Environmental co-contamination presents significant challenges. To tackle these, while microbial consortia offer advantages over single-strain, such as functional redundancy and synergistic degradation, rationally designing effective synthetic microbiomes for complex co-contamination scenarios remains a major challenge. Here, we utilize our advanced genome-scale metabolic modeling (GSMM) tool, SuperCC, to simulate the metabolic behavior of communities consisting of six isolated key strains under single- and multi-carbon source conditions, mimicking single-pollutant or co-contamination scenarios respectively. By integrating multi-omics data with metabolic modeling of cultures, we systematically elucidate key strain interaction networks and adaptive strategies under co-contamination. This reveal that the specific secretory products of broad-spectrum resource-utilizing bacteria serve as key metabolites driving cooperation and highlight the pivotal role of indigenous keystone strains in stabilizing and enhancing community function. Consequently, we propose an innovative and rational paradigm for consortium design: DHP-Com (Degrader-Helper-Potentiator Consortium). Potentiators are top species with stable habitat abundance. Synthetic microbiomes built on this framework exhibit enhanced ecological fitness and substantially improve remediation performance across diverse co-contamination scenarios. Our findings advanced the practical application of GSMM predictions to decipher intricate multi-pollutant/multi-strain interaction networks, offering a powerful rational framework and robust methodological tools for engineering multi-functional and effective synthetic microbiomes for complex environmental remediation. Microbial consortia offer advantages over single strains for the remediation of environmental co-contamination. Here, the authors introduce an Degrader-Helper-Potentiator Consortium framework to design synthetic microbiomes that enhance the remediation of environmental co-contamination.

Rising atmospheric CO₂ levels and their impact on climate change have intensified the need for innovative carbon capture and fixation strategies. The Calvin-Benson-Bassham (CBB) cycle, a central metabolic pathway in all photoautotrophic organisms and many autotrophic bacteria, plays a pivotal role in global carbon assimilation but is limited by the low catalytic efficiency of Rubisco. Here, we engineered a complete, functional CBB cycle in Escherichia coli, by heterologously expressing up to 13 genes encoding phosphoribulokinase, α-carboxysomes, and inorganic carbon pumps. This bioengineering approach allowed E. coli to utilize atmospheric CO2 and led to increased levels of sugars such as ribose (4.94-fold) and xylitol (8.94-fold). Detailed metabolomic profiling of central carbon metabolism using gas chromatography-mass spectrometry (GC-MS) demonstrated that installation of the CBB cycle has a notable impact on the metabolic landscape of E. coli, resulting in substantial alterations in central carbon and amino acid metabolism. These findings deepen our understanding of the natural biological carbon-fixation pathway and its engineering in heterotrophic hosts. Furthermore, this work provides a versatile platform for evaluating and selecting efficient carbon-fixation modules, as well as assessing metabolic bottlenecks in engineered systems. These advances offer practical guidance for rational metabolic engineering in diverse organisms for biotechnological applications, including carbon sequestration, sustainable bioproduction, and crop improvement. 

Cyanobacteria, as phototrophic organisms with low nutritional requirements and great metabolic versatility, are attractive for the sustainable production of value-added chemicals from CO2 and sunlight. One limitation of these strategies is that carbon is partitioned towards biomass synthesis rather than product synthesis. An alternative to conventional metabolic engineering approaches involves controlling regulatory circuits to enhance the flow of carbon towards the synthesis of desired compounds. The carbon-flow-regulator A (CfrA) is pivotal in redirecting carbon flux during nitrogen deficiency in cyanobacteria, promoting glycogen accumulation by inhibiting 2,3-phosphoglycerate mutase enzyme. The moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803 accumulates sucrose and glucosylglycerol (GG) as compatible solutes under salt stress. Sucrose is a valuable carbon source for heterotrophic organisms, whether they are cultivated independently or in co-cultures. In this context, we explored the potential biotechnological relevance of CfrA in redirecting carbon flow towards sucrose production.  A strain that overexpresses cfrA, independently of nitrogen growth conditions, and carries a plasmid that expresses sucrose-phosphate synthase (SPS) from Synechocystis sp. PCC 6803 and the heterologous sucrose permease CscB inducibly (Pars-cfrA/suc strain) was constructed and analysed. In this strain, cfrA expression increased sucrose production by 40% compared to non-induced levels. The fixed carbon was partially redirected towards sucrose production at the expense of glycogen accumulation and biomass generation. Furthermore, an improvement in the photosynthetic activity of this strain was observed due to the presence of this carbon sink. The effect of eliminating GG synthesis (ΔggpS/Pars-cfrA/suc strain) on sucrose production was also analyzed. Under high salinity conditions (400 mM NaCl), this strain exhibited a maximum sucrose accumulation of 2.72 g/L. Encapsulation of the Pars-cfrA/suc strain has also been studied.  Our results indicate that modulating carbon flow through CfrA overexpression can substantially boost sucrose production. Glycogen accumulation, mediated by CfrA, enhances sucrose production, which is partly derived from the use of stored glycogen. Furthermore, immobilising Synechocystis cells in alginate improves sucrose production and facilitates its utilisation. Given the widespread occurrence of the cfrA gene in cyanobacteria, its potential as a target in various biotechnological strategies that require the redirection of carbon flow should be considered.

Type IV secretion systems (T4SS) are versatile nanomachines responsible for the transfer of DNA and proteins across cell envelopes. From their ancestral role in conjugation, these systems have diversified into a superfamily with functions ranging from horizontal gene transfer to the delivery of toxins to eukaryotic and prokaryotic hosts. Recent structural and functional studies have uncovered unexpected architectural variations not only among Gram-negative systems but also between Gram-negative and Gram-positive systems. Despite this diversity, a conserved set of core proteins is maintained across the superfamily. To facilitate cross-system comparisons, we propose in this review a unified nomenclature for conserved T4SS subunits found in both Gram-negative and Gram-positive systems. We further highlight conserved and divergent mechanistic and architectural principles across bacterial lineages, and we discuss the diversity of emerging T4SSs whose unique structures and functions expand our understanding of this highly adaptable secretion superfamily..

Cells combat peroxide stress using peroxiredoxins and catalases. The paradigmatic H2O2-sensing OxyR or PerR transcription regulators typically control their expression in bacteria. Here, we report our discovery of a noncanonical mechanism for H2O2-signaled regulation of peroxiredoxin ahpC and catalase katB genes in Myxococcus xanthus by PexR, an ATP-binding bacterial enhancer binding protein that acts as a dual-function repressor-activator. PexR, a dimer capable of higher-order oligomerization, binds to dyad-symmetry repeats upstream of ahpC and katB, activating their H2O2-induced σ54-dependent expression. Under peroxide stress-free conditions, PexR downregulates housekeeping σA-dependent ahpC expression. Deleting ahpC causes pleiotropy, and synthetic lethality when eliminated with katB or pexR, indicating PexR-mediated co-regulation of AhpC and KatB as critical for normal growth. We show that resting state PexR is autoinhibited by its N-terminal Zn2+/Fe2+-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which senses H2O2 and releases its bound metal to trigger PexR-activated σ54-dependent expression. Our genomic analyses reveal conservation of PexR and its regulatory elements and, likely, mechanism across Myxococcota, frequently co-occurring with OxyR or PerR, showcasing this phylum’s remarkable diversity of potential peroxide stress response regulatory mechanisms. Moreover, PexR likely operates in phyla beyond Myxococcota, and its discovery expands the toolkit for genetically encoded H2O2 sensors.

Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5’-NNNGRY-3’). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. Seeking to further expand its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications. A compact Cas9 ortholog, Cj4Cas9, enables efficient genome editing in vivo compatibility.

With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates. The disruption of the rhomboid protease gene glpG in E. coli leads to defects in bacterial colonization. Here, the authors show that GlpG is essential for the assembly of type 1 pili, a virulence factor required for colonization of bacteria in eukaryotic cells.