Selenocysteine-independent suppression of UGA codons in the archaeon Methanococcus maripaludis.

BACKGROUND: Proteins containing selenocysteine (sec) are found in Bacteria, Eukarya, and Archaea. While selenium-dependence of methanogenesis from H(2)+CO(2) in the archaeon Methanococcus maripaludis JJ is compensated by induction of a set of cysteine-containing homologs, growth on formate is abrogated in the absence of sec due to the dependence of formate dehydrogenase (Fdh) on selenium. Despite this dependence, formate-dependent growth occurs after prolonged incubation of M. maripaludis mutants lacking sec. METHODS: To study this phenomenon, a M. maripaludis strain with only one Fdh isoform and an FdhA selenoprotein C-terminally tagged for affinity enrichment was constructed. Factors required for sec synthesis were deleted in this strain and translation of UGA in fdhA was analyzed physiologically, enzymatically, immunologically, and via mass spectrometry. RESULTS: M. maripaludis JJ mutants lacking sec synthesis grew at least five times more slowly than the wild type on formate due to a 20-35-fold reduction of Fdh activity. The enzyme in the mutant strains lacked sec but was still produced as a full-length protein. Peptide mass spectrometry revealed that both cysteine (cys) and tryptophan (trp) were inserted at the UGA encoding sec without apparent mutations in tRNA(cys) or tRNA(trp), respectively. CONCLUSIONS: We demonstrate that M. maripaludis has the inherent capacity to translate UGA with cys and trp; other mechanisms to replace sec with cys in the absence of selenium could thereby be ruled out. GENERAL SIGNIFICANCE: This study exemplifies how an organism uses the inherent flexibility in its canonical protein synthesis machinery to recover some activity of an essential selenium-dependent enzyme in the absence of sec.

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

Enzymes of anaerobic ethylbenzene and p-ethylphenol catabolism in 'Aromatoleum aromaticum': differentiation and differential induction.

The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.

Evaluation of antibody responses in healthcare workers before & after meningococcal vaccine and determination of meningococcal carriage rates.

The rates of nasopharyngeal meningococcal carriage in healthcare workers are unknown. Meningococcal vaccine is recommended for risk groups but healthcare workers are not included in risk groups for many countries. Herein, we aimed to investigate the nasopharyngeal meningococcal carriage rates, basal and after one dose of Men-ACWY-DT vaccine response on the 30th day by evaluating meningococcus IgG antibody levels and decolonization at month six after vaccination among the detected carriers. Nasopharyngeal swab samples were taken before vaccination to evaluate meningococcal carriage in healthcare workers. All participants received a single dose of Men-ACWY-DT vaccine. Serum samples were collected immediately before vaccination and again on day 30 post-vaccination. Antibodies in the stored sera were analyzed using the ELISA method. Participants who were determined to carry meningococci at the initial visit underwent another round of nasopharyngeal swab tests six months post-vaccination to check for decolonization. Between November 2020 and May 2021, we evaluated samples from 100 physicians [52 % females, 28.28 ± 4.45 (min: 24, max: 49)]. The majority of the physicians worked in the emergency department (45 %), followed by the infectious diseases clinic (14 %). Fifty-eight physicians had a history of at least one contact with a meningococcus-infected patient, and 53 (91.4 %) had used prophylactic antibiotics at least once due to this exposure. None of the study group nasopharyngeal swab cultures were positive for Neisseria meningitidis. Before the Men-ACWY-DT vaccine, anti-meningococcus IgG positivity was detected in the serum samples of only 3 (3 %) participants. By day 30 after vaccination, 48 % of participants showed positive for antibodies. As we didn't detect nasopharyngeal carriage in any participants, we didn't evaluate decolonization among carriers six months post-vaccination. Notably, detection of antibodies was evident in about half of the participants on day 30 after receiving a single dose of the Men-ACWY-DT vaccine.

Importance of Source Control in the Subgroup of Intra-Abdominal Infections for Septic Shock Patients: Analysis of 390 Cases.

