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Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to man. Nevertheless, its physiological role, and the underlying molecular mechanisms, are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5’-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics suggesting that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis showing increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone. 

ABSTRACT Gut bacteria influence human physiology by chemically modifying host-synthesized primary bile acids. These modified bile acids, known as secondary bile acids, can act as signaling molecules that modulate host lipid, glucose, and energy metabolism and affect gut microbiota composition via selective antimicrobial properties. However, knowledge regarding the bile acid-transforming capabilities of individual gut microbes remains limited. To help address this knowledge gap, we screened 72 bacterial isolates, spanning seven major phyla commonly found in the human gut, for their ability to chemically modify unconjugated bile acids. We found that 43 isolates, representing 41 species, were capable of in vitro modification of one or more of the three most abundant unconjugated bile acids in humans: cholic acid, chenodeoxycholic acid, and deoxycholic acid. Of these, 32 species have not been previously described as bile acid transformers. The most prevalent bile acid transformations detected were oxidation of 3α-, 7α-, or 12α-hydroxyl groups on the steroid core, a reaction catalyzed by hydroxysteroid dehydrogenases. In addition, we found 7α-dehydroxylation activity to be distributed across various bacterial genera, and we observed several other complex bile acid transformations. Finally, our screen revealed widespread bacterial conjugation of primary and secondary bile acids to glycine, a process that was thought to only occur in the liver, and to 15 other amino acids, resulting in the discovery of 44 novel microbially conjugated bile acids. IMPORTANCE Our current knowledge regarding microbial bile acid transformations comes primarily from biochemical studies on a relatively small number of species or from bioinformatic predictions that rely on homology to known bile acid-transforming enzyme sequences. Therefore, much remains to be learned regarding the variety of bile acid transformations and their representation across gut microbial species. By carrying out a systematic investigation of bacterial species commonly found in the human intestinal tract, this study helps better define the gut bacteria that impact composition of the bile acid pool, which has implications in the context of metabolic disorders and cancers of the digestive tract. Our results greatly expand upon the list of bacterial species known to perform different types of bile acid transformations. This knowledge will be vital for assessing the causal connections between the microbiome, bile acid pool composition, and human health. 

Living organisms need energy to stay alive; in cells, this energy is supplied in the form of a small molecule called adenosine triphosphate, or ATP, a nucleotide that stores energy in the bonds between its three phosphate groups. ATP is present in all living cells and is often referred to as the energy currency of the cell, because it can be easily stored and transported to where it is needed. However, it is unknown why cells rely so heavily on ATP when a highly similar nucleotide called guanosine triphosphate, or GTP, could also act as an energy currency. There are several examples of proteins that originally used GTP and have since evolved to use ATP, but it is not clear why this switch occurred. One suggestion is that ATP is the more readily available nucleotide in the cell. To test this hypothesis, Updegrove, Harke et al. studied a protein that helps bacteria transition into spores, which are hardier and can survive in extreme environments until conditions become favorable for bacteria to grow again. In modern bacteria, this protein uses ATP to provide energy, but it evolved from an ancestral protein that used GTP instead. First, Updegrove, Harke et al. engineered the protein so that it became more similar to the ancestral protein and used GTP instead of ATP. When this was done, the protein gained the ability to break down GTP and release energy from it, but it no longer performed its enzymatic function. This suggests that both the energy released and the source of that energy are important for a protein’s activity. Further analysis showed that the modern version of the protein has evolved to briefly hold on to ATP after releasing its energy, which did not happen with GTP in the modified protein. Updegrove, Harke et al. also discovered that the levels of GTP in a bacterial cell fall as it transforms into a spore, while ATP levels remain relatively high. This suggests that ATP may indeed have become the source of energy of choice because it was more available. These findings provide insights into how ATP became the energy currency in cells, and suggest that how ATP is bound by proteins can impact a protein’s activity. Additionally, these experiments could help inform the development of drugs targeting proteins that bind nucleotides: it may be essential to consider the entirety of the binding event, and not just the release of energy. 

Abstract The alarmone nucleotides guanosine tetraphosphate and pentaphosphate, commonly referred to as (p)ppGpp, regulate bacterial responses to nutritional and other stresses. There is evidence for potential existence of a third alarmone, guanosine-5′-monophosphate-3′-diphosphate (pGpp), with less-clear functions. Here, we demonstrate the presence of pGpp in bacterial cells, and perform a comprehensive screening to identify proteins that interact respectively with pGpp, ppGpp and pppGpp in Bacillus species. Both ppGpp and pppGpp interact with proteins involved in inhibition of purine nucleotide biosynthesis and with GTPases that control ribosome assembly or activity. By contrast, pGpp interacts with purine biosynthesis proteins but not with the GTPases. In addition, we show that hydrolase NahA (also known as YvcI) efficiently produces pGpp by hydrolyzing (p)ppGpp, thus modulating alarmone composition and function. Deletion of nahA leads to reduction of pGpp levels, increased (p)ppGpp levels, slower growth recovery from nutrient downshift, and loss of competitive fitness. Our results support the existence and physiological relevance of pGpp as a third alarmone, with functions that can be distinct from those of (p)ppGpp. 

ABSTRACT Biofilms are structured communities of tightly associated cells that constitute the predominant state of bacterial growth in natural and human-made environments. Although the core genetic circuitry that controls biofilm formation in model bacteria such as Bacillus subtilis has been well characterized, little is known about the role that metabolism plays in this complex developmental process. Here, we performed a time-resolved analysis of the metabolic changes associated with pellicle biofilm formation and development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. We report surprisingly widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. Most of these metabolic alterations were hitherto unrecognized as biofilm associated. For example, we observed increased activity of the tricarboxylic acid (TCA) cycle during early biofilm growth, a shift from fatty acid biosynthesis to fatty acid degradation, reorganization of iron metabolism and transport, and a switch from acetate to acetoin fermentation. Close agreement between metabolomic, transcriptomic, and proteomic measurements indicated that remodeling of metabolism during biofilm development was largely controlled at the transcriptional level. Our results also provide insights into the transcription factors and regulatory networks involved in this complex metabolic remodeling. Following upon these results, we demonstrated that acetoin production via acetolactate synthase is essential for robust biofilm growth and has the dual role of conserving redox balance and maintaining extracellular pH. This report represents a comprehensive systems-level investigation of the metabolic remodeling occurring during B. subtilis biofilm development that will serve as a useful road map for future studies on biofilm physiology. IMPORTANCE Bacterial biofilms are ubiquitous in natural environments and play an important role in many clinical, industrial, and ecological settings. Although much is known about the transcriptional regulatory networks that control biofilm formation in model bacteria such as Bacillus subtilis , very little is known about the role of metabolism in this complex developmental process. To address this important knowledge gap, we performed a time-resolved analysis of the metabolic changes associated with bacterial biofilm development in B. subtilis by combining metabolomic, transcriptomic, and proteomic analyses. Here, we report a widespread and dynamic remodeling of metabolism affecting central carbon metabolism, primary biosynthetic pathways, fermentation pathways, and secondary metabolism. This report serves as a unique hypothesis-generating resource for future studies on bacterial biofilm physiology. Outside the biofilm research area, this work should also prove relevant to any investigators interested in microbial physiology and metabolism.