Integration of Bacterial Nutritional Responses
- Michael Cashel, MD, PhD, Head, Section on Molecular Regulation
- Rajendran Harinarayanan, PhD, Visiting Fellow
- Christophe Penno, PhD, Visiting Fellow
- Katarzyna Potrykus, PhD, Visiting Fellow
- Helen Murphy, MS, Microbiologist
Remarkably, slowing bacterial growth by limiting almost any nutrient (nitrogen, carbon, amino acids, many vitamins, phosphate, iron, or oxygen) produces elevated levels of a regulatory nucleotide called ppGpp. The nucleotide signal in turn leads to positive or negative regulation of the expression of about one-third of the E. coli genome. We are interested in understanding this fundamental microbial regulatory mechanism. Although regulation by ppGpp occurs largely at the level of gene transcription by RNA polymerase, few molecular details are known. Many physiological changes accompanying ppGpp regulation may be viewed as coordinated to ensure cellular survival and adaptation to nutritional stress. Coordinated cellular responses are thought to be attributable in part to direct effects on promoter activity, in part to transcription regulation by alternative sigma factor activities, and in part to regulatory effects on metabolism. Our understanding of these phenomena is just beginning to take shape. It is also important to recognize that ppGpp may be correlated with virulence in pathogens. Nuclear genes for ppGpp synthesis from plants are functional in bacteria, chloroplasts being targeted for ppGpp production. The nucleotide mediates embryonic development and responses to environmental stress ranging from pathogens to wounding and nutrient stress.
DksA, GreA, GreB, and TraR proteins unexpectedly affect ppGpp transcription regulation
Potrykus, Vinella, Herman, Harinarayanan, Murphy, Cashel; in collaboration with Herman, Vinella
Regulation of transcription by ppGpp at the molecular level is unusual (for prokaryotes) in that it does not involve the typical interplay of sequence-specific DNA-binding repressor or inducer proteins. Instead, ppGpp is thought to modify RNA polymerase initiation kinetics in ways that inhibit or activate specific classes of promoters—ppGpp and RNA polymerase together targeting core sequences. Regulation occurs by a synergistic interaction between ppGpp and some member of a group of structurally similar proteins whose structural similarity parallels the group’s ability to probe the secondary channel of RNA polymerase; however, it remains unclear how secondary-channel binding triggers regulation. DksA was the first regulatory protein of this type to be discovered (in another laboratory). Over the past few years, we found that, in some respects, two proteins (GreA and mutant GreB) behave similarly to DksA. For example, wild-type GreA or a GreB mutant can alter phenotypes produced simply by the absence of DksA. When both DksA and ppGpp are absent, many phenotypic features are found in addition to those dependent on DksA alone. GreA as well as the same GreB mutant can also reverse a subset these additional changes. Both GreA and GreB also have a well-known function that is distinct from mimicking ppGpp and DksA regulation, namely, the ability to reverse transcription elongation arrest. We do not know why GreA and GreB mimic some features of regulation by DksA and ppGpp. A plausible explanation is that DksA can reverse arrest of transcription elongation, although researchers long ago ruled out such an explanation. One working hypothesis holds that the proteins induce a conformational change in RNA polymerase that leads to shared kinetic features for promoter recognition.
This year, in collaboration with Christophe Herman’s group, we discovered that TraR should be added to the list of DksA-, GreA-, and GreB-like proteins. The traR gene is found on the F′ episomal element that mediates conjugation. While the function of TraR was previously unknown, TraR was named for its position between genes traQ and traS. Even small amounts of TraR complement the amino acid requirements for growth in the absence of ppGpp, demonstrating an ability similar to that of GreA and GreB mutants. Unlike GreA and GreB but like ppGpp, TraR is an inhibitor of ribosomal RNA transcription. TraR shares the coiled-coil structural feature with DksA, GreA, and GreB that has been postulated to be associated with secondary-channel penetration. However, researchers have yet to demonstrate secondary-channel binding of TraR or solve TraR’s structure. Even so, a potentially important difference between regulation by ppGpp and by TraR is that inhibition of growth by even large amounts of TraR takes hours, whereas growth stops within minutes when ppGpp is increased. We believe that this difference indicates an additional regulatory activity for ppGpp that would be important to identify.
