in prokaryotes. These include those responsible for the ability of motile, aquatic magnetotactic bacteria (MTB) to align and swim along magnetic field lines and the biomineralization of magnetosomes that are responsible for this behaviour. There is some genomic evidence that HGT might be responsible for the distribution of magnetosome genes in different phylogenetic groups of bacteria. For example, in the genomes of a number of MTB, magnetosome genes are present as clusters within a larger structure known as the magnetosome genomic island surrounded by mobile elements such as insertion sequences and transposases as well as tRNA genes. Despite this, there is no strong direct proof of HGT between these organisms. Here we show that a phylogenetic tree based on magnetosome protein amino acid sequences from a number of MTB was congruent with the tree based on the organisms' 16S rRNA gene sequences. This shows that evolution and divergence of these proteins and the 16S rRNA gene occurred similarly. This suggests that magnetotaxis originated monophyletically in the Proteobacteria phylum and implies that the common ancestor of all Proteobacteria was magnetotactic.     

The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.     

From invagination to navigation: The story of magnetosome‐associated proteins in magnetotactic bacteria

Magnetotactic bacteria (MTB) are a group of Gram‐negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome‐chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome‐associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme.     

Molecular analysis of a subcellular compartment: the magnetosome membrane in<Emphasis Type="Italic"> Magnetospirillum gryphiswaldense</Emphasis>

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane. In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs). Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes. Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB. The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island. Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.Abbreviations MM Magnetosome membrane - MMP Magnetosome membrane protein - MTB Magnetotactic bacteria     

Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2)

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.     

Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria

Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe₃O₄) or/and greigite (Fe₃S₄) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.     

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.     

Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5 kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with DNA double-strand breaks (DSBs) generated by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and three clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at single-guide RNA target sites. Our results indicated that the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammalian cells, and also caused frequent mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.     

Mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics

The 5' nontranslated region (NTR) of pestiviruses functions as an internal ribosome entry site (IRES) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting RNA signals involved in crucial processes of the viral life cycle. Computer modeling suggests that the 5'-terminal 75 nucleotides preceding the IRES element form two stable hairpins, Ia and Ib. Spontaneous and engineered mutations located in the genomic region comprising Ia and Ib were characterized by using infectious cDNA clones of bovine viral diarrhea virus. Spontaneous 5' NTR mutations carrying between 9 and 26 A residues within the loop region of Ib had no detectable influence on specific infectivity and virus growth properties. After tissue culture passages, multiple insertions and deletions of A residues occurred rapidly. In contrast, an engineered mutant carrying 5 A residues within the Ib loop was genetically stable during 10 tissue culture passages. This virus was used as starting material to generate a number of additional mutants. The analyses show that (i) deletion of the entire Ib loop region resulted in almost complete loss of infectivity that was rapidly restored during passages in cell culture by insertions of variable numbers of A residues; (ii) mutations within the 5'-terminal 4 nucleotides of the genomic RNA severely impaired virus replication; passaging of the supernatants obtained after transfection resulted in the emergence of efficiently replicating mutants that had regained the conserved 5'-terminal sequence; (iii) provided the conserved sequence motif 5'-GUAU was retained at the 5' end of the genomic RNA, substitutions and deletions of various parts of hairpin Ia or deletion of all of Ia and part of Ib were found to support replication, but to a lower degree than the parent virus. Restriction of specific infectivity and virus growth of the 5' NTR mutants correlated with reduced amounts of accumulated viral RNAs.     

Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-lox-based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense. Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB, mms6, and mamGFDC operons. As expected, all mutants were Mag⁻ and some were Mot⁻; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.Magnetosomes are unique membrane-enveloped organelles that are formed by magnetotactic bacteria (MTB) for magnetic navigation (2, 37). The mechanism of magnetosome formation is within the focus of a multidisciplinary interest and has relevance for biotechnological applications (5). It has been recognized that the biomineralization of inorganic magnetite crystals and their assembly into highly ordered magnetosome chains are under strict genetic control. Recent studies combining proteomic and bioinformatic approaches suggested that the genetic determination of magnetosome formation is complex and may potentially involve 25 to 50 gene functions (15), with unknown numbers of accessory genes and those controlling signal transduction and motility to achieve effective magnetotaxis (8, 9, 12, 26, 27, 29). However, the functional characterization of these candidate genes has been lagging behind. This is due to technical difficulties and the lack of facile tools for genetic manipulation of MTB. Allelic replacement systems have been established for Magnetospirillum magneticum (18) and Magnetospirillum gryphiswaldense (39, 40), but so far, there are only few examples of these for magnetosome genes that were functionally characterized because of the tedious and cumbersome procedures required for mutant generation (11, 19, 28, 31-32). Most genes controlling magnetosome formation in these and other MTB are located within a genomic magnetosome island (MAI) (34), which is genetically instable during stationary growth (47) and more or less conserved in other MTB (12, 13, 35). Most known magnetosome genes are organized within several conserved operons, which are interspersed with large, poorly conserved genome sections of unknown functions that have been speculated to represent genetic junk irrelevant for magnetotaxis but to cause genetic instability by their high content of repeats and transposable elements (34, 47). Thus, for large-scale functional genome analysis and rearrangements of the MAI, there is a great need for additional and more efficient genetic methods.Artificial genome recombination systems have been described for a number of bacteria. Many of them are based on the Cre-loxP system of the P1 phage (42). The Cre-loxP recombination system is a simple two-component system that is recognized as a powerful genetic tool in a multitude of eukaryotic and prokaryotic organisms (4, 6, 48). The Cre protein belongs to the integrase family of site-specific recombinases and catalyzes reciprocal site-specific recombination of DNA at 34-bp loxP sites, resulting in either excision or inversion, depending on the parallel or antiparallel orientation of the loxP sites, respectively (21). It does not require any host cofactors or accessory proteins (7). Cre-lox deletion has several advantages over other methods, such as a high efficiency and the independency of the length of DNA located between the two lox sites. The utility of Cre-lox systems has been demonstrated in a wide variety of Gram-positive and Gram-negative bacteria (17, 22-23). In several studies, it was applied for the generation of large-scale deletions, as in for example, the Gram-positive Corynebacterium glutamicum (43-46) and Bacillus subtilis (49).In M. gryphiswaldense, the functionality of a Cre-loxP antibiotic marker recycling system (25) has been previously demonstrated by deletion of a single gene based on double-crossover insertion of two loxP sites, followed by subsequent Cre-mediated excision (31). In this study, we describe a novel strategy for Cre-loxP-mediated deletion of large genomic fragments which requires only two single crossovers. The system has been validated by the generation of three large deletions, two single and one combination within the MAI, which demonstrated that the total deleted region of approximately 87 kb is not essential for viability and growth in the laboratory.     

