A Genetic Strategy for Probing the Functional Diversity of Magnetosome Formation
[['Genetic analysis historically relied on model systems that were easy to manipulate and for which genetic maps , then later full genome sequences , were available .', 'Today many interesting organisms have their genomes sequenced , but cannot be easily manipulated due to their incompatibility with standard genetic tools .', 'One such organism is Desulfovibrio magneticus RS-1 ( RS-1 ) .', 'RS-1 is a member of the magnetotactic bacteria , a phylogenetically diverse group of gram-negative bacteria that synthesize magnetic iron oxide ( magnetite ) or iron sulfide ( greigite ) crystals within complex genetically encoded intracellular organelles .', 'Magnetotactic bacteria were independently discovered by Bellini in 1963 [1] , [2] and Blakemore in 1975 [3] .', 'Both investigators observed bacteria that aligned with a magnetic field .', 'It was hypothesized that this behavior allows the bacteria , which were isolated in the northern hemisphere , to easily swim northwards to the bottom of the water column where micro-oxic or anoxic conditions exist .', 'This behavior is due to intracellular single-domain magnetite or greigite crystals organized in one or more chains along the length of the cell [4] .', 'Since their discovery , magnetotactic bacteria have inspired studies in bacterial cell biology [5] , biomineralization [6] , and nanotechnology [7] .', 'Magnetotactic bacteria have been isolated all over the world , and belong to the α- , γ- , and δ-proteobacteria , as well as the Nitrospirae and candidate OP3 division ( Figure 1A ) .', 'Different species produce different types of magnetosomes , varying in their number , shape and size of crystal , organization within the cell , and mineral composition [8] , [9] .', 'In addition to the phenotypic diversity of the magnetosomes themselves , the magnetotactic bacteria are morphologically and physiologically diverse , with anaerobic and microaerophilic species; coccoids , vibrios , spirilla , and rod-shaped cells; and some obligate multicellular bacteria .', 'Bioinformatic analyses of magnetotactic bacterial genomes have identified a genomic island that contains the genes for constructing magnetosomes [10] , [11] , termed the Magnetosome Island ( MAI ) .', 'While the MAIs of diverse magnetotactic bacteria vary in content , they share core magnetosome forming ( mam ) genes [11]–[13] .', 'Though the magnetotactic bacteria are dispersed among many non-magnetotactic members of the Proteobacteria and related phyla , the mam genes suggest a monophyletic origin for the magnetosome trait .', 'The phylogenetic trees of individual mam genes are the same as the phylogenetic trees of the ribosomal 16S sequences for the bacteria that possess them [14] , suggesting either that the common ancestor of all Proteobacteria , Nitrospirae and the candidate OP3 division was a magnetotactic bacterium , or that the MAI was passed to some members of these groups via ancient horizontal gene transfer .', 'Mechanistic analyses of magnetosome formation have been conducted largely in two closely related model organisms , Magnetospirillum magneticum AMB-1 ( AMB-1 ) , and Magnetospirillum gryphiswaldense MSR-1 ( MSR-1 ) .', 'Through biochemical [15]–[17] and genetic [18] , [19] analyses , the mam and mms genes were identified as participating in magnetosome formation .', 'These genes are located within the MAI , but they make up only a small portion of it .', 'For example , the AMB-1 MAI is 98 kilobases ( kb ) long , but an 18 kb portion containing only the mam genes is able to partially substitute for the whole island [20] , and 26 kb of the 115 kb MSR-1 island can reconstitute magnetosome synthesis in a non-magnetotactic bacterium [21] .', 'Because much of the MAI encodes genes that are not required for magnetosome synthesis , it is not obvious which genes in an MAI from any given magnetotactic bacterium are participating in creating magnetosomes with different phenotypes .', 'AMB-1 and MSR-1 are α-proteobacteria , which produce cubo-octohedral shaped magnetite crystals , while the δ-proteobacteria , represented by RS-1 , produce elongated bullet or tooth shaped crystals ( Figure 1B ) .', 'The MAIs of δ-proteobacteria have homologs to some of the α-proteobacterial MAI genes , but not others .', 'Conspicuously absent are genes whose mutants have small and misshaped crystals , such as the mms genes and mamS ( amb0975 ) [12] .', 'For comparison , the AMB-1 and RS-1 islands are shown in Figure 1C .', 'In addition to missing some α-proteobacterial genes , RS-1 contains genes that are shared exclusively among the MAIs of magnetotactic δ-proteobacteria , designated mad genes [11] .', 'Microscopy studies have shown that AMB-1 and MSR-1 build membrane vesicles , called magnetosome membranes , which are then the site for magnetite synthesis [15] , [18] .', 'RS-1 contains numerous intracellular membranes [22] , which makes looking for magnetosome membranes difficult .', 'After release from iron starvation , RS-1 constructs another organelle composed of membrane-bound iron and phosphorus inclusions .', 'In the same imaging study membranes were observed around these particles but not around magnetosomes [22] .', 'These findings suggest that RS-1 may have a fundamentally different mechanism for constructing and maintaining its magnetic organelle .', 'These differences make a compelling argument for genetic and molecular analyses of magnetotactic δ-proteobacteria .', 'Understanding the biological control of magnetite crystal shape and size is of particular interest to those designing magnetic nano-tools for industry [23] or health care [24] .', 'The comparison of different systems that perform similar tasks has proved fruitful in other cases .', 'For example research on different bacterial CRISPR systems resulted in the discovery of the simplified type II CRISPR that has become a valuable tool for manipulating eukaryotic genomes [25]–[27] .', 'In this study we overcome the need for sophisticated genetic tools in RS-1 by taking advantage of modern sequencing methods to identify mutations from a classic forward genetic screen .', 'We identified non-magnetic phenotypes for mutants of homologs of mam and mad genes , as well as genes not previously identified as being involved with magnetosome synthesis .', 'These findings expand our understanding of magnetosome formation , both in the δ-proteobacteria and across all magnetotactic bacteria .', 'Additionally , our results highlight the broad utility of this approach for studying interesting organisms that have previously been intractable to genetic analysis .'], ['We chose RS-1 as a representative of the magnetotactic δ-proteobacteria because it can be grown as colonies on solid medium and in pure culture in liquid medium under conditions where magnetosomes are produced [22] .', 'It also has a sequenced genome [12] , consisting of a 5 . 2 megabase chromosome and two plasmids .', 'In order to conduct genetic experiments , we attempted to transform RS-1 with a plasmid , pBMK7 , that is designed for Desulfovibrio species [28] .', 'We were successfully able to obtain RS-1 cells carrying pBMK7 by conjugation with Escherichia coli .', 'However , the transconjugation rate was extremely low .', 'With pBMK7 , we obtained 10−7 transconjugates per recipient cell .', 'For comparison , conjugation of AMB-1 , an excellent genetic model system , yields 10−3 transconjugates per recipient cell [19] , which results in thousands of transposon insertions per conjugation and allows for efficient screening of the entire genome .', 'The most comprehensive genetic analyses of the α-proteobacteria have come from genetic dissections of the AMB-1 and MSR-1 MAIs [29]–[31] .', 'Systematic deletions have identified minimal sets of genes required to make magnetosomes , as well as assigned genes into groups that perform the functions of membrane remodeling , crystal nucleation , and crystal maturation .', 'However , the tools available to create targeted deletions rely on suicide vectors that cannot replicate in the target organism such that DNA uptake and integration on the chromosome , two rare events , are selected for in one step .', 'Attempts to move a suicide vector into RS-1 were unsuccessful , due to the low transconjugation efficiency and perhaps also a low frequency of homologous recombination .', 'An alternative is a genetic screen where genes are disrupted randomly and mutants with interesting phenotypes are selected from a large pool .', 'However , modern bacterial genetic screens are performed with transposon-based tools that allow for easy identification of the disrupted gene but are delivered on suicide vectors .', 'Attempts to obtain mutants with a range of mariner , Tn5 , Tn7 , and Tn10 based transposons were also unsuccessful .', 'To work around this inability to generate