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Super‐resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single‐molecule resolution on a millisecond time scale. Here we review recent applications of super‐resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action. [ABSTRACT FROM AUTHOR]

Nucleus-forming jumbo phages establish an intricate subcellular organization, enclosing phage genomes within a proteinaceous shell called the phage nucleus. During infection in Pseudomonas, some jumbo phages assemble a bipolar spindle of tubulin-like PhuZ filaments that positions the phage nucleus at midcell and drives its intracellular rotation. This facilitates the distribution of capsids on its surface for genome packaging. Here we show that the Escherichia coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of a mutant PhuZ protein strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes for cytoplasmic streaming and nucleus alignment, this work identifies a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

Abstract Background Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. Results We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term “secretion zones”. Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. Conclusions Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.

Background: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. Results: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. Conclusions: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus. [ABSTRACT FROM AUTHOR]

Many Gram-negative pathogens utilize the type III secretion system (T3SS) to translocate virulence-promoting effector proteins into eukaryotic host cells. The activity of this system results in a severe reduction of bacterial growth and division, summarized as secretion-associated growth inhibition (SAGI). In Yersinia enterocolitica, the T3SS and related proteins are encoded on a virulence plasmid. We identified a ParDE-like toxin--antitoxin system on this virulence plasmid in genetic proximity to yopE, encoding a T3SS effector. Effectors are strongly upregulated upon activation of the T3SS, indicating a potential role of the ParDE system in the SAGI or maintenance of the virulence plasmid. Expression of the toxin ParE in trans resulted in reduced growth and elongated bacteria, highly reminiscent of the SAGI. Nevertheless, the activity of ParDE is not causal for the SAGI. T3SS activation did not influence ParDE activity; conversely, ParDE had no impact on T3SS assembly or activity itself. However, we found that ParDE ensures the presence of the T3SS across bacterial populations by reducing the loss of the virulence plasmid, especially under conditions relevant to infection. Despite this effect, a subset of bacteria lost the virulence plasmid and regained the ability to divide under secreting conditions, facilitating the possible emergence of T3SS-negative bacteria in late acute and persistent infections. [ABSTRACT FROM AUTHOR]

Many Gram-negative pathogens utilize the type III secretion system (T3SS) to translocate virulence-promoting effector proteins into eukaryotic host cells. The activity of this system results in a severe reduction of bacterial growth and division, summarized as secretion-associated growth inhibition (SAGI). In Yersinia enterocolitica, the T3SS and related proteins are encoded on a virulence plasmid. We identified a ParDE-like toxin–antitoxin system on this virulence plasmid in genetic proximity to yopE, encoding a T3SS effector. Effectors are strongly upregulated upon activation of the T3SS, indicating a potential role of the ParDE system in the SAGI or maintenance of the virulence plasmid. Expression of the toxin ParE in trans resulted in reduced growth and elongated bacteria, highly reminiscent of the SAGI. Nevertheless, the activity of ParDE is not causal for the SAGI. T3SS activation did not influence ParDE activity; conversely, ParDE had no impact on T3SS assembly or activity itself. However, we found that ParDE ensures the presence of the T3SS across bacterial populations by reducing the loss of the virulence plasmid, especially under conditions relevant to infection. Despite this effect, a subset of bacteria lost the virulence plasmid and regained the ability to divide under secreting conditions, facilitating the possible emergence of T3SS-negative bacteria in late acute and persistent infections.

