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Bacterial persistence has been shown to be an underlying factor in the failure of antibiotic treatments. Although many pathways, among them the stringent response and toxin-antitoxin modules, have been linked to antibiotic persistence, a clear molecular mechanism for the growth arrest that characterizes persistent bacteria remained elusive. Here, we screened an expression library for putative targets of HipA, the first toxin linked to persistence, and a serine/threonine kinase. We found that the expression of GltX, the glutamyl-tRNA-synthetase, reverses the toxicity of HipA and prevents persister formation. We show that upon HipA expression, GltX undergoes phosphorylation at Ser239, its ATP-binding site. This phosphorylation leads to accumulation of uncharged tRNA(Glu) in the cell, which results in the activation of the stringent response. Our findings demonstrate a mechanism for persister formation by the hipBA toxin-antitoxin module and provide an explanation for the long-observed connection between persistence and the stringent response.

MazG is a homodimeric alpha-helical protein that belongs to the superfamily of all-alpha NTP pyrophosphatases. Its function has been connected to the regulation of the toxin-antitoxin module mazEF, implicated in programmed growth arrest/cell death of Escherichia coli cells under conditions of amino acid starvation. The goal of the first detailed biophysical study of a member of the all-alpha NTP pyrophosphatase superfamily, presented here, is to improve molecular understanding of the unfolding of this type of proteins. Thermal unfolding of MazG monitored by differential scanning calorimetry, circular dichroism spectroscopy, and fluorimetry at neutral pH in the presence of a reducing agent (dithiothreitol) can be successfully described as a reversible four-state transition between a dimeric native state, two dimeric intermediate states, and a monomeric denatured state. The first intermediate state appears to have a structure similar to that of the native state while the final thermally denatured monomeric state is not fully unfolded and contains a significant fraction of residual alpha-helical structure. In the absence of dithiothreitol, disulfide cross-linking causes misfolding of MazG that appears to be responsible for the formation of multimeric aggregates. MazG is most stable at pH 7-8, while at pH <6, it exists in a molten-globule-like state. The thermodynamic parameters characterizing each step of MazG denaturation transition obtained by global fitting of the four-state model to differential scanning calorimetry, circular dichroism, and fluorimetry temperature profiles are in agreement with the observed structural characteristics of the MazG conformational states and their assumed functional role.

We have previously reported that mazEF, the first regulatable chromosomal 'addiction module' located on the Escherichia coli chromosome, downstream from the relA gene, plays a crucial role in the programmed cell death in bacteria under stressful conditions. It consists of a pair of genes encoding a stable toxin, MazF, and MazE, a labile antitoxin interacting with MazF to form a complex. The cellular target of MazF toxin was recently described to be cellular mRNA, which is degraded by this toxin. On the same operon, downstream to the mazEF genes, we found another open reading frame, which was called mazG. Recently, it was shown that the MazG protein has a nucleotide pyrophosphohydrolase activity. Here we show that mazG is being transcribed in the same polycistronic mRNA with mazEF. We also show that the enzymatic activity of MazG is inhibited by MazEF proteins. When the complex MazEF was added, the enzymatic activity of MazG was about 70% inhibited. We demonstrate that the enzymatic activity of MazG in vivo causes depletion of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation conditions. Based on our results, we propose a model in which this third gene, which is unique for chromosomal addiction systems, has a function of limiting the deleterious activity of MazF toxin. In addition, MazG solves a frequently encountered biological problem: how to avoid the persistence of a toxic product beyond the time when its toxicity is useful to the survival of the population.

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

The discovery of toxin-antitoxin gene pairs (also called addiction modules) on extrachromosomal elements of Escherichia coli, and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures. We have reported on the E. coli mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and MazE is a labile antitoxin. Here we show that cell death mediated by the E. coli mazEF module can be triggered by several antibiotics (rifampicin, chloramphenicol, and spectinomycin) that are general inhibitors of transcription and/or translation. These antibiotics inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes cell death. Our results have implications for the possible mode(s) of action of this group of antibiotics.

The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.

The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA(+) cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752-5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.

In bacteria, programmed cell death is mediated through "addiction modules" consisting of two genes. The product of the second gene is a stable toxin, whereas the product of the first is a labile antitoxin. Here we extensively review what is known about those modules that are borne by one of a number of Escherichia coli extrachromosomal elements and are responsible for the postsegregational killing effect. We focus on a recently discovered chromosomally borne regulatable addiction module in E. coli that responds to nutritional stress and also on an antideath gene of the E. coli bacteriophage lambda. We consider the relation of these two to programmed cell death and antideath in bacterial cultures. Finally, we discuss the similarities between basic features of programmed cell death and antideath in both prokaryotes and eukaryotes and the possibility that they share a common evolutionary origin.

