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J Bacteriol. Sep 2011; 193(18): 4571–4573.
PMCID: PMC3165694

Unnecessary Signaling: Poorly Named? [down-pointing small open triangle]

TEXT

Perhaps due to my optimistic disposition, I like to think that there is a good reason why every little thing about Escherichia coli, and other bacteria, is the way it is. That is, I like to believe that countless generations of natural selection have resulted in an organism that is very highly evolved for survival and replicative efficiency in its natural niche(s). Thus, I do not expect to find that modern bacterial cells contain extensive genetic regulatory systems or elaborate signal transduction pathways that they never use. I would expect these to have been lost long ago. But what if this trust in the enormous power of natural selection is an overestimation, such that even the modern bacterium is a work in progress, littered with molecular fossils and remnants of extinct pathways that are no longer used under any circumstances but which have yet to be eliminated in the cause of replicative efficiency? In that case, one might expect to find genes and proteins that no longer have any useful function whatsoever, as well as remnants of signal transduction pathways that still retain rudimentary functions but which provide no selective advantage because a different system provides those same functions better or eliminates their necessity. This conundrum of distinguishing the significance of an elaborate signaling system that apparently is not necessary at all in E. coli by our current methods of assessing phenotypes is raised by the work of Reaves and Rabinowitz in this issue (15). Earlier work showed that null mutation of ptsN, encoding an EII protein designated EIIANtr, had a variety of phenotypes in E. coli, including suppression of the conditional lethality of the essential GTPase Era (14), and several nutrient utilization and growth phenotypes. For example, a ptsN null mutation renders the cells sensitive to leucine and leucine-containing peptides (6) and results in a growth defect on certain poor sources of both nitrogen and carbon, such as alanine, when a carbohydrate is present (14). Investigation of those growth phenotypes led to the remarkable demonstrations that IIANtr regulates intracellular K+ levels in two distinct ways, by controlling the activity of the low-affinity K+ transport system (7) and by signaling through a two-component regulatory system to control the transcription of the structural genes for a high-affinity K+ transport system (8). These studies suggested that intracellular K+ serves as a “second messenger” by controlling both the activity of AHASI and the expression of its structural genes (10). The absence of AHASI activity apparently provided for the nutrient utilization and growth phenotypes that were observed in a strain lacking EIIANtr. In their paper, Reaves and Rabinowitz point out that the strains used in earlier studies were ilvG mutants and thus lacked the enzyme AHASII, which is less sensitive to K+ regulation (15). When cells that were ilvG+ and ptsN mutants were examined, all of the known growth phenotypes caused by the ptsN mutation were gone! Presumably, the signal transduction events observed in earlier studies are still going on in cells that have ilvG+. IIANtr is still binding to TrkA to regulate the low-affinity K+ transport system, and it is still binding to KdpD to activate its kinase activity and inhibit its phosphatase activity to cause KdpE~P to accumulate and activate the expression of kdpFABC. The ptsP product, EINtr, is still using its GAF domain to sense a stimulatory effector and control the phosphorylation state of Npr (reviewed in reference 13). The phosphotransfer between Npr and IIANtr and the other cellular phosphotransferase system (PTS) components still is going on. It is just that none of that apparently matters as long as you have an AHASII enzyme. At least the ptsN null mutation had no nutrient utilization phenotype. Why go through all that signaling if there is no growth phenotype?

If it were my grant application at stake, I would consider pointing out that perhaps AHASII is inhibited or genetically repressed under some condition, such that the elaborate regulation of K+ levels mediated in part by IIANtr would affect the growth phenotype: the old “we just haven't found the condition yet” argument. Since such a complex regulatory system evolved in the presence of AHASII inhibition or its genetic repression, this inhibition or repression cannot have been a rare condition. The fact that IIANtr controls both K+ transport systems suggests physiological relevance, even in the face of the ptsN mutation producing no growth phenotype. Perhaps we need to rethink how we assess phenotypes. In nature, cells compete in mixed populations under constantly changing conditions; in the lab, we typically examine pure cultures under constant conditions. Let us not lose sight of the abstractions implicit in our experimental designs.

Alternatively, perhaps the role of IIANtr in controlling K+ uptake is just a molecular fossil from the days before the appearance of (relatively) K+-resistant AHASII. I am not an omics person, so I cannot speak to such things. Maybe IIANtr is still there working away, thinking it matters, but its function has become completely unnecessary. It happens.

