Unraveling the Biogenesis and Roles of Iron-sulfur Proteins during Nitrogen Fixation by Methanosarcina acetivorans

Iron-sulfur (Fe-S) clusters, the ancient cofactors found in proteins across all life forms, play vital roles in numerous cellular processes. Fe-S clusters exhibit structural diversity, ranging from simple to complex forms. Methanogens are anaerobic archaea that produce methane as a metabolic by-product through methanogenesis, a process dependent on numerous Fe-S proteins. Notably, methanogens are capable of fixing nitrogen (diazotrophy) facilitated by the enzyme complex called nitrogenase, which contains both simple and complex Fe-S clusters. While methanogens possess the largest number of Fe-S proteins, the factors involved in Fe-S cluster biogenesis remain largely unknown. Bacteria possess three distinct and well-characterized Fe-S cluster biogenesis systems - ISC, SUF, and NIF, with the SUF and ISC systems functioning in general Fe-S cluster biogenesis and the NIF system specific to Fe-S cluster biogenesis in nitrogenase. All methanogens encode homologs of SufBC, the core Fe-S cluster scaffold component of the SUF system and some species also encode homologs of the core components of the ISC system (IscS and IscU). No species contains homologs of the NIF system components, despite many methanogens containing nitrogenase. The SUF system is the most ancient of all Fe-S cluster biogenesis systems and since it is present in all methanogens, it has been postulated to serve as the primary and nitrogenase-specific Fe-S cluster biogenesis system in methanogens. This dissertation investigates the biogenesis and roles of Fe-S proteins during nitrogen fixation by Methanosarcina acetivorans, a model methanogen that contains all three types of nitrogenase. In chapter one, the recently developed CRISPRi-dCas9 and CRISPR-Cas9 systems were used to investigate the role of SufBC in M. acetivorans. Findings from gene repression and deletion studies reveal that the SUF system is not essential to M. acetivorans, is not the primary Fe-S cluster biogenesis system, nor is it required for nitrogenase Fe-S cluster biogenesis. In chapter two, the role of NifB in the biosynthesis of the complex Fe-S clusters in nitrogenases was investigated. NifB is a radical S-adenosy-L-methionine (SAM) enzyme required for the insertion of carbide into FeMo-co, FeV-co, and FeFe-co during the maturation of Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. Surprisingly, results for gene deletion studies revealed that nifB is essential to the viability of M. acetivorans and that NifB serves a distinct function apart from nitrogenase maturation, a result not seen in bacteria. In chapter three, potential factors involved in the electron transfer to nitrogenases were investigated. Specifically, a low-potential flavodoxin (FldA) and the Fe-S protein complex heterodisulfide reductase (HdrABC) were tested for the involvement in diazotrophy using the CRISPRi repression system. The absence of any observable phenotype in the fldA and hdrABC repression strains suggests that neither of these genes are involved in electron transfer to nitrogenase. Overall, these findings shed light on the roles of SufBC and NifB in the biosynthesis of simple and complex Fe-S clusters and provide new insights into the factors involved in electron transfer to nitrogenase during methanogenesis

Unraveling the Biogenesis and Roles of Iron-sulfur Proteins during Nitrogen Fixation by Methanosarcina acetivorans

