Gene regulatory networks enable bacteria to sense and respond in fluctuating environments and to control gene expression levels in constant conditions. Despite substantial advances in understanding their molecular biology, the evolutionary conditions that select for and maintain gene regulatory networks have remained elusive. Here, we investigate the evolutionary costs and benefits of gene regulation under metabolic fluctuations, and identify distinct fluctuating environments that select for or against gene regulation. Using barcode sequencing to track strain frequencies over time, we compete strains with perturbed expression dynamics to assess the strength of selection on gene expression levels. We reveal that expression levels can be shaped to enhance phenotypic memory of past environmental states, reducing the impact of lag phases on long-term population growth. By independently perturbing the ability to sense the environment and the control of expression levels, we discover sign epistasis between sensing and control in a gene regulatory network, and identify its molecular underpinnings. Due to this epistatic interaction, maintenance of sensing in gene regulation enhances the ability of evolution to tune gene expression in a fluctuating environment. Our work establishes a new basis for understanding how gene regulatory networks evolve in fluctuating environments.

Plant synthetic biology holds great promise for engineering plants to meet future demands. Genetic circuits are being designed, built, and tested in plants to demonstrate proof of concept. However, developing these components in monocots, which the world relies on for grain, lags behind dicot models, such as Arabidopsis thaliana and Nicotiana benthamiana. Here, we show the successful adaptation of a ligand-inducible sensor to activate an endogenous anthocyanin pathway in the C4 monocot model Setaria viridis. We identify two transcription factors sufficient to induce endogenous anthocyanin production in S. viridis protoplasts and whole plants in a constitutive or ligand-inducible manner. We also test multiple ligands to overcome physical barriers to ligand uptake, identifying triamcinolone acetonide (TA) as a highly potent inducer of this system. Using hyperspectral imaging and a discriminative target characterization method in a near-remote configuration, we can non-destructively detect anthocyanin production in leaves in response to ligands. This work demonstrates the use of inducible expression systems in monocots to manipulate endogenous pathways, stimulating plants to overproduce secondary metabolites with value to human health. Applying inducible pigmentation coupled with sensitive detection algorithms could enable crop plants to report on the status of field contamination or detect undesirable chemicals impacting agriculture, ushering in an era of agriculture-based sensor systems.

Nitrification significantly contributes to N2O emissions in wastewater treatment, typically enhanced at low dissolved oxygen (DO). The present study revealed that low DO (∼0.2 mg/L) enhanced the N2O emission factor (EF) from nitrification by 4.5 times in canonical ammonia-oxidizing bacteria (AOB)-dominated sludge, while it reduced N2O EF by 73% in comammox Nitrospira-dominated sludge. During nitrification, the accumulation of intermediate NH2OH in AOB-dominant sludge was much higher and increased more significantly by low DO (from 0.018 to 0.067 mg-N/L) compared to comammox Nitrospira-dominant sludge (from 0.004 to 0.009 mg-N/L). In AOB-dominant sludge, the increased NH2OH and upregulated AOB-NOR gene at low DO promoted NO reduction, thus increasing N2O EF while simultaneously decreasing NO emission. In the comammox Nitrospira-dominant sludge, N2O was primarily produced via abiotic pathways. Further investigation found that N2O production decreased significantly under low DO in inactivated sludge and pure water with the sole addition of NH2OH, suggesting that low DO inhibited N2O formation via abiotic NH2OH oxidation. Therefore, in comammox Nitrospira-dominant sludge, low DO decreased N2O production primarily due to the inhibition of low DO to N2O formation via abiotic NH2OH oxidation and the low NH2OH accumulation. These results imply that enriching comammox Nitrospira in the wastewater treatment process can ensure stably low N2O emissions regardless of the variations in DO.

Microbial growth is often described in terms of resource uptake rates, making the understanding and parameterisation of these rate-limiting processes critical for microbial modelling. In phototrophic plankton, theoretical studies suggest that nutrient uptake is limited by mechanistic processes involving membrane transporters, and it has been observed that the cell-specific maximum resource uptake rate (Vmax) follows a power-law relationship with cell size, as well as a trade-off between Vmax and the half-saturation constant (Km). These constraints may also apply to chemotrophic microorganisms; however, many datasets lack direct cell-size measurements. We therefore leveraged the assumption that prokaryotic cell sizes, Vmax, and Km each follow log-normal distributions, drawing parallels with established phytoplankton scaling laws. Our analysis suggests that chemotrophic organisms generally exhibit higher maximum uptake rate per dry weight (VmaxDW) and Km values than phototrophs, and that VmaxDW and Km are not strongly correlated when all chemotroph data are combined. Furthermore, the Bayesian-derived exponents for VmaxDW and Km exceeded those expected from allometric scaling relationships based on the membrane-transport capacity observed for phototrophs, implying that a range of additional factors likely affect observed kinetic parameters.