Eukaryotic cells transcribe ribosomal RNA and largely assemble ribosomes in a structure called the nucleolus, where chromosomal regions containing rRNA operons are clustered. In bacteria, many rRNA operons cluster close to the origin regions that are positioned on the outer borders of nucleoids, close to polar areas, where translating 70S ribosomes are located. Because outer regions of the nucleoids contain the highest accumulation of RNA polymerase, it has been hypothesized that bacteria contain “nucleolus-like” structures. However, ribosome subunits freely diffuse through the entire cells, and could thus be assembled and matured throughout the non-compartmentalized cell. By tracking single molecules of two GTPases that play an essential role in ribosomal folding and processing in Bacillus subtilis, we show that this process takes place at sites of translation, i.e., predominantly at the cell poles. Induction of the stringent response led to a change in the population of GTPases assumed to be active in maturation, but did not abolish nucleoid occlusion of ribosomes or of GTPases. Our findings strongly support the idea of the conceptualization of nucleolus-like structures in bacteria, i.e., rRNA synthesis, ribosomal protein synthesis and subunit assembly occurring in close proximity at the cell poles, facilitating the efficiency of ribosome maturation even under conditions of transient nutrient deprivation.

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion, and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated in Myxococcus xanthus , which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP-formation and T4aP-dependent motility in various mutants supports a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB to the T4aP machinery. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility., Importance: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in translocation across surfaces, surface adhesion, biofilm formation, and virulence. T4aP-dependent translocation crucially depends on the number of pili. To address how the number of T4aP is regulated, we focused on M. xanthus , which assembles T4aP at the leading cell pole and is a model organism for T4aP biology. Our results support a model whereby the four proteins MglA, SgmX, FrzS, and the newly identified SopA protein establish a highly intricate interaction network for orchestrating T4aP formation at the leading cell pole. This network allows for combinatorial regulation of the number of T4aP resulting in discrete levels of T4aP-dependent motility.

Antimicrobial resistance has emerged as a global threat to the treatment of infectious diseases. Antibacterial photodynamic therapy (aPDT) is a promising alternative approach and is highly suitable for the treatment of cutaneous bacterial infections through topical applications. aPDT relies on light-responsive compounds called photosensitizer (PS) dyes, which generate reactive oxygen species (ROS) when induced by light, thereby killing bacterial cells. Despite several previous studies in this area, the molecular details of targeting and cell death mediated by PS dyes are poorly understood. In this study, we further investigate the antibacterial properties of two water-soluble Sn(IV) tetrapyridylporphyrins that were quaternized with methyl and hexyl groups ( 1 and 2 ). In this follow-up study, we demonstrate that Sn(IV)-porphyrins can be photoexcited by blue light (a 427 nm LED) and exhibit various levels of bactericidal activity against both Gram-(+) and Gram-(-) strains of bacteria. Using localization studies through fluorescence microscopy, we show that 2 targets the bacterial membrane more effectively than 1 and exhibits comparatively higher aPDT activity. Using multiple fluorescence reporters, we demonstrate that photoactivation of 1 and 2 results in extensive collateral damage to the bacterial cells including DNA cleavage, membrane damage, and delocalization of central systems necessary for bacterial growth and division. In summary, this investigation provides deep insights into the mechanism of bacterial killing mediated by the Sn(IV)-porphyrins. Moreover, our approach offers a new method for evaluating the activity of PS, which may inspire the discovery of new PS with enhanced aPDT activity.

Actin and actin-like proteins form filamentous polymers that carry out important cellular functions in all domains of life. In this review, we sketch a map of the function and regulation of actin-like proteins across bacteria, archaea, and eukarya, marking some of the terra incognita that remain in this landscape. We focus particular attention on archaea because mapping the structure and function of cytoskeletal systems across this domain promises to help us understand the evolutionary relationship between the (mostly) mono-functional actin-like filaments found in bacteria and the multi-functional actin cytoskeletons that characterize eukaryotic cells. [ABSTRACT FROM AUTHOR]

Bdellovibrio bacteriovorus is a predatory bacterium that feeds upon and proliferates inside other Gram-negative bacteria. Upon entry into the periplasmic space of the prey envelope, B. bacteriovorus initiates an exquisite developmental program in which it digests the host resources and grows as a filament, which eventually divides in a non-binary manner, releasing a variable number of daughter cells. The progeny then escape from the prey ghost to encounter new victims and resume the predation cycle. Owing to its unique biology, B. bacteriovorus undoubtedly represents an attractive model to unravel novel mechanisms of bacterial cell cycle control and cellular organization. Yet, the molecular factors behind the sophisticated lifestyle of this micro-predator are still mysterious. In particular, the spatiotemporal dynamics of proteins that control key cellular processes such as transmission of the genetic information, cell growth and division remain largely unexplored. In this Perspective article, I highlight outstanding fundamental questions related to these aspects and arising from the original biology of this bacterium. I also discuss available insights and potential cell biology approaches based on quantitative live imaging techniques, in combination with bacterial genetics and biochemistry, to shed light on the intracellular organization of B. bacteriovorus in space and time.

Prokaryotic cells display a striking subcellular organization. Studies of the underlying mechanisms in different species have greatly enhanced our understanding of the morphological and physiological adaptation of bacteria to different environmental niches. The image analysis software tool BacStalk is designed to extract comprehensive quantitative information from the images of morphologically complex bacteria with stalks, flagella, or other appendages. The resulting data can be visualized in interactive demographs, kymographs, cell lineage plots, and scatter plots to enable fast and thorough data analysis and representation. Notably, BacStalk can generate demographs and kymographs that display fluorescence signals within the two‐dimensional cellular outlines, to accurately represent their subcellular location. Beyond organisms with visible appendages, BacStalk is also suitable for established, non‐stalked model organisms with common or uncommon cell shapes. BacStalk, therefore, contributes to the advancement of prokaryotic cell biology and physiology, as it widens the spectrum of easily accessible model organisms and enables highly intuitive and interactive data analysis and visualization. [ABSTRACT FROM AUTHOR]

Bacterial cell division is initiated by the midcell assembly of polymers of the tubulin-like GTPase FtsZ. The FtsZ ring (Z-ring) is a discontinuous structure made of dynamic patches of FtsZ that undergo treadmilling motion. Roughly a dozen additional essential proteins are recruited to the division site by the dynamic Z-ring scaffold and subsequently activate cell wall synthesis to drive cell envelope constriction during division. In this Cell Science at a Glance article and the accompanying poster, we summarize our understanding of the assembly and activation of the bacterial cell division machinery. We introduce polymerization properties of FtsZ and discuss our current knowledge of divisome assembly and activation. We further highlight the intimate relationship between the structure and dynamics of FtsZ and the movement and activity of cell wall synthases at the division site, before concluding with a perspective on the most important open questions on bacterial cell division. [ABSTRACT FROM AUTHOR]

Bdellovibrio bacteriovorus is a predatory bacterium that feeds upon and proliferates inside other Gram-negative bacteria. Upon entry into the periplasmic space of the prey envelope, B. bacteriovorus initiates an exquisite developmental program in which it digests the host resources and grows as a filament, which eventually divides in a non-binary manner, releasing a variable number of daughter cells. The progeny then escape from the prey ghost to encounter new victims and resume the predation cycle. Owing to its unique biology, B. bacteriovorus undoubtedly represents an attractive model to unravel novel mechanisms of bacterial cell cycle control and cellular organization. Yet, the molecular factors behind the sophisticated lifestyle of this micro-predator are still mysterious. In particular, the spatiotemporal dynamics of proteins that control key cellular processes such as transmission of the genetic information, cell growth and division remain largely unexplored. In this Perspective article, I highlight outstanding fundamental questions related to these aspects and arising from the original biology of this bacterium. I also discuss available insights and potential cell biology approaches based on quantitative live imaging techniques, in combination with bacterial genetics and biochemistry, to shed light on the intracellular organization of B. bacteriovorus in space and time. [ABSTRACT FROM AUTHOR]

