Unicellular microorganisms can make a transition to multicellular states that enhance survival under environmental fluctuations. In bacteria, one of these states is the biofilm, defined by the production of an extracellular matrix. Although biofilm maturation and dispersion have been extensively studied, where and how initial matrix production is induced within a growing population remains largely unknown. Here we show that production of colanic acid, an important matrix component, is initiated around topological defects, where cell orientation mismatches and growth-induced pressure builds up, in bacterial monolayers. Using E. coli reporting mechanically induced production of colanic acid in response to cell contact and deformation, we found matrix production accompanied by out-of-plane growth under agar-pad confinement. Controlling confinement geometry using microfluidic devices dictated the positions of topological defects and thereby localized regions of high matrix production. These findings reveal that the cell orientation patterning spatially organizes mechanical cues to induce matrix production for biofilm initiation of bacteria.

Bacterial surface polysaccharides are versatile structures that provide specificity to the behavior and interactions of a given bacterial strain. One surface polysaccharide displayed on the organism Bacteroides fragilis, Capsular Polysaccharide A, has been implicated as a potential therapeutic for autoimmune disorders. This polymer is composed of repeating units of the tetrasaccharide 2-acetamido-4-amino-2,4,6-trideoxygalactopyranose (AATGal), 4,6-O-pyruvate-galactopyranose (PyrGal), N-acetylgalactosamine (GalNAc), and galactofuranose (Galf). While this and other bacterial surface polysaccharides are attractive to study and apply to biomedicine, it can be difficult to acquire quick, inexpensive access to these pure materials. In this work, we developed a recombinant expression system in E. coli for the stepwise production of the CPSA polymer. A series of sequential plasmids were prepared, each incorporating successive genes required for CPSA biosynthesis. Using these iterative plasmids, we were able to observe production of the CPSA repeating unit and precursors by liquid chromatography mass spectrometry (LC-MS) analysis of cell lysates. We found that it was critical to include the CPSA polymerase but not the flippase, indicating that a native E. coli flippase could support polymer production. We also provide evidence that the CPSA polymer produced by E. coli can be ligated to LPS by the E. coli WaaL ligase, and deletion of this gene led to the formation of a water-soluble polymer. Overall, this work describes the first recombinant system for CPSA production and outlines a key strategy for the production of complex glycopolymers.

Transformer-based genomic sequence models represent an emerging frontier in computational biology. Yet, their embeddings have not yet shown the same level of predictive power as natural and protein language models, indicating a gap between current implementations and theoretical promise. Existing benchmarks for DNA language models primarily focus on classifying regulatory elements in eukaryotic genomes, leaving open the fundamental question of whether these models learn sequence-level features across whole genomes. We introduce LAMBDA, a benchmark designed to rigorously evaluate genome language model embeddings through phage-bacteria sequence discrimination across four categories of increasing complexity: probing tasks, fine-tuning assessments, diagnostic tests, and genome-wide prophage detection. Our comprehensive analysis of current genomic language models provides novel insights into the importance of training data quality relative to model size, the need for domain-specific training, and the application of genomic language models for detecting prophage sequences. This benchmark represents a challenging genomic annotation task in the bacterial domain and addresses a key computational problem with direct relevance to microbiology and medicine.

Protein expression is an important aspect of synthetic biology, where host selection critically impacts the production efficiency. V. natriegens has emerged as a promising prokaryotic host due to its exceptionally rapid growth rate and broad substrate metabolic spectrum. Benefiting from the advances in annotation on microbial genomes and metabolic pathways, as well as the development of molecular biology tools, continuous progress has been made in bioproduction using V. natriegens. In this Perspective, we provide a systematic introduction of the basic biological properties of V. natriegens and present the engineered V. natriegens chassis which are currently used for protein expression. We summarize the latest synthetic biology components and genetic tools specifically designed for the modification of V. natriegens. Especially, we focus on the most recent study and application achievements of V. natriegens in in vivo and in vitro protein expression, which has received limited attention in previous reviews. This review aims to present researchers with a comprehensive overview of using V. natriegens for protein expression, highlight its advantages and application prospects, and present some challenges and future perspectives in this field, so as to promote wider research in the synthetic biology field.