Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Colonies of F. perrara PEB0191 (wt) after 48 hr of growth on modified tryptone yeast glucose (mTYG) agar. Arrowhead points at a larger white colony in between many yellow colonies. The area in the white square is magnified. ( B ) Colony morphology of F. perrara wt and the isolated white ihfA* mutant after growth on mTYG for 48 hr. ( C ) Protein sequence comparison of IhfA and IhfB of F. perrara wt and E. coli wt. The outlined positions refer to the residues mutated in the three spontaneous ihfA mutants: (i) lysine (Lys) to serine (Ser) at position 38 of F. perrara IhfA, (ii) proline (Pro) to lysine (Lys) at position 83 of F. perrara IhfA, and (iii) proline (Pro) to lysine (Lys) at position 82 of F. perrara IhfB. Note that the numbers given on top of the alignment refer to alignment positions and not to positions in the individual sequences. Secondary structures are depicted above as ribbons (α-helix) and arrows (β-sheet) and are numbered according to their appearance in the protein and the structure shown in ( D ). ( D ) Three-dimensional structure of E. coli IhfA/B heterocomplex with DNA (source protein databank NDB: PDT040). DNA is depicted in blue and IhfB in dark gray. IhfA is colored according to secondary structure: α-helix orange, β-sheet light blue, and the rest in light gray. α-helices and β-sheets are numbered. The mutated Pro83 and Lys38 residues of F. perrara IhfA and the Pro82 residue of IhfB are marked with an orange and green circle, respectively.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Modification, Isolation, Mutagenesis, Sequencing, Comparison, Residue

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Growth curve of F. perrara PEB0191 wt and ihfA* in liquid tryptone yeast glucose (TYG). The optical density (OD 600 ) was measured every hour for 14 hr (permutation test, p=0.097). ( B ) Quantification of single-cell length of F. perrara wt PEB0191 and ihfA* from the single-cell microscopy experiment shown in ( C ). Significance was tested using Kolgomorov–Smirnov test, asterisks depict significance: ***p<0.0001. ( C ) Single-cell light microscopy of F. perrara wt and ihfA*. The scale bar (5 µm) is depicted in the right lower corner. contains the numeric values for the figures shown here. Figure 1—figure supplement 2—source data 1. Numeric data underlying the results shown in .

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Microscopy, Light Microscopy

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Comparison of gene synteny and sequence similarity of the genomic islands of F. perrara PEB0191 (top) and E. coli CFT073 (bottom) encoding the aryl polyene (APE) biosynthesis genes. Gray lines indicate homologous regions based on tblastx analysis. Plots were generated with genoplotR . Transcripts per million (TPMs) are shown on top of the genomic island for one RNAseq replicate of each F. perrara wt (blue) and the ihfA* mutant grown in vitro. Coverage plots were generated with the Integrated Genome Browser v9 . ( B ) Total ion chromatogram (TIC) and UV trace (λ = 420 nm) of wt and ihfA *. A peak highly abundant in the wt was discovered at 18.85 min. Its high UV absorbance at λ = 420 nm indicated a conjugated carbon double bond system. ( C ) The normalized mass spectrum at 18.85 min reveals the ion m/z = 323.1647 Da to be approximately 50-fold more abundant in the wt compared to ihfA *. ( D ) Enrichment of the ion containing fraction by HPLC followed by nuclear magnetic resonance (NMR) experiments suggest a structure identical to that reported by . Reported (red) and observed (blue) 1 H and 13 C chemical shifts are shown. Central methines could not be assigned.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Comparison, Sequencing, Generated, Mutagenesis, In Vitro, Nuclear Magnetic Resonance

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Total ion chromatogram of extracts of F. perrara wt. ( B ) Extracted ion chromatogram of m/z = 323.1640 Da (± 5 ppm). Storage led to the formation of an additional peak at 19.28 min, possibly by isomerization. ( C ) The UV spectrum at 18.94 min has the maximum around 415 nm, similar to that reported by . ( D ) The UV spectrum at 19.25 min shows an additional maximum at 315 nm, characteristic for a trans-cis conversion of a double bond.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques:

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Light microscopy pictures of pylorus region of bees colonized with F. perrara PEB0191wt or ihf A* 10 d post colonization. ( B ) Quantification of scab phenotype of bees 5 and 10 d post colonization with n = 18 and n = 36 per treatment, respectively. ( C ) Quantification of colonization levels is measured by colony-forming units (CFUs) at day 5 (n = 18) and day 10 (n = 36) post colonization. Wilcoxon rank-sum test was used to assess significant differences. ( D ) Time-course experiment of bees colonized with F. perrara wt or ihf A*. Colonization levels were measured by CFUs every second day until day 10 and then at day 14 and day 21. n = 12 bees per time point per treatment. Wilcoxon rank-sum test was used to assess significant differences per time point. Error bars represent median and interquartile range. Data from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. contains the numeric values for the figures shown here. Figure 3—source data 1. Numeric data underlying the results shown in and .

