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Escherichia coli adenylate cyclase homepage: Warning

Minst.org Warning Articles one is entitled not to read!
This section [Online March 20, 2009] lists Escherichia coli adenylate cyclase-connected articles which raise suspicion concerning the review process.  The data are either poor or not interpreted properly.  In some cases interpretation of data lacks understanding of prior work.  In worst cases data are manipulated, if not falsified, as to fit an agenda.  In any case these articles would not have appeared in their present form if reviewers had been both responsible and unbiased!

February 23, 2017
Nature Scientific Reports doi: 10.1038/srep25191 The lag-phase during diauxic growth is a trade-off between fast adaptation and high growth rate
Dominique Chu, David J. Barnes  
PubMed Commons

January 18, 2017
Journal of bacteriology doi: 10.1128/JB.00128-15 Acetate Exposure Determines the Diauxic Behavior of Escherichia coli during the Glucose-Acetate Transition
Enjalbert B, Cocaign-Bousquet M, Portais JC, Letisse F 
PubMed Commons

June 29, 2016
Nature Scientific Reports doi: 10.1038/srep24834 Glucose becomes one of the worst carbon sources for E. coli on poor nitrogen sources due to suboptimal levels of cAMP
Bren A, Park JO, Towbin BD, Dekel E, Rabinowitz JD, Alon U 
PubMed Commons

March 28, 2016
Journal of Bacteriology doi: 10.1128/JB.00008-16 Classic Spotlight: When Phenotypic Heterogeneity Met Carbon Catabolite Repression
Anke Becker  
PubMed Commons

November 23, 2015
Microbiology and Molecular Biology Reviews doi: 10.1128/MMBR.00038-15 The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite
Huergo LF, Dixon R 
PubMed Commons

July 21, 2015
Infection and Immunity doi: 10.1128/IAI.00411-15 A genome wide screen reveals that Vibrio cholerae phosphoenolpyruvate phosphotransferase system (PTS) modulates virulence gene expression
Wang Q, Millet YA, Chao MC, Sasabe J, Davis BM & Waldor MK 
PubMed Commons

April 29, 2015
Molecular Systems Biology DOI 10.15252/msb.20145537 A growth-rate composition formula for the growth of E. coli on co-utilized carbon substrates
Rutger Hermsen, Hiroyuki Okano, Conghui You, Nicole Werner & Terence Hwa 
PubMed Commons

February 3, 2015
Trends in Microbiology doi:10.1016/j.tim.2014.11.002 Understanding carbon catabolite repression in Escherichia coli using quantitative models
A. Kremling, J. Geiselmann, D. Ropers, and H. de Jong

Sorry, this review is not even worth commenting on.

June 14, 2014
Microbiology and Molecular Biology Reviews doi:10.1128/MMBR.00001-14 The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions.
Deutscher J, Aké FM, Derkaoui M, Zébré AC, Cao TN, Bouraoui H, Kentache T, Mokhtari A, Milohanic E, Joyet P.

The authors stated: "cAMP formation seems to affect the lag phase" and "addition of extracellular cAMP either shortens or entirely eliminates the lag phase".  In fact addition of cAMP does not always eliminate the lag phase, for example the glucose-galactose diauxie.

The authors also stated: "addition of extracellular cAMP cannot prevent CCR [Carbon Catabolite Repression]".  Of course not, as generally the cAMP-regulated transcription and inducer exclusion both contributes to CCR.  However the extent of these effects depends on the system considered.  Thus elimination of inducer exclusion cannot prevent CCR as well, as best exemplified by CCR of the glycerol regulon.  (Note: the reference to support the contention that exogenous cAMP cannot prevent CRR is not appropriate, the strain used in the referenced article expresses β-galactosidase constitutively).  Finally the contention that inducer exclusion is the major CCR mechanism in E. coli is based on a flawed paper by Inada et al. (see Diauxie correspondence)