In Escherichia coli, programmed cell death is mediated through "addiction modules" consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of lambdarexB, one of the few genes expressed in the lysogenic state of bacteriophage lambda, prevents cell death directed by each of two addiction modules, phd-doc of plasmid prophage P1 and the rel mazEF of E. coli, which is induced by the signal molecule guanosine 3',5'-bispyrophosphate (ppGpp) and thus by amino acid starvation. lambdaRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of lambdaRexB through its action on the ClpP proteolytic subunit. We also propose that the lambdarex operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the rex operon may be considered as the "survival operon" of phage lambda.

Most bacterial RNA consists of stable RNA which is composed of rRNA and tRNA. We have followed by primer extension analysis the level of ribosomal RNA synthesis along the growth phases of a cell culture. A sharp drop in rRNA synthesis was observed upon the transition from the exponential to the stationary phase of growth. Our results demonstrate that an effective shut-off of rRNA synthesis occurs also in the absence of ppGpp. Mutations in the host factors Fis and H-NS, which are known to regulate rrn P1 promoters, did not affect the shut-off process of ribosomal RNA promoters. We also tested the effect of RpoS, the sigma factor known to induce a number of genes in the stationary phase. It was shown that the host factors Fis, H-NS and RpoS do not play a major role in the regulation of the shut-off process of rRNA synthesis. The results presented demonstrate that the rate of rRNA synthesis provides a sensitive measure of the growth phase of the bacterial culture.

"Addiction modules" consist of two genes. In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect. Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module. Furthermore, the expression of mazEF is regulated by the cellular level of guanosine [corrected] 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation. These properties suggest that the mazEF system may be responsible for programmed cell death in E. coli and thus may have a role in the physiology of starvation.

ppGpp serves as an alarmon in prokaryotes, distributing and coordinating different cellular processes according to the nutritional potential of the growth medium. This work is interpreted as favoring the view that, in addition to its previously documented role in regulating the rate of ribosome synthesis, ppGpp participates in coordinating DNA replication and cell division. We studied the effects of ppGpp on the cell division cycle, using cells containing plasmid pSM11 that codes for the 55-kDa truncated RelA protein under the inducible Ptac promoter. In this system it was found that the rate of initiation of new rounds of DNA replication is inversely correlated with the intracellular level of ppGpp. Furthermore, ppGpp levels similar to those found during the activation of stringent control inhibited replication initiation, in a manner comparable to that resulting from inhibition of protein synthesis by amino acid starvation or by chloramphenicol addition. However, in contrast to chloramphenicol treatment, elevated ppGpp levels did not block septum formation, and, in fact, there is some evidence for enhanced septation. As a result, the residual cell division following elevation in ppGpp levels was higher than after chloramphenicol treatment, resulting in cells with a size similar to that of stationary phase cells.

Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth-phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the relA and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth-phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS. Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF-binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF-binding site was found upstream of the himD/hip promoter. All three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.

Ribosomal RNA synthesis in Escherichia coli is under stringent control. The seven rRNA operons are highly conserved and are each transcribed from two tandem promoters, P1 and P2, which are located about 120 base-pairs apart. In exponentially growing cells the majority of the transcripts are initiated at the P1 promoters. The P1 promoters are highly regulated, and are under stringent as well as growth rate controls. Here we demonstrate that transcription from the rrnA P1 promoter diminishes P2 expression. In the absence of P1, the P2 promoter acts as a rather strong promoter. Insertion of a transcription terminator between P1 and P2 eliminates the inhibition of P2 by P1, suggesting that the physical movement of RNA polymerase originating at P1 and progressing along the P2 promoter is necessary for the interference process to take place. Similarly to P1, the solitary P2 promoter is subject to stringent control.

Temperature downshifts of Escherichia coli throughout its growth range resulted in transient growth inhibition and a cold shock response consisting of transient induction of several proteins, repression of heat shock proteins, and, despite the growth lag, continued synthesis of proteins involved in transcription and translation. The paradoxical synthesis of the latter proteins, which are normally repressed when growth is arrested, was explored further. First, by means of a nutritional downshift, a natural stringent response was induced in wild-type cells immediately prior to a shift from 37 to 10 degrees C. These cells displayed decreased synthesis of transcriptional and translational proteins and decreased induction of cold shock proteins; also, adaptation for growth at 10 degrees C was delayed, even after restoration of the nutrient supplementation. Next, the contribution of guanosine 5'-triphosphate-3'-diphosphate and guanosine 5'-diphosphate-3'-diphosphate, collectively abbreviated (p)ppGpp, to the alteration in cold shock response was studied with the aid of a mutant strain in which overproduction of these nucleotides can be artificially induced. Induction of (p)ppGpp synthesis immediately prior to shifting this strain from 37 to 10 degrees C produced results differing only in a few details from those described above for nutritional downshift of the wild-type strain. Finally, shifting a relA spoT mutant, which cannot synthesize (p)ppGpp, from 24 to 10 degrees C resulted in a greater induction of the cold shock proteins, increased synthesis of transcriptional and translational proteins, decreased synthesis of a major heat shock protein, and faster adaptation to growth than for the wild-type strain. Our results indicate that the previously reported decrease in the (p)ppGpp level following temperature downshift plays a physiological role in the regulation of gene expression and adaptation for growth at low temperature.

