Decoding the impact of interspecies interactions on biofilm matrix components

Abstract

Multispecies biofilms are complex communities where extracellular polymeric substances (EPS) shape structure, adaptability, and functionality. However, characterizing the components of EPS, particularly glycans and proteins, remains a challenge due to the diverse bacterial species present and their interactions within the matrix. This study examined how interactions between different species affect EPS component composition and spatial organization. We analyzed a consortium of four bacterial soil isolates that have previously demonstrated various intrinsic properties in biofilm communities: Microbacterium oxydans, Paenibacillus amylolyticus, Stenotrophomonas rhizophila, and Xanthomonas retroflexus. We used fluorescence lectin binding analysis to identify specific glycan components and meta-proteomics to characterize matrix proteins in mono- and multispecies biofilms. Our results revealed diverse glycan structures and composition, including fucose and different amino sugar-containing polymers, with substantial differences between monospecies and multispecies biofilms. In isolation, M. oxydans produced galactose/N-Acetylgalactosamine network-like structures and influenced the matrix composition in multispecies biofilms. Proteomic analysis revealed presence of flagellin proteins in X. retroflexus and P. amylolyticus, particularly in multispecies biofilms. Additionally, surface-layer proteins and a unique peroxidase were identified in P. amylolyticus multispecies biofilms, indicating enhanced oxidative stress resistance and structural stability under these conditions. This study highlights the crucial role of interspecies interactions in shaping the biofilm matrix, as well as the production of glycans and proteins, thereby enhancing our understanding of biofilm complexity.

Keywords: Extracellular polymeric substances; Glycans; Interspecies interactions; Multispecies biofilms.

Fig. 1

Identification of matrix components in biofilms. a) Experimental approach including i) fluorescent lectin screening of multispecies biofilms to visualize cell associated/matrix glyconconjugates; ii) Matrix proteomics to identify membrane, surface, or extracellular proteins with unique/differential abundance in mono- or multispecies biofilms. b) Lectins displaying strong binding to multispecies biofilms. The selected lectins, the glycan residues they bind, and the type of structures they stain, are indicated on top of the images. Some examples of cell surface binding (¹) are highlighted as discontinued boxes, network-like structures (²) with white arrows, and cloud-like structures (³) with light blue arrows. PMT3 gain (green channel) is indicated at the bottom right corner of each picture. AAL: AAL- Alexa488. RCA: RCA-Fluorescein. VVA: VVA-FITC; WGA: WGA-FITC. IAA: IAA-Alexa488. Images are 123 × 123 μm and maximum intensity projection (MIP). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Maximum intensity projections of mono-, dual-, and multispecies biofilms stained with lectin IAA and SYTO60. MO: M. oxydans, PA: P. amylolyticus; SR: S. rhizophila, XR: X. retroflexus; XR + PA: co-culture of X. retroflesus and P. amylolyticus; Multispecies: all four species. 24-hour mono, dual- or multispecies biofilms were stained with the fluorescent lectin IAA-Alexa488 (top row) and SYTO60 (middle row) as cell biomass stain and imaged with a 63x water-immersion objective. Merged images in the bottom row show combined lectin and biomass channels. Examples of network-like structures are indicated with white triangles while raceme-like structures are depicted as discontinued pink shapes, respectively. Images are 123 × 123 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Maximum intensity projections of mono-, dual-, and multispecies biofilms with lectin RCA and SYTO60. MO: M. oxydans, PA: P. amylolyticus; SR: S. rhizophila, XR: X. retroflexus; XR + PA: co-culture of X. retroflesus and P. amylolyticus; Multispecies: all four species. 24-hour mono-, dual- or multispecies biofilms were stained with the fluorescent lectin RCA-Fluorescein (top row) and SYTO60 (middle row) as cell biomass stain and imaged with a 63x water-immersion objective. Pink arrows indicate examples of structures resembling adhesive scaffold. Merged images in the bottom row show combined lectin and biomass channels. Images are 123 × 123 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Maximum intensity projections of mono-, dual-, and multispecies biofilms with lectin WGA and SYTO60. MO: M. oxydans, PA: P. amylolyticus; SR: S. rhizophila, XR: X. retroflexus; XR + PA: co-culture of X. retroflesus and P. amylolyticus; Multispecies: all four species. 24-hour mono-, dual- or multispecies biofilms were stained with the fluorescent lectin WGA-FITC (top row) and SYTO60 (middle row) as cell biomass stain and imaged with a 63x water-immersion objective. Examples of cloud-like structures are indicated by blue arrows while globule-like structures are indicated by yellow arrows. Merged images in the bottom row show combined lectin and biomass channels. Images are 123 × 123 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Maximum intensity projections of X. retroflexus mono- and multispecies biofilms with lectin RCA and SYTO60. Biofilms of X. retroflexus (XR) tagged with gfp (a, green) in mono- or multispecies 24-h biofilms were stained with SYTO60 (b, blue) as cell biomass stain, and the fluorescent lectin RCA-Rhodamine (c, red), and imaged with a 63x water-immersion objective. Merged images in the fourth column show combined GFP, cell biomass, and RCA lectin channels (d and e). Images are 123 × 123 μm. e) Regions of interest highlighted from merged images (3x magnification). White arrows indicate examples of network-like structures. Images represent 41 × 41 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Matrix proteomics of mono- and multispecies samples using trimmed reference proteomes. MO: M. oxydans, PA: P. amylolyticus; SR: S. rhizophila, XR: X. retroflexus; XR + PA: co-culture of X. retroflesus and P. amylolyticus; Multispecies: all four species. All samples are biofilms, except for XR planktonic. a) Workflow of matrix proteomics analysis. b) Proteins identified per species, cellular localization and role category (COG). Cellular localization of identified proteins was predicted using the tool DeepLocPro 1.0. [47]. Role categories correspond to COG categories. c) Venn diagrams of mono- and multispecies samples per species. Proteins detected in each condition are indicated for each comparison.

Fig. 7

Proteins identified in the cell wall and motility categories. a) Heatmap of proteins identified in XR for cell wall/membrane biogenesis. Color indicates abundance difference, expressed as Log₂ fold change (Log2FC) of multispecies vs. XR biofilm (M vs. XR), multispecies vs. XR planktonic (M vs. XRp), and XR biofilm vs. XR planktonic (XR vs. XRp). Positive and negative values indicate higher protein abundance in the first or second group, respectively. Asterisks indicate the level of significance of the comparison (Welch's t-test; ∗: p < 0.05; ∗∗: p < 0.01; ∗∗∗: p < 0.001). b) Heatmap of XR proteins identified in the cell motility category, illustrated as in panel a). c) Unique proteins from the cell wall/membrane biogenesis or cell motility detected only in specific conditions. SR: detected in SR monospecies biofilms; XRp: detected in XR monospecies planktonic samples; Multispecies: detected in multispecies biofilm samples. Color indicates Log2 transformed protein intensity. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)