transposon insertions , we isolated non-magnetic mutants created by chemical and UV mutagenesis , then used whole genome sequencing to identify the causative genetic change .', 'RS-1 has a doubling time of 11 hours and is an obligate anaerobe , though in our hands it is somewhat aerotolerant , and there is some evidence that it can survive near the oxic-anoxic transition zone in a gradient [32] .', 'Screening large numbers of colonies is impractical , so we employed a two-step process .', 'First a selection in liquid increased the proportion of non-magnetic cells in the population , then single colonies were screened for non-magnetic phenotypes .', 'This strategy is similar to one previously used with AMB-1 [18] .', 'To ensure that mutants with magnetosome-synthesis defects would not be accidentally discarded as magnetic because they still possessed magnetic crystals synthesized prior to mutagenesis , we began with non-magnetic cells , obtained by passaging wild type ( WT ) cells without iron .', 'We next wanted to verify the loss of magnetic particles in our cells due to this growth condition .', 'The ability of RS-1 cells to synthesize magnetosomes can be measured by quantifying cellular alignment with a magnetic field rotated 90 degrees , using a spectrophotometer .', 'The ratio of optical densities is called the coefficient of magnetism , or Cmag [33] .', 'WT RS-1 has a Cmag between 1 . 4 and 1 . 6 .', 'After two passages without iron we found that the culture had a Cmag of 1 , indicating that the cells no longer responded to a magnetic field .', 'These WT , non-magnetic cells were then mutagenized with either ultraviolet radiation or ethyl methanesulfonate .', 'In order to ensure that a sufficient level of mutagenesis was occurring , each mutagen was provided at a dose that resulted in 50% cell survival .', 'As described in Table 1 , this dose resulted in some mutants with only one genetic change , and others with dozens of changes when compared to the WT .', 'After mutagenesis , the cells were inoculated into fresh growth medium with iron .', 'We expected the resultant culture to contain a mixture of mutant cells , most of which would be magnetic .', 'To isolate the rare non-magnetic mutants , we passed the culture over a magnetized column and collected the flow-through , which contained non-magnetic cells .', 'However , much of the flow-through was only transiently non-magnetic , and when inoculated into fresh media with iron resulted in a culture with a WT Cmag .', 'This suggests that a proportion of a WT RS-1 culture is either naturally non-magnetic , or lags in magnetosome formation after release from iron starvation .', 'To isolate those cells that had a true non-magnetic phenotype , we repeated the outgrowth and column selection several times .', 'With each repetition , the Cmag of the outgrowth decreased , indicating non-magnetic mutants were taking over the population .', 'After four rounds of growth and selection the column flow-through was plated on solid media and single colonies were picked for further analysis .', 'Those colonies with non-magnetic or low-magnetic phenotypes were analyzed by whole genome sequencing to determine the causative genetic change .', 'The screen strategy is described in Figure 2 .', 'Because of the number of outgrowths , we were concerned that identical mutant colonies could be isolated that all descended from one mutagenized cell .', 'To avoid this , the outgrowth process was performed on many independent cultures , and only one colony from each culture was analyzed .', 'After the mutation for each strain was identified , we used PCR and Sanger sequencing to check for this change in the other strains isolated from the same outgrowth .', 'We then analyzed those strains that were not clones by whole genome sequencing to determine their mutation .', 'One strain contained a different mutation in the same gene , and was included in this study without having its whole genome sequenced ( mamL ( DMR_41030 ) allele 3 ) .', 'In addition to deletions of the entire MAI , we characterized 29 mutants , which are summarized in Tables 1 and S1 .', 'They include three large deletions and mutations in ten single genes .', 'Two mutations were caused by the insertion of a transposon into coding sequences .', 'This transposon , consisting of a gene with similarity to the IS5 family transposases , exists naturally in RS-1 , and appears to have mobilized during the mutagenesis