In a recent paper by Shachrai at al. (which I discussed in detail here) a SpoT knock-out E. coli strain was reported, even though it is known that this strain is lethal both in E. coli and in Legionella pheumophilia. The good news is that now it is all cleared up.
The authors of the original paper wrote an erratum saying that yes, their SpoT knock-out was not just a SpoT knock-out; it has compensatory mutations. More specifically, RelA (which makes the brunt of the ppGpp in the cell) was compromised by mutations involved in the auto-regulation of its ppGpp-producing activity.
This incident brings up a question of validity of various knock-out strains that are so easy to make using a PCR product homologous to the target sequence in the genomic DNA, and therefore are so widely used.
First are the compensatory mutations, as it happened in the Shachrai paper. Here they had compensatory mutation in RelA, but it is the only one? May be there are more? P1 phage transduction in the clean background is the way to go, and as prices go down, whole-genome sequencing of the resulting construct is a good control.
Second are the downstream effects of the gene you knocked-out. In bacteria genes are arranged in operones, and knocking out one you mess up expression of the following one. In the case of SpoT knock-out in Shachrai at al. knocking out the whole SpoT ORF results in messing with rpoZ, the omega subunit of the RNA polymerase. This surely can not be good. Therefore the common practice is to insert something inside the ORF rather than substitute the whole thing altogether.
Tricky business.
References:
Shachrai, I., Zaslaver, A., Alon, U., & Dekel, E. (2010). Cost of Unneeded Proteins in E. coli Is Reduced after Several Generations in Exponential Growth Molecular Cell, 38 (5), 758-767 DOI: 10.1016/j.molcel.2010.04.015
Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, & Cashel M (1991). Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. The Journal of biological chemistry, 266 (9), 5980-90 PMID: 2005134
Datsenko KA, & Wanner BL (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97 (12), 6640-5 PMID: 10829079
Gropp M, Strausz Y, Gross M, & Glaser G (2001). Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. Journal of bacteriology, 183 (2), 570-9 PMID: 11133950
Gentry DR, & Burgess RR (1989). rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT. Journal of bacteriology, 171 (3), 1271-7 PMID: 2646273
Zusman T, Gal-Mor O, & Segal G (2002). Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. Journal of bacteriology, 184 (1), 67-75 PMID: 11741845
Mendeley group on stringent response
The authors of the original paper wrote an erratum saying that yes, their SpoT knock-out was not just a SpoT knock-out; it has compensatory mutations. More specifically, RelA (which makes the brunt of the ppGpp in the cell) was compromised by mutations involved in the auto-regulation of its ppGpp-producing activity.
This incident brings up a question of validity of various knock-out strains that are so easy to make using a PCR product homologous to the target sequence in the genomic DNA, and therefore are so widely used.
First are the compensatory mutations, as it happened in the Shachrai paper. Here they had compensatory mutation in RelA, but it is the only one? May be there are more? P1 phage transduction in the clean background is the way to go, and as prices go down, whole-genome sequencing of the resulting construct is a good control.
Second are the downstream effects of the gene you knocked-out. In bacteria genes are arranged in operones, and knocking out one you mess up expression of the following one. In the case of SpoT knock-out in Shachrai at al. knocking out the whole SpoT ORF results in messing with rpoZ, the omega subunit of the RNA polymerase. This surely can not be good. Therefore the common practice is to insert something inside the ORF rather than substitute the whole thing altogether.
Tricky business.
References:
Shachrai, I., Zaslaver, A., Alon, U., & Dekel, E. (2010). Cost of Unneeded Proteins in E. coli Is Reduced after Several Generations in Exponential Growth Molecular Cell, 38 (5), 758-767 DOI: 10.1016/j.molcel.2010.04.015
Xiao H, Kalman M, Ikehara K, Zemel S, Glaser G, & Cashel M (1991). Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. The Journal of biological chemistry, 266 (9), 5980-90 PMID: 2005134
Datsenko KA, & Wanner BL (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97 (12), 6640-5 PMID: 10829079
Gropp M, Strausz Y, Gross M, & Glaser G (2001). Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. Journal of bacteriology, 183 (2), 570-9 PMID: 11133950
Gentry DR, & Burgess RR (1989). rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT. Journal of bacteriology, 171 (3), 1271-7 PMID: 2646273
Zusman T, Gal-Mor O, & Segal G (2002). Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. Journal of bacteriology, 184 (1), 67-75 PMID: 11741845
Mendeley group on stringent response
yohoo!
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