Widespread bacterial protein flavinylation in functionally distinct extracytosolic redox biochemistries

Disparate redox biochemistries that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox biochemistries but the breadth of functions of this modification remains unknown. Here we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemistries. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that suggest functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of multi-flavinylated proteins that may resemble multiheme cytochromes in facilitating longer distance electron transfer. These findings provide mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

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Post-translational flavinylation is associated with diverse extracytosolic redox functionalities throughout bacterial life

eLife ◽

10.7554/elife.66878 ◽

2021 ◽

Vol 10 ◽

Author(s):

Raphaël Méheust ◽

Shuo Huang ◽

Rafael Rivera-Lugo ◽

Jillian F Banfield ◽

Samuel H Light

Keyword(s):

Electron Transfer ◽

Iron Reduction ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Redox Systems ◽

Bacterial Physiology ◽

Biochemical Processes ◽

Electron Transport Chains ◽

Protein Classes ◽

Functionally Diverse

Disparate redox activities that take place beyond the bounds of the prokaryotic cell cytosol must connect to membrane or cytosolic electron pools. Proteins post-translationally flavinylated by the enzyme ApbE mediate electron transfer in several characterized extracytosolic redox systems but the breadth of functions of this modification remains unknown. Here we present a comprehensive bioinformatic analysis of 31,910 prokaryotic genomes that provides evidence of extracytosolic ApbEs within ~50% of bacteria and the involvement of flavinylation in numerous uncharacterized biochemical processes. By mining flavinylation-associated gene clusters, we identify five protein classes responsible for transmembrane electron transfer and two domains of unknown function (DUF2271 and DUF3570) that are flavinylated by ApbE. We observe flavinylation/iron transporter gene colocalization patterns that implicate functions in iron reduction and assimilation. We find associations with characterized and uncharacterized respiratory oxidoreductases that highlight roles of flavinylation in respiratory electron transport chains. Finally, we identify interspecies gene cluster variability consistent with flavinylation/cytochrome functional redundancies and discover a class of 'multi-flavinylated proteins' that may resemble multiheme cytochromes in facilitating longer distance electron transfer. These findings provide key mechanistic insight into an important facet of bacterial physiology and establish flavinylation as a functionally diverse mediator of extracytosolic electron transfer.

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Draft Genome Sequence of Streptomyces sp. TP-A0874, a Catechoserine Producer

Genome Announcements ◽

10.1128/genomea.01163-16 ◽

2016 ◽

Vol 4 (5) ◽

Author(s):

Hisayuki Komaki ◽

Akira Hosoyama ◽

Natsuko Ichikawa ◽

Yasuhiro Igarashi

Keyword(s):

Genome Sequence ◽

Cell Invasion ◽

Draft Genome ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Biosynthetic Gene Cluster ◽

Tumor Cell Invasion ◽

Draft Genome Sequence ◽

Biosynthetic Gene ◽

Streptomyces Sp

We report the draft genome sequence of Streptomyces sp. TP-A0874 isolated from compost. This strain produces catechoserine, a new catecholate-type inhibitor of tumor cell invasion. The genome harbors at least six gene clusters for polyketide and nonribosomal peptide biosyntheses. The biosynthetic gene cluster for catechoserines was identified by bioinformatic analysis.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III) oxide, Mn(IV) oxide, or electrode reduction by Geobacter sulfurreducens

10.1101/169086 ◽

2017 ◽

Cited By ~ 1

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R. Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

AbstractAt least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c-type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and −0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an ΔomcBC background, such as extEFG, were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the ΔextABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens, depending on the available extracellular electron acceptor.

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Identification of different putative outer membrane electron conduits necessary for Fe(III) citrate, Fe(III)-oxide, Mn(IV)-oxide, or electrode reduction by Geobacter sulfurreducens .

10.1101/350553 ◽

2018 ◽

Author(s):

Fernanda Jiménez Otero ◽

Chi Ho Chan ◽

Daniel R Bond

Keyword(s):

Electron Transfer ◽

Outer Membrane ◽

Electron Acceptor ◽

Single Gene ◽

Gene Clusters ◽

Geobacter Sulfurreducens ◽

Transcriptional Analysis ◽

Growth Defect ◽

Redox Potentials ◽

Wild Type

At least five gene clusters in the Geobacter sulfurreducens genome encode putative ‘electron conduits’ implicated in electron transfer across the outer membrane, each containing a periplasmic multiheme c -type cytochrome, integral outer membrane anchor, and outer membrane redox lipoprotein(s). Markerless single gene cluster deletions and all possible multiple deletion combinations were constructed and grown with soluble Fe(III) citrate, Fe(III)- and Mn(IV)-oxides, and graphite electrodes poised at +0.24 V and -0.1 V vs. SHE. Different gene clusters were necessary for reduction of each electron acceptor. During metal oxide reduction, deletion of the previously described omcBC cluster caused defects, but deletion of additional components in an Δ omcBC background, such as extEFG , were needed to produce defects greater than 50% compared to wild type. Deletion of all five gene clusters abolished all metal reduction. During electrode reduction, only the Δ extABCD mutant had a severe growth defect at both redox potentials, while this mutation did not affect Fe(III)-oxide, Mn(IV)-oxide, or Fe(III) citrate reduction. Some mutants containing only one cluster were able to reduce particular terminal electron acceptors better than wild type, suggesting routes for improvement by targeting specific electron transfer pathways. Transcriptomic comparisons between fumarate and electrode-based growth showed all of these ext clusters to be constitutive, and transcriptional analysis of the triple-deletion strain containing only extABCD detected no significant changes in expression of known redox proteins or pili components. These genetic experiments reveal new outer membrane conduit complexes necessary for growth of G. sulfurreducens , depending on the available extracellular electron acceptor.

