A method for genome editing in the anaerobic magnetotactic bacterium Desulfovibrio magneticus RS-1

ABSTRACT Magnetosomes are complex bacterial organelles that serve as model systems for studying cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacterial Magnetospirillum spp. that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have also been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing Deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified ten genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed many interesting mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration has not been feasible due to low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria. IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form small, intracellular magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membrane, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization as well as potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply-branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from the cubooctahedral-shaped magnetite crystals of the genetically tractable Alphaproteobacteria MTB. Here, we present a method for genome editing in the anaerobic Deltaproteobacterium Desulfovibrio magneticus RS-1, the first cultured representative of the deeply-branching MTB. This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB.

genetic methods of suicide vector integration has not been feasible due to low 26 transconjugation efficiency. Here, we present a reverse genetic method for targeted 27 mutagenesis in D. magneticus using a replicative plasmid. To test this method, we 28 generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the 29 upp gene that encodes uracil phosphoribosyltransferase. We also used this method for 30 targeted marker exchange mutagenesis by replacing kupM, a gene identified in our 31 previous screen as a magnetosome formation factor, with a streptomycin resistance 32 cassette. Overall, our results show that targeted mutagenesis using a replicative 33 plasmid is effective in D. magneticus and may also be applied to other genetically 34 recalcitrant bacteria. MTB ideal models for the study of compartmentalization and biomineralization in 69 with pAK914 was passaged two times in liquid media containing no antibiotic and plated 165 on 1% sucrose. Individual sucrose resistant (Suc r ) colonies were inoculated and 166 screened for kanamycin sensitivity (Kan s ). All isolated colonies (n=16) were Kan s , 167 suggesting that the cells had lost the plasmid. These experiments demonstrate that 168 sacB is a suitable counterselection marker in D. magneticus. 169

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Construction of a Δupp strain by markerless deletion. To test our replicative 171 deletion method, we chose to target the upp gene, the mutation of which has a 172 selectable phenotype. The upp gene encodes uracil phosphoribosyltransferase 173 (UPRTase), a key enzyme in the pyrimidine salvage pathway that catalyzes the reaction 174 of uracil with 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to UMP and PP i (34) (Fig.  175   3A). When given the pyrimidine analog 5-fluorouracil (5-FU), UPRTase catalyzes the 176 production of 5-fluoroxyuridine monophosphate (5-FUMP). 5-FUMP is further 177 metablized and incorporated into DNA, RNA, and sugar nucleotides resulting in 178 eventual cell death (Fig. 3A)  functional as detected by the sensitivity of D. magneticus to 5-FU (Fig. 3B, Fig. 4A). To 183 show that the upp gene product confers 5-FU sensitivity and to validate our replicative 184 To construct a upp deletion vector, a markerless cassette containing the regions 188 upstream and downstream of the upp gene were inserted into plasmid pAK914 (Fig.  189   2B). The resulting plasmid (pAK1126) was transferred to WT D. magneticus and a non-190 magnetic strain (ΔMAI) (19) by conjugation and single, kanamycin resistant (Kan r ) 191 colonies were isolated and passaged in growth medium containing no antibiotic. After 192 the third passage, upp mutants that had lost the vector backbone were selected for with 193 5-FU and sucrose. Compared with a control plasmid (pAK914), over 10-fold more 5-FU 194 resistant (5-FU r ) mutants were obtained from pAK1126. PCR of the region flanking the 195 upp gene confirmed that the 5-FU r colonies from pAK1126 resulted in markerless 196 deletion of upp (Δupp) while 5-FU r colonies from pAK914 were likely the result of point 197 mutations (Fig. 3B, Fig. 3D). Similar to results obtained for D. vulgaris Hildenborough 198 (31), the Δupp mutant of D. magneticus was able to grow in the presence of 5-FU (Fig. 199 4B, Table 2). Complementation of the upp gene in trans restored UPRTase function 200 and cells were no longer able to grow with 5-FU (Fig. 2C, Fig. 4C 10 replace a gene with a known phenotype, kupM (DMR_40800), with a streptomycin-208 resistance gene cassette (strAB). kupM is located in the D. magneticus MAI and 209 encodes a functional potassium transporter (19). Mutant alleles in kupM, including 210 missense, nonsense, and frameshift mutations, were previously identified in our screen 211 for non-magnetic mutants (19). These kupM mutations resulted in cells that rarely 212 contained electron-dense particles and were unable to turn in a magnetic field, as 213 measured by the coefficient of magnetism or C mag (19). 214

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To mutate kupM, we inserted a marker-exchange cassette, with regions upstream and 216 downstream of kupM flanking strAB, into pAK914 ( Fig. 2D) to create the deletion 217 plasmid pAK941. Following conjugation, single colonies of D. magneticus with pAK941 218 were isolated by kanamycin selection. After three passages in growth medium without 219 selection, potential mutants were isolated on plates containing streptomycin and 220 sucrose. Single colonies (n=48) that were streptomycin resistant (Str r ) and Suc r were 221 inoculated into liquid medium and screened for Kan s . Of the ten isolates that were Kan s , 222 two had the correct genotype (ΔkupM::strAB) as confirmed by PCR and sequencing 223 ( Fig. 3C, Fig. 3E). 224 225 Similar to the phenotypes previously observed in kupM mutants (19), ΔkupM::strAB 226 cells were severely defective in magnetosome synthesis and ability to turn in a magnetic 227 field (Fig. 5). Though a slight C mag could be measured, few cells contained electron-228 dense particles or magnetosomes. Importantly, the WT phenotype was rescued by 229 expressing kupM on a plasmid in the ΔkupM::strAB mutant (Fig. 5). These results 230 11 confirm that the replicative deletion plasmid method described here can be used 231 successfully for marker exchange mutagenesis. 232

DISCUSSION 234
In this study, we expand the genetic toolbox of D. magneticus to include a replicative 235 plasmid method for targeted mutagenesis (Fig. 1C). We show the utility of this method 236 for markerless deletion of genes with a selectable phenotype and for marker exchange 237 mutagenesis. Some of the earliest examples of targeted mutagenesis in Gram-negative 238 bacteria used replicative plasmids, similar to the method described here. (33,38). 239 These studies, which predated the application of suicide vectors, relied on plasmid 240 instability by introducing a second plasmid of the same incompatibility group or by 241 limiting nutrients in the growth medium (33,38). 242 243 Because the D. magneticus genetic toolbox has a limited number of plasmids, antibiotic 244 markers, and narrow growth constraints, we used a replicative plasmid and established 245 sacB as a counterselection marker to generate and isolate mutants. While sacB 246 counterselection was ultimately successful, a large number of false-positives were also 247 isolated at the sucrose selection step. Mutations in sacB have been found to occur at a 248 high frequency in many bacteria (30,(39)(40)(41)(42). Indeed, we found that deletions and 249 mutations in P mamA _sacB are abundant in the false-positive Suc r Str r isolates (data not 250 shown). Alternative counterselection markers, including upp, have been shown to select 251 for fewer false-positives (31, 42-44). Since D. magneticus is sensitive to 5-FU only 252 when the upp gene is present (Fig. 4), the upp mutants generated in this study may be 12 used as the parent strains for future targeted mutagenesis using upp as a 254 counterselectable marker rather than sacB. Additionally, the combined use of upp and 255 sacB for counterselection could reduce the false-positive background that results from 256 the accumulation of mutations in these markers. 257

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The replicative deletion plasmid described here is designed to replace a target gene 259 with an antibiotic resistance marker. As such, the construction of strains with multiple 260 directed mutations will be complicated by the need for additional antibiotic-resistance 261 markers, which are limited in D. magneticus. These limitations may be overcome by 262 removing the chromosomal antibiotic marker in subsequent steps (33,45,46). To what extent are lipid membranes involved in forming these crystals? How is the 283 elongated and irregular crystal shape achieved? Finally, in addition to D. magneticus, 284 the method described here may extend to other bacteria that are not amenable to 285 targeted mutagenesis with suicide vectors but are able to accommodate replicative 286 plasmids. 287

