scholarly journals Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

2014 ◽  
Vol 81 (1) ◽  
pp. 250-259 ◽  
Author(s):  
Adrienne Zaprasis ◽  
Monika Bleisteiner ◽  
Anne Kerres ◽  
Tamara Hoffmann ◽  
Erhard Bremer
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Building Blocks ◽  
Compatible Solute ◽  
Uptake System ◽  
Content Type ◽  
Stress Protection ◽  
Metabolic Conversion ◽  
Entry Points ◽  
Stressed Cells

ABSTRACTThe data presented here reveal a new facet of the physiological adjustment processes through whichBacillus subtiliscan derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ1-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection ofB. subtilisby this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na+-coupled symporter, is the main uptake system for both glutamate and aspartate inB. subtilis. Collectively, our data show how effectivelyB. subtiliscan exploit environmental resources to derive osmotic-stress protection through physiological means.

scholarly journals Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

2012 ◽  
Vol 78 (16) ◽  
pp. 5753-5762 ◽  
Author(s):  
Tamara Hoffmann ◽  
Carsten von Blohn ◽  
Agnieszka Stanek ◽  
Susanne Moses ◽  
Helena Barzantny ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
High Salinity ◽  
De Novo ◽  
Compatible Solutes ◽  
Growth Conditions ◽  
Compatible Solute ◽  
Content Type ◽  
Cellular Defense ◽  
Stressed Cells ◽  
External Salinity

ABSTRACTBacillus subtilissynthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, anopuEmutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of theopuEmutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of theopuEmutant was considerably lower than that of itsopuE+parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels ofB. subtilisparticipated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesizedde novoand subsequently released by salt-stressedB. subtiliscells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

scholarly journals Two Ways To Convert a Low-Affinity Potassium Channel to High Affinity: Control of Bacillus subtilis KtrCD by Glutamate

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Larissa Krüger ◽  
Christina Herzberg ◽  
Robert Warneke ◽  
Anja Poehlein ◽  
Janina Stautz ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Potassium Channel ◽  
Living Cell ◽  
Potassium Uptake ◽  
Selectivity Filter ◽  
Uptake System ◽  
Content Type ◽  
Apparent Affinity ◽  
High Affinity ◽  
External Potassium

ABSTRACT Potassium and glutamate are the major cation and anion, respectively, in every living cell. Due to the high concentrations of both ions, the cytoplasm of all cells can be regarded as a potassium glutamate solution. This implies that the concentrations of both ions need to be balanced. While the control of potassium uptake by glutamate is well established for eukaryotic cells, much less is known about the mechanisms that link potassium homeostasis to glutamate availability in bacteria. Here, we have discovered that the availability of glutamate strongly decreases the minimal external potassium concentration required for the highly abundant Bacillus subtilis potassium channel KtrCD to accumulate potassium. In contrast, the inducible KtrAB and KimA potassium uptake systems have high apparent affinities for potassium even in the absence of glutamate. Experiments with mutant strains revealed that the KtrD subunit responds to the presence of glutamate. For full activity, KtrD synergistically requires the presence of the regulatory subunit KtrC and of glutamate. The analysis of suppressor mutants of a strain that has KtrCD as the only potassium uptake system and that experiences severe potassium starvation identified a mutation in the ion selectivity filter of KtrD (Gly282 to Val) that similarly results in a strongly glutamate-independent increase of the apparent affinity for potassium. Thus, this work has identified two conditions that increase the apparent affinity of KtrCD for potassium, i.e., external glutamate and the acquisition of a single point mutation in KtrD. IMPORTANCE In each living cell, potassium is required for maintaining the intracellular pH and for the activity of essential enzymes. Like most other bacteria, Bacillus subtilis possesses multiple low- and high-affinity potassium uptake systems. Their activity is regulated by the second messenger cyclic di-AMP. Moreover, the pools of the most abundant ions potassium and glutamate must be balanced. We report two conditions under which the low-affinity potassium channel KtrCD is able to mediate potassium uptake at low external potassium concentrations: physiologically, the presence of glutamate results in a severely increased potassium uptake. Moreover, this is achieved by a mutation affecting the selectivity filter of the KtrD channel. These results highlight the integration between potassium and glutamate homeostasis in bacteria.

scholarly journals Identification and Characterization of Mutations Conferring Resistance to d-Amino Acids in Bacillus subtilis

2015 ◽  
Vol 197 (9) ◽  
pp. 1632-1639 ◽  
Author(s):  
Sara A. Leiman ◽  
Charles Richardson ◽  
Lucy Foulston ◽  
Alexander K. W. Elsholz ◽  
Eric A. First ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Protein Biosynthesis ◽  
Trna Synthetase ◽  
Inhibitory Effects ◽  
Content Type ◽  
Growth Inhibitory ◽  
Spontaneous Mutants ◽  
Identification And Characterization

