The fundamental details of how nutritional stress leads to elevating (p)ppGpp are questionable. By common usage, the meaning of the stringent response has evolved from the specific response to (p)ppGpp provoked by amino acid starvation to all responses caused by elevating (p)ppGpp by any means. Different responses have similar as well as dissimilar positive and negative effects on gene expression and metabolism. The different ways that different bacteria seem to exploit their capacities to form and respond to (p)ppGpp are already impressive despite an early stage of discovery. Apparently, (p)ppGpp can contribute to regulation of many aspects of microbial cell biology that are sensitive to changing nutrient availability: growth, adaptation, secondary metabolism, survival, persistence, cell division, motility, biofilms, development, competence, and virulence. Many basic questions still exist. This review tries to focus on some issues that linger even for the most widely characterized bacterial strains.

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Strains of Escherichia coli which lack detectable guanosine 3',5'-bispyrophosphate (ppGpp) display a pleiotropic phenotype that in some respects resembles that of rpoS (katF) mutants. This led us to examine whether ppGpp is a positive regulator of sigma s synthesis. sigma s is a stationary-phase-specific sigma factor that is encoded by the rpoS gene. We found that a ppGpp-deficient strain is defective in sigma s synthesis as cells enter stationary phase in a rich medium, as judged by immunoblots. Under more-defined conditions we found that the stimulation of sigma s synthesis following glucose, phosphate, or amino acid starvation of wild-type strains is greatly reduced in a strain lacking ppGpp. The failure of ppGpp-deficient strains to synthesize sigma s in response to these starvation regimens could indicate a general defect in gene expression rather than a specific dependence of rpoS expression on ppGpp. We therefore tested the effect of artificially elevated ppGpp levels on sigma s synthesis either with mutations that impair ppGpp decay or by gratuitously inducing ppGpp synthesis with a Ptac::relA fusion. In both instances, we observed enhanced sigma s synthesis. Apparently, ppGpp can activate sigma s synthesis under conditions of nutrient sufficiency as well as during entry into stationary phase. This finding suggests that changes in ppGpp levels function both as a signal of imminent stationary phase and as a signal of perturbations in steady-state growth.

Summary CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR, and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

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Summary It is widely accepted that the DNA, RNA and protein content of Enterobacteriaceae is regulated as a function of exponential growth rates; macromolecular content increases with faster growth regardless of specific composition of the growth medium. This phenomenon, called growth rate control, primarily involves regulation of ribosomal RNA and ribosomal protein synthesis. However, it was uncertain whether the global regulator ppGpp is the major determinant for growth rate control. Therefore, here we re-evaluate the effect of ppGpp on macromolecular content for different balanced growth rates in defined media. We find that when ppGpp is absent RNA/protein and RNA/DNA ratios are equivalent in fast and slow growing cells. Moreover, slow growing ppGpp deficient cells with increased RNA content, display a normal ribosomal subunit composition although polysome content is reduced when compared to fast growing wild type cells. From this we conclude that growth rate control does not occur in the absence of ppGpp. Also, artificial elevation of ppGpp or introduction of stringent RNA polymerase mutants in ppGpp deficient cells restores this control. We believe these findings strongly argue in favor of ppGpp and against redundant regulation of growth rate control by other factors in E. coli and other enteric bacteria.

Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments. In addition, expression of E. coli GppA, a pppGpp 5′-gamma phosphate hydrolase that converts pppGpp to ppGpp, is manipulated to fine tune differential accumulation of ppGpp and pppGpp. In vivo and in vitro experiments show that pppGpp is less potent than ppGpp with respect to regulation of growth rate, RNA/DNA ratios, ribosomal RNA P1 promoter transcription inhibition, threonine operon promoter activation and RpoS induction. To provide further insights into regulation by (p)ppGpp, we have also determined crystal structures of E. coli RNA polymerase-σ70 holoenzyme with ppGpp and pppGpp. We find that both nucleotides bind to a site at the interface between β′ and ω subunits.

High levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), generated in response to amino acid starvation in Escherichia coli, lead to massive accumulations of inorganic polyphosphate (polyP). Inasmuch as the activities of the principal enzymes that synthesize and degrade polyP fluctuate only slightly, the polyP accumulation can be attributed to a singular and profound inhibition by pppGpp and/or ppGpp of the hydrolytic breakdown of polyP by exopolyphosphatase, thereby blocking the dynamic turnover of polyP. The K i values of 10 M for pppGpp and 200 M for ppGpp are far below the concentrations of these nucleotides in nutritionally stressed cells. In the complex metabolic network of pppGpp and ppGpp, the greater inhibitory effect of pppGpp (compared with ppGpp) leading to the accumulation of polyP, may have some significance in the relative roles played by these regulatory compounds.Inorganic polyphosphate (polyP), 1 a linear polymer of hundreds of phosphate residues linked by high-energy phosphoanhydride bonds, is ubiquitous having been found in all microbes, fungi, plants, and animals examined (1, 2). In Escherichia coli, polyP, which accumulates up to 20 mM (based on P i residues) in stationary-phase cells, 2 is produced from ATP by a membraneassociated enzyme, polyphosphate kinase (PPK) (3).Mutants lacking PPK are deficient in polyP, fail to adapt to stress, and do not survive in stationary phase (4,5). A regulatory function is one of the many possible effects of polyP that might account for this essential role. In this regard, the relationship of polyP levels to those of guanosine penta-and tetraphosphate ((p)ppGpp) deserves special attention. Levels of (p) ppGpp rise drastically in response to starvation for amino acids, carbon, or P i (6, 7). In response to nutritional stress, accompanied by increased levels of (p)ppGpp, large accumulations of polyP have been observed in E. coli, 2, 3 Myxococcus xanthus (8), and Pseudomonas aeruginosa 3 . E. coli mutants that fail to produce (p)ppGpp are also deficient in the accumulation of polyP. 3The present study explores the mechanism of polyP accumulation in nutritionally stressed E. coli and the relationships to (p)ppGpp. EXPERIMENTAL PROCEDURESReagents and Proteins-Sources were as follows: [␥-32 P]ATP was from Amersham Corp.; nonradiolabeled ATP and ADP and bovine serum albumin were from Sigma; polyethyleneimine-cellulose (PEI) TLC plate was from Merck; and creatine phosphate and creatine kinase were from Boehringer Mannheim.Bacterial Strains-The ⌬gppA::kan deletion-insertion allele in strain CF3376 was constructed as follows. A plasmid bearing the wild type gppA region was subcloned from phage 1039 (9). Using synthetic primers, DNA-encoding, amino acid residues 1-452 of the 496 total in GppA were deleted. A kanamycin resistance (Km-r) cassette derived from plasmid pUC4K (Pharmacia Biotech Inc.) was substituted for the deletion. The insertion-deletion allele was recombined into phage 1039 by phage growth on a plasmid-bearing strain and t...