A detailed look at 7 million years of genome evolution in a 439 kb contiguous sequence at the barley Hv-eIF4E locus: recombination, rearrangements and repeats

Six overlapping BAC clones covering the Hv-eIF4E gene region in barley were sequenced in their entire length, resulting in a 439.7 kb contiguous sequence. The contig contains only two genes, Hv-eIF4E and Hv-MLL, which are located in a small gene island and more than 88% of the sequence is composed of transposable elements. A detailed analysis of the repetitive component revealed that this chromosomal region was affected by multiple major duplication and deletion events as well as the insertion of numerous transposable elements, resulting in a complete reshuffling of genomic DNA. Resolving this highly complex pattern resulted in a model unraveling evolutionary events that shaped this region over an estimated 7 million years. Duplications and deletions caused by illegitimate recombination and unequal crossing over were major driving forces in the evolution of the Hv-eIF4E region, equaling or exceeding the effects of transposable element activities. In addition to a dramatic reshuffling of the repetitive portion of the sequence, we also found evidence for important contributions of illegitimate recombination and transposable elements to the sequence organization of the gene island containing Hv-eIF4E and Hv-MLL.     

FeoB2 Functions in magnetosome formation and oxidative stress protection in Magnetospirillum gryphiswaldense strain MSR-1

Magnetotactic bacteria (MTB) synthesize unique organelles, the magnetosomes, which are intracellular nanometer-sized, membrane-enveloped magnetite. The biomineralization of magnetosomes involves the uptake of large amounts of iron. However, the iron metabolism of MTB is not well understood. The genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense strain MSR-1 contains two ferrous iron transport genes, feoB1 and feoB2. The FeoB1 protein was reported to be responsible mainly for the transport of ferrous iron and to play an accessory role in magnetosome formation. To determine the role of feoB2, we constructed an feoB2 deletion mutant (MSR-1 ΔfeoB2) and an feoB1 feoB2 double deletion mutant (MSR-1 NfeoB). The single feoB2 mutation did not affect magnetite crystal biomineralization. MSR-1 NfeoB had a significantly lower average magnetosome number per cell (～65%) than MSR-1 ΔfeoB1, indicating that FeoB2 plays a role in magnetosome formation when the feoB1 gene is deleted. Our findings showed that FeoB1 has a greater ferrous iron transport ability than FeoB2 and revealed the differential roles of FeoB1 and FeoB2 in MSR-1 iron metabolism. Interestingly, compared to the wild type, the feoB mutants showed increased sensitivity to oxidative stress and lower activities of the enzymes superoxide dismutase and catalase, indicating that the FeoB proteins help protect bacterial cells from oxidative stress.     

A Second Actin-Like MamK Protein in Magnetospirillum magneticum AMB-1 Encoded Outside the Genomic Magnetosome Island

Magnetotactic bacteria are able to swim navigating along geomagnetic field lines. They synthesize ferromagnetic nanocrystals that are embedded in cytoplasmic membrane invaginations forming magnetosomes. Regularly aligned in the cytoplasm along cytoskeleton filaments, the magnetosome chain effectively forms a compass needle bestowing on bacteria their magnetotactic behaviour. A large genomic island, conserved among magnetotactic bacteria, contains the genes potentially involved in magnetosome formation. One of the genes, mamK has been described as encoding a prokaryotic actin-like protein which when it polymerizes forms in the cytoplasm filamentous structures that provide the scaffold for magnetosome alignment. Here, we have identified a series of genes highly similar to the mam genes in the genome of Magnetospirillum magneticum AMB-1. The newly annotated genes are clustered in a genomic islet distinct and distant from the known magnetosome genomic island and most probably acquired by lateral gene transfer rather than duplication. We focused on a mamK-like gene whose product shares 54.5% identity with the actin-like MamK. Filament bundles of polymerized MamK-like protein were observed in vitro with electron microscopy and in vivo in E. coli cells expressing MamK-like-Venus fusions by fluorescence microscopy. In addition, we demonstrate that mamK-like is transcribed in AMB-1 wild-type and ΔmamK mutant cells and that the actin-like filamentous structures observed in the ΔmamK strain are probably MamK-like polymers. Thus MamK-like is a new member of the prokaryotic actin-like family. This is the first evidence of a functional mam gene encoded outside the magnetosome genomic island.