Zebrafish (Danio rerio) larvae are widely recognized for studying host–pathogen interactions in vivo because of their optical transparency, genetic manipulability, and translational potential. The development of the zebrafish immune system is well understood, thereby use of larvae enables investigation solely in the context of innate immunity. As a result, infection of zebrafish with natural fish pathogens including Mycobacterium marinum has significantly advanced our understanding of bacterial pathogenesis and vertebrate host defense. However, new work using a variety of human pathogens (bacterial, viral, and fungal) has illuminated the versatility of the zebrafish infection model, revealing unexpected and important concepts underlying infectious disease. We propose that this knowledge can inform studies in higher animal models and help to develop treatments to combat human infection. Mycobacterium marinum infection in zebrafish is an important system for studying natural host–pathogen interactions in vivo. New work has shown that a wide variety of human pathogens can also be studied using a zebrafish infection model. Zebrafish larvae are highly valuable in the discovery and dissection of fundamental concepts underlying human infection. A better understanding of the biology of human pathogens in vivo will be necessary to develop new therapies to fight infection. [ABSTRACT FROM AUTHOR]

High‐throughput analyses of single‐cell microscopy data are a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase‐contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell‐to‐cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post‐processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a command‐line based R package that allows researchers to transform cell segmentation and spot detection data generated by different programs into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP complies with standard practice in R which enables the use of advanced statistical analysis tools, and its graphic output is compatible with ggplot2, enabling adjustable plot graphics in every operating system. User feedback will be used to create a fully automated Graphical User Interface version of BactMAP in the future. Using BactMAP, we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus, and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z‐ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap. [ABSTRACT FROM AUTHOR]

ABSTRACT Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.

Colonies are an abundant form of bacterial multicellularity; however, relatively little is known about the initial stages of their construction. We have previously described that colony development of the soil bacterium Bacillus subtilis is a highly ordered process, typically initiating with the formation of extending cell chains arranged in a Y shape structure. Furthermore, we demonstrated that Y arm extension is a key for defining the size of the future colony. Here we conducted a genetic screen surveying for mutants deficient in these early developmental stages, and revealed LtaS, the major lipoteichoic acid (LTA) synthase, to be crucial for execution of these events. We found that the ltaS mutant fails to produce proper Y shape structures, forming extremely elongated chains of cells with no evidence of chain breakage, necessary for Y shape formation. Furthermore, we show that frequent cell death at the tips of the cell chains is a major cause in limiting arm extension. Collectively, these perturbations lead to the production of a small sized colony by the mutant. Thus, deficiency in LTA synthesis causes a mechanical failure in executing the colony developmental program.

Colonies are an abundant form of bacterial multicellularity; however, relatively little is known about the initial stages of their construction. We have previously described that colony development of the soil bacterium Bacillus subtilis is a highly ordered process, typically initiating with the formation of extending cell chains arranged in a Y shape structure. Furthermore, we demonstrated that Y arm extension is a key for defining the size of the future colony. Here we conducted a genetic screen surveying for mutants deficient in these early developmental stages, and revealed LtaS, the major lipoteichoic acid (LTA) synthase, to be crucial for execution of these events. We found that the ltaS mutant fails to produce proper Y shape structures, forming extremely elongated chains of cells with no evidence of chain breakage, necessary for Y shape formation. Furthermore, we show that frequent cell death at the tips of the cell chains is a major cause in limiting arm extension. Collectively, these perturbations lead to the production of a small sized colony by the mutant. Thus, deficiency in LTA synthesis causes a mechanical failure in executing the colony developmental program. [ABSTRACT FROM AUTHOR]

First isolated and classified in the 1960s, Caulobacter crescentus has been instrumental in the study of bacterial cell biology and differentiation. C. crescentus is a Gram-negative alphaproteobacterium that exhibits a dimorphic life cycle composed of two distinct cell types: a motile swarmer cell and a nonmotile, division-competent stalked cell. Progression through the cell cycle is accentuated by tightly controlled biogenesis of appendages, morphological transitions, and distinct localization of developmental regulators. These features as well as the ability to synchronize populations of cells and follow their progression make C. crescentus an ideal model for answering questions relevant to how development and differentiation are achieved at the single-cell level. This review will explore the discovery and development of C. crescentus as a model organism before diving into several key features and discoveries that have made it such a powerful organism to study. Finally, we will summarize a few of the ongoing areas of research that are leveraging knowledge gained over the last century with C. crescentus to highlight its continuing role at the forefront of cell and developmental biology.

