A Tailored galK Counterselection System for Efficient Markerless Gene Deletion and Chromosomal Tagging in Magnetospirillum gryphiswaldense

Abstract

cMagnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.

Genetic Dissection of the mamAB and mms6 Operons Reveals a Gene Set Essential for Magnetosome Biogenesis in Magnetospirillum gryphiswaldense

“…Markerless single gene deletions within the mamAB, mms6, and mamGFDC operons were partially realized with the pORFM_galK plasmid (59). The vector was digested with BamHI and KpnI to insert the approximately 1-kb downstream and upstream fragments of mamI, -L, -N, -P, -Q, -R, -S, -T, and -U, mms36, mms48, and mmsF_mms6.…”

The Terminal Oxidase cbb 3 Functions in Redox Control of Magnetite Biomineralization in Magnetospirillum gryphiswaldense

“…First, the fused flanking sections of operons encoding cbb 3 , aa 3 , and bd oxidases were cloned into PstI/SpeIdigested pOR093 to yield pLYJ128, pLYJ129, and pLYJ130, respectively. Unmarked deletions of cbb 3 , aa 3 , and bd oxidase operons were performed by a two-step homologous recombination technique in the same manner as that described previously (41). After PCR screening, mutants were generated and finally designated the ⌬cbb 3 , ⌬aa 3 , and ⌬bd mutants, respectively.…”

Polarity of bacterial magnetotaxis is controlled by aerotaxis through a common sensory pathway

“…After digestion the fragment was ligated into pOR093 to obtain pFP11. This plasmid was used to obtain strain FP56 through a two-step homologous recombination method using galK-based counterselection 52 .…”