Single-probe, amplification-free multiplex mRNA PNA-FISH maps bacterial gene expression in intact three-dimensional biofilms

Authors

Barbosa, A., Smith, H. J., Parker, A., Jones, C. J., Goeres, D. M., Azevedo, N. F., Cerqueira, L.

Abstract

Biofilms are spatially structured communities in which local microenvironments drive physiological differentiation, yet mapping gene expression inside intact three-dimensional biofilms remains challenging. Bacterial spatial transcriptomics methods typically rely on tiled probe sets, sequential hybridization rounds, or signal amplification. Here, we show that a single short, directly labelled peptide nucleic acid (PNA) probe per target is sufficient to quantify bacterial mRNA in situ without amplification. With all probes designed for a common hybridization condition, a transcript panel is resolved in one hybridization on a standard confocal microscope, in unmodified cells while preserving biofilm architecture. Benchmarked against the biphasic cycle of Legionella pneumophila, multiplex mRNA PNA-FISH recovers the expected transcriptional switch and agrees quantitatively with RT-qPCR in planktonic and biofilm populations. In flow-cell biofilms, it shows that sessile populations do not recapitulate the planktonic program and that transcriptional differences follow cellular aggregation state rather than depth.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 03 Nov 2026.

Stats

1-terrible, 9-excellent. How would you rate this preprint? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.