The regulation of adenylate cyclase by phosphorylated Enzyme IIAGlc is poorly addressed as seen by the first reference to a paper by Krin et al.  That paper addresses the role of the histone-like protein H-NS on the cAMP levels, and its main conclusion is adenylate cyclase is not fully activated in a crp hns double mutant strain (lacking both CRP and H-NS) due to a reduced expression of crr which encodes EnzymeIIAGlc (a conclusion to be taken with caution considering the more than two-fold decrease in expression of the adenylate cyclase gene in the double mutant).  The statement that "low intracellular levels of cAMP are observed in E. coli cells grown on an efficiently metabolized carbon source" is erroneous, for example it does not apply to the PTS sugar fructose.  Also the contention that all PTS carbohydrates (one exception noted) are phosphorylated during their transport  might not be quite correct because certain sugars can be transported by facilitated diffusion without phosphorylation under specific conditions.

This review being released for the 50th anniversary of the PTS discovery by Kundig, Ghosh and Roseman, is disconcerting in that the pioneer's contributions was so poorly acknowledged.  In Conclusion and Perspectives, the authors stated: "The PTS was discovered 50 years ago in the laboratory of Saul Roseman at the University of Michigan, Ann Arbor, with the first article describing a PTS component, the HPr from E. coli, and its role in hexose phosphorylation being published in 1964".  In fact, the first article by Kundig, Ghosh and Roseman, besides characterizing the protein-bound phosphohistidine, remarkably indicated PEP (phosphoenolpyruvate) could act as the initial phosphoryl donor for their purified PTS proteins, D-sugars were PTS substrates and the product formed from glucose was glucose 6-phophate, and more than one Enzyme II could be synthesized by the cell.  Thus the landmark paper by Kundig, Ghosh and Roseman deserves recognition and should have been introduced with proper accolades.

November 23, 2013
Nature doi:10.1038/nature12446 Coordination of bacterial proteome with metabolism by cyclic AMP signalling
Conghui You, Hiroyuki Okano, Sheng Hui, Zhongge Zhang, Minsu Kim, Carl W. Gunderson, Yi-PingWang, Peter Lenz, Dalai Yan and Terence Hwa 
PubMed Commons

In an article by Doucette et al. [Nat Chem Biol], it is clearly demonstrated that the effect of α-ketoglutarate on cAMP synthesis is related to an inhibition of Enzyme I.  As a consequence phosphorylation of Enzyme IIAGlc is impaired which relates to a decrease in cAMP synthesis, and yet You et al. studied the effect of α-ketoglutarate in pts mutant strains.  Because "a strong transient repression was still observed upon the addition of α-ketoacids in strains with deletion of various PTS proteins" - as stated - You et al. basically conclude that the PTS proteins are no longer necessary for mediating the main effect by α-ketoglutarate but instead α-ketoglutarate directly affect the activity of adenylate cyclase.  Incidentally 10 mM α-ketoglutarate does not inhibit purified adenylate cyclase.

Be happy to know that "harsher treatment" - as stated - "may damage proteins" but "the results are meant to provide an alternative perspective"!  And the authors laughingly conclude: "Thus, the repression effect of α-ketoacids on AC [adenylate cyclase] activity is robust even in this harsh assay condition."  One may wonder, if using "harsh assay condition" does not make any difference, then the effect of α-ketoacids on AC activity seems to be quite questionable.  Next time the authors should try to boil their sample for a good amount of time, just to provide an 'alternative' control!

Some figures, for example figure S2, raise suspicion for their legitimacy considering the goodness of fit.  Supplemental material is often incomprehensible or erroneous.  Figure S4(c) gives a good example of such by ignoring data from Wanner et al. (1978).

The authors state: "…although PTS proteins do affect the degree of Crp-cAMP-mediated regulation of catabolic gene expression as is long known, they are not necessary for the existence of this response…".  Really?  The PTS proteins are not 'necessary' for the Crp-cAMP mediated regulation?  In other words, Enzyme IIAGlc, a PTS protein, is not 'necessary' for regulating the cAMP levels that is: is not 'necessary' for the cAMP-mediated transcriptional response via Crp.  This is contrary to well established data indicating that cAMP levels in Escherichia coli are generally dependent on the phosphorylation state of Enzyme IIAGlc which controls "the degree of Crp-cAMP-mediated regulation of catabolic gene expression as is long known".