Iron-sulfur (Fe-S) clusters, the ancient cofactors found in proteins across all life forms, play vital roles in numerous cellular processes. Fe-S clusters exhibit structural diversity, ranging from simple to complex forms. Methanogens are anaerobic archaea that produce methane as a metabolic by-product through methanogenesis, a process dependent on numerous Fe-S proteins. Notably, methanogens are capable of fixing nitrogen (diazotrophy) facilitated by the enzyme complex called nitrogenase, which contains both simple and complex Fe-S clusters. While methanogens possess the largest number of Fe-S proteins, the factors involved in Fe-S cluster biogenesis remain largely unknown. Bacteria possess three distinct and well-characterized Fe-S cluster biogenesis systems - ISC, SUF, and NIF, with the SUF and ISC systems functioning in general Fe-S cluster biogenesis and the NIF system specific to Fe-S cluster biogenesis in nitrogenase. All methanogens encode homologs of SufBC, the core Fe-S cluster scaffold component of the SUF system and some species also encode homologs of the core components of the ISC system (IscS and IscU). No species contains homologs of the NIF system components, despite many methanogens containing nitrogenase. The SUF system is the most ancient of all Fe-S cluster biogenesis systems and since it is present in all methanogens, it has been postulated to serve as the primary and nitrogenase-specific Fe-S cluster biogenesis system in methanogens. This dissertation investigates the biogenesis and roles of Fe-S proteins during nitrogen fixation by Methanosarcina acetivorans, a model methanogen that contains all three types of nitrogenase. In chapter one, the recently developed CRISPRi-dCas9 and CRISPR-Cas9 systems were used to investigate the role of SufBC in M. acetivorans. Findings from gene repression and deletion studies reveal that the SUF system is not essential to M. acetivorans, is not the primary Fe-S cluster biogenesis system, nor is it required for nitrogenase Fe-S cluster biogenesis. In chapter two, the role of NifB in the biosynthesis of the complex Fe-S clusters in nitrogenases was investigated. NifB is a radical S-adenosy-L-methionine (SAM) enzyme required for the insertion of carbide into FeMo-co, FeV-co, and FeFe-co during the maturation of Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. Surprisingly, results for gene deletion studies revealed that nifB is essential to the viability of M. acetivorans and that NifB serves a distinct function apart from nitrogenase maturation, a result not seen in bacteria. In chapter three, potential factors involved in the electron transfer to nitrogenases were investigated. Specifically, a low-potential flavodoxin (FldA) and the Fe-S protein complex heterodisulfide reductase (HdrABC) were tested for the involvement in diazotrophy using the CRISPRi repression system. The absence of any observable phenotype in the fldA and hdrABC repression strains suggests that neither of these genes are involved in electron transfer to nitrogenase. Overall, these findings shed light on the roles of SufBC and NifB in the biosynthesis of simple and complex Fe-S clusters and provide new insights into the factors involved in electron transfer to nitrogenase during methanogenesis

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans, which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2, respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1. In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens

Prosthodontic Management of Flabby Ridge - From Modified Impression to Denture

International audienc

Prosthodontic Management of Flabby Ridge - From Modified Impression to Denture

International audienc

Randomised controlled trial of central venous catheterisation through external jugular vein: A comparison of success with or without body manoeuvres

Background and Aims: The external jugular vein (EJV), often used for resuscitation, has been underutilised for central venous catheterisation (CVC) in view of an unpredictable success rate. There is an encouraging literature on the improved success rate of CVC through EJV with the inclusion of certain body manoeuvres. This prospective randomised controlled study was conducted with the aim of evaluating the efficacy of body manoeuvres in improving the success rate of CVC through EJV. Methods: One hundred patients aged 18–50 years, scheduled for elective surgery requiring CVC, were randomly assigned to either undergo CVC using Seldinger technique with body manoeuvres or a control group undergoing CVC without body manoeuvres. The primary outcome was the success rate of CVC, as observed in the post-procedure chest radiograph. Secondary outcomes included quality of central venous pressure waveform, catheterisation attempts, total time for CVC, complications. Results: CVC was achieved in 98% (49/50) of patients in study group and 80% (40/50) of patients in control group (P = 0.008). Mean catheterisation time was significantly lower in the study group (151.06 ± 40.50 s) compared to control group (173.50 ± 50.66 s) (P = 0.023). The incidence of catheter misplacement and failure to cannulate were lower in the study group (0%, 2% vs. 20%, 12.5%, respectively). Groups did not differ in a number of catheterisation attempts and incidence of haematoma. Conclusion: Inclusion of various body manoeuvres to Seldinger technique significantly improves the success rate of CVC through EJV

Proceedings of International Conference on Women Researchers in Electronics and Computing

This proceeding contains articles on the various research ideas of the academic community and practitioners presented at the international conference, “Women Researchers in Electronics and Computing” (WREC’2021). WREC'21 was organized in online mode by Dr. B R Ambedkar National Institute of Technology, Jalandhar (Punjab), INDIA during 22 – 24 April 2021. This conference was conceptualized with an objective to encourage and motivate women engineers and scientists to excel in science and technology and to be the role models for young girls to follow in their footsteps. With a view to inspire women engineers, pioneer and successful women achievers in the domains of VLSI design, wireless sensor networks, communication, image/ signal processing, machine learning, and emerging technologies were identified from across the globe and invited to present their work and address the participants in this women oriented conference. Conference Title: International Conference on Women Researchers in Electronics and ComputingConference Acronym: WREC'21Conference Date: 22–24 April 2021Conference Location: Online (Virtual Mode)Conference Organizers: Department of Electronics and Communication Engineering, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar, Punjab, INDI