Our knowledge of the regulation of genes involved in bacterial growth and stress responses is extensive; however, we have only recently begun to understand how environmental cues influence the dynamic, three-dimensional distribution of RNA polymerase (RNAP) in Escherichia coli on the level of single cell, using wide-field fluorescence microscopy and state-of-the-art imaging techniques. Live-cell imaging using either an agarose-embedding procedure or a microfluidic system further underscores the dynamic nature of the distribution of RNAP in response to changes in the environment. A general agreement between live-cell and fixed-cell images has validated the formaldehyde-fixing procedure, which is a technical breakthrough in the study of the cell biology of RNAP. In this review we use a systems biology perspective to summarize the advances in the cell biology of RNAP in E. coli, including the discoveries of the bacterial nucleolus, the spatial compartmentalization of the transcription machinery at the periphery of the nucleoid, and the segregation of the chromosome territories for the two major cellular functions of transcription and replication in fast-growing cells. Our understanding of the coupling of transcription and bacterial chromosome (or nucleoid) structure is also summarized. Using E. coli as a simple model system, co-imaging of RNAP with DNA and other factors during growth and stress responses will continue to be a useful tool for studying bacterial growth and adaptation in changing environment.

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolutionmicroscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while anotherMutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches. [ABSTRACT FROM AUTHOR]

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M. xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells. [Display omitted] • Cell contact-dependent prey killing involves two distinct protein secretion systems • Combined action of Tad- and type 3-like systems induces prey cell death and lysis • Components of both systems interdependently accumulate at the predator-prey interface • Respective M. xanthus mutants are impaired in powering motility with prey biomass Bacterial predation is abundant in different habitats, but mechanistic insight is lacking. Thiery et al. determine distinct functions of two atypical protein secretion systems in predatory Myxobacteria: a Tad-like complex and a rudimentary, needle-less type 3-like system act together to induce cell death and lysis of bacterial cells. [ABSTRACT FROM AUTHOR]

Eukaryotic cells transcribe ribosomal RNA and largely assemble ribosomes in a structure called the nucleolus, where chromosomal regions containing rRNA operons are clustered. In bacteria, many rRNA operons cluster close to the origin regions that are positioned on the outer borders of nucleoids, close to polar areas, where translating 70S ribosomes are located. Because outer regions of the nucleoids contain the highest accumulation of RNA polymerase, it has been hypothesized that bacteria contain "nucleolus-like" structures. However, ribosome subunits freely diffuse through the entire cells, and could thus be assembled and matured throughout the non-compartmentalized cell. By tracking single molecules of two GTPases that play an essential role in ribosomal folding and processing in Bacillus subtilis , we show that this process takes place at sites of translation, i.e., predominantly at the cell poles. Induction of the stringent response led to a change in the population of GTPases assumed to be active in maturation, but did not abolish nucleoid occlusion of ribosomes or of GTPases. Our findings strongly support the idea of the conceptualization of nucleolus-like structures in bacteria, i.e., rRNA synthesis, ribosomal protein synthesis and subunit assembly occurring in close proximity at the cell poles, facilitating the efficiency of ribosome maturation even under conditions of transient nutrient deprivation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stoll, Zegarra, Bange and Graumann.)