September 28, 2013
Nucleic Acids Research doi: 10.1093/nar/gkt655 A genome-wide screen for identifying all regulators of a target gene
Baptist G, Pinel C, Ranquet C, Izard J, Ropers D, de Jong H, Geiselmann J.

A ptsI mutant strain of Escherichia coli does not grow on minimal medium glucose after 48 hours.  Therefore the ptsI mutant strain from the Keio collection which is reported to grow on glucose with growth defect (see Supplementary Information, S3 and S7) is not adequate.  Therefore it is irrelevant to state "… the cyaA and crr mutants should behave as the ptsI strain" (see Section Confirmation of known regulators of acs expression) because, unlike ptsI strain, cyaA and crr mutant strains grow on glucose, even though they do not grow as well as a wild type strain (note it is also reported in S3 that gltA and icd mutant strains do not grow on glucose but they actually do when supplemented with glutamate).  In addition under the experimental conditions described, it is not clear to which extent glucose is used preferentially over acetate by certain mutant strains particularly in the presence of cAMP (absorbance should have been provided in Figure 4C, D, E and F).  Also the authors seem to ignore that any mutant strains lacking any of the TCA cycle enzymes fail to grow on acetate as they wrote "Interestingly we found that the above mutants [sucB and lpdA] fail to grow on acetate."

Researchers are better off reading the 2002 paper by James C. Liao and colleagues titled "Global expression profiling of acetate-grown Escherichia coli" which investigates the transcript profile of an E. coli strain grown on acetate as compared to the profile of the same strain grown on glucose (surprisingly none of the Liao papers are cited by the authors).

Finally, and most importantly, the authors should re-consider analyzing "connections between the metabolic state of the cell and gene expression" by using luciferase as a reporter system considering that luciferase strongly depletes the pool of cellular ATP.  No wonder mutant strains lacking genes involved in energy supply display the lowest luciferase activity!  And yet the authors used a second reporter system to detect "artifacts due to metabolic influences on luciferase activity", as stated in the Introduction.

April 27, 2013
Applied and Environmental Microbiology doi: 10.1128/AEM.03871-12 Glucose Triggers ATP Secretion from Bacteria in a Growth-Phase-Dependent Manner
Hironaka I, Iwase T, Sugimoto S, Okuda K, Tajima A, Yanaga K, Mizunoe Y.

The authors "assumed that enterococcal species and bacteria belonging to other genera might secrete ATP during growth" (sic), and claimed glucose is essential for ATP secretion.  However it is not clear what the glucose concentration used by the authors was in some of their experiments, particularly Figure 7.  Readers of this article may 'assume' the glucose concentration used in the culture medium of Escherichia coli MC4100 was not too high (so to speak) considering the growth curve presented in Figure 7.  Indeed figure 7 indicates an OD600 of about 0.5 was finally reached after a whopping 10 hours in an unspecified medium.  LB medium was used in their studies - as stated in Materials and Methods - and therefore one may presume it was used for Escherichia coli but there is no way of knowing, really.  And why was ATP detected in RPMI medium in Figure 2A and not in Figure 2C?  Possibly in Figure 2C the authors 'deleted' glucose from the RPMI medium, we may think!

January 24, 2013
Nucleic Acids Research doi: 10.1093/nar/gks1207 Structures of the Escherichia coli transcription activator and regulator of diauxie, XylR: an AraC DNA-binding family member with a LacI/GalR ligand-binding domain
Lisheng Ni, Nam K. Tonthat, Nagababu Chinnam and Maria A. Schumacher

Introduction states: "With the broader goal of generating an E. coli biocatalyst that can co-metabolize all biomass sugars, it would be necessary to also eliminate the diauxie between D-xylose and L-arabinose, as these two sugars comprise 95% of the total sugar hemicellulose (6,7)."  Xylose and arabinose are two class B sugars for E. coli, as defined by Jacques Monod, and there is no report of a diauxic growth between xylose and arabinose even though E. coli was reported to preferentially utilize arabinose when grown in a mixture of xylose and arabinose.  Reference 6 reports that, in the presence of glucose, xylose and arabinose were simultaneously consumed in E. coli mutants impaired in Carbon Catabolite Repression (CCR), and reference 7, which suffers many flaws, is irrelevant to E. coli utilization of xylose and arabinose.