and selection process .', 'There are 13 copies of this gene in the published genome sequence of RS-1 , including one located in the MAI region between groups I and II ( DMR_41190 ) .', 'The mutants had a variety of magnetic phenotypes , categorized into three groups: ( 1 ) a completely non-magnetic phenotype , ( 2 ) a very limited magnetic response with some replicate cultures displaying a Cmag near the limit of detection and others appearing to have no magnetic response , ( 3 ) a consistently measurable Cmag below that of WT .', 'These phenotypes are described as − , + , and ++ , respectively , in Table 1 .', 'Three genes have been named in this study .', "DMR_41280 has been named tauE , based on its gene product's membership in the Domain of Unknown Function ( DUF ) 81 TauE protein family .", 'DMR_41090 and DMR_41100 have previously been assigned names , but for reasons outlined in the discussion are renamed here fmpA and fmpB , respectively , for Fewer Magnetic Particles .', 'Many magnetotactic bacteria organize magnetosome synthesis genes into a genomic island , the MAI .', 'The MAI is frequently surrounded by repetitive sequence elements that enable its excision and loss from the chromosome by homologous recombination [10] , [34] .', 'In the laboratory model systems AMB-1 and MSR-1 spontaneous island deletions are often isolated during normal laboratory cultivation .', 'As with other magnetotactic bacteria , the genome sequence of RS-1 reveals a cluster of magnetosome-related genes surrounded by repetitive sequence elements .', 'The RS-1 MAI is an 82 kb region of DNA between nucleotides 4607747 and 4689778 [12] , [17] .', 'Flanking this region is a 2 . 4 kb direct repeat ( Figure 3C , pink regions ) that consists of three genes with homology to the IS66 family of insertion sequence elements .', 'There are a total of 12 of these IS66 operons located on the RS-1 chromosome and plasmids , including two other pairs that have 100% nucleotide identity to the pair that flanks the RS-1 MAI .', 'Approximately 90% of mutants recovered in this work were island deletions .', 'Island deletions had no coverage at positions within the MAI when analyzed by whole genome sequencing ( Figure S1 ) .', 'The deletion of the MAI was confirmed by the ability to obtain PCR products using primers just outside , but not just inside the border of the MAI .', 'RS-1 MAI deletions could be isolated by the non-magnetic enrichment and outgrowth method used in this screen even in the absence of mutagenesis , suggesting that island loss is occurring naturally in RS-1 .', 'RS-1 MAI deletions lack the electron-dense particles found in the WT ( Figure 3A and B ) and have no magnetic response ( Cmag\u200a=\u200a1 ) .', 'In addition to the 2 . 4 kb repeat , portions of the MAI consist of DNA transposases , recombinases and short repetitive sequences ( Figure 3C , dark grey regions ) .', 'These areas divide the remaining genes , which may function in magnetosome synthesis , into four groups , indicated in Figure 3C by Roman numerals .', 'The largest of these , group I , contains most of the genes that have homology to genes in other magnetotactic bacteria .', 'These include all the mam genes , which are common to magnetotactic bacteria [11]–[13] , and some of the mad genes , which are shared among the magnetic δ-proteobacteria [11] .', 'Groups II and III contain some mad genes ( mad5 ( DMR_41230 ) and mad12 ( DMR_41300 ) ) , as well as some genes that share homology with signaling or transcriptional regulation genes ( DMR_41220 , DMR_41310 ) .', 'Group IV contains genes associated with motility , such as a cheW homolog and genes encoding a methyl accepting chemotaxis protein and a GGDEF domain protein ( DMR_41440 , DMR_41450 , and DMR_41480 , respectively ) .', 'There is a copy of the 2 . 