•We have resolved the crystal structure of the decaheme OmcA to 2.7Å resolution.•A proposed heme binding motif is identified around heme 10.•Heme 5 is proposed as a site for intermolecular electron ...transfer.
The X-ray crystal structure of Shewanella oneidensis OmcA, an extracellular decaheme cytochrome involved in mineral reduction, was solved to a resolution of 2.7Å. The four OmcA molecules in the asymmetric unit are arranged so the minimum distance between heme 5 on adjacent OmcA monomers is 9Å, indicative of a transient OmcA dimer capable of intermolecular electron transfer. A previously identified hematite binding motif was identified near heme 10, forming a hydroxylated surface that would bring a heme 10 electron egress site to ∼10Å of a mineral surface.
OmcA and OmcAbind by X-ray crystallography(View interaction)
Summary
Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species ...electrons cross the outer membrane to extracellular substrates via ‘porin–cytochrome’ electron transport modules. The molecular structure of an outer‐membrane extracellular‐facing deca‐haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer‐membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as ‘nanowires’, or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram‐negative bacteria.
Many species of the bacterial Shewanella genus are notable for their ability to respire in anoxic environments utilizing insoluble minerals of Fe(III) and Mn(IV) as extracellular electron acceptors. ...In Shewanella oneidensis, the process is dependent on the decahaem electron-transport proteins that lie at the extracellular face of the outer membrane where they can contact the insoluble mineral substrates. These extracellular proteins are charged with electrons provided by an inter-membrane electron-transfer pathway that links the extracellular face of the outer membrane with the inner cytoplasmic membrane and thereby intracellular electron sources. In the present paper, we consider the common structural features of two of these outer-membrane decahaem cytochromes, MtrC and MtrF, and bring this together with biochemical, spectroscopic and voltammetric data to identify common and distinct properties of these prototypical members of different clades of the outer-membrane decahaem cytochrome superfamily.
The increase in resistance of bacterial strains to existing antibiotics represents a major public health concern. Antibiotics are an invaluable tool still frequently used in agriculture as they can ...both improve feed/energy conversion ratios in livestock and treat pathogenic infections. However, the misuse of these compounds has contributed to escalating antimicrobial resistance (AMR). The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. Research is currently being carried out to identify alternative antimicrobial compounds to use in animal production although to date, no effective alternatives to production-purpose antibiotics (PPAs) have been developed. Recent reports indicate that lysate of marine diatom Phaeodactylum tricornutum was able to significantly lower the maximal growth rate of Gram-negative bacteria. We have engineered P. tricornutum to accumulate antimicrobial recombinant proteins, S-thanatin and bovine lactoferricin (bLFcin). Transgenic P. tricornutum expressing heterologous S-thanatin and bLFcin demonstrated significantly enhanced antimicrobial activity against Gram-negative bacteria Enterobacter cloacae ATCC 13047 and Vibrio natriegens ATCC 14048 respectively. The lysate of transgenic strain Pt_LFcin1.15, expressing bLFcin, lowered maximal growth rate (μmax) of V. natriegens by 33.2 %, which was a 15.5 % further reduction than by wild type (WT) lysate. The lysate of the optimized bLFcin-expressing transgenic strain Pt_LFcin2.3 lowered V. natriegens μmax by 38.5 %, which was a 27.4 % further reduction than by WT lysate. The S-thanatin-expressing transgenic cell lysate of strain Pt_S-Tha1.11 lowered E. cloacae μmax by 61.3 %, which was a 38.1 % greater reduction than by WT lysate. These statistically significant results (p < 0.01) demonstrate that microalgae can be engineered to express a range of naturally occurring antimicrobial and anti-inflammatory compounds which can substitute for PPAs and treat serious bacterial infections.
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•S-thanatin and lactoferricin are antimicrobial peptides (AMP) with broad spectrum of antimicrobial activity.•The heterologous S-thanatin and bovine lactoferricin were functionally expressed in marine diatom Phaeodactylum tricornutum•The fusion proteins containing N-terminal AtpC leader sequence were correctly targeted to the chloroplast•Transgenic P. tricornutum cells expressing AMPs demonstrated enhanced antimicrobial activity against Gram-negative bacteria
Several Shewanella spp. are versatile in the respiratory substrates they use. A novel set of respiratory substrates implicated are insoluble Fe(III) and Mn(III,IV) oxides. OmcA from Shewanella ...oneidensis MR-1 plays a role in the terminal electron transfer during respiratory mineral reduction, but has minimal or contrasting properties in the literature. A suite of biophysical techniques confirmed the bis-histidine axial ligation of all OmcA’s haem content in the crystal structure and in solution. The paramagnetic resonance feature designated “LS3” was identified to be unique to OmcA. However this resonance signal is modelled to be produced spin-coupling and not unique haem ligation. OmcA’s electroactive coverage is comparable to UndA and the other major outer membrane multihaem cytochrome (OMMC) clades MtrC and MtrF (i.e. +0.08 V to -0.47 V vs S.H.E.). The crystal structure of OmcA shows domain fold, domain organisation and haem orientation conservation with MtrF and UndA. Comprehensive solution?structure studies of OmcA provided contrasting experimental data on the oligomeric state of OmcA, such that it is unresolved whether OmcA forms an ion-sensitive dimer. The crystal structure shows a predicted mineral interaction peptide (i.e. T725P726S727) is solvent exposed and would putatively bind substrate within electron tunneling distance of a terminal haem. Site-directed mutagenesis indicates Thr725 is significant to maintenance of the molecular environment of haem 10. Although T725G mutation produces a ≈80% decrease in whole cell reduction of synthesised hematite over 120 hours; secondary effects of change in haem reduction potentials or widespread conformational effects were ruled out. OmcA has thus been shown to share a common OMMC-fold and exist in S. oneidensis MR-1 outer membranes as a functioning mineral reductase cytochrome with unique paramagnetic resonance properties in this set of studies.
Several Shewanella spp. are versatile in the respiratory substrates they use. A novel set of respiratory substrates implicated are insoluble Fe(III) and Mn(III,IV) oxides. OmcA from Shewanella ...oneidensis MR-1 plays a role in the terminal electron transfer during respiratory mineral reduction, but has minimal or contrasting properties in the literature. A suite of biophysical techniques confirmed the bis-histidine axial ligation of all OmcA’s haem content in the crystal structure and in solution. The paramagnetic resonance feature designated “LS3” was identified to be unique to OmcA. However this resonance signal is modelled to be produced spin-coupling and not unique haem ligation. OmcA’s electroactive coverage is comparable to UndA and the other major outer membrane multihaem cytochrome (OMMC) clades MtrC and MtrF (i.e. +0.08 V to -0.47 V vs S.H.E.). The crystal structure of OmcA shows domain fold, domain organisation and haem orientation conservation with MtrF and UndA. Comprehensive solution?structure studies of OmcA provided contrasting experimental data on the oligomeric state of OmcA, such that it is unresolved whether OmcA forms an ion-sensitive dimer. The crystal structure shows a predicted mineral interaction peptide (i.e. T725P726S727) is solvent exposed and would putatively bind substrate within electron tunneling distance of a terminal haem. Site-directed mutagenesis indicates Thr725 is significant to maintenance of the molecular environment of haem 10. Although T725G mutation produces a ≈80% decrease in whole cell reduction of synthesised hematite over 120 hours; secondary effects of change in haem reduction potentials or widespread conformational effects were ruled out. OmcA has thus been shown to share a common OMMC-fold and exist in S. oneidensis MR-1 outer membranes as a functioning mineral reductase cytochrome with unique paramagnetic resonance properties in this set of studies.
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