We discovered AppppA, another surprising candidate for a regulatory substitute for ppGpp. The compound has been implicated in DNA replication in eukaryotes. We discovered AppppA with multicopy library screens in a search for genes that can complement the growth defect of ppGpp0 dksA double mutants. With preliminary evidence implicating AppppA as a substitute regulator for ppGpp and DksA, we are attempting to manipulate the activity of an AppppA hydrolase encoded by the apaH gene to control AppppA independently of synthesis, as occurs with our library isolates.
Synthetic ppGpp0 lethal mutants of tktA, aceE, and proB genes reveal that ppGpp regulates enzymes of intermediary metabolism
Harinarayanan, Murphy, Cashel
We have documented the basis for ppGpp regulation of intermediary metabolism by isolating tktA, aceE, and proB—mutants that grow poorly or not at all only when ppGpp is missing. Surprisingly, all encode enzymes of intermediary metabolism. The first example involves transketolases, key enzymes that form erythrose-3-phosphate in the pentose phosphate shunt. Erythrose-3-phosphate is an intermediate for the biosynthesis of aromatic amino acids and pyridoxine-derived vitamins. A ΔtktA/ΔtktB double mutant is completely deficient in transketolase. The synthetic lethal tktA ppGpp0 strain is only marginally less severe than a complete transketolase deficiency. Using a series of tktB::lacZ transcriptional fusions to localize the tktB promoter, we discovered that tktB expression was largely abolished in a ppGpp0 strain owing to an indirect effect—tktB expression depends on the presence of RpoS, an alternative sigma factor coordinating expression of stationary phase genes. Deletion of rpoS in a ppGpp+ background gives an almost complete loss of tktB::lacZ activity. The regulatory pathway deduced to relate ppGpp to tktB expression is ppGpp→rpoS→tktB. Weak tktB promoter activity is noticeable when rpoS is deleted but ppGpp is present. We proposed that this is a direct effect of ppGpp occurring at the level of RNA polymerase itself.
The second synthetic lethal is an aceE mutant. The aceE gene encodes a subunit of pyruvate dehydrogenase that functions in the pathway for acetyl CoA formation. We believe that a deficiency of acetyl CoA occurs in aceE ppGpp0 double mutants—we demonstrated that supplementation with acetate restores viability and that mutants blocking complementation of growth by acetate occur in genes needed to form acetyl CoA from acetate. A redundant pathway catalyzed by poxB can generate acetyl CoA when pyruvate dehydrogenase is inactive in aceE mutants. Again, the explanation for why aceE mutants are ppGpp0 synthetic lethal mutants lies in an rpoS requirement. Thus, aceE mutants become lethal in the absence of ppGpp because, similar to the synthetic lethality of tktA mutants described above, the mutants unveil a ppGpp requirement for poxB transcription that is exerted indirectly through RpoS. With isozymes quite common for glycolytic and hexose shunt pathways, we can surmise from these two examples that ppGpp0 synthetic lethals might occur more generally. Indeed, in solving the tktA/B puzzle, we discovered that a mutant of zwf is also a ppGpp0 synthetic lethal; zwf is a key enzyme in the branch point linking glycolysis and the pentose shunt.
The third ppGpp0 synthetic lethal mutant that we discovered does not involve intermediary carbon metabolism. This is a proB mutant that fails to grow at 42ºC on LB media in ppGpp0 strains. Such behavior seemed especially odd because the peptide-rich LB medium contains enough proline to allow the proA, proB, or proC mutants to grow when ppGpp is present. Raising proline concentrations in LB does cure the ppGpp0 growth defect, indicating that proline availability is likely to be the problem. As LB has proline-containing peptides but little free proline, we guessed that ppGpp might be needed in one of three ways: (1) to activate proteases for liberating free proline from peptides: (2) to facilitate chaperone functions already known to occur at very high intracellular proline levels; or (3) to transport low amounts of extracellular proline to achieve high intracellular pools. We then screened a multicopy random gene library for isolates that restored LB growth of proAB mutants. We did not find complementation by overexpression of genes encoding either proteases or chaperonins, thus arguing against the first or second explanation. We did find an isolate that suggests the third explanation—complementation by a multicopy gene, nhaA. NhaA is known to maintain the membrane proton gradient through a sodium-proton pump. Characterization of nhaA phenotypes for ppGpp0 and wild-type strains revealed that ppGpp is partly needed for a wild-type phenotype. Our working hypothesis posits that a partially defective sodium-proton pump in ppGpp0 cells creates a need for high external proline concentrations to achieve enough intracellular proline to allow growth. We will test the prediction that additional transport defects for compounds other than proline should also be apparent in the absence of ppGpp.