ABSTRACTBacteria produced-amino acids for incorporation into the peptidoglycan and certain nonribosomally produced peptides. However,d-amino acids are toxic if mischarged on tRNAs or misincorporated into protein. Common strains of the Gram-positive bacteriumBacillus subtilisare particularly sensitive to the growth-inhibitory effects ofd-tyrosine due to the absence ofd-aminoacyl-tRNA deacylase, an enzyme that prevents misincorporation ofd-tyrosine and otherd-amino acids into nascent proteins. We isolated spontaneous mutants ofB. subtilisthat survive in the presence of a mixture ofd-leucine,d-methionine,d-tryptophan, andd-tyrosine. Whole-genome sequencing revealed that these strains harbored mutations affecting tRNATyrcharging. Three of the most potent mutations enhanced the expression of the gene (tyrS) for tyrosyl-tRNA synthetase. In particular, resistance was conferred by mutations that destabilized the terminator hairpin of thetyrSriboswitch, as well as by a mutation that transformed a tRNAPheinto atyrSriboswitch ligand. The most potent mutation, a substitution near the tyrosine recognition site of tyrosyl-tRNA synthetase, improved enzyme stereoselectivity. We conclude that these mutations promote the proper charging of tRNATyr, thus facilitating the exclusion ofd-tyrosine from protein biosynthesis in cells that lackd-aminoacyl-tRNA deacylase.IMPORTANCEProteins are composed ofl-amino acids. Mischarging of tRNAs withd-amino acids or the misincorporation ofd-amino acids into proteins causes toxicity. This work reports on mutations that confer resistance tod-amino acids and their mechanisms of action.

scholarly journals GapMind: Automated Annotation of Amino Acid Biosynthesis

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Morgan N. Price ◽  
Adam M. Deutschbauer ◽  
Adam P. Arkin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Building Blocks ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Content Type ◽  
Alternative Pathways ◽  
Automated Annotation ◽  
A Genome ◽  
Minimal Media

ABSTRACT GapMind is a Web-based tool for annotating amino acid biosynthesis in bacteria and archaea (http://papers.genomics.lbl.gov/gaps). GapMind incorporates many variant pathways and 130 different reactions, and it analyzes a genome in just 15 s. To avoid error-prone transitive annotations, GapMind relies primarily on a database of experimentally characterized proteins. GapMind correctly handles fusion proteins and split proteins, which often cause errors for best-hit approaches. To improve GapMind’s coverage, we examined genetic data from 35 bacteria that grow in defined media without amino acids, and we filled many gaps in amino acid biosynthesis pathways. For example, we identified additional genes for arginine synthesis with succinylated intermediates in Bacteroides thetaiotaomicron, and we propose that Dyella japonica synthesizes tyrosine from phenylalanine. Nevertheless, for many bacteria and archaea that grow in minimal media, genes for some steps still cannot be identified. To help interpret potential gaps, GapMind checks if they match known gaps in related microbes that can grow in minimal media. GapMind should aid the identification of microbial growth requirements. IMPORTANCE Many microbes can make all of the amino acids (the building blocks of proteins). In principle, we should be able to predict which amino acids a microbe can make, and which it requires as nutrients, by checking its genome sequence for all of the necessary genes. However, in practice, it is difficult to check for all of the alternative pathways. Furthermore, new pathways and enzymes are still being discovered. We built an automated tool, GapMind, to annotate amino acid biosynthesis in bacterial and archaeal genomes. We used GapMind to list gaps: cases where a microbe makes an amino acid but a complete pathway cannot be identified in its genome. We used these gaps, together with data from mutants, to identify new pathways and enzymes. However, for most bacteria and archaea, we still do not know how they can make all of the amino acids.

scholarly journals Unusual Metabolism and Hypervariation in the Genome of a Gracilibacterium (BD1-5) from an Oil-Degrading Community

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Christian M. K. Sieber ◽  
Blair G. Paul ◽  
Cindy J. Castelle ◽  
Ping Hu ◽  
Susannah G. Tringe ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Surface Protein ◽  
Local Variation ◽  
Building Blocks ◽  
Pentose Phosphate ◽  
Stabilizing Selection ◽  
Repeat Region ◽  
Content Type ◽  
Colwellia Psychrerythraea

