Hayani Aji, I. | Search bioRxiv

bioRxiv2026

Single-cell imaging links antibiotic resistance gene copy number and bacterial host identity

Identifying the in situ microbial hosts of antibiotic resistance genes (ARGs) in complex environments remains a major challenge, as many of these genes are associated with mobile genetic elements. Here, we introduce combined-FISH, which integrates direct-geneFISH for ARG detection with catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) for rRNA labelling, thereby enabling the simultaneous visualization and copy number quantification of ARGs in taxonomically identified single cells within microbial samples. Using the sulfonamide resistance gene sul1 as a model target, we designed a polynucleotide probe set covering all non-redundant database sequences of this gene within 5% sequence mismatch. The method was validated using model organisms harboring plasmids with sul1, Escherichia coli K12 J53 with R388 and Acinetobacter defluvii with pOXA58_010030. Combined-FISH enabled quantitative determination of sul1 copy numbers at single-cell resolution, with E. coli harboring 1-8 copies and A. defluvii 1-4 copies per cell. Following plasmid-mediated transfer of sul1 from E. coli to Pseudomonas alloputida, recipient cells typically contained 2-6 sul1 copies, with up to 14 copies detected in individual cells, demonstrating the applicability of the approach for investigating horizontal gene transfer. The method was successfully applied to river biofilm samples containing sul1-harboring bacteria, enabling simultaneous tracking of the bacterial host and its associated ARG within a complex microbial community. Collectively, these results establish combined-FISH as a powerful tool for quantitatively linking ARGs and microbial hosts in situ, and for investigating plasmid copy number and gene transfer at single-cell resolution in technical and environmental systems.

molecular biology↗

bioRxiv2025

The infection cycle of the haloarchaeal virus HFTV1 is tightly regulated and strongly inhibits motility of its host

Although viruses have been shown to infect all domains of life, our understanding of the genetic program behind the exploitation of host resources to produce progeny virions is thus far limited to several bacterial viruses. Therefore, to elucidate the transcriptome of euryarchaeal viruses and their hosts, we employed RNAseq analysis of samples taken at different time points from Haloferax gibbonsii LR2-5 cultures infected with the lytic model virus Haloferax Tailed Virus 1 (HFTV1). While following the transcription of viral genes throughout the infective life cycle, we observed a tight temporal regulation of viral transcripts as well as differential expression from within viral gene clusters. Furthermore, anti-sense RNAs (asRNAs) appear to play an important role in support of the timing of late-expressed viral genes. Therefore, with many differentially expressed transcripts, including intragenic transcripts and asRNAs, the regulatory machinery employed by HFTV1 contrasts with viral model systems (based on phages), in which antitermination and/or alternative polymerases (seemingly lacking in HFTV1) are more widespread. When looking into differentially expressed host genes, we observed a strong downregulation of genes involved in motility, such as the archaellum and chemotaxis machinery, which was confirmed with swimming assays of HFTV1 infected cells. This might be a strategy of the virus to redirect energy flowing into movement towards the production of virions. In conclusion, this work thus provides a stepping stone for further exploration of the intriguing strategies of viral transcriptional regulation of their infection cycle across the domains of life. IMPORTANCEViruses infect members of all three domains of life, including Archaea. Euryarchaea are widespread microorganisms found in various environments such as the human gut and solar salterns. Due to the exceptional availability of cell biology and genetic tools of some salt-loving archaea, they are a model system to extrapolate from. Insights into the regulation of viral infections are of particular importance, especially since HFTV1, has been adopted as a model virus by the archaeal viral community. We found that, while harboring parallels with bacterial viruses, such as tight temporal regulation, HFTV1 harbors an impressive number of differentially expressed transcriptional units. Furthermore, anti-sense RNAs and intragenic regulatory elements seem to play a much more prominent role in HFTV1 gene expression. Thus, this work challenges current models and provides valuable new insights into the gene regulation of viral infection of archaea, which mark similarities and differences with viruses from other domains of life.

microbiology↗