Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: (A) Phylogenetic analysis of Flavobacterium isolates using a multi-locus approach with 10 single copy core genes (as previously described by ). Evolutionary distances were inferred using the maximum-likelihood method. The Brassica napus isolates (green circles) spread the breadth of terrestrial Flavobacterium strains and captured more diversity than DSM2064 and Israeli strains tested (green squares) or the Arabidopsis strains (olive green triangles). ( B) BRIG analysis of the oilseed rape (OSR) isolates, F52, and four root-associated Flavobacterium strains compared against F. johnsoniae DSM2064 revealed common patterns of genomic architecture among the strains.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques:

Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: Genomic comparison of selected open reading frames (ORFs) amongst the Flavobacterium strains used in this study. (A) ORFs representing housekeeping genes and core metabolic genes identified by were used to determine genome completeness and isolate similarity. All genes were compared against DSM2064 queries. (B) Genes encoding all the potential P acquisition proteins as well as the master regulator PhoBR were compared. The scale bar represents the percentage identity at the amino acid level of each orthologue. White squares indicate no orthologue in a given genome. (C) The presence of PHO regulon genes in >100 Flavobacterium genomes obtained from different environments. Flavobacterium genomes were partitioned based on their source of isolation. n= the number of genomes analysed from each environmental niche. The mean (light grey dashed line) and median (black dashed line) values for the percentage abundance of all PHO regulon are shown for each graph.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques: Isolation

Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: (A) The differential expression of SusCD complexes in the exoproteomes (n=3) of Flavobacterium strains , including Pi-insensitive (left column) and Pi-sensitive (right column). Red indicates an enrichment in Pi-deplete and blue indicates an enrichment in Pi-replete. For convenience, the difference in log2 LFQ intensities has been inversed compared to , i.e. a positive difference equals an enrichment in Pi-deplete. (B) Maximum-likelihood tree displaying the phylogeny of various Flavobacterium SusD/PusD homologs. Marine (MAR) sequences identified in were added to the alignment and highlighted by squares. Certain non Pi-responsive F. johnsoniae DSM2064 SusD homologs are highlighted (blue circles). Red, maroon and pink circles represent the various Pi-sensitive PusD homologs. Numbers 1-6 represent the various PusD homologs. (C) The genetic neighbourhood of all PusCD complexes detected through exoproteomics, co-located with various genes predicted to be involved in Pi-acquisition. (D) Occurrence of various pusD and pstS genes in Flavobacterium genomes outlined in Table S12. Yellow squares indicate the presence of the given gene in a genome. Abbreviations: phoA , alkaline phosphatase A-like; pusCD , Pi-sensitive outer membrane SusCD-like complexes; fecR , iron-like transcriptional regulator; phnX , phosphonoacetaldehyde hydrolase; dao , oxidoreductase; gdpd , glycerolphosphodiesterase-like; ctpA, protease-like; deoB , Phosphopentomutase; dgdA , 2,2-dialkylglycine decarboxylase; gpmI1 , 2,3-bisphosphoglycerate-independent phosphoglycerate mutase.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: A) Structural modelling of the abundantly secreted Pi-sensitive hypothetical exoproteins, FI, FII and FIII. SWISS homology modelling revealed that these hypothetical exoproteins share structural homology with pectate lyases/ characterised polygalacturonases found in Bacteria and Fungi. (B) Maximum-likelihood tree displaying the phylogeny of low-Pi inducible hypothetical exoproteins FI, FII, and FIII (light green represents DSM2064). The previously characterised classical polygalacturonases (Maroon, Dark Blue and Mustard) and other predicted polygalacturonases found in DSM2064 and F52 (Dark green) were also added to the analysis. (C) The genetic neighbourhood of the gene encoding FI, which was the most abundant protein in Pi deplete exoproteomes of DSM2064 and LAO5, illustrating co-localisation with various other low Pi-responsive genes in a genetic operon. (D) The gene encoding FII was co-localised with genes encoding the PHO regulon two-component regulatory system for the PHO regulon, phoBR , a Pi-sensitive tbdt/susC -like transporter, a putative outer membrane phosphate selective oprP . For OSR_004, the genes encoding the high affinity transport system, pstSABC were also co-localised in this region. For convenience, the difference in log2 LFQ intensities has been inversed compared to , i.e. a positive difference equals an enrichment in Pi-deplete.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques:

Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: (A) Phosphatase (monoesterase) activity (n=3) of F. johnsoniae DSM2064 genetic variants, including the wild type during Pi-replete and Pi-deplete growth conditions. Para-nitrophenol phosphate (PNPP) was used as the substrate. (B) Complementation of the ΔphoX Pseudomonas putida BIRD-1 mutant with different candidate phosphatase genes from F. johnsoniae DSM2064. phoX from Pseudomonas sp. BIRD-1 (PPUBIRD-1_1093) and phoA from E. coli were used as controls. Note that the phoX -like gene (Fjoh_2478) from F. johnsoniae DSM2064 did not restore APase activity in the mutant, whilst the two phoA -like (Fjoh_3249 and Fjoh_3187) genes did. Results presented are the mean of triplicate assays from three independent cultures. Error bars denote standard deviation.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques: Activity Assay, Mutagenesis, Standard Deviation

Journal: bioRxiv

Article Title: Novel mechanisms for phosphate acquisition in abundant rhizosphere-dwelling Bacteroidetes

doi: 10.1101/719427

Figure Lengend Snippet: Characterisation of the ( ΔpusCD1:ΔpusCD2 ) F. johnsoniae DSM2064 mutant. (A) Distribution of either genes encoding PstS or the various PusD homologs in 100 rhizobacterial genomes (Table S11) chosen from IMG/JGI. Genomes were categorised according to class to differentiate between Flavobacteria and Sphinogbacteria (B) Growth rates of either the mutant or wild type grown on glucose (20 mM) and various concentrations of Pi. (C) Competition experiments (n=3) between the mutant and wild type. Results plotted are the log2 of the ratio between wild type and mutant colony forming units (c.f.u.). Final cell counts varied from 106 to 109 c.f.u across the Pi concentrations gradients. Error bars denote standard deviation.

Article Snippet: Flavobacterium johnsoniae DSM2064 (UW101) was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ).

Techniques: Mutagenesis, Standard Deviation