bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.05.30.728972

Nitrogenase regulation in Vibrio natriegens differs from other γ-proteobacteria

Haas, N. W.·Wiesler, E. E.·Dalia, A. B.·Wang, X.·McKinlay, J. B.
View original source ↗Export metadata ↓

Abstract

Regulation of nitrogenase, which converts nitrogen gas (N2) into ammonium (NH4+), typically involves a conserved set of regulatory proteins across diverse N2-fixing (diazotrophic) bacteria. However, the interactions and relative influence of these regulators can vary between species. Thus, one cannot make assumptions about nitrogenase regulation when working with uncharacterized diazotrophs like Vibrio natriegens, a {gamma}-proteobacterium of growing interest for synthetic biology. Little is known about V. natriegens nitrogenase regulation, which could be used to exploit inexpensive N2 for various applications, including NH4+ production. Here, we characterized the roles of several annotated V. natriegens nitrogenase regulatory proteins in response to NH4+ versus N2. Using functional genomics, targeted mutations, and reporter assays, we identified a typical regulatory hierarchy where the two-component system NtrBC governs a nitrogen-scavenging regulon that includes NifA, the transcriptional activator of nitrogenase genes. Unlike other diazotrophic {gamma}-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N2 after a lag phase. Thus, NtrBC was dispensable, but still important for timely nitrogenase expression. Furthermore, NtrBC was negatively regulated by the nitrogen-responsive PII proteins GlnB and GlnK; disruption of both PII proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH4+ excretion. The redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic E. coli than other diazotrophic {gamma}-proteobacteria. Together, our findings provide a framework for V. natriegens nitrogenase regulation that can be leveraged for applications like NH4+ production. HIGHLIGHTSO_LIA genetic examination of Vibrio natriegens nitrogenase regulation is performed C_LIO_LINtrBC is important for early nitrogenase gene expression but is not essential C_LIO_LINifA autoactivation is sufficient for nitrogenase expression C_LIO_LIPII proteins GlnB and GlnK are redundant negative regulators of nitrogenase C_LIO_LIGenetic targets are identified that result in excretion of NH4+ C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/728972v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@aac4adorg.highwire.dtl.DTLVardef@1565c0corg.highwire.dtl.DTLVardef@b545a6org.highwire.dtl.DTLVardef@efb052_HPS_FORMAT_FIGEXP M_FIG C_FIG

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Haas, N. W., Wiesler, E. E., Dalia, A. B., Wang, X., McKinlay, J. B.. 2026-06-01. Nitrogenase regulation in Vibrio natriegens differs from other γ-proteobacteria. https://doi.org/10.64898/2026.05.30.728972

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

KEEP EXPLORING

Related preprints

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↗
bioRxiv2026

Matrix-controlled emergence of biofilm architecture shapes antimicrobial survival

Biofilms are structured microbial communities whose extracellular matrix is widely regarded as a basis of their protection against antimicrobial compounds. Yet how matrix production by individual bacteria gives rise to collective architecture and antimicrobial protection remains poorly understood. Here, we systematically varied expression of the master biofilm regulator csgD in Salmonella enterica and found that increasing matrix production reorganizes biofilms from dense, isotropic packings into sparse, nematically aligned communities by altering cell-cell interactions. By combining experimentally measured biofilm architectures with reaction-diffusion modeling, we show that these structural changes produce distinct patterns of antimicrobial killing, ranging from preferential killing near the liquid-biofilm interface to more uniform killing throughout the community. Consequently, increasing matrix production unexpectedly reduces antimicrobial survival by shifting the biofilm into different transport regimes, while strain-specific physiological differences further modulate antimicrobial depletion. Rather than acting as a passive barrier, EPS therefore shapes antimicrobial susceptibility by reorganizing biofilm architecture and its transport properties. EPS thus provides a physical link between molecular regulation, collective architecture and antimicrobial survival, providing a quantitative framework for understanding how cellular matrix production generates emergent biofilm function.

microbiology↗