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Light Microscopy

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Mono-colonization of bees with F. perrara wt and ihfA* . Colonization levels are measured by colony-forming units (CFUs) and quantitative PCR (qPCR) 10 d post colonization in the specified gut regions: pylorus and ileum. Wilcoxon rank-sum test was used to assess the statistical significance of differences. n = 6. contains the numeric values for the figure shown here.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Real-time Polymerase Chain Reaction

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Chromosomal localization of all genes significantly differentially expressed (2-log fold change = |2|, Fisher’s exact test p-value <0.05, false discovery rate [FDR] < 0.05) between F. perrara wt and the ihfA* mutant. Starting from outside, the first circle shows the scale of the genome representation of F. perrara in gray and white steps of 100 kb. The second and third circles (gray) depict the genes on the plus and minus strands of F. perrara . The fourth (beige) and fifth (light blue) circle depicts genes upregulated and downregulated in wt compared to ihfA* . Genomic islands are highlighted by coloration. ( B ) Bar plot of the genes differentially expressed between F. perrara wt and ihfA* with a log2-fold change > 2 (Fisher’s exact test p-value<0.05, FDR <0.05). ( C ) Comparison of the transcriptional profile of the genomic location encoding the colibactin biosynthetic gene cluster between F. perrara wt and the ihfA* mutant. Transcripts per million were visualized using the Integrative Genome Browser . The colibactin operon is schematically depicted below (green arrows). and contain the data used to produce the figure shown here. Figure 4—source data 1. List of F . perrara genes differentially expressed in vitro. Figure 4—source data 2. Clusters of orthologous groups for genes differentially expressed in vitro.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Mutagenesis, Comparison, In Vitro

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Significantly differentially expressed genes with a |log2-fold change| >|2| of F. perrara PEB0191 wt at day 5 ( A ) and day 10 ( B ) post colonization in comparison to growth in vitro. Genes are colored according to their category. The experiment was performed in quadruplicates, all genes displayed were significantly differentially expressed with p<0.05 and a false discovery rate (FDR) < 0.05 (exact test).

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Comparison, In Vitro

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Transcripts per million were calculated for all replicates of the in vitro and the in vivo RNAseq experiments. For the in vitro experiment (Exp. 1), all three replicates of the wt and the ihfA* mutant are shown. For the in vivo experiment (Exp. 2), the four replicates of the day 10 time point and the in vitro reference condition are shown. Data for day 5 time point in comparison to day 10 time point is shown in . and contain the data used to generate the figure shown here. ( A-F ) The expression of Flp pili genes ( A ), genes involved in the synthesis of Arylpolyene (B) and Colibactin (C) and genes involved in the function of T6SSs ( D-F ) is shown. Figure 5—source data 1. List of F . perrara genes differentially expressed in vivo. Figure 5—source data 2. Clusters of orthologous groups for genes differentially expressed in vivo.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: In Vitro, In Vivo, Mutagenesis, Comparison, Expressing

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Same as , but only time point day 5 and day 10 are depicted in comparison to F. perrara ihfA* in vitro. ( A-F ) The expression of Flp pili genes ( A ), genes involved in the synthesis of Arylpolyene ( B ) and Colibactin ( C ), and genes involved in the function of T6SSs ( D-F ) are shown.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Comparison, In Vitro, Expressing

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) The six gene-deletion mutants, ihfA* , and wt F. perrara strains were grown in liquid Brain Heart Infusion (BHI), diluted to OD 600 = 0.1, plated in agar patches and imaged using a Nikon Ti inverted light microscope. Images were taken with a ×100 objective. Scale bar indicates 20 µm. ( B ) Cell length was quantified using the MicrobeJ plugin of ImageJ. ( C ) Electron microscopy images were obtained for the wt, ihfA* , and ΔpilE strains. Scale bar indicates 200 nm. contains the numeric values for the figures shown here.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Light Microscopy, Electron Microscopy