4 kb repeat that has 94% nucleotide identity to those surrounding the MAI and is located directly upstream of group IV , suggesting that these genes might compose a motility module that could be lost by recombination , though this was not observed during this study .', 'Three mutants with deletions of a portion of the island were recovered ( Figure 3C , Table S1 ) .', 'In each case the region between groups I and II formed one edge of the deletion .', 'This region contains many 46 base , 12 base , and 10 base direct repeats; however these repeats do not border the deletions , suggesting they were not created by homologous recombination with the repeats .', 'In deletion 1 , a portion of group I is missing , removing many mam genes , such as mamB ( DMR_41110 ) and mamQ ( DMR_41130 ) , whose deletions in the α-proteobacteria result in non-magnetic cells [29] , [30] .', 'The other two deletions remove areas outside of group I . One includes most of group II , and the other includes the entire right side of the island , groups II , III , and IV .', 'All three deletions have non-magnetic phenotypes , with a Cmag of 1 and no particles visible by transmission electron microscopy ( TEM , Figure S2 ) .', 'These results show that there are genes in addition to the mam and mad genes of group I that are necessary for RS-1 to form magnetite crystals .', 'RS-1 cells are filled with intracellular membranes [22] .', 'Intracellular membranes are rare among bacteria , often being involved in photosynthesis or energy production [35] .', 'As RS-1 does not photosynthesize , the function of its extensive intracellular membrane network remains mysterious .', 'In our previous work we were not able to see membranes surrounding RS-1 magnetite crystals , even though many intracellular membranes are visible throughout the cell and surrounding the iron-phosphorus inclusions that RS-1 transiently produces upon release from iron starvation [22] .', 'However , it is possible that membranes do surround RS-1 crystals , but that they are so closely associated that they are not visible by microscopy .', 'Indeed , Matsunaga and coworkers observed some material by TEM around purified magnetosomes that was removed by detergent treatment , which they interpreted as magnetosome membranes [17] .', 'Some of the intracellular membranes of RS-1 have an elongated shape that vaguely resembles the elongated shape of RS-1 magnetite crystals although their dimensions are significantly different .', 'In previous work no magnetite crystals were observed within these intracellular membranes yet a connection between them and magnetite formation could not be ruled out [22] .', 'In the α-proteobacteria , the genes for remodeling the inner membrane to produce magnetosome membranes are encoded within the MAI , and in the absence of these specific genes or of the entire MAI no magnetosome membranes are synthesized [29] .', 'To determine if the intracellular membranes of RS-1 depend on the MAI , we compared an MAI deletion strain to WT by preparing the cells using cryo-ultramicrotomy and imaging them with TEM .', 'As shown in Figure 4A , both strains contain elongated intracellular membrane structures , suggesting that RS-1 magnetosomes and intracellular membranes are unrelated .', 'It is possible , however , that RS-1 synthesizes crystals using membranes that were native to the bacterium before the acquisition of the MAI , or that the genes responsible for creating and maintaining the intracellular membranes were originally part of the MAI but have since been moved to a different part of the genome .', 'In addition to making magnetosomes , RS-1 makes an iron and phosphorus containing organelle [22] .', 'In the hours after release from iron starvation , hundreds of intracellular nanometer-sized iron phosphorus particles surrounded by membranes appear throughout the cell , and then disappear over the course of three days [22] .', 'As their disappearance corresponds with the synthesis of magnetosomes , we initially hypothesized that they were magnetosome precursors .', 'However , a pulse-chase experiment showed that the iron in the iron-phosphorus organelles did not end up in magnetosome crystals [22] , suggesting that they are unrelated to magnetosome synthesis .', 'To determine if the iron-phosphorus organelles require magnetosome genes , we subjected WT and MAI deletion cells to iron starvation , and then followed them by TEM after the re-introduction of iron .', 'As shown in Figure 4B , both strains produced the iron-phosphorus organelles , confirming our original finding that these are unrelated processes that occur in RS-1 .', 'Some of the isolated mutants differed from the WT genome sequence at only one position , while others contained many changes ( Tables 1 , S2 ) .', 'In these cases , we predicted that the change in a gene located in the MAI was responsible for the observed phenotype .', 'To test this , we complemented each of the mutants by expressing a WT copy of the mutated MAI gene from plasmid pBMK7 .', 'Some genes were expressed from the RS-1 mamA ( DMR_41160 ) promoter , while