[Mn(CO)₃(tpa-κ³N)]Br was prepared as a novel photoactivatable CO-releasing molecule (PhotoCORM) from [MnBr(CO)₅] and tris(2-pyridylmethyl)amine (tpa) for the delivery of carbon monoxide to biological systems, with the κ³N binding mode of the tetradentate tpa ligand demonstrated by X-ray crystallography. The title compound is a CORM prodrug stable in solution in the dark for up to 16 h. However, photoactivation at 365 nm leads to CO release from the metal coordination sphere and transfer to haem proteins, as demonstrated by the standard myoglobin assay. Different iCORM intermediates could be detected with solution IR spectroscopy and assigned using DFT vibrational calculations. The antibacterial activity of the complex was studied on Escherichia coli. No effects were observed when the cultures were either kept in the dark in the presence of PhotoCORM or illuminated in the absence of metal complex. However, photoactivation of [Mn(CO)₃(tpa-κ³N)]Br at 365 nm led to the appearance of the spectral signatures of CO-coordinated haems in the terminal oxidases of the bacterial electron transport chain in whole-cell UV/Vis absorption spectra. Significant internalization of the PhotoCORM was demonstrated by ICP-MS measurement of the intracellular manganese concentration. In particular when using medium with succinate as the sole carbon source, a very pronounced and concentration-dependent decrease in the E. coli growth rate could be observed upon illumination in the presence of metal complex, which is attributed to the constrained energy metabolism under these conditions and a strong indicator of terminal oxidase inhibition by carbon monoxide delivered from the PhotoCORM.

Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence. We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism’s ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.

Peroxynitrite is formed in macrophages by the diffusion-limited reaction of superoxide and nitric oxide. This highly reactive species is thought to contribute to bacterial killing by interaction with diverse targets and nitration of protein tyrosines. This work presents for the first time a comprehensive analysis of transcriptional responses to peroxynitrite under tightly controlled chemostat growth conditions. Up-regulation of the cysteine biosynthesis pathway and an increase in S-nitrosothiol levels suggest S-nitrosylation to be a consequence of peroxynitrite exposure. Genes involved in the assembly/repair of iron-sulfur clusters also show enhanced transcription. Unexpectedly, arginine biosynthesis gene transcription levels were also elevated after treatment with peroxynitrite. Analysis of the negative regulator for these genes, ArgR, showed that post-translational nitration of tyrosine residues within this protein is responsible for its degradation in vitro. Further up-regulation was seen in oxidative stress response genes, including katG and ahpCF. However, genes known to be up-regulated by nitric oxide and nitrosating agents (e.g. hmp and norVW) were unaffected. Probabilistic modeling of the transcriptomic data identified five altered transcription factors in response to peroxynitrite exposure, including OxyR and ArgR. Hydrogen peroxide can be present as a contaminant in commercially available peroxynitrite preparations. Transcriptomic analysis of cells treated with hydrogen peroxide alone also revealed up-regulation of oxidative stress response genes but not of many other genes that are up-regulated by peroxynitrite. Thus, the cellular responses to peroxynitrite and hydrogen peroxide are distinct.

Purified recombinant proteins from Synechocystis PCC6803 were used to show that the magnesium chelatase ChlH subunit stimulates magnesium protoporphyrin methyltransferase (ChlM) activity. Steady-state kinetics demonstrate that ChlH does not significantly alter the K_m for the tetrapyrrole substrate. However, quenched-flow analysis reveals that ChlH dramatically accelerates the formation and breakdown of an intermediate in the catalytic cycle of ChlM. In light of the profound effect that ChlH has on the methyltransferase catalytic intermediate, the pre steady-state analysis in the current study suggests that ChlH is directly involved in the reaction chemistry. The kinetic coupling between the chelatase and methyltransferase has important implications for regulation of chlorophyll biosynthesis and for the availability of magnesium protoporphyrin for plastid-to-nucleus signalling.