Microbial bioproduction is an important approach to realizing green biomanufacturing. However, poor bioproduction stability caused by genetic heterogeneity is one of the important factors limiting its industrial-scale applications. Here, two methods have been developed to reduce genetic heterogeneity in Bacillus subtilis. SiteMuB (the site-dependent mutation bias) was proposed to enable stable genome integration expression by analyzing the spontaneous mutation rate of the same DNA sequences integrated at different genome sites. Additionally, robustly growing chassis with low mutation rates (ChassisLMR) were developed by deleting unstable elements and enhancing DNA repair. These methods were then employed to improve the production stability of small molecule metabolites and proteins. In N-acetylneuraminic acid production, after 76 generations of cell division, corresponding to the number of cell generations required for > 200-m3 industrial-scale production, strains with SiteMuB and ChassisLMR achieved 15.9-fold and 11.1-fold higher titres than that of the starting strain, respectively. Moreover, by improving the genetic stability of burdensome T7RNAP, combining SiteMuB with ChassisLMR stably maintained the T7 expression system for up to 74 generations, representing a 2.1-fold improvement. Furthermore, ChassisLMR improved the production stability of GFP on the plasmids by 1.38-fold. Overall, SiteMuB and ChassisLMR provide broadly applicable and highly efficient ways to achieve stable bioproduction by reducing genetic heterogeneity.

Corynebacterium glutamicum serves as a pivotal industrial chassis for biomanufacturing and an ideal model for studying the phylogenetically related pathogen Mycobacterium tuberculosis. Oxidative stress poses a critical challenge to microorganisms during aerobic industrial processes and immune cell-mediated antibacterial killing by perturbing cellular redox homeostasis, affecting central metabolism, and damaging the integrity of biomacromolecules. However, the intricate mechanisms underlying the dynamic defence of C. glutamicum, despite previous transcriptomic studies on acute and adaptive responses to oxidative stresses, remain largely unclear, hindering strain engineering for industrial applications and the development of effective antimicrobial treatments. In this study, the susceptibility of C. glutamicum to hydrogen peroxide (H2O2) was evaluated, and the inhibitory dynamics of H2O2 were characterized through viable cell counting. RNA sequencing (RNA-seq) was employed to analyse gene expression changes after exposure to 720 mM H2O2. The treatment induced differential expression of 966 and 787 genes at 2 and 6 h, respectively, reflecting perturbations across a broad array of pathways, including (i) enhanced H2O2 and peroxide scavenging, mycothiol biosynthesis, and iron chelation; (ii) repressed central metabolism and enhanced anaplerosis; (iii) elevated sulphur assimilation; (iv) altered amino acid biosynthesis; and (v) altered transcriptional regulation in response to oxidative stress. Further validation by overexpression of ahpD, cysN, and exogenous supplementation with l-methionine and l-cysteine significantly enhanced bacterial tolerance to H2O2. Overall, this study provides the most comprehensive analysis to date of temporal cellular adaptation to H2O2 stress in C. glutamicum, establishing a foundation for future applications in both biomanufacturing and antimicrobial research.

Microorganisms live in close association with plants, forming ecological interaction webs and providing beneficial traits such as nutrition, growth, and tolerance to biotic and abiotic stresses. Via the rhizosphere, plants recruit bacteria which colonize internal plant tissues, creating a spatial gradient between the rhizosphere and the endosphere. This study presents a high throughput in planta endophyte enrichment scheme designed for the identification of 'super'-endophytic bacteria which can serially colonise the rice root endosphere. Oryza sativa (rice) plants were grown in bulk soil, and endophytes were then recovered from roots. The recovered endophyte mixture was used as inoculum for the first generation of rice plantlets, which were then grown under no stress or nitrogen (N) depletion. The total endophytic community was then purified and used as a second inoculum for a new set of plants; this procedure was repeated for four generations. Enrichment patterns of root bacterial endophytes were observed, such as Kosakonia in the non-stressed plants and Ferrovibrio in plants grown under nitrogen starvation. This enrichment method proved to be suitable for the identification of endophytes which can efficiently colonise the root endosphere.

Phenazines are bioactive secondary metabolites with antifungal, anticancer, and insecticidal properties, while hydroxylated derivatives often exhibit enhanced bioactivity. 2-hydroxyphenazine (2-OH-PHZ), which is synthesised by the flavin-dependent monooxygenase PhzO from phenazine-1-carboxylic acid (PCA), shows better bioactivity against the pathogenic fungus Gaeumannomyces graminis vars. tritici. However, the low catalytic efficiency (10%–20% conversion) of PhzO limited 2-OH-PHZ production. To boost PhzO activity, engineering flavin reductase (Fre)-mediated FADH2 regeneration was applied to Pseudomonas chlororaphis LX24AE. Remarkably, this approach improved catalytic efficiency from 25% to 40% and increased the production of a novel dihydroxylated derivative. Then, it was first characterised by UPLC-MS and NMR, and identified as 3,4-dihydroxyphenazine-1-carboxylic acid (3,4-OH-PCA). Next, the Fre-PhzO module through heterologous co-expression in P. putida KT2440 demonstrated a 4.5-fold enhancement in hydroxylation efficiency relative to the PhzO mono-component system, which also confirmed that PhzO and flavin reductase are essential for 3,4-OH-PCA biosynthesis. Moreover, in vitro assays further verified that PhzO exhibits FAD-dependent catalytic promiscuity, simultaneously generating 2-OH-PCA and 3,4-OH-PCA. Furthermore, in vitro and in vivo assays demonstrated that substrate concentration affected the distribution of products. Finally, cytotoxicity evaluation of the isolated 3,4-OH-PCA was performed, and it showed substantial inhibition against oesophageal cancer TE-1 cells and human cervical cancer HeLa cells with an IC50 value of 8.55 μM and 17.69 μM, respectively. This work redefines PhzO as a promiscuous biocatalyst capable of dual hydroxylation, offering a modular platform for engineering bioactive phenazine derivatives.