others were expressed from their own promoters ( Tables S3 , S4 ) .', 'Though complementation efficiency differed , expression of the candidate MAI gene increased the Cmag in all cases compared to the same mutant that was carrying the empty vector ( Figure 5 ) .', 'In some cases different mutant alleles of the same gene exhibited varying levels of complementation .', 'For instance , unlike other alleles of mamB , allele 1 was complemented at a very low level .', 'This allele is a transposon insertion that may have polar effects on downstream genes .', 'By contrast , the transposon insertion in tauE allele 3 complemented to the same level as the other tauE alleles .', 'The gene downstream of tauE is on the opposite strand , so in this case no polar effects would be predicted .', 'mamL allele 1 ( E63K ) , and fmpB allele 2 ( a frameshift ) could not be complemented even though the other alleles of mamL and fmpB could , suggesting that these are dominant negative alleles .', 'Alternatively , one or a combination of the three or two additional mutations in these respective strains could be responsible for the phenotype .', 'Surprisingly , fmpB allele 2 contains a frameshift at the same amino acid as fmpB allele 1 , which is complementable .', 'The alleles are the gain ( allele1 ) or loss ( allele', '2 ) of an extra cytosine in a poly-cytosine tract , and encode proteins that end with different polypeptides .', 'These may impart different biochemical properties on the FmpB mutant proteins , perhaps resulting in degradation of one and toxic build up of the other .', 'tauE was the only single gene we found outside of group I whose mutants have a non-magnetic phenotype .', 'The location of tauE could account for the phenotypes of large deletions 2 and 3 , both of which lack tauE .', 'We investigated whether tauE was sufficient to complement the loss of most of group II or the loss of groups II–IV .', 'We found that tauE alone was able to complement deletion 2 to a similar level as single tauE mutations but had little effect on the magnetic response of deletion 3 ( Figure 5 ) .', 'This indicates that while much of group II besides tauE is dispensable for magnetosome formation , there is another factor or factors in groups III and IV that are required for magnetosome synthesis in RS-1 but were not identified in this screen .', 'Due to their functional importance in the α-proteobacteria and their conservation in every magnetotactic bacterium investigated , we expected to find some mam gene mutants with non-magnetic phenotypes .', 'We isolated mutations in mamB , mamL , and mamQ .', 'MamB is a member of the cation diffusion facilitator ( CDF ) family , which exports divalent cations from cells .', 'In magnetosome synthesis MamB and another CDF protein , MamM , are thought to be involved in the transport of iron [36] .', 'MamB is also required for magnetosme membrane formation in AMB-1 [29] .', 'Five alleles of mamB were isolated , four with non-magnetic phenotypes .', 'As shown in Figure 6A , G20 is conserved among the δ-proteobacterial MamBs .', 'MamBG20D has a less severe phenotype than the other alleles of mamB suggesting that this residue may not be required for MamB function , even among the δ-proteobacteria .', 'MamBC9Y is of particular interest as the mutated cysteine , along with neighboring residues C6 and K2 , is conserved across all magnetosome MamB proteins , but varies among CDF family homologs from other species ( Figure 6A ) .', 'Together with the non-magnetic phenotype of MamBC9Y , this conservation pattern suggests the amino-terminus of MamB is important to its role in magnetosome formation .', 'We isolated three mamL alleles , which initially appeared to have no magnetic response , as shown in Figure 5 .', 'However , occasional cultures of all three mamL mutants had a Cmag of about 1 . 