Crosstalk between (p)ppGpp and other nucleotide second messengers.

In response to environmental cues, bacteria produce intracellular nucleotide messengers to regulate a wide variety of cellular processes and physiology. Studies on individual nucleotide messengers, such as (p)ppGpp or cyclic (di)nucleotides, have established their respective regulatory themes. As research on nucleotide signaling networks expands, recent studies have begun to uncover various crosstalk mechanisms between (p)ppGpp and other nucleotide messengers, including signal conversion, allosteric regulation, and target competition. The multiple layers of crosstalk implicate that (p)ppGpp is intricately linked to different nucleotide signaling pathways. From a physiological perspective, (p)ppGpp crosstalk enables fine-tuning and feedback regulation with other nucleotide messengers to achieve optimal adaptation.

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris (XccSAH) and found that it metabolizes both cellular alarmones and cofactors. In vitro, XccSAH displays abilities to hydrolyze multiple nucleotides, including pppGpp, ppGpp, pGpp, pppApp, and NADPH. In vivo, X. campestris pv. campestris cells lacking sah accumulated higher levels of cellular (pp)pGpp and NADPH compared to wild-type cells upon amino acid starvation. In addition, X. campestris pv. campestris mutants lacking sah were more sensitive to killing by Pseudomonas during interbacterial competition. Interestingly, loss of sah also resulted in reduced growth in amino acid-replete medium, a condition that did not induce (pp)pGpp or pppApp accumulation. Further metabolomic characterization revealed strong depletion of NADH levels in the X. campestris pv. campestris mutant lacking sah, suggesting that NADPH/NADH regulation is an evolutionarily conserved function of both bacterial and metazoan SAHs and Mesh1. Overall, our work demonstrates a regulatory role of bacterial SAHs as tuners of stress responses and metabolism, beyond functioning as antitoxins. IMPORTANCE Small alarmone hydrolases (SAHs) comprise a widespread family of alarmone metabolizing enzymes. In metazoans, SAHs have been reported to control multiple aspects of physiology and stress resistance through alarmone and NADPH metabolisms, but their physiological functions in bacteria is mostly uncharacterized except for a few reports as antitoxins. Here, we identified an SAH functioning independently of toxins in the phytopathogen Xanthomonas campestris pv. campestris. We found that XccSAH hydrolyzed multiple alarmones and NADPH in vitro, and X. campestris pv. campestris mutants lacking sah displayed increased alarmone levels during starvation, loss of interspecies competitive fitness, growth defects, and strong reduction in NADH. Our findings reveal the importance of NADPH hydrolysis by a bacterial SAH. Our work is also the first report of significant physiological roles of bacterial SAHs beyond functioning as antitoxins and suggests that SAHs have far broader physiological roles and share similar functions across domains of life.

Bacillus subtilis produces (p)ppGpp in response to the bacteriostatic antibiotic chloramphenicol to prevent its potential bactericidal effect.

Antibiotics combat bacteria through their bacteriostatic (by growth inhibition) or bactericidal (by killing bacteria) action. Mechanistically, it has been proposed that bactericidal antibiotics trigger cellular damage, while bacteriostatic antibiotics suppress cellular metabolism. Here, we demonstrate how the difference between bacteriostatic and bactericidal activities of the antibiotic chloramphenicol can be attributed to an antibiotic-induced bacterial protective response: the stringent response. Chloramphenicol targets the ribosome to inhibit the growth of the Gram-positive bacterium Bacillus subtilis. Intriguingly, we found that chloramphenicol becomes bactericidal in B. subtilis mutants unable to produce (p)ppGpp. We observed a similar (p)ppGpp-dependent bactericidal effect of chloramphenicol in the Gram-positive pathogen Enterococcus faecalis. In B. subtilis, chloramphenicol treatment induces (p)ppGpp accumulation through the action of the (p)ppGpp synthetase RelA. (p)ppGpp subsequently depletes the intracellular concentration of GTP and antagonizes GTP action. This GTP regulation is critical for preventing chloramphenicol from killing B. subtilis, as bypassing (p)ppGpp-dependent GTP regulation potentiates chloramphenicol killing, while reducing GTP synthesis increases survival. Finally, chloramphenicol treatment protects cells from the classical bactericidal antibiotic vancomycin, reminiscent of the clinical phenomenon of antibiotic antagonism. Taken together, our findings suggest a role of (p)ppGpp in the control of the bacteriostatic and bactericidal activity of antibiotics in Gram-positive bacteria, which can be exploited to potentiate the efficacy of existing antibiotics.

