During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

ppGpp: magic beyond RNA polymerase

Like for all microbes, the goal of every pathogen is to survive and replicate. However, to overcome the formidable defenses of their hosts, pathogens are also endowed with traits commonly associated with virulence, such as surface attachment, cell or tissue invasion, and transmission. Numerous pathogens couple their specific virulence pathways with more general adaptations, like stress resistance, by integrating dedicated regulators with global signaling networks. In particular, many of nature's most dreaded bacteria rely on nucleotide alarmones to cue metabolic disturbances and coordinate survival and virulence programs. Here we discuss how components of the stringent response contribute to the virulence of a wide variety of pathogenic bacteria.

ppGpp Conjures Bacterial Virulence

https://www.cell.com/trends/microbiology/pdf/S0966-842X(13)00013-9.pdf

Bacterial lifestyle shapes stringent response activation

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.

Differential regulation by ppGpp versus pppGpp in Escherichia coli

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 A wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit 'closure', providing insights into the dynamics of high-fidelity tRNA decoding.

/pdf/ribosome-rela-structures-reveal-the-mechanism-of-stringent-375t9w8zj5.pdf

Ribosome•RelA structures reveal the mechanism of stringent response activation

Summary
To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (σ28) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co-ordinate L. pneumophila differentiation.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-2958.2010.07094.x

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation.

Given increasing numbers of people experiencing transitions in health insurance due to declines in employer-sponsored insurance and changes in health policy, the understanding and application of health insurance terms and concepts (health insurance literacy) may be important for navigating use of health care. The study objective was to systematically review evidence on the relationship between health insurance literacy and health care utilization. Medline, SCOPUS, Web of Science, CINAHL, PsychInfo, Cochrane Library, and reference lists of published literature were searched in August 2019. Quantitative, qualitative, and intervention studies that assessed the association of health insurance literacy as the exposure and health care utilization as the outcome were identified, without language or date restrictions. Outcomes were independently assessed by 2–3 reviewers. Twenty-one studies including a total of 62,416 subjects met inclusion criteria: three interventional trials, two mixed-methods studies, and sixteen cross-sectional studies. Ten of thirteen preventive care studies suggested that higher health insurance literacy was associated with greater utilization of primary care and other preventive services. Eight of nine studies of care avoidance demonstrated that individuals with lower health insurance literacy were more likely to delay or avoid care. A few studies had mixed results regarding the utilization of emergency department, inpatient, and surgical care. The emerging literature in this area suggests that health insurance literacy is an important factor that can enable effective utilization of health care, including primary care and preventive services. However, the literature is limited by a paucity of studies using validated tools that broadly measure health insurance literacy (rather than testing knowledge of specific covered services). Improving health insurance literacy of the general public and increasing plain language communication of health insurance plan features at the point of health care navigation may encourage more effective and cost-conscious utilization.

/pdf/association-of-health-insurance-literacy-with-health-care-4k7v0t8jzf.pdf

Association of Health Insurance Literacy with Health Care Utilization: a Systematic Review

Appropriate for the Protection of the Public Health: Why We Need Electronic Nicotine Delivery System Product Standards

This Viewpoint reviews recent challenges in electronic nicotine delivery system product regulation and the potential benefit of modeling product user fees after pharmaceutical product and device user fees. 

Brian F. Yagi

Papers

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation.

Association of Health Insurance Literacy with Health Care Utilization: a Systematic Review

Appropriate for the Protection of the Public Health: Why We Need Electronic Nicotine Delivery System Product Standards

Why We Need User Fees for Electronic Nicotine Delivery System Products.

The 510(K) Third Party Review Program: Promise and Potential