September 20, 2012
Molecular Microbiology DOI: 10.1111/j.1365-2958.2012.08161.x Corrigendum: Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2
Catarina S. Pereira, António J. M. Santos, Michal Bejerano-Sagie, Paulo B. Correia, Joao C. Marques and Karina B. Xavier

The corrigendum to 'the article one is entitled not to read' (see below June 01, 2012) brings a corrected reference [Xavier et al., 1996, J Bacteriol] in support of the contention that "trehalose is one of the most abundant carbohydrates in LB media".  The 1996 article by Xavier et al. does not however establish that trehalose is the 'most abundant' sugar in LB medium but establishes the presence of trehalose in yeast extract (the presence of other sugars in yeast extract was not analyzed) therefore the trehalose permease may not be one of the "most induced" PTS permease in LB medium - as stated in their article.  Furthermore, indicating that trehalose is the most abundant sugar in LB medium is misleading considering the carbon sources for E. coli in LB medium are catabolizable amino acids, not sugars [J Bacteriol].  The presence of trehalose in yeast extract solely indicates that, in LB medium, intracellular trehalose-6-phosphate, which results from the transport of trehalose by the phosphotransferase system, may act as an inducer under certain experimental conditions.

June 01, 2012
Molecular Microbiology DOI: 10.1111/j.1365-2958.2012.08010.x Phosphoenolpyruvate phosphotransferase system regulates detection and processing of the quorum sensing signal autoinducer-2
Catarina S. Pereira, António J. M. Santos, Michal Bejerano-Sagie, Paulo B. Correia, Joao C. Marques and Karina B. Xavier

Transcription of the lsrACDBFGE operon, which encodes a quorum sensing signal autoinducer-2 (AI-2) uptake and modification system, is positively regulated by CRP-cAMP.  The flanking lsrRK operon encoding the repressor LsrR and AI-2 kinase is regulated as well by CRP-cAMP.  Also, uptake of AI-2 occurs during entry into stationary phase (referred to as '4 hours post inoculation' by the authors) which is known to be related to a rise in cAMP.  However by using a cya crp* strain (supposedly insensitive to catabolite repression) to "avoid selecting mutants affected in lsr-lacZ regulation due to altered cAMP levels" the authors circumvent addressing the regulatory effects of CRP-cAMP which are most likely controlling AI-2 uptake and modification.  It is therefore not surprising that they conclude, in view of their unsuccessful attempts to explain their observations, that "the mechanism enabling PTS-dependent AI-2 uptake remains unknown".  Furthermore Pereira et al. apparently ignore cAMP regulations linked to catabolite repression are not fully 'restored' in the pts mutants they isolated, particularly because the typical increase in cAMP observed in stationary phase does not occur in such mutants.  So much for avoiding the selection of mutants affected in lsr-lacZ regulation due to altered cAMP levels!

All isolated mutants have growth defects, but as stated, "… these [growth defects] are insufficient to explain the strong phenotypes observed with respect to AI-2 internalization observed" and in the same sentence "but can explain the slight delay in AI-2 accumulation when compared with the lsrK mutant strain"!  And, even better, because "AI-2 internalization observed in a EI mutant cannot be exclusively explained by the loss of any single permease" the authors conclude "multiple permeases, regulated by the PTS, contribute additively to the transport of AI-2".  And if you want to know what the PTS is you’ll find out that it provides a mechanism for 'intracellular sequestration' of the so-called PTS-sugars.  Who said 'Research is what I'm doing when I don't know what I'm doing'?

