All data in this paper should be discarded simply because the strains used by the authors are not what the authors think they are, as explained further. An Escherichia coli strain lacking PEP synthase does not grow on pyruvate. In fact, the crucial role of PEP synthase during growth on pyruvate is well documented. In brief, mutant strains were isolated which could grow on glucose or acetate, but not on pyruvate; it was found they lacked PEP synthase (see Cooper RA, 1967 for an early paper). Furthermore, because the PEP synthase gene (ppsA) is transcriptionally positively regulated by the fructose repressor FruR (Geerse RH, 1986, also known as Cra), fruR mutant strains are routinely checked for their inability to grow on pyruvate. Therefore, data in supplementary Fig. 1 indicating wild type and ppsA strains grow equally well on pyruvate are incorrect; the strain used by the authors is not a ppsA strain. The ptsI strain also does not appear to be a ptsI strain, as it grows on xylose as well as a wild type strain (Figure 3b), it should not; growth on xylose requires cAMP, which requires the phosphorylated form of Enzyme IIAGlc.
Proc Natl Acad Sci U S A. 2017The cya crp* mutant strain CA-8404 isolated by L. Soll (Sabourin D, 1975), which has been widely used for transduction of its crp* gene (also used in the present work), was finally characterized by Karimova G, 2004 as containing three mutations in the crp gene. Karimova G, 2004 further reported this CRP* was indeed capable of responding to cAMP and therefore was still sensitive to Carbon Catabolite Repression (CCR). Of interest to the present study, and any other studies aimed at releasing CCR in Escherichia coli, Karimova G, 2004 also characterized a novel CRP* with two mutations which totally relieved CCR as compared to the three-mutation CRP*.
Here cAMP-dependent CCR may not be the main culprit for preventing xylose utilization by the E. coli wild type W strain (the Waksman’s strain). Generally, an increase in cAMP upon glucose depletion allows utilization of less-preferred sugar like xylose. Figure S7-A indicates that upon glucose exhaustion, E. coli W was still unable to use xylose after 96 hours, even though it could use xylose quite efficiently in the absence of glucose (Figure 2-D). In addition, the CRP* isolated by the authors (G141D) for specifically increasing xylose catabolism (Figure 1), and which doubled xylose utilization in the parent strain XW043, did not improve E. coli W xylose consumption at all in the presence of glucose (Figure 4-A). Thus, based on current knowledge, the inability of the Waksman’s strain to use xylose in the presence of a large excess glucose does not appear to relate to cAMP-dependent CCR. Interestingly, E. coli B, unlike W, is unable to use glucose fully when grown in excess glucose, and the typical increase in cAMP does not occur after cessation of growth (Figure 2 in Peterkofsky A, 1971). Thus, if some glucose remains unused in the medium, cells may fail to use xylose.
J Bacteriol. 2017The authors have previously reported that very little SgrT is made in Escherichia coli as compared to Salmonella typhimurium, which led them to conclude that E. coli K12 has "lost the need for SgrT" (Wadler CS, 2009). Later on, the rationale for using S. typhimurium instead of E. coli for studying SgrT was reinforced (Balasubramanian D, 2013). In the present work, the authors use E. coli sgrS mutant strains overproducing SgrT. Therefore, the present work does not establish a 'physiological' role for SgrT in preventing the E. coli PTS-transport of glucose, thus the title of the article is misleading.
The authors’ interpretation of Figure 1 does not agree with the following data. E. coli mutant strains lacking Enzyme IICBGlc (PtsG) do grow on glucose (see Curtis SJ, 1975; Table VIII in Stock JB, 1982). Mutant strains lacking both the glucose and mannose enzyme II grow very slowly on glucose. In other words, because growth had been observed on mannose, growth should have been observed on glucose. Furthermore, the authors should have been aware that an increased level of cAMP from overexpressing SgrT further impairs growth on glucose.
PtsG is not "comprised of three main functional domains", as the authors state. PtsG has two functional domains (IIB and IIC) connected by a flexible linker. In the nomenclature for PTS proteins (Saier MH Jr, 1992), PtsG translates into Enzyme IICBGlc which is informative (and therefore should be preferred to any other designations) because it indicates a two-domain structure, a specificity for glucose, and the order of the domains (from N to C terminus).
Kosfeld A, 2012 clearly established, by cross-linking experiments, the interaction between SgrT and Enzyme IICBGlc in the presence of glucose. They also visualized the recruitment of SgrT to the membrane by in vivo fluorescence microscopy. It is therefore unwarranted for the authors to 'hypothesize' an interaction and localization to the membrane, and to state: "Once we established that SgrT inhibits PtsG specifically and its localization to the membrane …". In addition, the demonstration by Kosfeld A, 2012, the motif KTPGRED (in the flexible linker) is the main target for SgrT, is rather convincing.
Finally, the statement "SgrT-mediated relief of inducer exclusion may allow cells experiencing glucose-phosphate stress to utilize alternative carbon sources" is inaccurate because it ignores the positive effect of cAMP on the utilization of alternative carbon sources like lactose.
Sci Rep. 2017The terminology 'induction prevention' does not apply to cAMP-dependent Carbon Catabolite Repression (CCR). It has been used to illustrate CCR in Bacillus subtilis, see Figure 2 in Görke B, 2008. Escherichia coli cAMP-dependent CCR does not cause induction prevention. It is therefore incorrect to state: "The current model for glucose preference over other sugars involves inducer exclusion and induction prevention, both of which are strictly dependent on the phosphorylation state of EIIAGlc in E. coli". Moreover, the major player in induction prevention is HPr, not EnzymeIIAGlc.
Some referenced papers were misinterpreted by the authors. Lengeler J, 1972 reported, in wild type cells, induction of the mannitol operon 'is not prevented' by glucose. Lengeler J, 1978 reported expression of the mtl operon, in both constitutive and inducible strains, is 'resistant to CCR' caused by glucose. This expression was nearly insensitive to cAMP addition, even though expression of the mtl operon is dependent on cAMP (a cya mutant strain does not grow on mannitol). Hence, the level of cAMP in glucose-growing cells is probably sufficient for expression of the mannitol operon. It is unclear how Lengeler J, 1978 monitored CCR with their inducible strains, as data were not shown.
It was proposed induction of the mannitol operon may take place in the absence of PTS transport as follows. In the unphosphorylated state, transport of mannitol by Enzyme IICBAMtl (MtlA) occurred by facilitated diffusion, upon high-affinity binding of mannitol to the IIC domain (Lolkema JS, 1990). Thus, the IIC domain appears as a transporter by itself translocating mannitol at a slow rate. This provides an explanation for the observations that (1) mutant strains lacking Enzyme I and/or HPr were still inducible by mannitol which originally led to the proposal mannitol may be the inducer of the mannitol operon (Solomon E, 1972), and (2) mutant strains lacking Enzyme IICBAMtl could not be induced unless mannitol was artificially generated in the cytoplasm. It was therefore concluded mannitol was the inducer of the mannitol operon. Interestingly, in the phosphorylated state, transport of free mannitol by Enzyme IICBAMtl can be detected on the condition that the transporter has a poor phosphorylation activity (Otte S, 2003).