Regulatory Themes and Variations by the Stress-Signaling Nucleotide Alarmones (p)ppGpp in Bacteria.

Bacterial stress-signaling alarmones are important components of a protective network against diverse stresses such as nutrient starvation and antibiotic assault. pppGpp and ppGpp, collectively (p)ppGpp, have well-documented regulatory roles in gene expression and protein translation. Recent work has highlighted another key function of (p)ppGpp: inducing rapid and coordinated changes in cellular metabolism by regulating enzymatic activities, especially those involved in purine nucleotide synthesis. Failure of metabolic regulation by (p)ppGpp results in the loss of coordination between metabolic and macromolecular processes, leading to cellular toxicity. In this review, we document how (p)ppGpp and newly characterized nucleotides pGpp and (p)ppApp directly regulate these enzymatic targets for metabolic remodeling. We examine targets' common determinants for alarmone interaction as well as their evolutionary diversification. We highlight classical and emerging themes in nucleotide signaling, including oligomerization and allostery along with metabolic interconversion and crosstalk, illustrating how they allow optimized bacterial adaptation to their environmental niches.

Combating Health Care Fraud and Abuse: Conceptualization and Prototyping Study of a Blockchain Antifraud Framework.

BACKGROUND: An estimated US $2.6 billion loss is attributed to health care fraud and abuse. With traditional health care claims verification and reimbursement, the health care provider submits a claim after rendering services to a patient, which is then verified and reimbursed by the payer. However, this process leaves out a critical stakeholder: the patient for whom the services are actually rendered. This lack of patient participation introduces a risk of fraud and abuse. Blockchain technology enables secure data management with transparency, which could mitigate this risk of health care fraud and abuse. OBJECTIVE: The aim of this study is to develop a framework using blockchain to record claims data and transactions in an immutable format and to enable the patient to act as a validating node to help detect and prevent health care fraud and abuse. METHODS: We developed a health care fraud and abuse blockchain technical framework and prototype using key blockchain tools and application layers including consensus algorithms, smart contracts, tokens, and governance based on digital identity on the Ethereum platform (Ethereum Foundation). RESULTS: Our technical framework maps to the claims adjudication process and focuses on Medicare claims, with the US Centers for Medicare and Medicaid Services (CMS) as the central authority. A prototype of the framework system was developed using the blockchain platform Ethereum (Ethereum Foundation), with its design features, workflow, smart contract functions, system architecture, and software implementation outlined. The software stack used to build the system consisted of a front-end user interface framework, a back-end processing server, and a blockchain network. React was used for the user interface framework, and NodeJS and an Express server were used for the back-end processing server; Solidity was the smart contract language used to interact with a local Ethereum blockchain network. CONCLUSIONS: The proposed framework and the initial prototype have the potential to improve the health care claims process by using blockchain technology for secure data storage and consensus mechanisms, which make the claims adjudication process more patient-centric for the purposes of identifying and preventing health care fraud and abuse. Future work will focus on the use of synthetic or historic CMS claims data to assess the real-world viability of the framework.

Small Alarmone Synthetase SasA Expression Leads to Concomitant Accumulation of pGpp, ppApp, and AppppA in Bacillus subtilis.

(p)ppGpp is a highly conserved bacterial alarmone which regulates many aspects of cellular physiology and metabolism. In Gram-positive bacteria such as B. subtilis, cellular (p)ppGpp level is determined by the bifunctional (p)ppGpp synthetase/hydrolase RelA and two small alarmone synthetases (SASs) YjbM (SasB) and YwaC (SasA). However, it is less clear whether these enzymes are also involved in regulation of alarmones outside of (p)ppGpp. Here we developed an improved LC-MS-based method to detect a broad spectrum of metabolites and alarmones from bacterial cultures with high efficiency. By characterizing the metabolomic signatures of SasA expressing B. subtilis, we identified strong accumulation of the (p)ppGpp analog pGpp, as well as accumulation of ppApp and AppppA. The induced accumulation of these alarmones is abolished in the catalytically dead sasA mutant, suggesting that it is a consequence of SasA synthetase activity. In addition, we also identified depletion of specific purine nucleotides and their precursors including IMP precursors FGAR, SAICAR and AICAR (ZMP), as well as GTP and GDP. Furthermore, we also revealed depletion of multiple pyrimidine precursors such as orotate and orotidine 5'-phosphate. Taken together, our work shows that induction of a single (p)ppGpp synthetase can cause concomitant accumulation and potential regulatory interplay of multiple alarmones.

