Intra-colony channels in E. coli function as a nutrient uptake system

Abstract

The ability of microorganisms to grow as aggregated assemblages has been known for many years, however their structure has remained largely unexplored across multiple spatial scales. The development of the Mesolens, an optical system which uniquely allows simultaneous imaging of individual bacteria over a 36 mm² field of view, has enabled the study of mature Escherichia coli macro-colony biofilm architecture like never before. The Mesolens enabled the discovery of intra-colony channels on the order of 10 μm in diameter, that are integral to E. coli macro-colony biofilms and form as an emergent property of biofilm growth. These channels have a characteristic structure and re-form after total mechanical disaggregation of the colony. We demonstrate that the channels are able to transport particles and play a role in the acquisition of and distribution of nutrients through the biofilm. These channels potentially offer a new route for the delivery of dispersal agents for antimicrobial drugs to biofilms, ultimately lowering their impact on public health and industry.

Fig. 1. Visualising the intra-colony channel system of E. coli macro-colony biofilms.

A deconvolved 36-μm-thick transverse sub-stack of a mature E. coli macro-colony biofilm acquired using widefield mesoscopy. An axial colour-coded LUT has been applied, which indicates the relative position of each cell within the context of the biofilm. A magnified ROI is presented where individual cells can be clearly resolved. Channel structures are seen to permeate throughout the biofilm and present a 3D topography within the context of the biofilm.

Fig. 2. Characterising the structure of intra-colony channels.

a Maximum intensity projection of an unlabelled JM105 colony acquired using reflection confocal mesoscopy, with a single isolated optical section shown. Reflection imaging determined that intra-colony channels were not occupied by material of differing refractive index to the biomass. The colony-medium interface can be observed clearly, while there is no evident structure within the colony. b Signal from non-viable cells (yellow) was subtracted from viable cells to negate any spectral overlap in the emission of Sytox Green and HcRed1. A composite maximum intensity projection of the entire colony is presented. Intra-colony channels in the viable cell population (cyan) did not contain any non-viable cells. c Alexa594-WGA-stained EPS residues (magenta) were not present in the intra-colony channels when compared with elsewhere in the biofilm, meaning channels were not composed of an EPS-based matrix. The high background signal in the surrounding agar is likely owed to non-specific binding of the WGA dye with gycan components of the agar substrate. d Nile red-stained lipids (red) clustered in the centre of E. coli biofilms while intra-colony channels remain unstained by Nile Red. Therefore, intra-colony channels were not composed of lipids. e Emission of SYPRO Ruby-stained extracellular proteins (magenta) mimicked the spatial patterns of intra-colony channels, showing that channels were filled by a protein-based matrix.

Fig. 3. Intra-colony channels form as an emergent property of biofilm formation.

a An unmixed, naïve control biofilm of JM105-miniTn7-gfp with established intra-colony channels. b A macro-colony JM105-miniTn7-gfp biofilm that was initially grown for 10 h before mechanical disruption and subsequent recovery and regrowth at 37 °C for a further 10 h. Regrowth was accompanied with the re-emergence of intra-colony channels in the outgrown region of the disrupted colony, showing that channel formation is an emergent property of macro-colony biofilm development. The purple circle indicates the boundary of the juvenile colony at the time of disaggregation, where channels have not reformed in the disrupted region.

Fig. 4. Intra-colony channels are confined within clonal populations and unable to cross strain boundaries.

A mixed culture of isogenic JM105 strains which express either GFP (cyan) or HcRed1 (magenta). Each strain sectored into segregated clonal populations, which have propagated from a single colony-forming unit, and cells from each sector were unable to cross the strain boundary. The intra-colony channels present within each sector were also unable to cross the strain boundary and were therefore not shared by opposing isogenic colonies.

Fig. 5. Intra-colony channels facilitate transport of microscopic particles.

A single optical section ~25 μm above the base of the colony shows a mature JM105-miniTn7-gfp biofilm (green) and a lawn of 200 nm fluorescent microspheres (magenta). The fluorescent microspheres were transported from a confluent lawn at the base of the colony into the intra-colony channels and directed towards the centre of the colony. Two ROIs are presented from different regions of the colony where fluorescent microspheres were transported into the colony via intra-colony channels.

Fig. 6. Intra-colony channels play a functional role in nutrient acquisition and transport to the centre of bacterial biofilms.

A deconvolved image of a JM105-pJM058 macro-colony biofilm grown on M9 minimal medium with L-arabinose as the sole carbon source. This arabinose biosensor expresses GFP only in the presence of L-arabinose. GFP emission intensity was higher in cells, which line the intra-colony channels compared with cells elsewhere within the biofilm, which shows that the channel structures have a higher concentration of L-arabinose compared with elsewhere within the biofilm. This provides evidence of a functional role in nutrient acquisition and transport for the intra-colony channel system.