Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera

© 2021 by the authors.Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The “plant-associated” group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.This research was funded by the BBSRC-Newton Fund (grant numbers BB/N013476/1 and BB/N003608/1). MC was funded by grant BIO2016-79736-R from the Ministry of Economy and Competitiveness of Spain.Peer reviewe

The Signal Recognition Particle: noncanonical functions and drug discovery

The Signal Recognition Particle (SRP) is an essential ribonucleoprotein complex responsible for co-translational delivery of membrane and secretory proteins to the plasma membrane in prokaryotes and to the endoplasmic reticulum in eukaryotes. In Eubacteria, SRP consists of the GTPase Ffh and the small 4.5S RNA; in eukaryotes, the system is more complex and SRP comprises six proteins (the heterodimer SRP 9/14, SRP 19, SRP 54, and the heterodimer SRP 68/72) along with a large RNA moiety, the 7SL RNA. SRP has been reported to be involved in many cellular processes outside the SRP-targeting cycle. Two layers of the SRP interactome, the SRP proteome and SRP transcriptome, were analysed by LC-MS/MS and RIP-seq, respectively. The majority of identified RNA and protein targets of the SRP subunits have nucleic-acid, chromatin and protein-binding functions and are involved in ribonucleoprotein particles (RNPs) formation, RNA processing and protein transport. Confocal microscopy studies showed that SRP subunits localizes in a dynamic manner during the cell cycle, indicating a spatial-temporal regulation of SRP-binding partners. Truncations or mutations on any of the bacterial components of the SRP system have proven to be either lethal or severely impact cell viability. Here, we propose the bacterial SRP and its interactions with the cognate SRP receptor, FtsY, as an ideal target for the development of novel antibiotics. Using a Fragment-Based Drug Design approach we have identified three fragments from a commercial library that bind to FtsY. We have crystallized FtsY in an Apo form and soaked GTP analogues as well as the three fragments and analogues and determined their X-ray crystal structures at resolution ranging from 1.221.9 Å. Despite the low affinity of the compounds, we were able to identify unambiguously their binding site and show that soaking of fragments, even at low affinity, is possible to aid in our drug discovery project

A chimeric Potato virus X encoding a heterologous peptide affects Nicotiana benthamiana chloroplast structure

The cytopathology of a Potato virus X (PVX) recombinant variant (encoding as fusion of an epitope of immunological interest with the N-terminus of the coat protein, PVXSmaP18DD) has been compared with that induced by the wild-type virus (PVX wt) in Nicotiana benthamiana plants. Both PVX wt and PVXSmaP18DD caused similar ultrastructural alterations, characterized by the presence of laminated inclusion components and bulk virus accumulations in mesophyll cells. However, some striking differences were observed not only in the morphology of these accumulations (typically ordered in PVX wt infection and disordered in PVXSmaP18DD infection) but also because the chimeric virus caused peculiar alterations in chloroplasts structure. Abbreviations: CP, coat protein; d.p.i., days post inoculation; LIC, laminated inclusion components; PVX, Potato virus X

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat proteinpeptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time

FtsY_NG bound with fragments.

<p>(A) FtsY_NG bound to Fragment 1 (shown in green) in the Trp343 and Phe365 binding sites. (B and C) FtsY_NG:Fragment 1 interaction profile in (B) Trp343 binding site and (C) Phe365 binding site. (D) FtsY_NG bound to Fragment 2 (shown in orange) in the Trp343 and Phe365 binding sites. (E and F) FtsY_NG:Fragment 2 interaction profile in (E) Trp343 binding site and (F) Phe365 binding site. (G) FtsY_NG bound to Fragment 3 (shown in pink) in the Trp343 and Phe365 binding sites. (H and I) FtsY_NG:Fragment 3 interaction profile in (H) Trp343 binding site and (I) Phe365 binding site. Fragments are displayed as sticks with Fragment 1 shown in green, Fragment 2 in orange, Fragment 3 in pink and the amino acids interacting with them shown as blue sticks. Interactions are water bridge (grey line), hydrophobic (red dashed line), hydrogen bond (blue line) and π-stacking (green dashed line). Distances for interactions are indicated in the figure. mF_o-DF_c fragment electron density map is shown in grey and contoured at 3σ level in (A), (D) and (G).</p

STD, WATERlogsy and CPMG ligand-detected experiments carried out on Fragment 2 in the absence (green traces) and presence (orange traces) of FtsY_NG.

<p>The top (black) trace is the ¹H 1D NMR spectrum of Fragment 2 for reference. (B−D) ¹⁵N-TROSY-HSQC spectrum of ¹⁵N-FtsY_NG alone (blue) and following addition of (B) Fragment 2 (red), (C) GTP analogue (red) and (D) 4.5S RNA (red). Arrows indicate the peaks that have shifted during the titration with green arrows highlighting the same peaks that have shifted in the fragment titration and the 4.5S RNA titration. (E) Chemical structures of Fragments 1, 2 and 3.</p

Discovery of fragments that target key interactions in the signal recognition particle (SRP) as potential leads for a new class of antibiotics

<div><p>Given the increasing incidence of antibiotic resistance, antibiotics that employ new strategies are urgently needed. Bacterial survival is dependent on proper function of the signal recognition particle (SRP) and its receptor (FtsY). A unique set of interactions in FtsY:SRP-RNA represents a promising candidate for new antibiotic development as no antibiotic targets this complex and these interactions are functionally replaced by protein:protein interactions in eukaryotes. We used a Fragment Based Drug Design (FBDD) approach to search for new compounds that can bind FtsY, and have identified three lead fragments. <i>In vitro</i> and <i>in vivo</i> analyses have shown that despite a high micromolar binding affinity, one fragment has some antimicrobial properties. X-ray structures of <i>E</i>. <i>coli</i> FtsY:fragments reveal the fragments bind in the targeted RNA interaction site. Our results show that FBDD is a suitable approach for targeting FtsY:SRP-RNA for antibiotic development and opens the possibility of targeting protein:RNA interactions in general.</p></div

Chemical environment surrounding the fragment-binding sites.

<p>In Trp343 binding site: (A) Fragment 1 (green), (B) Fragment 2 (orange) and (C) Fragment 3 (magenta). (D−F) In Phe365 binding site: (D) Fragment 1, (E) Fragment 2 and (F) Fragment 3. FtsY_NG is shown as surface with amino acids that form the surrounding fragment binding site labelled and shown as blue sticks. Fragment 1 is shown in green, Fragment 2 in orange and Fragment 3 in magenta. R represents positions to be modified according to the Phe365 binding site.</p

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.publishe