February 28, 2012
Applied Microbiology and Biotechnology DOI: 10.1007/s00253-011-3857-3 A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli
Kuo-Chih Lin, Chih-Yuan Chen, Chih-Wei Chang, Kuo-Jien Huang, Shih-Pin Lin, Shih-Hung Lin, Ding-Kwo Chang, Meei-Ru Lin and David Shiuan

Connection with Escherichia coli adenylate cyclase: The dodecapeptide is reported to block HPr, one of the phosphotransferase system general proteins which indirectly regulates adenylate cyclase by phosphorylating Enzyme IIAGlc, See Ecach Chapter II ›››

Escherichia coli strain DH5α grows very poorly in minimal media [Appl Environ Microbiol].  It is therefore inappropriate to use this strain "to determine the effect of selected peptides on bacterial cell growth" - as stated.  In addition the contention by the authors that "binding of [the dodecapeptide] AP1 with HPr may block phosphotransfer and further phosphorylated enzymeIIA, thereby activate adenylate cyclase" is erroneous.  Blocking the phosphotransfer cannot result in adenylate cyclase activation.  And how a peptide which inhibits "the growth of Escherichia coli cells efficiently" can induce cell aggregation in the conditions described by the authors?

February 19, 2011
Journal of Bacteriology 2011, 193(3): 649 Novel Members of the Cra Regulon Involved in Carbon Metabolism in Escherichia coli
Tomohiro Shimada, Kaneyoshi Yamamoto, and Akira Ishihama

Connection with Escherichia coli adenylate cyclase: In E. coli the fructose repressor (FruR also known as Cra) indirectly controls cAMP levels under specific conditions, see Ecach chapter III ›››

Introduction section reads: "The induction of the fructose operon takes place when the repressor Cra [FruR] is inactivated after interactions with inducers such as D-fructose-1-phosphate and D-fructose-1, 6-biphosphate. … In the presence of glucose, the intracellular concentration of the inducers increase, which interact with Cra [FruR] to prevent its binding to the target promoters".  The intracellular concentration of the inducer fructose-1-phosphate does not increase in the presence of glucose.  It 'increases' in the presence of fructose which enters the cell as fructose-1-phosphate via the phosphotransferase system (PTS).  Furthermore the synthesis of 1-phosphofructokinase is specifically induced by fructose, and inhibited by fructose-1, 6-biphosphate.  Therefore in the presence of glucose (or mannose or mannitol) the intracellular concentration of the inducer fructose-1-phosphate cannot possibly 'increase'.  It increases in the presence of fructose.

January 31, 2011
Journal of Bacteriology 2011, 193(5): 1086 Escherichia coli exports cyclic AMP via TolC
Klaus Hantke, Karin Winkler, and Joachim E. Schultz

November 22, 2015 Update: This article was retracted in 2015 [RETRACTION]

Results section reads: "Mutants like BTH2 cya grow as colorless colonies [on MacConkey agar plates].  Application of cAMP via filter paper discs resulted in a narrow red growth zone only around the disc which was soaked with 40 mM cAMP indicating cAMP dependent utilization of maltose" and same section further on: "Next, induction of β-galactosidase was assayed as a function of cAMP concentration.  The parent strain BTH2 cya was rather insensitive to cAMP addition.  Even at 10 mM cAMP in the medium a full response was not elicited indicating the presence of a highly effective system for cAMP export from the cells".  Both data indicates the BTH2 cya strain is unlike any other cya strains of E. coli.  Therefore the conclusion that E. coli exports cAMP via TolC must be taken with caution. 

December 17, 2010
FEBS Letters 2010, 584(22): 4537 A mammalian insulysin homolog is regulated by enzyme IIAGlc of the glucose transport system in Vibrio vulnificus
You-Jin Kim, Yangkyun Ryu, Byoung-Mo Koo, Na Yeon Lee, Se-Jin Chun, Soon-Jung Park, Kyu-Ho Lee, and Yeong-Jae Seok