NAMES MATTER

What's in a name? That which we call a rose by any other name would smell as sweet. I wish that the sentiment expressed in that passage from Romeo and Juliet were true in science, but it is not. Names matter because they help to shape, and limit, our concepts, and they influence our sense of worth. If this were not true, there would be no such thing as “branding.” So, it is important to try to get names right. Of course, that is easy for me to say, since I have spent my whole career working on genes and proteins that other people named. These genes and proteins already had multiple names, none of which were particularly useful. But I digress. In a perfect world, we would name things in a logical way, according to their function, at both the gene and protein levels. Unfortunately, we live in an imperfect world, one consequence of which is that we do not really know what the functions are, and our opinions on that subject are constantly changing. Thus, naming things based upon functions, appealing as I personally find it to be, inevitably results in a bunch of bad names, conveying anachronistic conclusions and thus limiting learning and understanding. It seems that things are starting to break badly for the name “PTSNtr” after just a 16-year run, which still has to be considered pretty impressive considering the thinness of the evidence used to justify the “Ntr” designation in the first place.

There never really was any good evidence that IIANtr, or any of the so-called Ntr (nitrogen-related) PTSs for that matter, had anything to do with the genetic regulatory system that has come to be known as the Ntr system, which controls nitrogen assimilation. It is of interest to go back and see where the name came from. Merrick and Coppard published a paper in 1989, the title of which suggested that mutations in genes downstream from rpoN (i.e., ptsN) affect expression from σ54-dependent promoters in Klebsiella pneumoniae (9). However, in Table 2 of that paper, where the results of lacZ fusions to various promoters were reported, we can see that the mutation actually had a very modest effect on all of the promoters examined, with the exception of the nifH promoter fusion, and in that case only when the cells were provided with an intermediate level of ammonium as the sole nitrogen source (9), a growth condition that has more recently been called into question (1). In 1992, J. Reizer and colleagues showed that one of the open reading frames (ORFs) studied by Merrick and Coppard was a IIA protein, and since it was known to be involved in controlling expression from some of the nitrogen-regulated promoters in K. pneumoniae, it could be a IIANtr that provides regulation (16). A quote from that paper, “a recent study has indicated that the product of ORF162 regulates the activity of σ54 in K. pneumoniae…” shows the influence of the Merrick paper. Based on the fact that ptsN encoded a IIA protein, they suggested that PTS-catalyzed protein phosphorylation provides a regulatory link between carbon and nitrogen assimilation (16). Finally, in 1995, the name IIANtr was used in the title of a paper by Powell and colleagues where the phenotype of a ptsN null mutant was closely studied (14). In Results, they showed that the ptsN mutation “does not affect classical nitrogen regulation” and only acts to regulate the utilization of certain poor nitrogen sources under certain conditions, such as when carbohydrates are present. They were actually very conservative in their hypotheses, suggesting that IIANtr might have something to do with coordinating the trichloroacetic acid (TCA) cycle with nitrogen metabolism. But the use of the name IIANtr in the title seems to have obscured the sober realities in the Results and Discussion sections. IIANtr had arrived.

BEYOND E. COLI

There is an extensive base of knowledge regarding the functions of ptsN in other bacteria, which will be only briefly noted here, along with a few citations to help the interested reader get started (26, 11, 12, 15, 17). A two-dimensional gel electrophoresis study of the effects of the ptsN mutation in Pseudomonas putida showed that ~9% of its genes were affected, most of these were not regulated by σ54 (2). In general, IIANtr regulates polysaccharide synthesis in Pseudomonas (polyhydroxyalkanoic acid), Azotobacter (β-hydroxybutyrate), and Vibrio (Vibrio polysaccharide), biofilm production, and carbohydrate utilization; it also affects genes necessary for the utilization of certain sulfur sources. Interestingly, in Brucella melitensis, the so-called Ntr PTS and an IIAmannose are present but PTS permeases (EIIs) are missing from the organism, so the function must be strictly regulatory (3). It has been hypothesized, based on some evidence, that the Ntr PTS in this organism is involved in the regulation of α-ketoglutarate dehydrogenase by α-ketoglutarate levels; that is, it is involved in controlling flux through the TCA cycle.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 July 2011.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

REFERENCES

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15. Reaves M. L., Rabinowitz J. D. 2011. Characteristic phenotypes associated with ptsN-null mutants in Escherichia coli K-12 are absent in strains with functional ilvG. J. Bacteriol. 193:4576–4581 [PMC free article] [PubMed]
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