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Discovery and Biosynthesis of Natural Products from New Zealand Soil Metagenome Libraries

10.26686/wgtn.17147699.v1 ◽

2021 ◽

Author(s):

◽

Luke Stevenson

Keyword(s):

Natural Products ◽

New Zealand ◽

Heterologous Expression ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Coli Strain ◽

Functional Screening ◽

Functional Difference ◽

Biosynthetic Pathways ◽

New Members

<p>Antibiotic discovery rates dramatically declined following the “golden age” of the 1940’s to the 1960’s. The platforms that underpinned that age of discovery rested upon laboratory cultivation of a small clade of bacteria, the actinomycetes, primarily isolated from soil environments. Fermentation extracts of these isolated bacteria have provided the majority of antibiotics and anticancer small molecules still used today. By applying modern genetic analysis techniques to these same environmental sources that have previously yielded such success, we can uncover new biosynthetic pathways, and bioactive compounds. The work described in this thesis investigated New Zealand soil metagenomes for this purpose. Four large metagenome libraries were constructed from the microbiomes of diverse soil environments. These were then interrogated by a functional screening approach in a knockout Escherichia coli strain, to recover a large collection of the biosynthetic gene clusters responsible for bacterial secondary metabolite production. Using different modes of bioinformatic analysis, these gene clusters were demonstrated to have both phylogenetic divergence, and functional difference from bacterial biosynthesis pathways previously discovered from culture based studies. Two additional biosynthetic pathways were recovered from one of these metagenome libraries, and in each case found to have novel genetic features. These gene clusters were further studied by heterologous expression within Streptomyces albus production hosts. One of these gene clusters produced small aromatic polyketide compounds, the structure of one of which was solved by chemical analytic techniques, and found to be a new chemical entity. The second gene cluster was demonstrated to have similarity to known aureolic acid biosynthesis gene clusters – a class of potent anticancer natural products. Heterologous expression resulted in the production of many metabolites, two of which were characterised and found to be new members of this chemical class. The research in this thesis both validates the use of metagenomic analysis for future natural product discovery efforts, and adds to a growing body of evidence that understudied clades of bacteria have an untapped biosynthetic potential that can be accessed by metagenomic methods.</p>

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Ferric reducing reactivity assay with theoretical kinetic modeling uncovers electron transfer schemes of metallic-nanoparticle-mediated redox in water solutions

Environmental Science Nano ◽

10.1039/c9en00258h ◽

2019 ◽

Vol 6 (6) ◽

pp. 1791-1798 ◽

Cited By ~ 2

Author(s):

Xiangyu Bi ◽

Paul Westerhoff

Keyword(s):

Electron Transfer ◽

Kinetic Modeling ◽

Metallic Nanoparticles ◽

Metallic Nanoparticle ◽

Water Solutions ◽

Mediate Electron Transfer

We probed that metallic nanoparticles (NPs) can mediate electron transfer in water by different schemes.

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Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin

Applied and Environmental Microbiology ◽

10.1128/aem.01752-19 ◽

2019 ◽

Vol 85 (23) ◽

Cited By ~ 5

Author(s):

Juan Pablo Gomez-Escribano ◽

Jean Franco Castro ◽

Valeria Razmilic ◽

Scott A. Jarmusch ◽

Gerhard Saalbach ◽

...

Keyword(s):

Natural Products ◽

Heterologous Expression ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Bioinformatic Analysis ◽

Growth Media ◽

Content Type ◽

Lasso Peptide ◽

Lasso Peptides ◽

New Family

ABSTRACT Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides. IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

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Yosshi: a web-server for disulfide engineering by bioinformatic analysis of diverse protein families

Nucleic Acids Research ◽

10.1093/nar/gkz385 ◽

2019 ◽

Vol 47 (W1) ◽

pp. W308-W314 ◽

Cited By ~ 6

Author(s):

Dmitry Suplatov ◽

Daria Timonina ◽

Yana Sharapova ◽

Vytas Švedas

Keyword(s):

Disulfide Bonds ◽

Web Server ◽

Bioinformatic Analysis ◽

Protein Families ◽

Sequence Alignments ◽

Interactive Analysis ◽

Functionally Diverse ◽

Available Information ◽

Related Proteins

Abstract Disulfide bonds play a significant role in protein stability, function or regulation but are poorly conserved among evolutionarily related proteins. The Yosshi can help to understand the role of S–S bonds by comparing sequences and structures of homologs with diverse properties and different disulfide connectivity patterns within a common structural fold of a superfamily, and assist to select the most promising hot-spots to improve stability of proteins/enzymes or modulate their functions by introducing naturally occurring crosslinks. The bioinformatic analysis is supported by the integrated Mustguseal web-server to construct large structure-guided sequence alignments of functionally diverse protein families that can include thousands of proteins based on all available information in public databases. The Yosshi+Mustguseal is a new integrated web-tool for a systematic homology-driven analysis and engineering of S–S bonds that facilitates a broader interpretation of disulfides not just as a factor of structural stability, but rather as a mechanism to implement functional diversity within a superfamily. The results can be downloaded as a content-rich PyMol session file or further studied online using the HTML5-based interactive analysis tools. Both web-servers are free and open to all users at https://biokinet.belozersky.msu.ru/yosshi and there is no login requirement.

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