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remain largely unaddressed. Here, we used fluorescent fusion proteins introduced at the chromosomal CRISPR-Cas locus to study the localization of the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. When lacking a target in the cell, the Cascade complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. When targeted to an integrated prophage, however, the CRISPR RNA (crRNA)-guided type I-F Cascade complex and a majority of Cas3 molecules in the cell are recruited to a single focus. Nucleoid association of the Csy proteins that form the Cascade complex is crRNA dependent and specifically inhibited by the expression of anti-CRISPR AcrIF2, which blocks protospacer adjacent motif (PAM) binding. The Cas9 nuclease is also nucleoid localized, only when single guide RNA (sgRNA) bound, which is abolished by the PAM-binding inhibitor AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex. IMPORTANCE CRISPR-Cas systems, the prokaryotic adaptive immune systems, are largely understood using structural biology, biochemistry, and genetics. How CRISPR-Cas effectors are organized within cells is currently not well understood. By investigating the cell biology of the type I-F CRISPR-Cas system, we show that the surveillance complex, which "patrols" the cell to find targets, is largely nucleoid bound, while Cas3 nuclease is cytoplasmic. Nucleoid localization is also conserved for class 2 CRISPR-Cas single protein effector Cas9. Our observation of differential localization of the surveillance complex and Cas3 reveals a new layer of posttranslational spatiotemporal regulation to prevent autoimmunity.

Despite its fundamental nature, bacterial chromosome segregation remains poorly understood. Viewing segregation as a single process caused multiple proposed mechanisms to appear in conflict and failed to explain how asymmetrically dividing bacteria break symmetry to move only one of their chromosomes. Here, we demonstrate that the ParA ATPase extends from one cell pole and pulls the chromosome by retracting upon association with the ParB DNA-binding protein. Surprisingly, ParA disruption has a specific effect on chromosome segregation that only perturbs the latter stages of this process. Using quantitative high-resolution imaging, we demonstrate that this specificity results from the multistep nature of chromosome translocation. We propose that Caulobacter chromosome segregation follows an ordered pathway of events with distinct functions and mechanisms. Initiation releases polar tethering of the origin of replication, distinction spatially differentiates the two chromosomes, and commitment irreversibly translocates the distal centromeric locus. Thus, much as eukaryotic mitosis involves a sequence of distinct subprocesses, Caulobacter cells also segregate their chromosomes through an orchestrated series of steps. We discuss how the multistep view of bacterial chromosome segregation can help to explain and reconcile outstanding puzzles and frame future investigation. [ABSTRACT FROM AUTHOR]

Spirochaetes constitute a unique phylum of bacteria, many of which cause severe clinical diseases. Borrelia burgdorferi sensu lato (B. burgdorferi s.l.)—the primary agent of Lyme borreliosis (LB)—is a quintessential member of this poorly understood phylum and the leading cause of tick-borne illness throughout most of the northern hemisphere. Despite its importance in human health, we lack a fundamental understanding of how B. burgdorferi s.l. is able to accomplish basic physiological tasks, such as DNA replication/segregation, and cell elongation or division. Recent advances in molecular tools to probe these essential cellular processes are great strides forward but require genetic manipulation. The latter is important since not all agents of LB are genetically tractable. Here, we describe a single method that is capable of fluorescently labeling B. burgdorferi s.l. proteins in different sub-cellular compartments. A comparative analysis of six different methods indicates that our optimized procedure outperforms all others and is the first to localize a cytoplasmic protein in B. burgdorferi s.l. by immunofluorescence. We contend that this strategy could be easily adapted to study the localization of any protein, in many Borrelia genospecies, information that will yield functional insights into the complex biology of this fascinating group of bacteria. In addition, it may provide new avenues of research in both in situ studies and in Lyme diagnostics. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that the global organization of the bacterial chromosome is defined by its physical map. This architectural understanding has been gained mainly by observing the localization and dynamics of specific chromosomal loci. However, the spatial and temporal organization of the entire mass of newly synthesized DNA remains elusive. To visualize replicated DNA within living cells, we developed an experimental system in the bacterium Bacillus subtilis whereby fluorescently labeled nucleotides are incorporated into the chromosome as it is being replicated. Here, we present the first visualization of replication morphologies exhibited by the bacterial chromosome. At the start of replication, newly synthesized DNA is translocated via a helical structure from midcell toward the poles, where it accumulates. Next, additionally synthesized DNA forms a second, visually distinct helix that interweaves with the original one. In the final stage of replication, the space between the two helices is filled up with the very last synthesized DNA. This striking geometry provides insight into the three-dimensional conformation of the replicating chromosome. [ABSTRACT FROM AUTHOR]

