Role of Pseudomonas aeruginosa Dnr-regulated denitrification in oxic conditions

Balsam, S. S.; Mould, D. L.; Jean-Pierre, F.; Hogan, D. A.

doi:10.1101/2025.03.31.646406

Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.03.31.646406

Role of Pseudomonas aeruginosa Dnr-regulated denitrification in oxic conditions

Balsam, S. S.·Mould, D. L.·Jean-Pierre, F.·Hogan, D. A.

View original source ↗Export metadata ↓

Abstract

Pseudomonas aeruginosa causes acute and chronic infections such as those that occur in the lungs of people with cystic fibrosis (CF). In infection environments, oxygen (O2) concentrations are often low. The transcription factor Anr responds to low O2 by upregulating genes necessary for P. aeruginosa fitness in microoxic and anoxic conditions. Anr regulates dnr, a gene encoding a transcriptional regulator that promotes the expression of genes required for using nitrate as an alternative electron acceptor during denitrification. In CF sputum, transcripts involved in denitrification are highly expressed. While Dnr is necessary for the anoxic growth of P. aeruginosa in CF sputum and artificial sputum media (ASMi), the contribution of denitrification to P. aeruginosa fitness in oxic conditions has not been well described. Here we show that P. aeruginosa requires dnr for fitness in ASMi and the requirement for dnr is abolished when nitrate is excluded from the media. Additionally, we show that P. aeruginosa consumes nitrate in lysogeny broth (LB) under microoxic conditions. Furthermore, strains without a functioning quorum sensing regulator LasR, which leads to elevated Anr activity, consume nitrate in LB even in normoxia. There was no growth advantage for P. aeruginosa when nitrate was present at concentrations from 100 {micro}M to 1600 {micro}M. However, P. aeruginosa consumption of nitrate in oxic conditions created a requirement for Dnr and Dnr-regulated NorCB likely due to the need to detoxify nitric oxide. These studies suggest that Anr- and Dnr-regulated processes may impact P. aeruginosa physiology in many common culture conditions. ImportancePseudomonas aeruginosa is an opportunistic pathogen commonly isolated from low-oxygen environments such as the lungs of people with cystic fibrosis. While the importance of P. aeruginosa energy generation by denitrification is clear in anoxic environments, the effects of denitrification in oxic cultures is not clear. Here, we show that nitrate is consumed even in oxic environments and while it does not appear to stimulate growth, it does impact fitness. Further, we report that two regulators that are best known for their roles in anoxic conditions also contribute to P. aeruginosa fitness in commonly- used laboratory media in presence of oxygen.

Explore related subjects

microbiology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Balsam, S. S., Mould, D. L., Jean-Pierre, F., Hogan, D. A.. 2025-03-31. Role of Pseudomonas aeruginosa Dnr-regulated denitrification in oxic conditions. https://doi.org/10.1101/2025.03.31.646406

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗

bioRxiv2026

Rapid and largely reversible shifts in the canine fecal metabolome during dietary change

Diet can rapidly change the fecal metabolome, but less is known about recovery after the original diet is restored. We used untargeted UPLC-MS metabolomics to analyze 72 fecal samples from nine Pumi dogs during an owner-managed switch from dry food to raw food and back to dry food. Diet phase accounted for a large proportion of variation in both ionization modes. More than 13,000 LC-MS features changed at the first sampling point after the switch to raw food, with a similarly large response after return to dry food. Among features significant in both comparisons, more than 99% changed in opposite directions. At the final sampling point, no positive-mode (ESI+) features and only 13 negative-mode (ESI-) features differed from the second dry-food baseline under the same threshold. BARF-associated patterns persisted in analyses excluding individual dogs and in pedigree-adjusted candidate models, although individual feature effects depended on normalization. Putative metabolites from several biochemical classes differed in their response and recovery. The fecal metabolome therefore changed rapidly and returned largely toward baseline, with differences among dogs.

microbiology↗

bioRxiv2026

Taxonomic and functional concordance between full-length ONT 16S and ONT shotgun metagenomics in the canine gut microbiome

Background: Full-length Oxford Nanopore Technologies (ONT) 16S rRNA sequencing provides a scalable view of microbial community composition and can support phylogeny-based functional prediction, but it is not equivalent to shotgun metagenomics. We asked which biological conclusions are preserved when the same canine fecal specimens are profiled by full-length ONT 16S and ONT whole-genome shotgun (WGS) sequencing, and how their agreement depends on analytical scale, reference representation and classifier. Methods: Ninety-seven fecal specimens from 51 dogs were profiled with both assays from the same DNA extract. Functional profiles predicted from NanoASV/NanoPredict with PICRUSt2 were compared with WGS-supported KEGG Ortholog (KO) profiles generated by Kadath. Taxonomy was benchmarked in a source-genome-matched RefSeq universe and in a host-specific DogMAG universe using minitax and Kraken2. Agreement was evaluated at whole-profile, feature-abundance, detection, between-sample structure and biological-inference scales. Age-associated transfer was assessed with dog-aware continuous mixed models, grouped signed-score analyses and paired/dog-blocked PERMANOVA. Results: Functional whole-profile concordance was high: median within-sample CLR Spearman correlations ranged from 0.781 to 0.860 across developmental strata, while between-sample functional structure remained significant by Mantel (rho=0.543) and Procrustes (r=0.693; both p=0.001). Feature-wise transfer was substantially weaker (median KO-wise CLR Spearman=0.318). Continuous age-associated KO slopes showed substantial cross-assay concordance (Spearman=0.727; signed-score Spearman=0.753; direction agreement=77.9%), although 1,290/5,258 eligible KOs retained significant assay-by-age interactions. Taxonomically, exact genus/species abundance agreement was much lower than agreement in between-sample ecological structure. Host-specific DogMAG improved species-level median Spearman from 0.261 to 0.656 for minitax SpeciesEstimate and from 0.181 to 0.512 for Kraken2. The classifier effect was independent of reference choice: under both RefSeq and DogMAG, minitax yielded stronger 16S-WGS concordance than Kraken2, with all eight prespecified RefSeq paired genus/species endpoints and all 10 DogMAG primary paired endpoints significant after BH correction. The same ordering extended to developmental inference, with DogMAG genus/species age-slope concordance of 0.795/0.799 for SpeciesEstimate versus 0.693/0.702 for Kraken2. Taxonomic Aitchison PERMANOVA detected age-associated structure in every assay/reference/classifier/rank combination, whereas age-by-assay interactions were consistently significant but small (R2 approximately 1.1 to 2.2%). Stricter NanoASV identity thresholds removed substantial 16S abundance without improving species-level agreement. Conclusions: The extent of cross-assay agreement depends on the level of analysis. Full-length ONT 16S preserves broad functional organization, ecological structure and much of the direction of age-associated change, but exact fine-rank composition, individual-feature abundance and effect magnitude remain assay dependent. Host-specific reference representation substantially narrows the taxonomic gap, and classifier choice exerts an additional independent effect: within the same matched reference set, minitax consistently yields stronger 16S-WGS concordance than Kraken2 across abundance, detection, ecological-distance and developmental-inference endpoints. Full-length ONT 16S is therefore well suited to broad ecological screening and hypothesis generation, whereas WGS remains preferable when conclusions depend on quantitative fine-rank composition, directly supported gene content or precise feature-level effect estimates.

microbiology↗