Los factores ambientales, luz y temperatura, modifican la formación de la biopelícula en Pseudomonas syringae pv. syringae.

Comunicación a congreso en formato pósterLas biopelículas bacterianas están formadas por agregados celulares embebidos en una matriz extracelular de producción propia, formada principalmente por exopolisacáridos (EPS), proteínas y ADN extracelular. Existen datos recientes que sugieren que entre el 40-80% de las bacterias se encuentran formando biopelículas en la naturaleza, y que la formación de las biopelículas se ve afectada por factores ambientales. Pseudomonas syringae pv. syringae (Pss) es una bacteria fitopatógena causante de la necrosis apical del mango (NAM), enfermedad limitante de la producción de este cultivo en la región mediterránea. En Pss se ha descrito una conexión entre factores ambientales y la incidencia y severidad de la NAM, jugando los EPS celulosa y uno tipo-Psl, un papel relevante en la transición entre un estilo de vida epífito o patogénico. En este trabajo, se ha analizado el papel de algunos factores ambientales relevantes como son la luz y la temperatura en la formación de la biopelícula in vitro en una colección de cepas de Pss aisladas de mango. Los resultados obtenidos sugieren que la temperatura y particularmente la luz pueden influir en la formación de las biopelículas a través de la biosíntesis de los EPS, principalmente celulosa. Además, también se ha observado como niveles más bajos de producción de celulosa podrían estar asociados con una mayor virulencia. Estos resultados indican que los EPS, y principalmente celulosa juegan un papel clave en la ecología de Pss sobre la planta de mango a través de la formación de la biopelícula.Este trabajo ha sido financiado por Proyectos de Excelencia de la Junta de Andalucía (P12- AGR-1473), cofinanciado con ayudas a proyectos I+D+i en el marco del Programa Operativo FEDER Andalucía (UMA20-FEDERJA-060), y la Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Functional and evolutionary significance of unknown genes from uncultivated taxa

25 Pág.Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.This project has received funding from the National Programme for Fostering Excellence in Scientific and Technical Research (grant no. PGC2018-098073-A-I00 MCIU/AEI/FEDER, UE) and, partially, by MCIN/AEI/10.13039/501100011033/ and FEDER Una manera de hacer Europa (grant no. PID2021-127210NB-I00). A.R.d.R. was supported by a fellowship from la Caixa Foundation (ID 100010434, fellowship code no. LCF/BQ/DI18/11660009), cofunded by the European Union’s Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement no. 713673. C.P.C., S.S.-H. and Z.D. acknowledge support by Severo Ochoa Centres of Excellence Programme from the State Research Agency of Spain (grant nos. SEV-2016-0672 (2017–2021) and CEX2020-000999-S). J.B. acknowledges support by a grant from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (no. 2020-218584). A.H.-P. was supported by Research Technical Support Staff Aid (no. PTA2019-017593-I/AEI/10.13039/501100011033). M.M.-P., J.J.R.-H. and E.L.-S. acknowledge support from Ministerio de Ciencia e Innovación, MCIN/AEI/10.13039/501100011033 (grant no. PID2021-125673OB-I00). S.S. acknowledges support from the Swiss National Science Foundation project (grant no. 205321_184955) and NCCR Microbiomes (no. 51NF40_180575), and thanks the staff at ETH Zurich IT Services and HPC facilities.Peer reviewe

Identification of a Novel Calcium Binding Motif Based on the Detection of Sequence Insertions in the Animal Peroxidase Domain of Bacterial Proteins

<div><p>Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of <em>Pseudomonas putida</em> KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca²⁺ coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33–79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca²⁺ binding with a <em>K_D</em> of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.</p> </div

Section of the sequence alignment of ANP-like domains from 12 two-domain bacterial proteins.

<p>ANP-like domains as defined by PS50292 were aligned with MEGA 4.0. The numbers indicate the protein residues. The complete alignment is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s001" target="_blank">figure S1</a>. A region with two insertions (in boxes) matching the G-x-D-G-x(5)-D-D/E consensus and interrupting the major alignment are shown. The individual ANP-like domains are of <i>Fulvimarina pelagi</i> HTCC2506 (Q0G341), Manganese-oxidizing bacterium (strain SI85-9A1) (Q1YMS2), <i>Methylobacterium chloromethanicum</i> CM4 / NCIMB 13688 (B7KW13), <i>M. extorquens</i> DSM 5838 / DM4 (A9W3A5), <i>M. extorquens</i> PA1 (C7CGY0), <i>Pseudomonas putida</i> F1 (A5W572), <i>P. putida</i> GB1 (B0KJL7), <i>P. putida</i> KT2440 (Q88JT6), <i>Rhodopseudomonas palustris</i> BisA53 (Q07SX1), <i>Rhodopseudomonas palustris</i> BisB5 (Q13AU2), <i>Roseobacter</i> sp. MED193 (A3XF15) and <i>Roseovarius</i> sp. TM1035 (A6E280). Letters -A and -B refer to N- and C-terminal ANP-like domains, respectively.</p

Neighbor-joining phylogenetic tree of bacterial ANP-like domains contained in animal heme peroxidases with G-x-D-G-x(5)-D-D.

<p>The domain of human myeloperoxidase, which does not contain any hit, is included as an out-group member. The alignment shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s002" target="_blank">figure S2</a> was cut at the position which corresponds to the sequence boundaries of the human myeloperoxidase domain. The tree is based on this reduced alignment. The bootstrap consensus tree inferred from 500 replicates is shown. Letters –A and -B refer to N- and C-terminal ANP-like domains, respectively. All the 23 sequences here considered presented at least one insert with the consensus of PERCAL (G-x-D-G-x(2)-G/N-T/N-x-D-D) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s005" target="_blank">Table S1</a>). The column on the right indicates the number of insertions in each of the domains. Bar: 0.2 substitutions per amino acid position.</p

Isothermal titration calorimetry studies of the interaction of the BACHEMP-Cons peptide with CaCl₂.

<p>(A) Upper panel: raw data for the titration of 50 µM peptide with 3.2 µl aliquots of 5 mM CaCl₂. Experiments were conducted in polybuffer at the pH values indicated. Lower panel: Integrated, dilution-corrected and concentration-normalized raw data. Data were fitted with the “One binding site model” of the MicroCal (Northampton, MA) version of ORIGIN. pH 6.0 (○), pH 7.0 (□), pH 8.0 (Δ). The derived thermodynamic parameters are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone-0040698-t004" target="_blank">Table 4</a>. (B) Dependence of <i>K</i>_D on the pH. Experiments were conducted in polybuffer which was adjusted to the pH indicated by the addition of concentrated HCl or NaOH. Shown are means and standard errors derived from three individual experiments.</p

ITC analysis of Ca binding to the N-terminal ANP-like domain of PepA.

<p>Upper panel: Raw titration data for the injection of 6.4 µl aliquots of 1 mM CaCl₂ into 11.2 µl of recombinant protein. Ligand and protein were in buffer Tris-HCl 10 mM, NaCl 50 mM, Glycerol 10%, pH 7.5. Experiments were carried out at 25°C. Lower panel: Integrated, dilution-corrected and concentration-normalized peak areas of titration raw data. Shown is the fit with the “one binding site model” of the MicroCal version of ORIGIN.</p

Presence of the different versions of the PERCAL calcium binding motif in bacteria and eukaryotes.

<p>An expanded table with more motifs and the details required for the generation of this table are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s006" target="_blank">Table S2</a>.</p>a<p>Percentage of insertions (of a total of 74) within ANP-like domains recognized by the corresponding motif.</p>b<p>Number of total entries in the database 18215214</p>c<p>Entries from Bacteria (63%)</p>d<p>Entries from Eukaryota (28%)</p

Zoom at the calcium-binding region of the pseudopilin structure.

<p>The amino acids which establish contacts with the calcium ion are shown in ball-and-stick mode and the corresponding distances are indicated (in Å). The structure is deposited at the protein data bank under the code 3G20. The figure was produced using the program WebLabViewer (<a href="http://www.marcsaric.de/index.php/WebLab_Viewer_Lite" target="_blank">http://www.marcsaric.de/index.php/WebLab_Viewer_Lite</a>).</p

Sequence logo of insertions interrupting the ANP-like domains of bacterial proteins.

<p>The figure was generated at <a href="http://weblogo.berkeley.edu" target="_blank">http://weblogo.berkeley.edu</a> based on an alignment of 74 domain insertions matching the motif x-x-x-G-x-D-x(6)-D/E-D/E-x-x-x from 24 bacterial proteins. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s005" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040698#pone.0040698.s008" target="_blank">Text S2</a> for more details.</p