Blue-Light-Activated Water-Soluble Sn(IV)-Porphyrins for Antibacterial Photodynamic Therapy (aPDT) against Drug-Resistant Bacterial Pathogens.

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.

trans-translation system is important for maintaining genome integrity during DNA damage in bacteria.

DNA integrity in bacteria is regulated by various factors that act on the DNA. trans-translation has previously been shown to be important for the survival of Escherichia coli cells exposed to certain DNA-damaging agents. However, the mechanisms underlying this sensitivity are poorly understood. In this study, we explored the involvement of the trans-translation system in the maintenance of genome integrity using various DNA-damaging agents and mutant backgrounds. Relative viability assays showed that SsrA-defective cells were sensitive to DNA-damaging agents, such as nalidixic acid (NA), ultraviolet radiation (UV), and methyl methanesulfonate (MMS). The viability of SsrA-defective cells was rescued by deleting sulA, although the expression of SulA was not more pronounced in SsrA-defective cells than in wild-type cells. Live cell imaging using a Gam-GFP fluorescent reporter showed increased double-strand breaks (DSBs) in SsrA-defective cells during DNA damage. We also showed that the ribosome rescue function of SsrA was sufficient for DNA damage tolerance. DNA damage sensitivity can be alleviated by partial uncoupling of transcription and translation by using sub-lethal concentrations of ribosome inhibiting antibiotic (tetracycline) or by mutating the gene coding for RNase H (rnhA). Taken together, our results highlight the importance of trans-translation system in maintaining genome integrity and bacterial survival during DNA damage.

Roles for the Synechococcus elongatus RNA-Binding Protein Rbp2 in Regulating the Circadian Clock.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

Distinct Subcellular Localization of a Type I CRISPR Complex and the Cas3 Nuclease in Bacteria.

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.

Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration.

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

Bacterial alginate regulators and phage homologs repress CRISPR-Cas immunity.

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection1. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases2. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB-AlgB), previously characterized in the regulation of Pseudomonas aeruginosa alginate biosynthesis3,4, as a regulator of the expression and activity of the P. aeruginosa Type I-F CRISPR-Cas system. Downstream of KinB-AlgB, activators of alginate production AlgU (a σE orthologue) and AlgR repress CRISPR-Cas activity during planktonic and surface-associated growth5. AmrZ, another alginate regulator6, is triggered to repress CRISPR-Cas immunity upon surface association. Pseudomonas phages and plasmids have taken advantage of this regulatory scheme and carry hijacked homologs of AmrZ that repress CRISPR-Cas expression and activity. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

Multi-bit Boolean model for chemotactic drift of Escherichia coli.

Dynamic biological systems can be modelled to an equivalent modular structure using Boolean networks (BNs) due to their simple construction and relative ease of integration. The chemotaxis network of the bacterium Escherichia coli (E. coli) is one of the most investigated biological systems. In this study, the authors developed a multi-bit Boolean approach to model the drifting behaviour of the E. coli chemotaxis system. Their approach, which is slightly different than the conventional BNs, is designed to provide finer resolution to mimic high-level functional behaviour. Using this approach, they simulated the transient and steady-state responses of the chemoreceptor sensory module. Furthermore, they estimated the drift velocity under conditions of the exponential nutrient gradient. Their predictions on chemotactic drifting are in good agreement with the experimental measurements under similar input conditions. Taken together, by simulating chemotactic drifting, they propose that multi-bit Boolean methodology can be used for modelling complex biological networks. Application of the method towards designing bio-inspired systems such as nano-bots is discussed.

A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases.

All viruses require strategies to inhibit or evade the immune pathways of cells that they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid immune pathways that target nucleic acids, such as CRISPR-Cas and restriction-modification systems, to replicate efficiently1. Here we show that jumbo phage ΦKZ segregates its DNA from immunity nucleases of its host, Pseudomonas aeruginosa, by constructing a proteinaceous nucleus-like compartment. ΦKZ is resistant to many immunity mechanisms that target DNA in vivo, including two subtypes of CRISPR-Cas3, Cas9, Cas12a and the restriction enzymes HsdRMS and EcoRI. Cas proteins and restriction enzymes are unable to access the phage DNA throughout the infection, but engineering the relocalization of EcoRI inside the compartment enables targeting of the phage and protection of host cells. Moreover, ΦKZ is sensitive to Cas13a-a CRISPR-Cas enzyme that targets RNA-probably owing to phage mRNA localizing to the cytoplasm. Collectively, we propose that Pseudomonas jumbo phages evade a broad spectrum of DNA-targeting nucleases through the assembly of a protein barrier around their genome.

OxyS small RNA induces cell cycle arrest to allow DNA damage repair.

To maintain genome integrity, organisms employ DNA damage response, the underlying principles of which are conserved from bacteria to humans. The bacterial small RNA OxyS of Escherichia coli is induced upon oxidative stress and has been implicated in protecting cells from DNA damage; however, the mechanism by which OxyS confers genome stability remained unknown. Here, we revealed an OxyS-induced molecular checkpoint relay, leading to temporary cell cycle arrest to allow damage repair. By repressing the expression of the essential transcription termination factor nusG, OxyS enables read-through transcription into a cryptic prophage encoding kilR The KilR protein interferes with the function of the major cell division protein FtsZ, thus imposing growth arrest. This transient growth inhibition facilitates DNA damage repair, enabling cellular recovery, thereby increasing viability following stress. The OxyS-mediated growth arrest represents a novel tier of defense, introducing a new regulatory concept into bacterial stress response.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Correction: Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

[This corrects the article DOI: 10.1371/journal.pgen.1005975.].

Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters.

Although the list of proteins that localize to the bacterial cell poles is constantly growing, little is known about their temporal behavior. EI, a major protein of the phosphotransferase system (PTS) that regulates sugar uptake and metabolism in bacteria, was shown to form clusters at the Escherichia coli cell poles. We monitored the localization of EI clusters, as well as diffuse molecules, in space and time during the lifetime of E. coli cells. We show that EI distribution and cluster dynamics varies among cells in a population, and that the cluster speed inversely correlates with cluster size. In growing cells, EI is not assembled into clusters in almost 40% of the cells, and the clusters in most remaining cells dynamically relocate within the pole region or between the poles. In non-growing cells, the fraction of cells that contain EI clusters is significantly higher, and dispersal of these clusters is often observed shortly after exiting quiescence. Later, during growth, EI clusters stochastically re-form by assembly of pre-existing dispersed molecules at random time points. Using a fluorescent glucose analog, we found that EI function inversely correlates with clustering and with cluster size. Thus, activity is exerted by dispersed EI molecules, whereas the polar clusters serve as a reservoir of molecules ready to act when needed. Taken together our findings highlight the spatiotemporal distribution of EI as a novel layer of regulation that contributes to the population phenotypic heterogeneity with regard to sugar metabolism, seemingly conferring a survival benefit.

Where are things inside a bacterial cell?

Bacterial cells are intricately organized, despite the lack of membrane-bounded organelles. The extremely crowded cytoplasm promotes macromolecular self-assembly and formation of distinct subcellular structures, which perform specialized functions. For example, the cell poles act as hubs for signal transduction complexes, thus providing a platform for the coordination of optimal cellular responses to environmental cues. Distribution of macromolecules is mostly mediated via specialized transport machineries, including the MreB cytoskeleton. Recent evidence shows that RNAs also specifically localize within bacterial cells, raising the possibility that gene expression is spatially organized. Here we review the current understanding of where things are in bacterial cells and discuss emerging questions that need to be addressed in the future.

Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries.

While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein.

The general phosphotransferase system proteins localize to sites of strong negative curvature in bacterial cells.

UNLABELLED: The bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near the Escherichia coli cell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shaped E. coli cells are comparable, implying that negatively curved "pole-like" sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein from Bacillus subtilis at strongly curved sites in both B. subtilis and E. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness. IMPORTANCE: Despite their tiny size and the scarcity of membrane-bounded organelles, bacteria are capable of sorting macromolecules to distinct subcellular domains, thus optimizing functionality of vital processes. Understanding the cues that organize bacterial cells should provide novel insights into the complex organization of higher organisms. Previously, we have shown that the general proteins of the phosphotransferase system (PTS) signaling system, which governs utilization of carbon sources in bacteria, localize to the poles of Escherichia coli cells. Here, we show that geometric cues, i.e., strong negative membrane curvature, mediate positioning of the PTS proteins. Furthermore, localization to negatively curved regions seems to support the PTS functionality.

Compartmentalization and spatiotemporal organization of macromolecules in bacteria.

For many years, the bacterial cells were regarded as tiny vessels lacking internal organization. This view, which stemmed from the scarcity of membrane-bounded organelles, has changed considerably in recent years, mainly due to advancements in imaging capabilities. Consequently, despite the rareness of conventional organelles, bacteria are now known to have an intricate internal organization, which is vital for many cellular processes. The list of bacterial macromolecules reported to have distinct localization patterns is rapidly growing. Moreover, time-lapse imaging revealed the spatiotemporal dynamics of various bacterial macromolecules. Although the regulatory mechanisms that underlie macromolecules localization in bacterial cells are largely unknown, certain strategies elucidated thus far include the establishment of cell polarity, the employment of cytoskeletal proteins, and the use of the membrane properties, that is, curvature, electric potential, and composition, as localization signals. The most surprising mechanism discovered thus far is targeting of certain mRNAs to the subcellular domains where their protein products are required. This mechanism relies on localization features in the mRNA itself and does not depend on translation. Localization of other mRNAs near their genetic loci suggests that the bacterial chromosome is involved in organizing gene expression. Taken together, the deep-rooted separation between cells with nucleus and without is currently changing, highlighting bacteria as suitable models for studying universal mechanisms underlying cell architecture.

Subcellular localization of RNA and proteins in prokaryotes.

The field of bacterial cell biology has been revolutionized in the last decade by improvements in imaging capabilities which have revealed that bacterial cells, previously thought to be non-compartmentalized, possess an intricate higher-order organization. Many bacterial proteins localize to specific subcellular domains and regulate the spatial deployment of other proteins, DNA and lipids. Recently, the surprising discovery was made that bacterial RNA molecules are also specifically localized. However, the mechanisms that underlie bacterial cell architecture are just starting to be unraveled. The limited number of distribution patterns observed thus far for bacterial proteins and RNAs, and the similarity between the patterns exhibited by these macromolecules, suggest that the processes that underlie their localization are inextricably linked. We discuss these spatial arrangements and the insights that they provide on processes, such as localized translation, protein complex formation, and crosstalk between bacterial machineries.