05 , nearing our limit of detection .', 'We interpret this to mean that these mutants are so impaired in magnetosome formation that only rare cultures had enough magnetic cells to produce a measureable Cmag .', 'This is supported by the rare particles observed in some cells of mamL allele 1 by TEM ( Figure S2 ) .', "Though no particles were observed for the other alleles of mamL , these strains' occasional magnetic response implies that crystals can be present , though not observed in our experiments .", 'In the α-proteobacteria , mamL deletion results in complete loss of crystals and magnetosome membranes [29] .', 'Together , this suggests that either all three alleles isolated for mamL retain partial function , or mamL is not required for the synthesis of magnetite in RS-1 .', 'The mamL alleles are all point mutations .', 'As shown in Figure 6B , the I37 position contains mainly aliphatic amino acids , but in mamLI37F an aromatic phenylalanine is at this position .', 'RS-1 E63 is in a group of acidic amino acids that are present only in δ-proteobacterial MamLs , but in mamLE63K this acidic stretch is disrupted with a basic lysine residue .', "The conservation pattern of this acidic stretch and its phenotype in RS-1 points to this area of the protein as being important for MamL's ability perform δ-proteobacteria-specific functions .", 'The mad genes were recently identified using comparative genomics .', 'They are defined as being conserved uniquely in the MAI regions of magnetotactic δ-proteobacteria [11] .', 'It was hypothesized that due to this conservation pattern they must be important for magnetosome formation in the δ-proteobacteria .', 'However , no mad gene phenotype has been observed and nothing is known about their activity or function .', '31 mad genes were identified , and RS-1 has homologs of 25 of them [6] , [11] .', 'We were interested in whether the screen uncovered mutations in any mad genes that resulted in non-magnetic phenotypes .', 'Indeed , mutants of mad1 ( DMR_41150 ) , mad2 ( DMR_41120 ) , and mad6 ( DMR_41050 ) were isolated .', 'This is the first demonstration of phenotypes for mad genes .', 'The mad6 mutant had a limited magnetic response .', 'Mad6 consists mostly of a NapH nitrate reductase domain , though RS-1 does not reduce nitrate [37] .', 'In MSR-1 , deletions of genes affecting nitrate reduction have effects on magnetite biomineralization [38] , [39] .', 'Mutations of mad1 and mad2 resulted in cells with no magnetic response .', 'Mad1 and Mad2 have no obvious homology to any characterized proteins .', 'Mad1 contains three CXXCH heme-binding motifs .', 'Two adjacent CXXCH motifs is a feature common to a number of magnetosome proteins , and has been termed the magnetochrome domain [40] , [41] .', 'The sequences of Mad1 and Mad6 suggest they both perform redox functions , a feature found in a number of magnetosome proteins from the α-proteobacteria .', 'This aspect of the δ-proteobacterial genes mad1 and mad6 , as well as fmpB ( described in the discussion ) , suggest that redox activity is just as important to magnetosome synthesis , if not more so , in the δ-proteobacteria .', 'Given the unique morphology of RS-1 crystals , we were particularly interested in mutants that produced misshaped magnetic particles .', 'Thus , we examined the mutants with whole-cell TEM .', 'As described in Table 1 and shown in Figures 7 and S2 , some non-magnetic mutants contained no electron-dense particles .', 'This suggests that MamB , MamQ , and TauE are absolutely required for magnetite synthesis .', 'As expected , mutants with low Cmag values were found to contain electron-dense particles consistent with magnetite .', 'As observed above for mamL allele 1 , the shape , size , and organization of these particles appear similar to WT; however the number of particles per cell was much lower ( Figures 7 , S2 ) .', 'In mutant cultures with Cmag values above one , most cells had no particles .', 'Those mutant cells that had particles contained between one and four per cell , while WT RS-1 cells have 10+/−4 crystals per cell .', 'These results indicate that Kup , MamL , Mad6 and FmpB contribute to efficient magnetite formation , but that WT-like crystals can be produced at a lower rate if they are impaired .', 'In contrast , mad1 and mad2 mutants and cells with fmpA allele 2 contained rare , electron-dense particles even though they have no magnetic response .', 'These particles appeared less elongated than in the WT ( Figure 7 mad1 ) , and they sometimes had sharp corners or jagged-looking edges ( Figure 7 mad2 allele 3 ) .', 'We wondered why some mutants , such as the mad1 mutant , have no magnetic response despite the presence of electron-dense particles .', 'To detect if the particles in mad1 mutants are magnetite , we performed high-resolution transmission electron microscopy ( HRTEM ) on these samples , finding small , multi-domain crystals ( Figure 8A ) .', 'The different angles of the lattice lines in Figure 8A indicate that the crystal is composed of multiple domains , and the crystal lattice line spacing indicates that the crystal is magnetite .', 'This is reminiscent of the phenotype of a deletion of mamS , a gene found in AMB-1 but absent in RS-1 , where many small crystals are found together in one magnetosome membrane [29] .', 'These results suggest that Mad1 may regulate crystal nucleation , limiting each developing crystal to one nucleus , or that it functions to cull multi-nucleate crystals down to one as the crystals grow in size .', 'The multi-domain nature of the magnetite crystals in the mad1 mutant offers an explanation for why these cells are unable to turn in a magnetic field .', 'It is thought that the many small , aligned magnetic moments of the magnetite crystals in magnetotactic bacteria combine to produce a magnetic moment strong enough to turn the cells in a magnetic field [42] .', 'As the crystals of mad1 mutant cells are multi-domain , each domain may be too small to hold a stable dipole moment .', 'Or , if large enough , the unaligned magnetic moments may work against each other .', 'To observe the effect this has on the magnetic moment of the entire cell , we employed a wide-field optical magnetic microscope using nitrogen-vacancy ( NV ) color centers in diamond [43] to observe the magnetic field produced by WT and mutant cells .', 'This technique relies on the fluorescence produced by NV centers in diamond that have been spectroscopically probed by microwaves in order to visualize local magnetic fields under ambient conditions .'
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Model genetic systems are invaluable , but limit us to understanding only a few organisms in detail , missing the variations in biological processes that are performed by related organisms .', 'One such diverse process is the formation of magnetosome organelles by magnetotactic bacteria .', 'Studies of model magnetotactic α-proteobacteria have demonstrated that magnetosomes are cubo-octahedral magnetite crystals that are synthesized within pre-existing membrane compartments derived from the inner membrane and orchestrated by a specific set of genes encoded within a genomic island .', 'However , this model cannot explain all magnetosome formation , which is phenotypically and genetically diverse .', 'For example , Desulfovibrio magneticus RS-1 , a δ-proteobacterium for which we lack genetic tools , produces tooth-shaped magnetite crystals that may or may not be encased by a membrane with a magnetosome gene island that diverges significantly from those of the α-proteobacteria .', 'To probe the functional diversity of magnetosome formation , we used modern sequencing technology to identify hits in RS-1 mutated with UV or chemical mutagens .', 'We isolated and characterized mutant alleles of 10 magnetosome genes in RS-1 , 7 of which are not found in the α-proteobacterial models .', 'These findings have implications for our understanding of magnetosome formation in general and demonstrate the feasibility of applying a modern genetic approach to an organism for which classic genetic tools are not available .']
['Model organisms are used to understand biological processes in genetic and molecular detail .', 'However , some interesting processes lack model organisms , and many would benefit from their study in diverse organisms .', 'Bacteria are an enormously diverse domain of life that contains only a few well-studied model systems , making much of their interesting biology inaccessible to genetic or molecular research .', 'Here , we employ a method for performing genetic experiments on non-model organisms that takes advantage of new DNA sequencing technology to work around a lack of genetic tools .', 'We use this method on a non-model organism , Desulfovibrio magneticus RS-1 , a member of the magnetotactic bacteria , which construct intracellular nano-sized magnets .', 'The current understanding of magnetotactic bacteria comes from two model organisms that differ from RS-1 in the shape of their magnets and the manner in which the particles are produced .', 'Genetic control over the size and shape of minerals is a poorly understood process and its elucidation has implications for nanotechnological applications .', 'Here , without the need to develop extensive genetic tools for a fastidious organism , we have extended the understanding of the molecular basis of bacterial nano-magnet production .']
['genetics', 'cell biology', 'biology and life sciences', 'microbiology']