To establish a growth curve, 50 ml batch cultures of wild type and mutant were maintained in air and in air supplemented with 5% CO2.
To determine the secretion requirements for TseL, V52 and an isogenic tap-1 mutant were maintained in LB broth until they reached the mid-logarithmic phase of growth.
Virginia wild-type and omp10:: himutantutant were maintained in tick ISE6 cells derived from embryonated eggs of the blacklegged tick, Ixodes scapularis at 34°C in non-vented 25-cm cell culture flasks (NUNC).
The RMSDs of both the wild-type and M185A mutant were maintained at 2.8 and 3.1 Å for 20 ns, respectively, indicating that (i) both sequences were considerably stable and (ii) replacement of Met185 with Ala has a little influence on the overall stability of dimeric CTDs.
We determined a 'cheating' mutant to be a non-producer which (1) had lower fitness (grew less well) than the producer genotype in monoculture, and (2) gained a growth advantage in mixed culture, such that it grew as well or better than the wild type, that is, the mutant was maintained in the population and may have increased in frequency.
Interestingly, at these two pH values, the Asp282-Cys283 peptide bond in the H251A mutant was maintained in cis, but in the H284A mutant, this peptide bond was in trans, indicating that the H251A mutant still retained GlcNAc binding activity, but the H284A mutant has lost its binding activity for GlcNAc (Table 2).
When the tlc1 mutant was maintained on solid plates, cells displayed sensitivity to bleomycin from the first streak, and they showed much lower viability on bleomycin plates for the following restreaks (Figure 3A, top), even after Y' amplification was generated at the 3rd restreak, as it was evident by Southern blot analysis (Figure 3B, top).
Molecular analysis of reisolated ben1-1 lineshoweded that the unstable state of the ben1-1 mutatinn in the triple mutant is maintained in an otherwise wild-type background.
The mutant was maintained as a triple homozygous, single heterozygous stock: two different parental genotypes were used, heterozygous for either LUT5/CYP97A3 or CHY2, with similar results (Additional file 1: Figure S2).
This is consistent with previous studies [ 17, 23, 29] which also identified that the I130T mutant retained the ability to form gap junction plaques with residual coupling activity, whereas the G60S mutant was maintained mainly in an intracellular profile and functionally dead.
The parent strain and mutants were maintained on potato dextrose agar (HiMedia Mumbai, India) supplemented with 2 g l−l yeast extract.
