Fig 5. Engineering of the M. genitalium MG491 mutant strains.

(a) Schematic representation of the different MG491 alleles under the control of the MG438 promoter. These alleles were introduced in a plasmid containing a mini-transposon using the indicated restriction sites and electroporated into Δmg491 cells. Coding sequences for helices α1, α2 and α3 and loops L1 and L2 are indicated. The Cys87 to serine mutation is labeled with an asterisk (*) in the MG_491C87S allele. A double asterisk (**) is pointing to the F157A and F158A mutations in the MG491-F157A-F158A allele. Deletion of the 463–480 bp region from MG_491 coding for residues 155 to 160 is labeled with lines in the MG491-loopL2 allele. (b) Western blot analysis of G37 wild type cells, Δmg491 cells and cells from transformant colonies containing the alleles in (a) using a heterologous polyclonal serum anti-P41 from M. pneumoniae. Expression of MG491 is detected in wild type G37 cells and in the transformant cells except for the Δmg491 strain. A lower amount of MG491 was observed in the Δmg491-C87S strain and a band with an approximate molecular weight of 40 kDa was detected in the mg491-ΔNt strain.