On the basis of protein homology, nhaR has previously been shown to belong to a large family of regulatory proteins, the LysR family (Henikoff, S., Haughn, G.W., Calvo, J.M., and Wallace, J.C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6602-6606). In this work we show that nhaR is a regulator of nhaA, a gene encoding a Na+/H+ antiporter in Escherichia coli. Multicopy plasmid bearing nhaR enhances the Na(+)-dependent induction of a chromosomal nhaA'-'lacZ fusion. Extracts derived from cells overexpressing nhaR exhibit specific DNA binding capacity to the upstream sequences of nhaA. Construction of an nhaR deletion mutant (OR100) shows that nhaR is required in addition to nhaA to tolerate the extreme conditions under which nhaA is indispensable. Whereas OR100 grows like the wild type at neutral pH even at high Na+ concentrations (700 mM), it becomes much more sensitive to Na+ (greater than 300 mM) at pH 8.5; furthermore, OR100 is more sensitive to Li+ (100 mM) than the wild type. Nevertheless, the phenotype of OR100, which is more resistant to Na+, Li+, and alkaline pH than a delta nhaA strain (NM81), implies that the regulation exerted by nhaR is not complete and that some expression of nhaA exists in OR100. Accordingly, the effect of nhaR in cells is dependent on the level of nhaA. OR200, a nhaA and nhaR deletion mutant, has the same phenotype as NM81. Multicopy plasmid bearing nhaR does not change the phenotype of either OR200 or NM81. On the other hand, multicopy nhaA renders the cells Li(+)- and and Na(+)-resistant even without nhaR.

Regulation of expression of nhaA, the gene which encodes a Na+/H+ antiporter in Escherichia coli has been studied. Two promoters have been identified in the upstream sequence of the gene and the corresponding start point of transcription mapped by primer extension. Monitoring the beta-galactosidase activity of a chromosomal translation fusion of nhaA'-'lacZ show that at pH 7.5 the gene is induced, within 1 h, by 100 mM of either Li+ or Na+. Change of pH between 6.5 and 8.5 by itself does not increase expression of the gene but it markedly increases the sensitivity of the expression system to the ions. At pH 7.5 maximal induction is obtained by 100 mM NaCl, whereas at pH 8.6, 10 mM NaCl elicit similar response. The pattern of regulation of nhaA reflects its importance in adaptation to high salinity and alkaline pH in E. coli.

Intracellular levels of guanosine 3',5'-bispyrophosphate (ppGpp) governed by the relA gene are normally regulated by aminoacyl-tRNA availability for protein synthesis. An experimental system is described in which cellular levels of ppGpp are controlled instead by induction of plasmid pKK223-3 derivatives with the relA structural gene, or portions thereof, under control of the Ptac promoter. In amino acid-rich media, isopropyl-1-thio-beta-D-galactopyranoside induction of transcription of the wild type relA gene in pSM10 yields about a 100-fold overexpression of a metabolically stable, full length (743 amino acid) RelA protein to levels approximating the number of cellular ribosomes. This overexpression is accompanied by a roughly parallel and relC-dependent elevation of ppGpp levels. Induction of a relA gene deletion mutant in pSM11 containing 455 amino-terminal amino acids results in much lower levels of expression of a metabolically unstable 55-kDa protein and elevated ppGpp levels that are almost equivalent to induced pSM10 and are relC-independent. Induction of a larger deletion in pSM12 containing 331 amino-terminal amino acids does not provoke ppGpp accumulation. We are able to elicit high levels of ppGpp without changing nutritional abundance and without massive overexpression of the RelA protein by inducing the metabolically unstable, truncated RelA protein. We find the effects of elevated ppGpp levels to include a slowing of growth, an inhibition of stable RNA accumulation, an inhibition of cellular rrn P1 promoter activities as measured by primer extension, and changes in the pattern of gene expression viewed by two-dimensional electrophoresis of cellular proteins.

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.

The relA gene product of Escherichia coli is known to be responsible for the synthesis of guanosine 3',5'-bispyrophosphate (ppGpp) during the stringent response to amino acid starvation. This report presents the sequence of the relA gene region and assignment of its 743-codon open reading frame by the following criteria: 1) genetic complementation of ppGpp synthesis in a relaxed (relA1) mutant during the stringent response; 2) changes in 3-aminotriazole resistance during growth to mimic a relA+ phenotype; 3) verification of the presence of an amber codon at the normal carboxyl terminus of the relA gene; and 4) immunological assays of expression of the RelA protein. The apparent molecular mass of the cloned relA gene product is calculated to be 83,856 daltons and as visualized by immunoblotting is identical to that of the previously characterized protein. A promoter has been identified that directs relA gene transcription towards the pyrG gene, in a counterclockwise direction on the E. coli chromosome. Genomic Southern blot analyses verify that the relA regions cloned and subjected to nucleotide sequence analysis correspond to homologous regions on the E. coli chromosome.

The functional structure of the promoter region of a bacterial ribosomal operon is analyzed by in vitro transcription of linear DNA fragments derived from the hybrid Col EI plasmid pGG1. This plasmid contains the promoter region of the rrn B ribosomal cistron present in the lambdarifd18. When transcripts arising from this promoter region are terminated by restriction endonuclease cleavage of DNA, two RNA chains are resolved by gel electrophoresis that differ in length by about 100 bases. Evidence is presented indicating that these two transcripts arise from different initiation sites, each directing tandem transcription on the sense strand of the early portion of the ribosomal cistron.

The Escherichia coli chromosome carries seven cistrons encoding ribosomal RNA sequences. In all cases studied, in vitro and in vivo, it has been established that transcription is initiated from two tandem promoters. The expression of the rRNA cistrons is regulated in response to growth rate as well as to aminoacyl tRNA availability. In the present study, a plasmid (pPS1) carrying the promoter region of the rrnA cistron fused to the terminator region of rrnB has been used for in vitro transcription experiments. The presence of the terminators (T1 and T2) together with the fact that supercoiled DNA is found to be a highly efficient template, provide an ideal in vitro system in which to study the functional interrelationship between the two tandem promoters of E. coli rRNA cistrons. The results suggest that the rate of rRNA synthesis in E. coli cells growing in various conditions, as reflected by the availability of RNA polymerase, is primarily dependent on the properties of the two tandem rRNA promoters.

A Lambda phage was constructed in which the structural gene for beta galactosidase is fused to a DNA segment carrying the ribosomal promoter rrnB of E. coli. In this hybrid operon beta galactosidase synthesis in vitro is repressed by ppGpp. Repression of beta galactosidase synthesis by cAMP is reported.

relA and spoT are designations for two unlinked Escherichia coli genes whose products function in the synthesis and degradation of guanosine 3',5'-bispyrophosphate during the stringent regulatory response to amino acid deprivation. The RelA protein catalyzes an ATP:GTP 3'-pyrophosphoryl group transfer reaction, and the SpoT protein has a guanosine 3',5'-bispyrophosphate 3'-pyrophosphohydrolyase activity. Both genes have been sequenced recently; the relA gene produces an 84-kDa protein, and the spoT gene is deduced to encode a 79-kDa protein. We report here that the protein sequences of the relA and spoT genes are extensively interrelated.

A sequence located upstream to the E. coli rrnA P1 promoter is required for optimal promoter activity. Deletion of this sequence reduces in vivo transcription by 90%. Substitution of this upstream activating sequence with the unrelated bent DNA sequence of the kinetoplast of Crithidia fasciculata, restores in vivo expression to high levels. Cellular proteins which are present only in exponentially growing cells bind specifically to intact rrnA P1, but do not bind to the promoter missing the upstream activating sequence. These proteins are associated with the 30S ribosomal subunits but can be washed off with concentrated salt. The correlation between the binding activity and cell growth rate suggests a role for these proteins in the transcriptional control of rRNA synthesis.

The most widely studied "relaxed" mutant of the relA locus, the relA1 allele, is shown here to consist of an IS2 insertion between the 85th and 86th codons of the otherwise wild-type relA structural gene, which normally encodes a 743-amino acid (84 kDa) protein. The RelA protein is a ribosome-dependent ATP:GTP (GDP) pyrophosphoryltransferase that is activated during the stringent response to amino acid starvation and thereby occasions the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp). We propose that the IS2 insertion functionally splits the RelA protein into two (alpha and beta) peptide fragments which can complement each other in trans to yield residual ppGpp synthetic activity; neither fragment shows this activity when expressed alone. Cell strains with a single copy relA null allele show physiological behavior that is much the same as relA1 mutant strains. Both relA1 and relA null strains accumulate ppGpp during glucose starvation and do not accumulate ppGpp during the stringent response. The presence of ppGpp in verifiable relA null strains is interpreted as unequivocal evidence for an alternate route of ppGpp synthesis that exists in addition to the relA-dependent reaction.