Background: This study aimed to evaluate the epidemiology of septic shock (SS) associated with intraabdominal infections (IAI) as well as associated mortality and efficacy of early source control in a tertiary-care educational hospital. Methods: Patients who had SS with IAI and consulted by Infectious Diseases consultants between December 2013 and October 2022 during night shifts in our centre were analyzed retrospectively. Results: A total number of 390 patients were included. Overall, 30-day mortality was 42.5% on day 3, while day 14 and 30 mortality rates were 63.3% and 71.3%, respectively. Source control by surgical or percutaneous operation was performed in 123 of 390 cases (31.5%), and the mortality rate was significantly lower in cases that were performed source control at any time during SS (65/123-52.8% vs 213/267-79.8%, p<0.001). In 44 of 123 cases (35.7%), source control was performed during the first 12 hours, and mortality was significantly lower in this group versus others (24/44-54.5% vs 254/346-73.4%, p=0.009). On the other hand, female gender (p<0.001, odds ratio(OR)= 2.943, 95%CI=1.714-5.054), diabetes mellitus (p= 0.014, OR=2.284, 95%CI=1.179-4.424), carbapenem-resistant Gram-negative etiology (p=0.011, OR=4.386, 95%CI=1.398-13.759), SOFA≥10 (p<0.001, OR=3.036, 95%CI=1.802-5.114), lactate >3 mg/dl (p<0.001, OR=2.764, 95%CI=1.562-4.891) and lack of source control (p=0.001, OR=2.796, 95%CI=1.523-5.133) were significantly associated with 30-day mortality in logistic regression analysis. Conclusion: Source control has a vital importance in terms of mortality rates for IAI-related septic shock patients. Our study underscores the need for additional research, as the present analysis indicates that early source control does not manifest as a protective factor in logistic regression.

Disruption and complementation of the selenocysteine biosynthesis pathway reveals a hierarchy of selenoprotein gene expression in the archaeon Methanococcus maripaludis.

Proteins containing selenocysteine are found in members of all three domains of life, Bacteria, Eukarya and Archaea. A dedicated tRNA (tRNA(sec)) serves as a scaffold for selenocysteine synthesis. However, sequence and secondary structures differ in tRNA(sec) from the different domains. An Escherichia coli strain lacking the gene for tRNA(sec) could only be complemented with the homologue from Methanococcus maripaludis when a single base in the anticodon loop was exchanged demonstrating that this base is a crucial determinant for archaeal tRNA(sec) to function in E. coli. Complementation in trans of M. maripaludis JJ mutants lacking tRNA(sec) , O-phosphoseryl-tRNA(sec) kinase or O-phosphoseryl-tRNA(sec) :selenocysteine synthase with the corresponding genes from M. maripaludis S2 restored the mutant's ability to synthesize selenoproteins. However, only partial restoration of the wild-type selenoproteome was observed as only selenocysteine-containing formate dehydrogenase was synthesized. Quantification of transcripts showed that disrupting the pathway of selenocysteine synthesis leads to downregulation of selenoprotein gene expression, concomitant with upregulation of a selenium-independent backup system, which is not re-adjusted upon complementation. This transcriptional arrest was independent of selenophosphate but depended on the 'history' of the mutants and was inheritable, which suggests that a stable genetic switch may cause the resulting hierarchy of selenoproteins synthesized.

Selenium-dependent gene expression in Methanococcus maripaludis: Involvement of the transcriptional regulator HrsM.

BACKGROUND: The archaeon Methanococcus maripaludis strain JJ employs several selenocysteine (Sec)-containing proteins in its primary energy metabolism, methanogenesis. Upon selenium deprivation, or when the pathway for selenoprotein synthesis is disrupted, they are replaced by cysteine (Cys)-containing isoforms, thus allowing for selenium-independent growth. METHODS: Expression of a fusion of the promoter region of frcA (encoding a subunit of the selenium-independent hydrogenase Frc) and bla [encoding ß-lactamase (Bla)] in M. maripaludis JJ was assessed in response to the selenium supply, growth substrate, and growth phase. Random transposon mutants of the reporter strain were screened for deregulated bla expression, which identified HrsM, a LysR-type transcriptional regulator (LTTR). Its involvement in selenium-dependent gene regulation was further assessed by analyzing in vivo transcription, synthesis of selenoproteins and of HrsM, and by analyzing in vitro binding of HrsM to DNA. RESULTS: HrsM, which is not required for selenoprotein synthesis, acts as a positive effector of selenoprotein gene expression and as a negative effector of Cys-encoding isogene expression, but its own expression is independent of the selenium availability. Specific binding in vitro of HrsM to a promoter region under in vivo HrsM control verified its role in selenium-dependent gene regulation. CONCLUSIONS: HrsM exerts a key role in regulating expression of selenoprotein genes and their Cys-encoding isogenes in M. maripaludis in a selenium-dependent fashion. However, this activity is not achieved via autoregulation but probably by a mechanism, which modulates the DNA-binding of HrsM. GENERAL SIGNIFICANCE: Although LTTRs are abundant in Bacteria, HrsM represents only the second characterized member of this group in Archaea.

Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes.

The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield (R)-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting p-cresol, 2-methylnaphthalene or n-alkanes.