MATERIALS AND METHODS 289
Strains, media, and growth conditions. The bacterial strains used in this study are 290 listed in Table 1. All E. coli strains were cultured aerobically with continuous shaking at 291 250 RPM at 37ºC in lysogeny broth (LB). D. magneticus strains were grown 292 anaerobically at 30ºC in sealed Balch tubes with a N 2 headspace containing RS-1 293 Growth Medium (RGM) that was degassed with N 2 , unless otherwise stated (50). 294 Sodium pyruvate (10 mM) was used as an electron donor with fumaric acid disodium 295 (10 mM) as the terminal electron acceptor. RGM was buffered with Hepes and the pH 296 was adjusted to 6.7 with NaOH ( and GoTaq (Promega, USA) DNA polymerases were used with the primers listed in 312 Table S1. All upstream and downstream homology regions were amplified from D. 313 magneticus genomic DNA. strAB and P npt were amplified from pBMS6 and pLR6, 314 respectively, and subcloned into pBMC7 to make pAK920 which served as the template 315 for amplifying P npt _strAB for the deletion vectors. sacB was amplified from pAK0 and 316 inserted into pLR6 digested with SalI and XbaI to create pAK914. To construct a 317 plasmid for the targeted deletion of upp (DMR_08390), 991 bp upstream and 1012 bp 318 downstream of upp were amplified and inserted into pAK914 digested with XbaI and 319 SacI using a 3-piece Gibson assembly. To create the upp complementation plasmid, 320 pAK914 was digested with BamHI and SacI and the upp gene, with its promoter, were 321 PCR amplified from D. magneticus genomic DNA. To construct pAK941 for marker 322 exchange mutagenesis of kupM, a cassette of 1064 bp upstream region and 1057 bp 323 downstream region flanking P npt _strAB was assembled using Gibson cloning. The 324 cassette was amplified and inserted into pAK914 digested with XbaI using a two-piece 325 Gibson assembly. 326 327 upp and kup mutant generation and complementation. Replicative deletion 328 plasmids were transformed into E. coli WM3064 by heat shock and transferred to D. 329 magneticus by conjugation, as described previously (19). Single colonies of Kan r D. 330 magneticus were isolated and inoculated into RGM containing no antibiotic. Cultures 331 were passaged three times and spread on 1% agar RGM plates containing either 50 332 μg/ml streptomycin and 1% sucrose or 2.5 μg/ml 5-FU and 1% sucrose. Single colonies 333 were screened for Kan s and by PCR using the primers listed in Table 2 (2.5 μg/ml in 0.1% DMSO) or DMSO (0.1%) and growth was measured for WT and 344 Δupp strains with an empty vector (pAK914) and for the Δupp strain with the 345 to minimum absorbances was calculated as the C mag (10). Whole-cell transmission 350 electron microscopy (TEM) was performed as previously described (50) Double recombination can occur in one step after plasmids are linearized by 526 24 endogenous restriction enzymes. Mutants are selected using the marker (e.g. strAB) 527 that was exchanged with the target gene. (B) Two-step double recombination is 528 possible when suicide vectors integrate into the chromosome in the first homologous 529 recombination event and then recombine out after the second homologous 530 recombination event. The first step and second step are selected for with antibiotic 531 resistance markers (e.g. npt) and counterselectable markers (e.g. sacB), respectively. 532 (C) A replicative deletion plasmid designed to target genes for deletion may undergo 533 double recombination in one or two steps as shown in A and B, respectively. After 534 passaging the cells without antibiotic, mutants are selected with an antibiotic resistance 535 cassette (e.g. strAB) and a counterselectable marker (e.g. sacB). mob, mobilization 536 genes (mobA', mobB, mobC) and oriT; npt, kanamycin-resistance gene; ori Dm , origin of 537 replication for D. magneticus; ori Ec , origin of replication for E. coli.   Mutants are selected using the marker (e.g. strAB) that was exchanged with the target gene. (B) Two-step double recombination is possible when suicide vectors integrate into the chromosome in the first homologous recombination event and then recombine out after the second homologous recombination event. The first step and second step are selected for with antibiotic resistance markers (e.g. npt) and counterselectable markers (e.g. sacB), respectively. (C) A replicative deletion plasmid designed to target genes for deletion may undergo double recombination in one or two steps as shown in A and B, respectively. After passaging the cells without antibiotic, mutants are selected with an antibiotic resistance cassette (e.g. strAB) and a counterselectable marker (e.g. sacB). mob, mobilization genes (mobA', mobB, mobC) and oriT; npt, kanamycin-resistance gene; oriDm, origin of replication for D. magneticus; oriEc, origin of replication for E. coli.