Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense

Abstract

Background: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain.

Results: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content.

Conclusions: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Keywords: Genome reduction; Magnetosomes; Magnetospirillum gryphiswaldense.

Fig. 1

Overview over generated MAI and MAI-adjacent mutants in M. gryphiswaldense. Regions R1, R3, R5 and R7 indicate the five key operons (brown, green, violet, red, grey) for magnetosome biosynthesis while R2, R4, R6 and R8 represent intervening and MAI-adjacent regions. Grey bars show the extensions of successful deletions, while connecting lines indicate non-deleted parts. Magenta color highlights the strain ∆A13 from Lohße et al. (2011) which served as parental strain for ∆M01 and ∆M03. Dashed bars show attempted deletions which failed. Genes with irrelevant functions for magnetosome biosynthesis are shown as black arrows, and transposable elements are shown in blue. The yellow bar indicates the extent of deletion (~ 12.8 kb) that had not been covered by previous approaches [28, 29]. The genotype of the five contiguous magnetosome biosynthesis operons compact cassette pTpsMAG1 is shown in the lower line containing regions R1, R3, R5 and R7 (brown, green, violet, red, grey)

Fig. 2

Results of genome re-sequencing of a typical false positive clone isolated during attempts to delete a ~ 68 kb region (M08). The red box indicates an unintended deletion of ~ 47 kb located between MSR1_03260 and MSR1_03780 (nt position 360,736). Brackets indicate parts of the remaining suicide vector with the Km^r marker and the galK gene inactivated by a spontaneous insertion of a tandem IS element (green arrows), as well as an unintended insertion of several mam genes (mamH, mamI and parts of mamE) next to the remnants of the suicide vector

Fig. 3

Phenotypes of non-magnetic mutant strains with largest deletion extents and their respective complemented strains with restored magnetosome biosynthesis. Mutants ∆M04 and ∆M13 are non-magnetic, while complemented mutants show WT-like magnetosome formation. Arrows indicate electron dense particles (EDPs) in mutant strains. Scale bars: left column, 500 nm; right column, 100 nm

Fig. 4

Growth characteristics of different mutants. Provided are the growth curves of non-magnetic mutant strains with largest deletion extents and their respective complemented strains with restored magnetosome biosynthesis. Growth curves show ∆M04 (a), ∆M13 (b) and its respective complemented mutants under different growth conditions in comparison to the WT. Growth of the WT is shown in diagrams for both mutants. Each strain was analyzed in technical triplicates, and growth curves represent the average while standard deviation was below 5%

Fig. 5

XEDS spectra and TEM micrographs (insets) of individual EDPs. EDPs were found in deletion strains ∆M01–∆M05 and ∆M13. Exemplary the mutants ∆M03 and ∆M05 are shown. Spectra indicate that EDPs are rich in potassium, phosphorus and oxygen, while no significant amounts of iron could be detected

Fig. 6

Schematic overview over the chromosomal positions of single deletions in this study. The yellow circle shows genes or gene sets targeted for deletion. Grey: MAI; red: M13 deletion; green: putative candidate genes for magnetosome biosynthesis outside the MAI [31, 32]