Following decades of research in genetics and biochemistry, the basic metabolism of bacteria is now well understood. In addition to core metabolism, however, bacterial cells also perform a number of mechanical tasks such as maintaining a characteristic shape, moving within their environment, segregating their genome, and dividing. Major advances in imaging technologies including fluorescence light microscopy (fLM) and electron cryotomography (ECT) have provided new insight into the bacterial ultrastructures that accomplish these tasks. It is now clear, for instance, that bacteria are highly organized, possessing cytoskeletons, specifically arranged genomes, internal compartments, and carefully positioned macromolecular machines. These structures and their functions are reviewed here in the form of a progress report toward a complete biomechanical understanding of a generalized bacterial cell. The goal of eventually integrating genetic, biochemical, imaging, and biophysical data into spatially explicit, mechanically predictive models of whole cells is highlighted. [ABSTRACT FROM AUTHOR]

High-throughput analyses of single-cell microscopy data are a critical tool within the field of bacterial cell biology. Several programs have been developed to specifically segment bacterial cells from phase-contrast images. Together with spot and object detection algorithms, these programs offer powerful approaches to quantify observations from microscopy data, ranging from cell-to-cell genealogy to localization and movement of proteins. Most segmentation programs contain specific post-processing and plotting options, but these options vary between programs and possibilities to optimize or alter the outputs are often limited. Therefore, we developed BactMAP (Bacterial toolbox for Microscopy Analysis & Plotting), a command-line based R package that allows researchers to transform cell segmentation and spot detection data generated by different programs into various plots. Furthermore, BactMAP makes it possible to perform custom analyses and change the layout of the output. Because BactMAP works independently of segmentation and detection programs, inputs from different sources can be compared within the same analysis pipeline. BactMAP complies with standard practice in R which enables the use of advanced statistical analysis tools, and its graphic output is compatible with ggplot2, enabling adjustable plot graphics in every operating system. User feedback will be used to create a fully automated Graphical User Interface version of BactMAP in the future. Using BactMAP, we visualize key cell cycle parameters in Bacillus subtilis and Staphylococcus aureus, and demonstrate that the DNA replication forks in Streptococcus pneumoniae dissociate and associate before splitting of the cell, after the Z-ring is formed at the new quarter positions. BactMAP is available from https://veeninglab.com/bactmap., (© 2019 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The type VI secretion system (T6SS) inhibits the growth of neighboring bacterial cells through a contact-mediated mechanism. Here, we describe a detailed characterization of the protein localization dynamics in the Pseudomonas aeruginosa T6SS. It has been proposed that the type VI secretion process is driven by a conformational-change-induced contraction of the T6SS sheath. However, although the contraction of an optically resolvable TssBC sheath and the subsequent localization of ClpV are observed in Vibrio cholerae, coordinated assembly and disassembly of TssB and ClpV are observed without TssB contraction in P. aeruginosa. These dynamics are inconsistent with the proposed contraction sheath model. Motivated by the phenomenon of dynamic instability, we propose a new model in which ATP hydrolysis, rather than conformational change, generates the force for secretion. [ABSTRACT FROM AUTHOR]

Summary β-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient “L-form” bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic. [ABSTRACT FROM AUTHOR]

Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them., (Copyright © 2018 Wenzel et al.)

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.