Evidence for quinol oxidase activity of ImoA, a novel NapC/NirT family protein from the neutrophilic Fe(II) oxidizing bacterium Sideroxydans lithotrophicus ES-1

The freshwater chemolithoautotrophic Gram-negative bacterium Sideroxydans lithotrophicus ES-1 oxidizes Fe(II) at the cell surface. In this organism, it is proposed that the monoheme cytochrome MtoD from the Mto pathway transfer electrons across the periplasm to an inner membrane NapC/NirT family tetraheme cytochrome encoded by Slit_2495, for which we propose the name ImoA (inner membrane oxidoreductase). ImoA has been proposed to function as the quinone reductase, receiving electrons from iron oxidizing extracellular electron uptake pathway to reduce the quinone pool. In this study, ImoA was cloned on a pBAD plasmid vector and overexpressed in Escherichia coli. Biochemical and spectroscopic characterization of the purified ImoA reveals that this 26.5 kDa cytochrome contains one high-spin and three low-spin hemes. Our data show that ImoA can function as a quinol oxidase and is able to functionally replace CymA, a related NapC/NirT family tetraheme cytochrome required for anaerobic respiration of a wide range of substrates by Shewanella oneidensis. We demonstrate that ImoA can transfer electrons to different periplasmic proteins from S. oneidensis including STC and FccA, but in a manner that is distinct from that of CymA. Phylogenetic analysis shows that ImoA is clustered closer to NirT sequences than to CymA. This study suggests that ImoA functions as a quinol oxidase in S. oneidensis and raises questions about the directionality and/or reversibility of electron flow through the Mto pathway in S. lithotrophicus ES-1.

The influence of Shewanella oneidensis MR-1 on the transformation of iron oxides and phosphorus in a red soil

In this study, Shewanella oneidensis MR-1, an iron (Fe)-reducing bacterium, was inoculated to a red soil, which was then incubated. Soil samples were taken regularly to analyse the variation of iron oxides and phosphorus (P) fractions. The results showed that the MR-1 inoculation increased the content of the free iron oxides, but decreased the activity of the iron oxides in the soil, and had no significant influence on the amorphous iron oxides. The MR-1 inoculation increased the resin-P and residual-P, decreased the NaHCO₃-extracted inorganic P (NaHCO₃-P_i) and NaOH-extracted inorganic P (NaOH-P_i), but did not significantly influence the diluted HCl-extracted inorganic P (D.HCl-P_i) and concentrated HCl-extracted inorganic P (C.HCl-P_i). The presence of MR-1 influenced the correlation between the free iron oxides and NaOH-P_i. In the CK where deactivated MR-1 was applied, there was a significant positive correlation between the free iron oxides and the NaOH-P_i; in the treatment with the live MR-1 inoculation, there was no correlation between them. In addition, there was a significant positive correlation between the free iron oxides and the C.HCl-P_i, and there was a significant negative correlation between the NaHCO₃-P_i, resin-P, and residual-P. Therefore, the MR-1 inoculation improved the P availability by decreasing the activity of the iron oxides and consequently improved the P use efficiency in the red soil.

Diverse Protein Architectures and α-N-Methylation Patterns Define Split Borosin RiPP Biosynthetic Gene Clusters

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1,600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.

Experimental Validation of Stability and Applicability of Start Growth Time Method For High-Throughput Bacterial Ecotoxicity Assessment.

Ecotoxicity assessments based on bacteria as model organisms is widely used for routine toxicity screening because it has advantages of time-saving, high sensitivity, cost-effectiveness, and less ethical responsibility. Determination of ecotoxicity effect via bacterial growth can avoid the restriction of model bacteria selection and unique equipment requirement, but traditional viable cell count methods are relatively labor- and time-intensive. The Start Growth Method (SGT) is a high-throughput and time-conserving method to determine the amount of viable bacterial cells. However, its usability and stability for ecotoxicity assessment are rarely studied. This study confirmed its applicability in terms of bacterial types (gram-positive and gram-negative), growth phases (middle exponential and early stationary phases), and simultaneous existence of dead cells (adjustment by flow cytometry). Our results verified that the stability of establishing SGT correlation is independent of the bacterial type and dead-cell portion. Moreover, we only observed the effect of growth phases on the slope value of established SGT correlation in Shewanella oneidensis, which suggests that preparing inoculum for the SGT method should be consistent in keeping its stability. Our results also elucidate that the SGT values and the live cell percentages meet the non-linear exponential correlation with high correlation coefficients from 0.97 to 0.99 for all the examined bacteria. The non-linear exponential correlation facilitates the application of the SGT method on the ecotoxicity assessment. Finally, applying the exponential SGT correlation to evaluate the ecotoxicity effect of copper ions on E. coli was experimentally validated. The SGT-based method would require about 6 to 7 hours to finish the assessment and obtained an estimated EC50 at 2.27 ± 0.04 mM. This study demonstrates that the exponential SGT correlation can be a high-throughput, time-conversing, and wide-applicable method for bacterial ecotoxicity assessment.

Light-induced Patterning of Electroactive Bacterial Biofilms

Electroactive bacterial biofilms can function as living biomaterials that merge the functionality of living cells with electronic components. However, the development of such advanced living electronics has been challenged by the inability to control the geometry of electroactive biofilms relative to solid-state electrodes. Here, we developed a lithographic strategy to pattern conductive biofilms of Shewanella oneidensis by controlling aggregation protein CdrAB expression with a blue light-induced genetic circuit. This controlled deposition enabled S. oneidensis biofilm patterning on transparent electrode surfaces and measurements demonstrated tunable biofilm conduction dependent on pattern size. Controlling biofilm geometry also enabled us, for the first time, to quantify the intrinsic conductivity of living S. oneidensis biofilms and experimentally confirm predictions based on simulations of a recently proposed collision-exchange electron transport mechanism. Overall, we developed a facile technique for controlling electroactive biofilm formation on electrodes, with implications for both studying and harnessing bioelectronics.

Obtención de membrana de grafeno a partir de Shewanella oneidensis

En las últimas décadas la crisis del agua potable ha incrementado en México, la mayor cantidad de agua en el planeta es salina, un método para la desalinización es la osmosis inversa, donde se utiliza una membrana la cual es costosa y tiene una corta vida útil por el tipo de material que está hecha. El grafeno es un material derivado del carbono, con estructura 2D, ofrece la posibilidad de fabricar una nueva membrana mediante superposición de nano-láminas. La producción de grafeno genera vapores altamente tóxicos. Esta investigación tiene como objetivo “Obtener una membrana de grafeno a partir de Shewanella oneidensis para la desalinización de agua”. Metodología. 1. Síntesis de grafeno por el método Hummers modificado. 2. Incubación la bacteria S. oneidensis. 3. Reducción de OG, mediante S.oneidensis. 4. Obtención de una membrana para la desalinización por osmosis inversa. El material obtenido se caracterizó por IR, XDR y SEM para su identificación.

Insights into the Neutralization and DNA Binding of Toxin–Antitoxin System ParESO-CopASO by Structure-Function Studies

ParESO-CopASO is a new type II toxin–antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParESO severely inhibits cell growth, while CopASO functions as an antitoxin to neutralize ParESO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParESO-CopASO complex that adopted an open V-shaped heterotetramer with the organization of ParESO-(CopASO)2-ParESO. The structure showed that upon ParESO binding, the intrinsically disordered C-terminal domain of CopASO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParESO. Thermodynamics analysis showed the DNA-binding affinity of CopASO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParESO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParESO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParESO-CopASO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.