In one of my experiment with wild type (WT), mutant strain and complemented strain (CS) of M. smegmatis, glycopeptidolipid (GPL) profile of mutant and CS are exactly matching, leaving a single band difference with WT, in thin layer chromatography (TLC) experiment. But surprisingly, the few relevant physiological features like, biofilm formation, pellicle formation and motility, are similar in WT and CS. That shows, the gene is ectopically expressing in the CS, but its corresponding GPL which is present in WT is missing in CS on TLC. Any suggestion please?
I have no experience with Mycobacterium, but when a complementation is not working, you should be aware that a secondary mutation might be present in the mutant. We observed this phenomenon in Pseudomonas aeruginosa.
Dear Pierre Cornelis, can you please send me a reference of P. aeruginosa. My GPL profiling of CS on TLC showing the same pattern as mutant strain which should ideally go with the WT. But the other physiological characteristics like, biofilm formation, pellicle formation, motility etc. are same as in WT. That means, complementation is working but somehow the ectopically expressed gene in CS or an influenced product from this gene is not printing on TLC.
Dear Satya,
We did not publish this, but when analyzing the transcriptome of a WT and an oxyR mutant of P. aeruginosa, we were surprised to see that many genes known to the quorum sensing dependent were also differentially expressed in the oxyR mutant. It appeared that we got a secondary mutation in the lasR egene which encodes the master regulator of the quorum sensing. Some publications also mention mutations in the LysR regulator MexT gene, which we described in a recent publication (Vercammen et al., 2015, J Bacteriology).
Dear Pierre Cornelis,
Although it looks like, mine and your observations in our respective fields are not similar. Mine is related to the complemented strain disparity with the WT in terms of TLC band but the physiological activities are same as WT. But your study was of course surprising but possible because one regulator may have role in various pathways. And that too a regulator like oxyR (if it is the same oxyR as in mycobacteria) which has wide role in AhpC (oxidative stress) regulation and antibiotic susceptibility. My question is still persist please assist me if find any clue. Thank you very much.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC107510/
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