Discovery of GraL small RNA in the greA leader transcript arising from dual promoters
Potrykus, Cashel
Our studies surveying regulation of greA expression have led to the finding that the greA leader transcript generates small RNA molecules (which we call GraL) arising from two strong promoters. The upstream P1 promoter uses a housekeeping Sigma-70 promoter, yielding a start site 149 nt away from the AUG of the greA orf. The downstream P2 promoter begins only 11 nt beyond the P1 start and requires the alternative sigma factor SigmaE, which is known to activate a class of promoters in response to cell-wall and cell-membrane stress. The activation of SigmaE itself requires ppGpp by pathways that involve direct interaction of ppGpp with RNA polymerase as well as an indirect mechanism that controls an inactive SigmaE precursor complex to release an active protein. From both P1- and P2-promoted RNA chains, GraL is generated by a shared terminator that releases 50- to 60-nt-long transcripts with two unusual features. The first such feature is that the end of the transcript chain length is variable, giving frayed ends with size differences ranging from 1–10 nt rather than precise stops at one or two positions. We have genetic evidence suggesting that the frayed ends do not result from ribonuclease cleavage, as supported by evidence from an array of antisense oligonucleotides showing that the frayed ends reflect actual termination sites. The second unusual feature is a (weak) P3 greA promoter that is found within the run of uridylic acid residues comprising the termination site for GraL. We have demonstrated the resulting GraL with in vitro transcripts as well as by Northern blots with samples isolated from cells. LacZ reporter activities somewhat surprisingly revealed that GraL had no effect on cellular greA expression. We therefore assessed the more general biological importance of this small RNA by determining whether GraL conferred a selective advantage over cells not overexpressing GraL during repeated cycles of overnight growth in LB media. We inoculated cultures with equal admixtures of cells containing a GraL-overproducing plasmid (or vector) with one or the other marked with a malT mutant. In one admixture the cell with the plasmid was marked; in the other admixture the cell with the vector was marked; in this way, we could be sure that the marker itself (a malT mutant) was not the cause of the fitness advantage. We could score the proportion of maltose-fermenting cells at any time of culture as the portion of red and white colonies on maltose indicator plates. We found that cells overexpressing GraL reproducibly and systematically outcompete wild-type cells. After four cycles of overnight growth, 95 percent of the culture consisted of GraL overexpressors whether or not the malT marker mutation was present together with the GraL plasmid.
We have also performed microarray analyses. When GraL was overproduced for 15 minutes, we found no effect in wild-type strains. However, in ppGpp0 over 100 genes were affected. Specifically, many genes affected by GraL are known to be repressed by Fur, a major regulator of iron metabolism. Our finding is intriguing given that we long ago showed that ppGpp is also involved in iron regulation. On the other hand, flagellar genes comprise a general class inhibited by GraL—ppGpp and DksA have been implicated in regulation of flagellar genes. Computer searches relying on RNA-RNA hybrids as mediators of small RNA regulation did not yield meaningful results. Genetic evidence also excluded RNaseIII and Hfq involvement in regulation exerted by this small RNA. GraL apparently functions in a manner dramatically different from more typical small RNA species.
- Harinarayanan R, Murphy H, Cashel M. Synthetic growth phenotypes of E. coli lacking ppGpp and transketolase A (tktA) are due to ppGpp-mediated transcriptional regulation of tktB. Mol Microbiol 2008;69:882-894.
- Potrykus K, Cashel M. (p)ppGpp: still magical? Annu Rev Microbiol 2008;62:35-51.
- Rhee H-W, Lee C-R, Cho S-H, Song M-R, Cashel M, Choy HE, Seok Y-J, Hong J-L. Selective fluorescent chemosensor for the bacterial alarmone (p)ppGpp. J Am Chem Soc 2008;130:784-785.
Collaborators
- Richard D’Ari, PhD, Institut Jacques Monod, CNRS, Universitá Paris 7, Paris, France
- Christophe Herman, PhD, Baylor College of Medicine, Houston, TX
- Agnieszka Szalewska-Palasz, PhD, University of Gdansk, Gdansk, Poland
- Daniel Vinella, PhD, Centre National de la Recherche Scientifique, Marseille, France
For more information, contact mcashel@nih.gov or visit http://smr.nichd.nih.gov.