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: For each F. perrara strain, a bacterial solution at OD 600 = 0.1 was prepared, serially diluted, and plated in BHIA medium. The number of CFUs present in 5 µl of solution at OD 600 = 0.1 was calculated based on the counts obtained from the serial dilutions. Statistics were calculated using a linear model with negative binomial distribution: ***p<0.0001, **p<0.001. contains the numeric values for the figures shown here.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques:

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: ( A ) Colonization levels were assessed 10 d after inoculation by counting colony-forming units (CFUs) in dilutions of homogenized bee guts plated on Brain Heart Infusion (BHI) agar. Only the pylorus and ileum section of the gut were analyzed. Limit of detection (LOD) corresponds to the lowest colonization level detectable in our assay, that is, points below the LOD correspond to bees for which no CFUs were detected. Statistically significant differences of the colonization levels of each mutant relative to the wt of F. perrara were determined using the Wilcoxon rank-sum test with BH correction. Bees were inoculated with an OD 600 of 0.1. Data come from two independent experiments. shows the data points by experiments. *p<0.05, **p<0.01, ***p<0.001. Filled circle colors indicate whether a scab was detected during dissection (green = scab; yellow = no scab). ( B ) Location within the pylorus was assessed using FISH microscopy. Bees were inoculated with different F. perrara genotypes at OD 600 = 0.1, guts were dissected at day 10 after inoculation and sectioned using a microtome. Hybridizations were done with probes specific for F. perrara (magenta). DAPI counterstaining of host nuclei and bacteria is shown in blue. Images were generated by merging brightfield, F. perrara and DAPI images that were obtained for the same section of the gut. The composite images here shown were obtained by merging the images of each channel presented in and . These were obtained using the ×5 and ×40 objectives of the Zeiss LSM900. Scale bar for images obtained with ×5: 100 µm, for ×40: 20 µm. contains the numeric values used to generate ( A ). Figure 6—source data 1. Numeric data underlying the results shown in and – .

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Mutagenesis, Dissection, Microscopy, Bacteria, Generated

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Composite images are the same as in . These were obtained by merging the brightfield, DAPI, and F. perrara probe individual images. Images were obtained with the ×5 objective of the Zeiss LSM900. Hybridizations were done with probes specific for F. perrara (magenta). DAPI counterstaining of host nuclei and bacteria is shown in blue. The scale bar correspons to 100 µm.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Bacteria

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Composite images are the same as in . These were obtained by merging the brightfield, DAPI and F. perrara probe images. Images were obtained with the ×40 objective of the Zeiss LSM900. Hybridizations were done with probes specific for F. perrara (magenta). DAPI counterstaining of host nuclei and bacteria is shown in blue. The scale bar corresponds to 20 µm.

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Bacteria

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet: Bees were inoculated with F. perrara alone (mono) or in the presence of a defined bacterial community representing core members of the bee gut microbiota (BeeCom_002). Colonization levels were assessed 10 d after inoculation by qPCR. Only the pylorus and ileum section of the gut were analyzed. The dashed gray line refers to the limit of detection (LOD) and corresponds to the lowest colonization level detectable in our assay, that is, points below the LOD correspond to bees for which no F. perrara was detected. Statistically significant differences between the colonization levels of each mutant in mono-association compared to in the presence of the defined microbial community were determined using the Wilcoxon rank-sum test with BH correction. Data comes from two independent experiments. shows the data points colored by experiments. *p<0.05, **p<0.01, ***p<0.001. Filled circle colors indicate whether a scab was detected during dissection (black = scab; yellow = no scab). contains the numeric values used to generate . Figure 7—source data 1. Numeric data underlying the results shown in and .

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Mutagenesis, Dissection

Journal: eLife

Article Title: Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

doi: 10.7554/eLife.76182

Figure Lengend Snippet:

Article Snippet: The following datasets were generated: Schmid K Santos-Matos G Leopold-Messer S El-Chazli Y Emery O Steiner T Piel J Engel P 2023 Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara Zenodo 10.5281/zenodo.7778751 Engel P Schmidt K 2021 RNA sequencing of Frischella perrara NCBI Gene Expression Omnibus GSE189728

Techniques: Isolation, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Assessing the performance of commercial reagent antibodies

doi: 10.1101/2023.06.01.543292

Figure Lengend Snippet: An antibody RRID can be used to search characterization studies across various databases, such as vendor page, the Antibody Registry and on the YCharOS community page on ZENODO. AB_2037651 is given as an example.

Article Snippet: Our studies are rapidly shared via the open platform ZENODO, called the YCharOS (“Antibody Characterization through Open Science”) community and selected studies were published on the F1000 publication platform ( https://f1000research.com/ycharos ).

Techniques: