scholarly journals Cyclic-di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions

2017 ◽  
Author(s):  
Merve S. Zeden ◽  
Christopher F. Schuster ◽  
Lisa Bowman ◽  
Qiyun Zhong ◽  
Huw D. Williams ◽  
...  
Keyword(s):  
Amino Acid ◽  
Adenosine Monophosphate ◽  
Defined Medium ◽  
Growth Defect ◽  
Uptake System ◽  
Osmotic Regulation ◽  
Bacterial Cells ◽  
Rich Medium ◽  
Anaerobic Conditions

ABSTRACTCyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such asStaphylococcus aureus. However, the exact role of this molecule has not been identified.dacA, theS. aureusgene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that anS. aureus dacAmutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality ofdacAwere identified inalsTandopuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system inS. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained inhepS, hemB, ctaAandqoxB, coding for proteins required for respiration. Furthermore, we show thatdacAis dispensable for growth in anaerobic conditions. Together, these finding reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration inS. aureus.

Role of Methionine in the Initiation of the Biosynthesis of Bovine Proinsulin

1973 ◽  
Vol 51 (6) ◽  
pp. 783-788 ◽  
Author(s):  
C. C. Yip ◽  
C. C. Liew
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Defined Medium ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Tissue Slices ◽  
Fetal Bovine ◽  
Bovine Pancreas

Slices of fetal bovine pancreas were used to study the initiation of proinsulin biosynthesis. The pancreatic slices were incubated with radioactive methionine, phenylalanine, or leucine, in a defined medium. The incorporation of amino acid into peptides in the tissue slices was measured for 2–3 h. Two types of radioactive peptides, "free" and "blocked," were identified by ion-exchange chromatography. Most of the radioactive "blocked" peptides labelled with [3H]phenylalanine and [35S]methionine were hydrolyzed by proteases, except for about 20% of those labelled with [35S]methionine, which also showed higher resistance to acid hydrolysis.Microsomes were isolated from the tissue slices after incubation and were extracted with acid alcohol. The radioactive proteins in the extract were reacted with a solid immunosorbant against insulin. Analysis of the immunoadsorbed radioactive peptides by Edman degradation showed the presence of both methionine and phenylalanine as the N-termini. It was concluded that methionine was an initiating amino acid in the biosynthesis of bovine proinsulin.

scholarly journals Functional Analysis of luxS in Staphylococcus aureus Reveals a Role in Metabolism but Not Quorum Sensing

2006 ◽  
Vol 188 (8) ◽  
pp. 2885-2897 ◽  
Author(s):  
Neil Doherty ◽  
Matthew T. G. Holden ◽  
Saara N. Qazi ◽  
Paul Williams ◽  
Klaus Winzer
Keyword(s):  
Staphylococcus Aureus ◽  
T Cells ◽  
Stationary Phase ◽  
Defined Medium ◽  
Growth Patterns ◽  
Transcriptional Unit ◽  
Growth Defect ◽  
Wild Type ◽  
Intracellular Growth ◽  
Anaerobic Conditions

ABSTRACT The function of AI-2 in many bacteria and the physiological role of LuxS, the enzyme responsible for its production, remain matters of debate. Here, we show that in Staphylococcus aureus the luxS gene forms a monocistronic transcriptional unit under the control of a σ70-dependent promoter. The gene was transcribed throughout growth under a variety of conditions, including intracellular growth in MAC-T cells. AI-2 was produced in rich media under aerobic and anaerobic conditions, peaking during the transition to stationary phase, but was hardly detectable in a sulfur-limited defined medium. In the presence of glucose or under anaerobic conditions, cultures retained considerable AI-2 activity after entry into stationary phase. Inactivation of luxS in various S. aureus strains did not affect virulence-associated traits, such as production of hemolysins and extracellular proteases, biofilm formation, and the agr signaling system. Conversely, AI-2 production remained unchanged in an agr mutant. However, luxS mutants grown in a sulfur-limited defined medium exhibited a growth defect. When grown together with the wild type in mixed culture, luxS mutants of various S. aureus strains showed reduced ability to compete for growth under these conditions. In contrast, a complemented luxS mutant grew as well as the parent strain, suggesting that the observed growth defect was of an intracellular nature and had not been caused by either second-site mutations or the lack of a diffusible factor. However, the LuxS/AI-2 system does not appear to contribute to the overall fitness of S. aureus RN6390B during intracellular growth in epithelial cells: the wild type and a luxS mutant showed very similar growth patterns after their internalization by MAC-T cells.

scholarly journals The yeast translational allosuppressor, SAL6: a new member of the PP1-like phosphatase family with a long serine-rich N-terminal extension.

Genetics ◽  
1994 ◽  
Vol 138 (3) ◽  
pp. 597-608 ◽  
Author(s):  
A Vincent ◽  
G Newnam ◽  
S W Liebman
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Amino Acid Position ◽  
Cold Sensitivity ◽  
Growth Defect ◽  
Physical Analysis ◽  
Acid Protein ◽  
Slight Growth ◽  
Amino Acid Protein

Abstract The allosuppressor mutation, sal6-1, enhances the efficiency of all tested translational suppressors, including codon-specific tRNA suppressors as well as codon-nonspecific omnipotent suppressors. The SAL6 gene has now been cloned by complementation of the increased suppression efficiency and cold sensitivity caused by sal6-1 in the presence of the omnipotent suppressor sup45. Physical analysis maps SAL6 to chromosome XVI between TPK2 and spt14. The SAL6 gene encodes a very basic 549-amino acid protein whose C-terminal catalytic region of 265 residues is 63% identical to serine/threonine PP1 phosphatases, and 66% identical to yeast PPZ1 and PPZ2 phosphatases. The unusual 235 residue N-terminal extension found in SAL6, like those in the PPZ proteins, is serine-rich. The sal6-1 mutation is a frameshift at amino acid position 271 which destroys the presumed phosphatase catalytic domain of the protein. Disruptions of the entire SAL6 gene are viable, cause a slight growth defect on glycerol medium, and produce allosuppressor phenotypes in suppressor strain backgrounds. The role of the serine-rich N terminus is unclear, since sal6 phenotypes are fully complemented by a SAL6 allele that contains an in-frame deletion of most of this region. High copy number plasmids containing wild-type SAL6 cause antisuppressor phenotypes in suppressor strains. These results suggest that the accuracy of protein synthesis is affected by the levels of phosphorylation of the target(s) of SAL6.

scholarly journals Influence of Amino Acid Feeding on Production of Calcimycin and Analogs in Streptomyces chartreusis

2021 ◽  
Vol 18 (16) ◽  
pp. 8740
Author(s):  
Kirstin I. Arend ◽  
Julia E. Bandow
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nitrogen Sources ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Defined Medium ◽  
Glutamine Supplementation ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters

Streptomyces chartreusis NRRL 3882 produces the polyether ionophore calcimycin and a variety of analogs, which originate from the same biosynthetic gene cluster. The role of calcimycin and its analogs for the producer is unknown, but calcimycin has strong antibacterial activity. Feeding experiments were performed in chemically defined medium systematically supplemented with proteinogenic amino acids to analyze their individual effects on calcimycin synthesis. In the culture supernatants, in addition to known calcimycin analogs, eight so far unknown analogs were detected using LC-MS/MS. Under most conditions cezomycin was the compound produced in highest amounts. The highest production of calcimycin was detected upon feeding with glutamine. Supplementation of the medium with glutamic acid resulted in a decrease in calcimycin production, and supplementation of other amino acids such as tryptophan, lysine, and valine resulted in the decrease in the synthesis of calcimycin and of the known intermediates of the biosynthetic pathway. We demonstrated that the production of calcimycin and its analogs is strongly dependent on amino acid supply. Utilization of amino acids as precursors and as nitrogen sources seem to critically influence calcimycin synthesis. Even amino acids not serving as direct precursors resulted in a different product profile regarding the stoichiometry of calcimycin analogs. Only slight changes in cultivation conditions can lead to major changes in the metabolic output, which highlights the hidden potential of biosynthetic gene clusters. We emphasize the need to further study the extent of this potential to understand the ecological role of metabolite diversity originating from single biosynthetic gene clusters.

scholarly journals A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

2004 ◽  
Vol 186 (9) ◽  
pp. 2757-2765 ◽  
Author(s):  
Eiji Masai ◽  
Miyuki Sasaki ◽  
Yasunori Minakawa ◽  
Tomokuni Abe ◽  
Tomonori Sonoki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Extract ◽  
Growth Defect ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Growth Deficiency ◽  
Glycine Cleavage ◽  
Derivative Strain

ABSTRACT Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed.

Roles and regulation of the glutamate racemase isogenes, racE and yrpC, in Bacillus subtilis

Microbiology ◽  
2004 ◽  
Vol 150 (9) ◽  
pp. 2911-2920 ◽  
Author(s):  
Keitarou Kimura ◽  
Lam-Son Phan Tran ◽  
Yoshifumi Itoh
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Minimal Medium ◽  
Degradation Products ◽  
Acid Synthesis ◽  
Rich Medium ◽  
Amino Acid Synthesis ◽  
Double Mutation ◽  
Glutamate Racemase

Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase. This enzyme catalyses the formation of d-glutamate, which is necessary for cell wall peptidoglycan synthesis. However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC. Since racE appears to be indispensable for growth in rich medium, the role of yrpC in d-amino acid synthesis is vague. Experiments with racE- and yrpC-knockout mutants confirmed that racE is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE. LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium. Neither racE nor yrpC was required for B. subtilis cells to synthesize poly-γ-dl-glutamate (γ-PGA), a capsule polypeptide of d- and l-glutamate linked through a γ-carboxylamide bond. Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, d-glutamate and l-glutamate, in the medium. In contrast, racE or yrpC mutant cells accumulated significant amounts of d- but not l-glutamate. Exogenous d-glutamate utilization was somewhat defective in the mutants and the double mutation of race and yrpc severely impaired d-amino acid utilization. Thus, both racemase genes appear necessary to complete the catabolism of exogenous d-glutamate generated from γ-PGA.

scholarly journals Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

2020 ◽  
Author(s):  
Dorothea Höpfner ◽  
Joel Fauser ◽  
Marietta S. Kaspers ◽  
Christian Pett ◽  
Christian Hedberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Recent Progress ◽  
Regulatory Mechanism ◽  
Target Identification ◽  
Adenosine Monophosphate ◽  
Post Translational Modification ◽  
Highly Sensitive ◽  
Amino Acid Side Chains

AbstractAMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

scholarly journals Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation inStaphylococcus aureusbut dispensable for viability in anaerobic conditions

2018 ◽  
Vol 293 (9) ◽  
pp. 3180-3200 ◽  
Author(s):  
Merve S. Zeden ◽  
Christopher F. Schuster ◽  
Lisa Bowman ◽  
Qiyun Zhong ◽  
Huw D. Williams ◽  
...  
Keyword(s):  
Adenosine Monophosphate ◽  
Osmotic Regulation ◽  
Anaerobic Conditions

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

2019 ◽  
Author(s):  
Bella Grigorenko ◽  
Igor Polyakov ◽  
Alexander Nemukhin
Keyword(s):  
Adenylyl Cyclase ◽  
Bond Cleavage ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Md Simulations ◽  
Coordination Shell ◽  
Energy Profile ◽  
One Step ◽  
Type V

<p>We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of Mg<sub>A</sub><sup>2+ </sup>are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of Mg<sub>A</sub><sup>2+ </sup>does not include the O<sub>δ1</sub> atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O<sup>3'</sup>H<sup>3' </sup>group in ATP to Asp440 via a shuttling water molecule and P<sup>A</sup>-O<sup>3A</sup> bond cleavage and O<sup>3'</sup>-P<sup>A</sup> bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, Mg<sub>A</sub><sup>2+</sup> is bound to the O<sub>δ1</sub>(Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O<sup>3'</sup>H<sup>3' </sup>group via shuttling water molecules. </p>

Sign in / Sign up

Export Citation Format

Share Document