SummaryThe transcription factor Rex has been implicated in regulation of the expression of genes important for fermentative growth and for growth under conditions of low oxygen tension in several Gram-positive bacteria. Rex senses the redox poise of the cell through changes in the NADH/NAD + ratio. The crystal structures of two essentially identical Rex proteins, from Thermus aquaticus and T. thermophilus, have previously been determined in complex with NADH. Here we present the crystal structure of the Rex protein from Bacillus subtilis, as well as extensive studies of its affinity for nucleotides and DNA, using surface plasmon resonance, isothermal titration calorimetry and electrophoretic mobility shift assays. We show that Rex has a very high affinity for NADH but that its affinity for NAD + is 20 000 times lower. However, the NAD + affinity is increased by a factor of 30 upon DNA binding, suggesting that there is a positive allosteric coupling between DNA binding and NAD + binding. The crystal structures of two pseudoapo forms (from crystals soaked with NADH and cocrystallized with ATP) show a very different conformation from the previously determined Rex:NADH complexes, in which the N-terminal domains are splayed away from the dimer core. A mechanism is proposed whereby conformational changes in a C-terminal domain-swapped helix mediate the transition from a flexible DNA binding form to a locked NADH-bound form incapable of binding DNA.

Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca 2+ ions (log K 1 = 7.1 + − 0.4, log K 2 = 4.7 + − 0.6, log K 3 = 3.6 + − 0.7 and log K 4 = 4.6 + − 0.6 in physiological salt buffers) with a

SummaryIn the soil bacterium Bacillus subtilis Spx is a key regulator that controls expression, positively or negatively, of several genes in response to certain oxidative stresses that lead to the formation of unwanted disulphide bonds. Here we characterized the yjbH gene and show that it encodes a novel effector of Spx. The yjbH gene is part of the yjbIH operon that encodes a truncated haemoglobin (YjbI) and a predicted 34 kDa cytosolic protein of unknown function (YjbH). Deletion of yjbIH or yjbH has pleiotropic effects and affects growth, sporulation and competence development. Cells lacking yjbIH display a reduced sensitivity to the thiol oxidant diamide and show an apparent down-or upregulation of several transcripts that belong to the Spx regulon. Twentytwo suppressor mutations that bypass the defects conferred by yjbH were isolated. These mutations were identified as six deletions, three nonsense and 11 missense substitutions in the spx gene. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that mutations in yjbIH or yjbH do not affect the level of spx transcription. The combined data from the present work show that strains lacking yjbIH or yjbH overproduce Spx under unperturbed growth. The elevated Spx concentration cannot be attributed to an increased spx expression but is likely to result from control at the post-transcriptional level. YjbH is proposed to affect the cellular concentration of Spx by modulating proteolysis via the ClpXP protease.

A variety of pathways for electron and carbon flow in the soil bacterium Bacillus subtilis are differentially expressed depending on whether oxygen is present in the cell environment. This study characterizes the regulation of the respiratory oxidase cytochrome bd and the NADH-linked fermentative lactate dehydrogenase (LDH). Transcription of the cydABCD operon, encoding cytochrome bd, is highly regulated and only becomes activated at low oxygen availability. This induction is not dependent on the gene encoding the redox regulator Fnr or the genes encoding the ResDE two-component regulatory system. The DNA-binding protein YdiH was found to be a principal regulator that controls cydABCD expression. Transcription from the cyd promoter is stimulated 15-fold by a region located upstream of the core promoter. The upstream region may constitute a binding site for an unidentified transcription activator that is likely to influence the level of transcription but not its timing, which is negatively controlled by YdiH. This report provides evidence that YdiH also functions as a repressor of the ldh gene encoding LDH and of a gene, ywcJ, which encodes a putative formate-nitrite transporter. Based on the similarity between YdiH and the Rex protein of Streptomyces coelicolor, it is proposed that YdiH serves as a redox sensor, the activity of which is regulated by cellular differences in the free levels of NAD+ and NADH. It is suggested that ydiH be renamed as rex.

Spx is a global regulator that is widespread among the low-G؉C-content Gram-positive bacteria. Spx has been extensively studied in Bacillus subtilis, where it acts as an activator and a repressor of transcription in response to disulfide stress. Under nonstress conditions, Spx is rapidly degraded by the ClpXP protease. This degradation is enhanced by the YjbH adaptor protein. Upon disulfide stress, the amount of Spx rapidly increases due to a decrease in degradation. In the opportunistic pathogen Staphylococcus aureus, Spx is a global regulator influencing growth, biofilm formation, and general stress protection, and cells lacking the spx gene exhibit poor growth also under nonstress conditions. To investigate the mechanism by which the activity of Spx is regulated, we identified a homolog in S. aureus of the B. subtilis yjbH gene. The gene encodes a protein that shows approximately 30% sequence identity to YjbH of B. subtilis. Heterologous expression of S. aureus yjbH in a B. subtilis yjbH mutant restored Spx to wild-type levels both under nonstress conditions and under conditions of disulfide stress. From these studies, we conclude that the two YjbH homologues have a conserved physiological function. Accordingly, inactivation of yjbH in S. aureus increased the level of Spx protein and transcription of the Spx-regulated gene trxB. Notably, the yjbH mutant exhibited reduced growth and increased pigmentation, and both phenotypes were reversed by complementation of the yjbH gene.

Bacteria use a number of mechanisms for coping with the toxic effects exerted by nitric oxide (NO) and its derivatives. Here we show that the flavohemoglobin encoded by the hmp gene has a vital role in an adaptive response to protect the soil bacterium Bacillus subtilis from nitrosative stress. We further show that nitrosative stress induced by the nitrosonium cation donor sodium nitroprusside (SNP) leads to deactivation of the transcriptional repressor NsrR, resulting in derepression of hmp. Nitrosative stress induces the sigma Bcontrolled general stress regulon. However, a sigB null mutant did not show increased sensitivity to SNP, suggesting that the sigma B-dependent stress proteins are involved in a nonspecific protection against stress whereas the Hmp flavohemoglobin plays a central role in detoxification. Mutations in the yjbIH operon, which encodes a truncated hemoglobin (YjbI) and a predicted 34-kDa cytosolic protein of unknown function (YjbH), rendered B. subtilis hypersensitive to SNP, suggesting roles in nitrosative stress management.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing Coronavirus Disease 19 (COVID-19), emerged at the end of 2019 and quickly spread to cause a global pandemic with severe socio-economic consequences. The early sequencing of its RNA genome revealed its high similarity to SARS, likely to have originated from bats. The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3′-to-5′ exoribonuclease and the 2′-O-methlytransferase activities of nsps 14 and 16, respectively. Here, we report the biophysical characterization and 1.6 Å resolution structure of the unbound form of nsp10 from SARS-CoV-2 and compare it to the structures of its SARS homologue and the complex-bound form with nsp16 from SARS-CoV-2. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication–transcription complex and virus replication.

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and...