Glucose does not trigger dephosphorylation of Enzyme IIAGlc in absence of the glucose permease (Enzyme IICBGlc).  Therefore glucose does not ensure complete dephosphorylation of Enzyme IIAGlc in the experimental conditions described by the authors in four instances.  (1) Material and Methods section reads: "To test the phosphorylation dependence of the interaction between IIAGlc and the vIDE, vIDE [V. vulnificus insulin-degrading enzyme] was incubated with EI, HPr and His-IIAGlc in a total volume of 500 µl of buffer A containing 2 mM DTT and 2 mM MgCl2 in the presence of 1 mM glucose (to ensure complete dephosphorylation of His-IIAGlc)…"  (2) Results section reads: "In one set of reactions, glucose was added, along with E. coli EI and HPr, to maintain IIAGlc in dephosphorylated form" and (3) same section further on: "To confirm whether IIAGlc is required for activity of vIDE, and to test whether this activation depends on the phosphorylation state of IIAGlc, the reaction mixture containing vIDE, EI, HPr and IIAGlc was pre-incubated with either PEP or glucose before the reaction was initiated by the addition of insulin"  (4) Legend of Figure 1(B) reads: "vIDE was incubated with EI and HPr in the presence or absence of His-IIAGlc.  Mixtures designated Glc and PEP were supplemented with 1 mM glucose and PEP to ensure complete dephosphorylation and phosphorylation of the added IIAGlc, respectively" (idem Figure 4).

May 22, 2009
Journal of Bacteriology 2009, 191(9): 3041 Pyruvate kinase-deficient Escherichia coli exhibits increased plasmid copy number and cyclic AMP levels
Drew S. Cunningham, Zhu Liu, Nathan Domagalski, Richard R. Koepsel, Mohammad M. Ataai, and Michael M. Domach

Discussion reads: 'Prior work also indicated that PL8UV5 is about fourfold stronger than Plac when both are compared for growth on glucose (21)'.  Reference (21) [J Bacteriol] does indeed describe lac up-promoter mutants including PUV5 but none of the mutants carry the L8 mutation.  And in a repeat performance: 'As a second control, Plac in PB25 was mutated to PL8UV5, which is cAMP insensitive and about fourfold stronger than Plac when these two promoters are compared for growth on either glucose-6-phosphate (37) or glucose (21)'.  In yet another example of citation violation, Discussion reads: '… glucose- 6-phosphate … is similar to glucose in repressive strength (29)'.  Not quite, see Table 1 in Reference (29) [Science Magazine].

April 17, 2009
Journal of Bacteriology 2009, 191(7): 2069 Involvement of the Cra global regulatory protein in the expression of the iscRSUA operon, revealed during studies of tricarballylate catabolism in Salmonella enterica
Jeffrey A. Lewis, Jeffrey M. Boyd, Diana M. Downs and Jorge C. Escalante-Semerena

Connection with Escherichia coli adenylate cyclase: In E. coli the fructose repressor (FruR also known as Cra) indirectly controls cAMP levels under specific conditions, see Ecach chapter III ›››

Material and Methods section reads: 'Skovran et al. previously reported that an isc mutant required nicotinic acid and thiamine for growth. The requirements for these nutrients were bypassed by using overnight cultures grown in LB, without washing the cells, to inoculate fresh medium'.  This is incorrect experimental procedure which leads to false data.

March 20, 2009
Nature Reviews Microbiology 2009, 7(3): 250 cAMP does not have an important role in carbon catabolite repression of the Escherichia coli lac operon
Atul Narang 
PubMed Commons

It is unethical for Atul Narang to use the data from an article by Wanner et al. and ignore their statement that 'Much of the variation [in β-galactosidase synthesis] was eliminated by growing E. coli in the presence of cAMP, and this component we call cAMP-mediated catabolite repression' [J Bacteriol].  It is fraudulent to use the data by Wanner et al. to let the reader believe that data in Figure 1A are best fitted with a curve showing that 'to a first approximation, the β-galactosidase activity of exponentially growing cells is inversely proportional to the specific growth rate'.  Readers of the correspondence are urged to read the article by Wanner et al. and compare their data to Atul Narang's re-illustration of the same data.  Finally it is irresponsible for Atul Narang to ignore the reasoning of a previous correspondence [PubMed] to discard the role of cAMP in the glucose-lactose diauxie.


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