ABSTRACT The candidate phyla radiation (CPR) comprises a large monophyletic group of bacterial lineages known almost exclusively based on genomes obtained using cultivation-independent methods. Within the CPR, Gracilibacteria (BD1-5) are particularly poorly understood due to undersampling and the inherent fragmented nature of available genomes. Here, we report the first closed, curated genome of a gracilibacterium from an enrichment experiment inoculated from the Gulf of Mexico and designed to investigate hydrocarbon degradation. The gracilibacterium rose in abundance after the community switched to dominance by Colwellia. Notably, we predict that this gracilibacterium completely lacks glycolysis, the pentose phosphate and Entner-Doudoroff pathways. It appears to acquire pyruvate, acetyl coenzyme A (acetyl-CoA), and oxaloacetate via degradation of externally derived citrate, malate, and amino acids and may use compound interconversion and oxidoreductases to generate and recycle reductive power. The initial genome assembly was fragmented in an unusual gene that is hypervariable within a repeat region. Such extreme local variation is rare but characteristic of genes that confer traits under pressure to diversify within a population. Notably, the four major repeated 9-mer nucleotide sequences all generate a proline-threonine-aspartic acid (PTD) repeat. The genome of an abundant Colwellia psychrerythraea population has a large extracellular protein that also contains the repeated PTD motif. Although we do not know the host for the BD1-5 cell, the high relative abundance of the C. psychrerythraea population and the shared surface protein repeat may indicate an association between these bacteria. IMPORTANCE CPR bacteria are generally predicted to be symbionts due to their extensive biosynthetic deficits. Although monophyletic, they are not monolithic in terms of their lifestyles. The organism described here appears to have evolved an unusual metabolic platform not reliant on glucose or pentose sugars. Its biology appears to be centered around bacterial host-derived compounds and/or cell detritus. Amino acids likely provide building blocks for nucleic acids, peptidoglycan, and protein synthesis. We resolved an unusual repeat region that would be invisible without genome curation. The nucleotide sequence is apparently under strong diversifying selection, but the amino acid sequence is under stabilizing selection. The amino acid repeat also occurs in a surface protein of a coexisting bacterium, suggesting colocation and possibly interdependence.

scholarly journals Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Irina A. Rodionova ◽  
Brian M. Schuster ◽  
Kristine M. Guinn ◽  
Leonardo Sorci ◽  
David A. Scott ◽  
...  
Keyword(s):  
Amino Acids ◽  
Drug Resistance ◽  
Drug Targets ◽  
Metabolic Flux ◽  
Building Blocks ◽  
Bactericidal Effect ◽  
New Drugs ◽  
Gas Chromatography Mass Spectrometry ◽  
Content Type ◽  
Target Enzyme

ABSTRACT Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.

scholarly journals Role of Branched-Chain Amino Acid Transport in Bacillus subtilis CodY Activity

2015 ◽  
Vol 197 (8) ◽  
pp. 1330-1338 ◽  
Author(s):  
Boris R. Belitsky
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Growth Medium ◽  
Amino Acid Transport ◽  
Transcriptional Regulator ◽  
Acid Transport ◽  
Content Type ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACTCodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units inBacillus subtilis. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases—BcaP, BraB, and BrnQ—that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.IMPORTANCEBacillus subtilisCodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

scholarly journals Chiroptical activity of hydroxycarboxylic acids with implications for the origin of biological homochirality

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jana Bocková ◽  
Nykola C. Jones ◽  
Uwe J. Meierhenrich ◽  
Søren V. Hoffmann ◽  
Cornelia Meinert
Keyword(s):  
Amino Acids ◽  
Lactic Acid ◽  
Organic Molecules ◽  
Building Blocks ◽  
Aliphatic Chain ◽  
Monocarboxylic Acids ◽  
Hydroxycarboxylic Acids ◽  
Polarised Light ◽  
Suitable Target ◽  
Anisotropy Spectra

AbstractCircularly polarised light (CPL) interacting with interstellar organic molecules might have imparted chiral bias and hence preluded prebiotic evolution of biomolecular homochirality. The l-enrichment of extra-terrestrial amino acids in meteorites, as opposed to no detectable excess in monocarboxylic acids and amines, has previously been attributed to their intrinsic interaction with stellar CPL revealed by substantial differences in their chiroptical signals. Recent analyses of meteoritic hydroxycarboxylic acids (HCAs) – potential co-building blocks of ancestral proto-peptides – indicated a chiral bias toward the l-enantiomer of lactic acid. Here we report on novel anisotropy spectra of several HCAs using a synchrotron radiation electronic circular dichroism spectrophotometer to support the re-evaluation of chiral biomarkers of extra-terrestrial origin in the context of absolute photochirogenesis. We found that irradiation by CPL which would yield l-excess in amino acids would also yield l-excess in aliphatic chain HCAs, including lactic acid and mandelic acid, in the examined conditions. Only tartaric acid would show “unnatural” d-enrichment, which makes it a suitable target compound for further assessing the relevance of the CPL scenario.

Sign in / Sign up

Export Citation Format

Share Document