Study of the Expression of Bacterial Multidrug Efflux Pumps in Anaerobic Conditions.

Bacterial multidrug efflux pumps belong to a class of membrane transporter proteins that dedicate to the extrusion of a diverse range of substances out of cells including all classes of currently available antibiotics. They constitute an important mechanism of bacterial antibiotic and multidrug resistance. Since many ecological niches of bacteria and the infection foci in animal host display low oxygen tension under which condition bacterial pathogens undergo fundamental changes on their metabolic modes, it is necessary to study the expression profiles of drug efflux pumps under these physiologically and clinically relevant conditions. In this chapter, we first introduce procedures to culture bacteria under anaerobic conditions, which is achieved using screw-capped Pyrex culture tubes without agitation. We then introduce ß-galactosidase activity assay using promoter-lacZ (encoding the ß-galactosidase enzyme) fusion to measure the expression of efflux pumps at transcriptional level, and Western blot using chromosomal FLAG-tagged construct to examine the expression of these proteins at translational level. Applications of these gene expression studies to reveal the regulatory mechanisms of efflux genes expression as well as their physiological functions are also discussed.

Signaling by the heavy-metal sensor CusS involves rearranged helical interactions in specific transmembrane regions.

Two-component systems (TCSs) play important roles in the adaptation of bacteria to stress. Despite their increasingly well understood mechanistic features, it remains poorly understood how TCSs transduce signals across membranes. Here, we use the E. coli Cu/Ag-responsive CusSR TCS as a model to investigate the roles of CusS transmembrane (TM) residues. Proline scanning of TM1 domain led to identification of the T17P, F18P, and S21P variants, which display higher kinase activities relative to wild type. A single point mutation, V202G, in the adjacent TM2 domain specifically suppresses the hyperactivities of these mutants. Disulfide crosslinking analysis demonstrated that T17 and V202 are situated in close proximity, and Cys residues substituted at those two positions form exclusive intramolecular crosslinks when CusS is in the signaling-inactive state. In the signaling-active variant of CusS, however, only intermolecular crosslinking between the two Cys residues could be observed, suggesting that destabilization of an intramolecular constraint and a subsequent rearrangement of helical interactions in this TM region is involved in the activation of CusS. An analogous TM helical interface in the P. aeruginosa heavy metal sensor kinase CzcS is also observed. Together, these results suggested a conserved transmembrane signal transduction mechanism in the heavy metal sensing TCSs.

Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect iron-sulfur cluster enzymes and biogenesis.

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P(cusC)-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.

Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development.

This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.

High-level gentamicin resistance mediated by a Tn4001-like transposon in seven nonclonal hospital isolates of Streptococcus pasteurianus.

We report on the first occurrence of high-level gentamicin resistance (MICs > or = 512 microg/ml) in seven clinical isolates of Streptococcus pasteurianus from Hong Kong. These seven isolates were confirmed to be the species S. pasteurianus on the basis of nucleotide sequencing of the superoxide dismutase (sodA) gene. Epidemiological data as well as the results of pulse-field gel electrophoresis analysis suggested that the seven S. pasteurianus isolates did not belong to the same clone. Molecular characterization showed that they carried a chromosomal, transposon-borne resistance gene [aac(6')Ie-aph(2'')Ia] which was known to encode a bifunctional aminoglycoside-modifying enzyme. The genetic arrangement of this transposon was similar to that of Tn4001, a transposon previously recovered from Staphylococcus aureus and other gram-positive isolates. Genetic linkage with other resistance elements, such as the ermB gene for erythromycin resistance, was not evident. On the basis of these findings, we suggest that routine screening for high-level gentamicin resistance should be recommended for all clinically significant blood culture isolates. This is to avoid the inadvertent use of short-course combination therapy with penicillin and gentamicin, which may lead to the failure of treatment for endocarditis, the selection of drug-resistant Streptococcus pasteurianus and other gram-positive organisms, as well as the unnecessary usage of gentamicin, a drug with potential toxicity.