Summary Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a post-transcriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

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DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over ∼1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization.

Adaptation to fluctuations in nutrient availability is a fact of life for single-celled organisms in the 'wild'. A decade ago our understanding of how bacteria adjust cell cycle parameters to accommodate changes in nutrient availability stemmed almost entirely from elegant physiological studies completed in the 1960s. In this Opinion article we summarize recent groundbreaking work in this area and discuss potential mechanisms by which nutrient availability and metabolic status are coordinated with cell growth, chromosome replication and cell division.The life of a bacterial cell is feast or famine. To survive the bacterium must rapidly adapt to changing environmental conditions. Colonization of the mammalian gut provides an enteric organism with an abundant source of carbohydrates, whereas a flash flood instantly depletes the nutrient supply for a soil bacterium. Nutrient-rich conditions lead to a decrease in mass doubling time and an increase in cell size, whereas nutrient-poor conditions curtail growth and reduce cell size 1,2 . Changes in growth rate must be accompanied by changes in the cell cycle to ensure that cell division stays coordinated with mass doubling, chromosome replication and chromosome segregation.How organisms adjust their cell cycle dynamics to compensate for changes in nutritional conditions is an important outstanding question in bacterial physiology. Recent work, reviewed here, suggests that multiple signalling pathways transmit nutritional and growth rate information directly to the cell cycle machinery. Multiple signalling pathways permit cells to constantly sample their environments and fine-tune cell cycle processes, a substantial advantage under challenging conditions. The bacterial cell cycleThe bacterial cell cycle is traditionally divided into three stages: the period between division (cell 'birth') and the initiation of chromosome replication (known as the B period); the period required for replication (known as the C period); and the time between the end of replication and completion of division (known as the D period) (FIG. 1). In the enteric organism

Preface DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separations of these two processes leads to their conflict. Failure to deal with these conflicts can result in genome alterations and reduced fitness. In recent years, significant advances have been made in understanding how cells avoid conflicts between replication and transcription, and how conflicts are resolved when they do occur. In this review, we summarize these findings, which shed light on the significance of the problem and on how cells deal with unwanted encounters between the replication and transcription machineries.

Nutrients—and by extension biosynthetic capacity—positively impact cell size in organisms throughout the tree of life. In bacteria, cell size is reduced three-fold in response to nutrient starvation or accumulation of the alarmone ppGpp, a global inhibitor of biosynthesis. However, whether biosynthetic capacity as a whole determines cell size or if particular anabolic pathways are more important than others remains an open question. Here we identify fatty acid synthesis as the primary biosynthetic determinant of Escherichia coli size and present evidence supporting a similar role for fatty acids as a positive determinant of size in the Gram-positive bacterium Bacillus subtilis and the single celled eukaryote, Saccharomyces cerevisiae. Altering fatty acid synthesis recapitulated the impact of altering nutrients on cell size and morphology, while defects in other biosynthetic pathways had either a negligible or fatty acid-dependent effect on size. Together our findings support a novel “outside-in” model in which fatty acid availability sets cell envelope capacity, which in turn dictates cell size. In the absence of ppGpp, limiting fatty acid synthesis leads to cell lysis, supporting a role for ppGpp as a linchpin linking expansion of cytoplasmic volume to growth of the cell envelope to preserve cellular integrity.

The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for “Rel SpoT homologue”; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the ∆rsh ∆relQ [(p)ppGpp0] strain, suggesting that a lack of basal (p)ppGpp places the cell in a “transcriptionally relaxed” state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp0 strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions.