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Recent advances in microfluidic technology have made it possible to image live bacterial cells with a high degree of precision and control. In particular, single-cell microfluidic designs have created new opportunities to study phenotypic variation in bacterial populations. However, the development and use of microfluidic devices require specialized resources, and these can be practical barriers to entry for microbiologists. With this review, our intentions are to help demystify the design, construction, and application of microfluidics. Our approach is to present design elements as building blocks from which a multitude of microfluidics applications can be imagined by the microbiologist.

Microbial communities exhibit complex behaviors driven by species interactions and individual characteristics. In this study, we delve into the dynamics of a mixed bacterial population comprising two distinct species with different morphology and motility aspects. Employing agent-based modeling and computer simulations, we analyze the impacts of size ratios and packing fractions on dispersal patterns, aggregate formation, clustering, and spatial ordering. Notably, we find that motility and anisotropy of elongated bacteria significantly influence the distribution and spatial organization of nonmotile spherical species. Passive spherical cells display a superdiffusive behavior, particularly at larger size ratios in the ballistic regime. As the size ratio increases, clustering of passive cells is observed, accompanied by enhanced alignment and closer packing of active cells in the presence of higher passive cell area fractions. In addition, we identify the pivotal role of passive cell area fraction in influencing the response of active cells toward nematicity, with its dependence on size ratio. These findings shed light on the significance of morphology and motility in shaping the collective behavior of microbial communities, providing valuable insights into complex microbial behaviors with implications for ecology, biotechnology, and bioengineering., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

The origin of the eukaryotic cell, with its compartmentalized nature and generally large size compared with bacterial and archaeal cells, represents a cornerstone event in the evolution of complex life on Earth. In a process referred to as eukaryogenesis, the eukaryotic cell is believed to have evolved between approximately 1.8 and 2.7 billion years ago from its archaeal ancestors, with a symbiosis with a bacterial (proto-mitochondrial) partner being a key event. In the tree of life, the branch separating the first from the last common ancestor of all eukaryotes is long and lacks evolutionary intermediates. As a result, the timing and driving forces of the emergence of complex eukaryotic features remain poorly understood. During the past decade, environmental and comparative genomic studies have revealed vital details about the identity and nature of the host cell and the proto-mitochondrial endosymbiont, enabling a critical reappraisal of hypotheses underlying the symbiotic origin of the eukaryotic cell. Here we outline our current understanding of the key players and events underlying the emergence of cellular complexity during the prokaryote-to-eukaryote transition and discuss potential avenues of future research that might provide new insights into the enigmatic origin of the eukaryotic cell., (© 2024. Springer Nature Limited.)

Bacteria employ quorum sensing as a remarkable mechanism for coordinating behaviors and communicating within their communities. In this study, we introduce a MATLAB Graphical User Interface (GUI) that offers a versatile platform for exploring the dynamics of quorum sensing. Our computational framework allows for the assessment of quorum sensing, the investigation of parameter dependencies, and the prediction of minimum biofilm thickness required for its initiation. A pivotal observation from our simulations underscores the pivotal role of the diffusion coefficient in quorum sensing, surpassing the influence of bacterial cell dimensions. Varying the diffusion coefficient reveals significant fluctuations in autoinducer concentration, highlighting its centrality in shaping bacterial communication. Additionally, our GUI facilitates the prediction of the minimum biofilm thickness necessary to trigger quorum sensing, a parameter contingent on the diffusion coefficient. This feature provides valuable insights into spatial constraints governing quorum sensing initiation. The interplay between production rates and cell concentrations emerges as another critical facet of our study. We observe that higher production rates or cell concentrations expedite quorum sensing, underscoring the intricate relationship between cell communication and population dynamics in bacterial communities. While our simulations align with mathematical models reported in the literature, we acknowledge the complexity of living organisms, emphasizing the value of our GUI for standardizing results and facilitating early assessments of quorum sensing. This computational approach offers a window into the environmental conditions conducive to quorum sensing initiation, encompassing parameters such as the diffusion coefficient, cell concentration, and biofilm thickness. In conclusion, our MATLAB GUI serves as a versatile tool for understanding the diverse aspects of quorum sensing especially for non-biologists. The insights gained from this computational framework advance our understanding of bacterial communication, providing researchers with the means to explore diverse ecological contexts where quorum sensing plays a pivotal role., (© 2024. The Author(s).)

Ranging from subcellular organelle biogenesis to embryo development, the formation of self-organized structures is a hallmark of living systems. Whereas the emergence of ordered spatial patterns in biology is often driven by intricate chemical signalling that coordinates cellular behaviour and differentiation 1-4 , purely physical interactions can drive the formation of regular biological patterns such as crystalline vortex arrays in suspensions of spermatozoa 5 and bacteria 6 . Here we discovered a new route to self-organized pattern formation driven by physical interactions, which creates large-scale regular spatial structures with multiscale ordering. Specifically we found that dense bacterial living matter spontaneously developed a lattice of mesoscale, fast-spinning vortices; these vortices each consisted of around 10 4 -10 5  motile bacterial cells and were arranged in space at greater than centimetre scale and with apparent hexagonal order, whereas individual cells in the vortices moved in coordinated directions with strong polar and vortical order. Single-cell tracking and numerical simulations suggest that the phenomenon is enabled by self-enhanced mobility in the system-that is, the speed of individual cells increasing with cell-generated collective stresses at a given cell density. Stress-induced mobility enhancement and fluidization is prevalent in dense living matter at various scales of length 7-9 . Our findings demonstrate that self-enhanced mobility offers a simple physical mechanism for pattern formation in living systems and, more generally, in other active matter systems 10 near the boundary of fluid- and solid-like behaviours 11-17 ., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

This personal story recounts the accidental observation, the struggles, the breakthroughs, and the collaborative spirit of a few individuals that led to the discovery that bacterial cells expend energy to effectively fluidize their otherwise "glass-like" cytoplasm and promote the dispersal of large cytoplasmic components. This adventure, which led us into an uncharted world at the intersection of cell biology and condensed matter physics about ten years ago, forever transformed the way I view cells and conduct research., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Determining the repertoire of a microbe's molecular functions is a central question in microbial biology. Modern techniques achieve this goal by comparing microbial genetic material against reference databases of functionally annotated genes/proteins or known taxonomic markers such as 16S rRNA. Here, we describe a novel approach to exploring bacterial functional repertoires without reference databases. Our Fusion scheme establishes functional relationships between bacteria and assigns organisms to Fusion-taxa that differ from otherwise defined taxonomic clades. Three key findings of our work stand out. First, bacterial functional comparisons outperform marker genes in assigning taxonomic clades. Fusion profiles are also better for this task than other functional annotation schemes. Second, Fusion-taxa are robust to addition of novel organisms and are, arguably, able to capture the environment-driven bacterial diversity. Finally, our alignment-free nucleic acid-based Siamese Neural Network model, created using Fusion functions, enables finding shared functionality of very distant, possibly structurally different, microbial homologs. Our work can thus help annotate functional repertoires of bacterial organisms and further guide our understanding of microbial communities., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Complex glycans serve essential functions in all living systems. Many of these intricate and byzantine biomolecules are assembled employing biosynthetic pathways wherein the constituent enzymes are membrane-associated. A signature feature of the stepwise assembly processes is the essentiality of unusual linear long-chain polyprenol phosphate-linked substrates of specific isoprene unit geometry, such as undecaprenol phosphate (UndP) in bacteria. How these enzymes and substrates interact within a lipid bilayer needs further investigation. Here, we focus on a small enzyme, PglC from Campylobacter, structurally characterized for the first time in 2018 as a detergent-solubilized construct. PglC is a monotopic phosphoglycosyl transferase that embodies the functional core structure of the entire enzyme superfamily and catalyzes the first membrane-committed step in a glycoprotein assembly pathway. The size of the enzyme is significant as it enables high-level computation and relatively facile, for a membrane protein, experimental analysis. Our ensemble computational and experimental results provided a high-level view of the membrane-embedded PglC/UndP complex. The findings suggested that it is advantageous for the polyprenol phosphate to adopt a conformation in the same leaflet where the monotopic membrane protein resides as opposed to additionally disrupting the opposing leaflet of the bilayer. Further, the analysis showed that electrostatic steering acts as a major driving force contributing to the recognition and binding of both UndP and the soluble nucleotide sugar substrate. Iterative computational and experimental mutagenesis support a specific interaction of UndP with phosphoglycosyl transferase cationic residues and suggest a role for critical conformational transitions in substrate binding and specificity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nearly 100 years ago, Winogradsky published a classic communication in which he described two groups of microbes, zymogenic and autochthonous. When organic matter penetrates the soil, zymogenic microbes quickly multiply and degrade it, then giving way to the slow combustion of autochthonous microbes. Although the text was originally written in French, it is often cited by English-speaking authors. We undertook a complete translation of the 1924 publication, which we provide as Supporting information. Here, we introduce the translation and describe how the zymogenic/autochthonous dichotomy shaped research questions in the study of microbial diversity and physiology. We also identify in the literature three additional and closely related dichotomies, which we propose to call exclusive copiotrophs/oligotrophs, coexisting copiotrophs/oligotrophs and fast-growing/slow-growing microbes. While Winogradsky focussed on a successional view of microbial populations over time, the current discussion is focussed on the differences in the specific growth rate of microbes as a function of the concentration of a given limiting substrate. In the future, it will be relevant to keep in mind both nutrient-focussed and time-focussed microbial dichotomies and to design experiments with both isolated laboratory cultures and multi-species communities in the spirit of Winogradsky's direct method., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Hygroscopic biological matter in plants, fungi and bacteria make up a large fraction of Earth's biomass 1 . Although metabolically inert, these water-responsive materials exchange water with the environment and actuate movement 2-5 and have inspired technological uses 6,7 . Despite the variety in chemical composition, hygroscopic biological materials across multiple kingdoms of life exhibit similar mechanical behaviours including changes in size and stiffness with relative humidity 8-13 . Here we report atomic force microscopy measurements on the hygroscopic spores 14,15 of a common soil bacterium and develop a theory that captures the observed equilibrium, non-equilibrium and water-responsive mechanical behaviours, finding that these are controlled by the hydration force 16-18 . Our theory based on the hydration force explains an extreme slowdown of water transport and successfully predicts a strong nonlinear elasticity and a transition in mechanical properties that differs from glassy and poroelastic behaviours. These results indicate that water not only endows biological matter with fluidity but also can-through the hydration force-control macroscopic properties and give rise to a 'hydration solid' with unusual properties. A large fraction of biological matter could belong to this distinct class of solid matter., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The rise of β-lactam resistance among pathogenic bacteria, due to the horizontal transfer of plasmid-encoded β-lactamases, is a current global health crisis. Importantly, β-lactam hydrolyzation by β-lactamases, not only protects the producing cells but also sensitive neighboring cells cooperatively. Yet, how such cooperative traits affect plasmid transmission and maintenance is currently poorly understood. Here we experimentally show that KPC-2 β-lactamase expression and extracellular activity were higher when encoded on plasmids compared with the chromosome, resulting in the elevated rescue of sensitive non-producers. This facilitated efficient plasmid transfer to the rescued non-producers and expanded the potential plasmid recipient pool and the probability of plasmid transfer to new genotypes. Social conversion of non-producers by conjugation was efficient yet not absolute. Non-cooperative plasmids, not encoding KPC-2, were moderately more competitive than cooperative plasmids when β-lactam antibiotics were absent. However, in the presence of a β-lactam antibiotic, strains with non-cooperative plasmids were efficiently outcompeted. Moreover, plasmid-free non-producers were more competitive than non-producers imposed with the metabolic burden of a plasmid. Our results suggest that cooperative antibiotic resistance especially promotes the fitness of replicons that transfer horizontally such as conjugative plasmids., (© 2023. The Author(s).)

There is growing evidence showing that many critical biological processes are driven by biomolecule condensates through liquid-liquid phase separation (LLPS). Although the qualitative observation and description of LLPS have been well documented, quantitative simulations of the time-dependent progression of LLPS in live cells are generally lacking. In this work, we build a stochastic Monte Carlo model to simulate the dynamic LLPS process during the formation of bacterial aggresomes. We demonstrate that the size distribution of the protein condensates evolves from an exponential-like to a bimodal-like pattern, and the number of condensates increases at the beginning and then decreases after reaching a maximum. Incorporating diffusion and collision, our simplified model recapitulates the two-step LLPS process in which many smaller condensates are formed in the first step and then merged into a few larger ones. We further reveal that the condensation speed, which can be defined by the condensates formed in unit time during the first step, is mainly determined by both the collision energy barrier and the initial protein density, while the number of condensates at the equilibrium is mainly associated with the dissociation energy barrier. Moreover, the LLPS process is not sensitive to temperature changes ranging around physiological conditions. Additionally, we consider the effect of the nucleation energy barrier on LLPS. We find that a higher nucleation energy barrier brings a slower condensation speed. Overall, we simulate the spatiotemporal dynamics of the LLPS process and provide qualitative guidance for understanding the dynamics of LLPS in bacterial cells, which can faithfully recapitulate experimental observations and facilitate the design of future experimental tests.

A search for the organisms responsible for anaerobic betaine degradation in soda lakes resulted in isolation of a novel bacterial strain, designated Z-7014 T . The cells were Gram-stain-negative, non-endospore-forming rods. Growth occurred at 8-52 °C (optimum 40-45 °C), pH 7.1-10.1 (optimum pH 8.1-8.8) and 1.0-3.5 M Na + (optimum 1.8 M), i.e. it can be regarded as a haloalkaliphile. The strain utilized a limited range of substrates, mostly peptonaceous but not amino acids, and was able to degrade betaine. Growth on betaine occurred only in the presence of peptonaceous substances which could not be replaced by vitamins. The G + C content of the genomic DNA of strain Z-7014 T was 36.1 mol%. The major cellular fatty acids (>5% of the total) were C 16:0 DMA, C 18: 0 DMA, C 16:1 ω8, C 16:0 , C 18:1 DMA, C 16:1 DMA, C 18:1 ω9, and C 18:0 . Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Z-7014 T formed a distinct evolutionary lineage in the order Halanaerobiales with the highest similarity to Halarsenitibacter silvermanii SLAS-1 T (83.6%), Halothermothrix orenii H168 T (85.6%), and Halocella cellulosilytica DSM 7362 T (85.6%). AAI and POCP values between strain Z-7014 T and type strains of the order Halanaerobiales were 51.7-57.8%, and 33.8-58.3%, respectively. Based on polyphasic results including phylogenomic data, the novel strain could be distinguished from other genera, which suggests that strain Z-7014 T represents a novel species of a new genus, for which the name Halonatronomonas betaini gen. nov., sp. nov. is proposed. The type strain is Z-7014 T (=KCTC 25237 T  = VKM B-3506 T ). On the basis of phylogenomic data, it is also proposed to evolve two novel families Halarsenitibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the current order Halanaerobiales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

The bacterial MinE and MinD division regulatory proteins form a standing wave enabling MinC, which binds MinD, to inhibit FtsZ polymerization everywhere except at the midcell, thereby assuring correct positioning of the cytokinetic septum and even distribution of contents to daughter cells. The MinE dimer undergoes major structural rearrangements between a resting six-stranded state present in the cytoplasm, a membrane-bound state, and a four-stranded active state bound to MinD on the membrane, but it is unclear which MinE motifs interact with the membrane in these different states. Using NMR, we probe the structure and global dynamics of MinE bound to disc-shaped lipid bicelles. In the bicelle-bound state, helix α1 no longer sits on top of the six-stranded β-sheet, losing any contact with the protein core, but interacts directly with the bicelle surface; the structure of the protein core remains unperturbed and also interacts with the bicelle surface via helix α2. Binding may involve a previously identified excited state of free MinE in which helix α1 is disordered, thereby allowing it to target the membrane surface. Helix α1 and the protein core undergo nanosecond rigid body motions of differing amplitudes in the plane of the bicelle surface. Global dynamics on the sub-millisecond time scale between a ground state and a sparsely populated excited state are also observed and may represent a very early intermediate on the transition path between the resting six-stranded and active four-stranded conformations. In summary, our results provide insights into MinE structural rearrangements important during bacterial cell division., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Many bacterial species are helical in shape, including the widespread pathogen H. pylori . Motivated by recent experiments on H. pylori showing that cell wall synthesis is not uniform [J. A. Taylor, et al ., eLife , 2020, 9 , e52482], we investigate the possible formation of helical cell shape induced by elastic heterogeneity. We show, experimentally and theoretically, that helical morphogenesis can be produced by pressurizing an elastic cylindrical vessel with helical reinforced lines. The properties of the pressurized helix are highly dependent on the initial helical angle of the reinforced region. We find that steep angles result in crooked helices with, surprisingly, a reduced end-to-end distance upon pressurization. This work helps explain the possible mechanisms for the generation of helical cell morphologies and may inspire the design of novel pressure-controlled helical actuators.

Oxidized bacterial nanocellulose (OBC) is reported to prevent microbial growth, but its antibacterial characteristics and mechanism are still unclear. Here, the antibacterial mechanism of OBC is explored by detecting and assessing the interaction of OBC with different carboxyl content on Staphylococcus aureus and Escherichia coli. The results show that OBC has strong antibacterial activity and antibiofilm activity against S. aureus and E. coli, which is positively correlated with the carboxyl content of OBC. After OBC treatment, the bacteria adhesion is inhibited and the cell membrane is destroyed leading to increased permeability. Further investigation reveals that the concentration of cyclic diguanosine monophosphate (c-di-GMP) that induced biofilm formation is significantly decreased to 1.81 pmol mg -1 after OBC treatment. In addition, OBC inactivates mature biofilms, with inactivation rates up to 79.3%. This study suggests that OBC has excellent antibacterial and antiadhesion properties, which can increase the cell membrane permeability and inhibit c-di-GMP formation. In addition, OBC also has a strong inactivation effect on mature biofilm, which can be used as an effective antibiofilm agent., (© 2023 Wiley-VCH GmbH.)

Background: Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.Methods: We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.Results: The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines' metastatic ability.Conclusions: The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes. [ABSTRACT FROM AUTHOR]

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis. [ABSTRACT FROM AUTHOR]

The article discusses the widespread use of Ibrutinib since its initial U.S. Food and Drug Administration approval for relapsed CLL 4 years ago. It notes that around 20% of treatment discontinuations in both the treatment-naive and relapsed or refractory cohorts were attributed to AEs over 5-year follow-up, which compares favorably to the 42% discontinuation rate at a median of 17 months in a real-world report of ibrutinib use.

Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks ( BacNet15 ) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria. [ABSTRACT FROM AUTHOR]

New bacterial detection and imaging methods are desirable for diagnostics and healthcare applications, as well as in basic scientific research. We present a simple analytical platform for bacterial detection and imaging based upon attachment of amphiphilic carbon dots (CDs) to bacterial cells. We show that CDs functionalized with hydrocarbon chains readily bind to bacterial cells following short incubation and enable detection of bacteria through both fluorescence spectroscopy and microscopy. Importantly, we demonstrate that the intensity and spectral position of the carbon dots’ fluorescence depend upon bacterial species, providing a tool for distinguishing among bacteria even in cases of mixed bacterial populations. Moreover, bacterial labelling with the amphiphilic CDs enables visualization of physiological processes such as cell division. [ABSTRACT FROM AUTHOR]

The functional consequences of trait associated SNPs are often investigated using expression quantitative trait locus (eQTL) mapping. While trait-associated variants may operate in a cell-type specific manner, eQTL datasets for such cell-types may not always be available. We performed a genome-environment interaction (GxE) meta-analysis on data from 5,683 samples to infer the cell type specificity of whole blood cis-eQTLs. We demonstrate that this method is able to predict neutrophil and lymphocyte specific cis-eQTLs and replicate these predictions in independent cell-type specific datasets. Finally, we show that SNPs associated with Crohn’s disease preferentially affect gene expression within neutrophils, including the archetypal NOD2 locus. [ABSTRACT FROM AUTHOR]

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs. [ABSTRACT FROM AUTHOR]

Summary Over 30 years ago, ZTP (5-aminoimidazole-4-carboxamide riboside 5′-triphosphate), a modified purine biosynthetic intermediate, was proposed to signal 10-formyl-tetrahydrofolate (10f-THF) deficiency in bacteria. However, the mechanisms by which this putative alarmone or its precursor ZMP (5-aminoimidazole-4-carboxamide ribonucleotide, also known as AICAR) brings about any metabolic changes remain unexplained. Herein, we report the existence of a widespread riboswitch class that is most commonly associated with genes related to de novo purine biosynthesis and one-carbon metabolism. Biochemical data confirm that members of this riboswitch class selectively bind ZMP and ZTP with nanomolar affinity while strongly rejecting numerous natural analogs. Indeed, increases in the ZMP/ZTP pool, caused by folate stress in bacterial cells, trigger changes in the expression of a reporter gene fused to representative ZTP riboswitches in vivo. The wide distribution of this riboswitch class suggests that ZMP/ZTP signaling is important for species in numerous bacterial lineages. [ABSTRACT FROM AUTHOR]

Exopolymers are known to be useful in improving sand aggregation and the development of biological soil crusts (BSCs). A facultative bacterium KLBB0001 was isolated from BSCs in the Gurbantunggut Desert in northwestern China. With the strong effective production of extracellular polymeric substances (EPS), this strain exhibits a multifunctional role for sand stabilization and maintenance of water under laboratory conditions. Practical testing of the feasibility of its inoculation to speed up BSC recovery in the field was also conducted in this experiment. This strain stimulated the heterotrophic community assembly in the topsoil layer (0-2 cm) before the commencement of autotrophic cyanobacteria, while also significantly increasing the number of bacteria, actinomycetes, and content of total phosphorus, available nitrogen, and available phosphorus. However, the low nitrogenase activity (NA) (0.57 µmol/h) that was observed caused us to doubt the previous identification as Azotobacter Beijerinck that was based on physiological and biochemical properties. A phylogenetic analysis based on 16S rRNA gene sequences revealed that this strain was a member of the genus Paenibacillus. It exhibited the closest phylogenetic affinity and highest sequence similarity to the strain Paenibacillus mucilaginosus VKPM B-7519 (sequence similarity 99.698%), which is well known as a typical silicate-weathering bacteria that releases lots of nutritional ions from minerals and the soil. Because P. mucilaginosus can excrete carbonic anhydrase (CA) to capture atmospheric CO2 through hydration of CO2, it is possible that KLBB0001 might use a similar strategy for heterotrophs in the BSCs to sequester CO2 from the air. Because of its potential role in the reestablishment of the BSC ecosystem due to its ability to improve water relations, sand stabilization, and chemical erosion, EPS-producing bacterial inoculation was concluded to be a suitable and effective treatment for BSC recovery, especially in environments with limited water and nutrients. [ABSTRACT FROM AUTHOR]

Bacillus subtilis has been used for over 50 years as a model organism for biochemistry, genetic, molecular biology and cell biology studies. More recently, its spore has been proposed as a platform to display heterologous proteins and as a vehicle for mucosal vaccination. We characterize here the spore surface of four human intestinal strains of B. subtilis, previously identified as able to grow anaerobically and form biofilm. These properties, lost in laboratory strains, are relevant for the colonization of human mucosal sites and likely to improve the efficiency of strains to be used for mucosal delivery. Our characterization is an essential preliminary step for the development of these intestinal strains as display systems and has indicated that spores of at least one of them are more efficient than the laboratory strain for the non-recombinant display of two model heterologous proteins. [ABSTRACT FROM AUTHOR]

Abstract: The impact of multi-well plate automation on bacterial flow cytometric analyses was investigated. Cell concentrations in up to 96 samples can be measured accurately, as long as a reproducible staining protocol and a total measurement time of below 80min is used. Fluorescence distribution in the samples may, however, display some variability. [Copyright &y& Elsevier]

Background: Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. Results: We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Conclusions: Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins. [ABSTRACT FROM AUTHOR]

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance 1-3 . Teixobactin 4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan 5 . Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin 4 . The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates., (© 2022. The Author(s).)

Bacteria encode myriad defences that target the genomes of infecting bacteriophage, including restriction-modification and CRISPR-Cas systems 1 . In response, one family of large bacteriophages uses a nucleus-like compartment to protect its replicating genomes by excluding host defence factors 2-4 . However, the principal composition and structure of this compartment remain unknown. Here we find that the bacteriophage nuclear shell assembles primarily from one protein, which we name chimallin (ChmA). Combining cryo-electron tomography of nuclear shells in bacteriophage-infected cells and cryo-electron microscopy of a minimal chimallin compartment in vitro, we show that chimallin self-assembles as a flexible sheet into closed micrometre-scale compartments. The architecture and assembly dynamics of the chimallin shell suggest mechanisms for its nucleation and growth, and its role as a scaffold for phage-encoded factors mediating macromolecular transport, cytoskeletal interactions, and viral maturation., (© 2022. The Author(s).)

Biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N -linked glycans) necessitates the transport of lipid-linked oligosaccharides (LLO) across membranes from their cytosolic site of synthesis to their sites of utilization. Much of our current understanding of LLO transport comes from genetic, biochemical, and structural studies of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily protein MurJ, which flips the peptidoglycan precursor lipid II. MurJ plays a pivotal role in bacterial cell wall synthesis and is an emerging antibiotic target. Here, we review the mechanism of LLO flipping by MurJ, including the structural basis for lipid II flipping and ion coupling. We then discuss inhibition of MurJ by antibacterials, including humimycins and the phage M lysis protein, as well as how studies on MurJ could provide insight into other flippases, both within and beyond the MOP superfamily.

The components of the bacterial division machinery assemble to form a dynamic ring at mid-cell that drives cytokinesis. The nature of most division proteins and their assembly pathway is known. Our knowledge about the biochemical activities and protein interactions of some key division elements, including those responsible for correct ring positioning, has progressed considerably during the past decade. These developments, together with new imaging and membrane reconstitution technologies, have triggered the ‘bottom-up’ synthetic approach aiming at reconstructing bacterial division in the test tube, which is required to support conclusions derived from cellular and molecular analysis. Here, we describe recent advances in reconstituting Escherichia coli minimal systems able to reproduce essential functions, such as the initial steps of division (proto-ring assembly) and one of the main positioning mechanisms (Min oscillating system), and discuss future perspectives and experimental challenges. [ABSTRACT FROM AUTHOR]

Summary: Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest. [ABSTRACT FROM AUTHOR]

Partition systems are responsible for the process whereby large and essential plasmids are accurately positioned to daughter cells during bacterial division. They are typically made of three components: a centromere-like DNA zone, an adaptor protein, and an assembling protein that is either a Walker-box ATPase (type I) or an actin-like ATPase (type II). A recently described type III segregation system has a tubulin/FtsZ-like protein, called TubZ, for plasmid movement. Here, we present the 2.3 Å structure and dynamic assembly of a TubZ tubulin homolog from a bacteriophage and unravel the Clostridium botulinum phage c-st type III partition system. Using biochemical and biophysical approaches, we prove that a gene upstream from tubZ encodes the partner TubR and localize the centromeric region (tubS), both of which are essential for anchoring phage DNA to the motile TubZ filaments. Finally, we describe a conserved fourth component, TubY, which modulates the TubZ-R-S complex interaction. [ABSTRACT FROM AUTHOR]

In this issue of Developmental Cell , Isabella and Horne-Badovinac (2016) show that Rab10 directs site-specific secretion of basement membrane components, which assemble into fibrils that spool out to elongate the Drosophila egg chamber. These findings establish the basement membrane’s active role in tissue sculpting. [ABSTRACT FROM AUTHOR]

With the increase in scientific activity globally, the geographical focus of basic research is shifting away from the West. At the same time, multidisciplinary approaches are uncovering new layers in our understanding of how cells work. How will these trends affect cell biology in the near future? [ABSTRACT FROM AUTHOR]

Small membrane proteins represent a subset of recently discovered small proteins (≤100 amino acids), which are a ubiquitous class of emerging regulators underlying bacterial adaptation to environmental stressors. Until relatively recently, small open reading frames encoding these proteins were not designated genes in genome annotations. Therefore, our understanding of small protein biology was primarily limited to a few candidates associated with previously characterized larger partner proteins. Following the first systematic analyses of small proteins in Escherichia coli over a decade ago, numerous small proteins across different bacteria have been uncovered. An estimated one-third of these newly discovered proteins in E. coli are localized to the cell membrane, where they may interact with distinct groups of membrane proteins, such as signal receptors, transporters, and enzymes, and affect their activities. Recently, there has been considerable progress in functionally characterizing small membrane protein regulators aided by innovative tools adapted specifically to study small proteins. Our review covers prototypical proteins that modulate a broad range of cellular processes, such as transport, signal transduction, stress response, respiration, cell division, sporulation, and membrane stability. Thus, small membrane proteins represent a versatile group of physiology regulators at the membrane and the whole cell. Additionally, small membrane proteins have the potential for clinical applications, where some of the proteins may act as antibacterial agents themselves while others serve as alternative drug targets for the development of novel antimicrobials.

Improvements in microscopy software and hardware have dramatically increased the pace of image acquisition, making analysis a major bottleneck in generating quantitative, single-cell data. Although tools for segmenting and tracking bacteria within time-lapse images exist, most require human input, are specialized to the experimental set up, or lack accuracy. Here, we introduce DeLTA 2.0, a purely Python workflow that can rapidly and accurately analyze images of single cells on two-dimensional surfaces to quantify gene expression and cell growth. The algorithm uses deep convolutional neural networks to extract single-cell information from time-lapse images, requiring no human input after training. DeLTA 2.0 retains all the functionality of the original version, which was optimized for bacteria growing in the mother machine microfluidic device, but extends results to two-dimensional growth environments. Two-dimensional environments represent an important class of data because they are more straightforward to implement experimentally, they offer the potential for studies using co-cultures of cells, and they can be used to quantify spatial effects and multi-generational phenomena. However, segmentation and tracking are significantly more challenging tasks in two-dimensions due to exponential increases in the number of cells. To showcase this new functionality, we analyze mixed populations of antibiotic resistant and susceptible cells, and also track pole age and growth rate across generations. In addition to the two-dimensional capabilities, we also introduce several major improvements to the code that increase accessibility, including the ability to accept many standard microscopy file formats as inputs and the introduction of a Google Colab notebook so users can try the software without installing the code on their local machine. DeLTA 2.0 is rapid, with run times of less than 10 minutes for complete movies with hundreds of cells, and is highly accurate, with error rates around 1%, making it a powerful tool for analyzing time-lapse microscopy data., Competing Interests: The authors have declared that no competing interests exist.

The deep biosphere is an energy constrained ecosystem yet fosters diverse microbial communities that are key in biogeochemical cycling. Whether microbial communities in deep biosphere groundwaters are shaped by infiltration of allochthonous surface microorganisms or the evolution of autochthonous species remains unresolved. In this study, 16S rRNA gene amplicon analyses showed that few groups of surface microbes infiltrated deep biosphere groundwaters at the Äspö Hard Rock Laboratory, Sweden, but that such populations constituted up to 49% of the microbial abundance. The dominant persisting phyla included Patescibacteria, Proteobacteria, and Epsilonbacteraeota. Despite the hydrological connection of the Baltic Sea with the studied groundwaters, infiltrating microbes predominantly originated from deep soil groundwater. Most deep biosphere groundwater populations lacked surface representatives, suggesting that they have evolved from ancient autochthonous populations. We propose that deep biosphere groundwater communities in the Fennoscandian Shield consist of selected infiltrated and indigenous populations adapted to the prevailing conditions., (© 2022. The Author(s).)

As our understanding of the importance of the human microbiota in health and disease grows, so does our need to carefully resolve and delineate its genomic content. 16S rRNA gene-based analyses yield important insights into taxonomic composition, and metagenomics-based approaches reveal the functional potential of microbial communities. However, these methods generally fail to directly link genetic features, including bacterial genes and mobile genetic elements, to each other and to their source bacterial genomes. Further, they are inadequate to capture the microdiversity present within a genus, species, or strain of bacteria within these complex communities. Here, we present a method utilizing fluorescence-activated cell sorting for isolation of single bacterial cells, amplifying their genomes, screening them by 16S rRNA gene analysis, and selecting cells for genomic sequencing. We apply this method to both a cultured laboratory strain of Escherichia coli and human stool samples. Our analyses reveal the capacity of this method to provide nearly complete coverage of bacterial genomes when applied to isolates and partial genomes of bacterial species recovered from complex communities. Additionally, this method permits exploration and comparison of conserved and variable genomic features between individual cells. We generate assemblies of novel genomes within the Ruminococcaceae family and the Holdemanella genus by combining several 16S rRNA gene-matched single cells, and report novel prophages and conjugative transposons for both Bifidobacterium and Ruminococcaceae . Thus, we demonstrate an approach for flow cytometric separation and sequencing of single bacterial cells from the human microbiota, which yields a variety of critical insights into both the functional potential of individual microbes and the variation among those microbes. This method definitively links a variety of conserved and mobile genomic features, and can be extended to further resolve diverse elements present in the human microbiota.

An extracellular electron transfer (EET) process between an electroactive biofilm and an electrode is a crucial step for the performance of microbial fuel cells (MFCs), which is highly related to the enrichment of exoelectrogens and the electrocatalytic activity of the electrode. Herein, an efficient N- and Fe-abundant carbon cloth (CC) electrode with the comodification of iron porphyrin (FePor) and polyquaternium-7 (PQ) was synthesized using a facile solvent evaporation and immersion method and developed as an anode (named FePor-PQ) in MFCs. The surface structural characterizations confirmed the successful introduction of N and Fe atoms, whereas FePor-PQ achieved the N content of 9.59 at %, which may offer various active sites for EET. The introduction of PQ contributed to improving the surface hydrophilicity, providing the composite electrode good biocompatibility for bacterial attachment and colonization as well as substrate diffusion. Based on the advantages, the MFC with the FePor-PQ anode produced a maximum power density of 2165.7 mW m -2 , strikingly higher than those of CC (1124.0 mW m -2 ), PQ (1668.8 mW m -2 ), and FePor (1978.9 mW m -2 ). Furthermore, with the EET mediated by the binding of flavins and c-type cytochromes on the outer membrane was enhanced prominently, the typical exoelectrogen Geobacter was enriched up to 55.84% in the FePor-PQ anode biofilm. This work reveals a synergistic effect from heteroatom coating and surface properties tailoring to boost both the EET efficiency and exoelectrogen enrichment for enhancing MFC performance, which also provides valuable insights for designing electrodes in other bio-electrochemical systems.

Uptake and concentration of inorganic ions are part of the complex cellular processes required for cell homeostasis, as well as for mineral formation by organisms. These ion transport mechanisms include distinct cellular compartments and chemical phases that play various roles in the physiology of organisms. Here, the prominent cases of dense ion pools in unicellular organisms are briefly reviewed. The specific observations that were reported for different organisms are consolidated into a wide perspective that emphasizes general traits. It is suggested that the intracellular ion pools can be divided into three types: a high cytoplasmic concentration, a labile storage compartment that hosts dense ion-rich phases, and a mineral-forming compartment in which a stable long-lived structure is formed. Recently, many labile pools were identified in various organisms using advanced techniques, bringing many new questions about their possible roles in the formation of the stable mineralized structures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Abstract: We designed and tested equipment to wash plugs following cell lysis in pulsed-field gel electrophoresis (PFGE). Our system can wash 30 plugs simultaneously in 1h using 15L of Tris–EDTA buffer, which makes plug washing for PFGE less labor-intensive. [Copyright &y& Elsevier]

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, nonsoil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone Spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of 13 distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g., FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine learning approaches based on a set of 634 genes informative of social lifestyle predicted a nonsocial behavior for Zodletone Myxococcota . Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities but carried genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 million years ago (Mya) in response to the rise of soil as a distinct habitat on Earth. IMPORTANCE The phylum Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of nonsoil, predominantly anaerobic habitats, and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA, that represent novel, yet-uncultured, orders and families in the Myxococcota . The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of 13 pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on Earth.

Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using next-generation 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems., (© 2021. The Microbiological Society of Korea.)

Correct decision making is fundamental for all living organisms to thrive under environmental changes. The patterns of environmental variation and the quality of available information define the most favourable strategy among multiple options, from randomly adopting a phenotypic state to sensing and reacting to environmental cues. Cellular memory-the ability to track and condition the time to switch to a different phenotypic state-can help withstand environmental fluctuations. How does memory manifest itself in unicellular organisms? We describe the population-wide consequences of phenotypic memory in microbes through a combination of deterministic modelling and stochastic simulations. Moving beyond binary switching models, our work highlights the need to consider a broader range of switching behaviours when describing microbial adaptive strategies. We show that memory in individual cells generates patterns at the population level coherent with overshoots and non-exponential lag times distributions experimentally observed in phenotypically heterogeneous populations. We emphasise the implications of our work in understanding antibiotic tolerance and, in general, bacterial survival under fluctuating environments., Competing Interests: The authors have declared that no competing interests exist.

Herein, we demonstrate that the use of index-matching materials (IMMs) allows direct visualization of microbial cells maintained at a solid-liquid interface through confocal reflection microscopy (CRM). The refractive index mismatch induces a background reflection at the solid-liquid interface that dwarfs the reflection signals from the cells and results in low-contrast images. We found that the IMMs sufficiently suppressed the background reflection at the solid-liquid interface, facilitating the imaging of microbes at the solid surface using CRM. The use of IMMs allowed quantitative analysis of the morphology of the mesh-like structure of Pseudomonas aeruginosa biofilms formed under denitrifying conditions, which led us to propose a novel structural model of the highly porous biofilm structure. These results indicate that the use of CRM coupled with an IMM offers a unique and promising tool for probing the dynamics of biofilm formation, along with visualization of environmental organisms and newly isolated bacteria, for which transformation methods are difficult to establish., (© 2021. The Author(s).)