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Soilborne Ralstonia solanacearum species complex (RSSC) pathogens disrupt microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped the T6SS of these bacterial pathogens, we analyzed the T6SS gene content and architecture across the RSSC and their evolutionary relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem-pathogenic RSSC and Paracidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,066 auxiliary T6SS toxin clusters ("aux" clusters) across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were located in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Although the single, ancestral T6SS was broadly conserved in the RSSC, the T6SS has been convergently lost in atypical, non-soilborne lineages. Overall, our data suggest dynamic interplay between the lifestyle of RSSC lineages and the evolution of T6SSes with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.IMPORTANCEWe explored the eco-evolutionary dynamics that shape the inter-microbial warfare mechanisms of a globally significant plant pathogen, the Ralstonia solanacearum species complex. We discovered that most Ralstonia wilt pathogens have evolved extensive and diverse repertoires of type VI secretion system-associated antimicrobial toxins. These expansive toxin arsenals potentially enhance the ability of Ralstonia pathogens to invade plant microbiomes, enabling them to rapidly colonize and kill their host plants. We devised a classification system to categorize the Ralstonia toxins. Interestingly, many of the toxin gene clusters are encoded on mobile genetic elements, including prophages, which may be mutualistic symbionts that enhance the inter-microbial competitiveness of Ralstonia wilt pathogens. Moreover, our findings suggest that the convergent loss of this multi-gene trait contributes to genome reduction in two vector-transmitted lineages of Ralstonia pathogens. Our findings demonstrate that the interplay between microbial ecology and pathogen lifestyle shapes the evolution of a genetically complex antimicrobial weapon.

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target.

Importance: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.

Mononuclear phagocytes facilitate the dissemination of the obligate intracellular parasite Toxoplasma gondii. Here, we report how a set of secreted parasite effector proteins from dense granule organelles (GRA) orchestrates dendritic cell-like chemotactic and pro-inflammatory activation of parasitized macrophages. These effects enabled efficient dissemination of the type II T. gondii lineage, a highly prevalent genotype in humans. We identify novel functions for effectors GRA15 and GRA24 in promoting CCR7-mediated macrophage chemotaxis by acting on NF-κB and p38 mitogen-activated protein kinase signaling pathways, respectively, with contributions by GRA16/18 and counter-regulation by effector TEEGR. Furthermore, GRA28 boosted chromatin accessibility and GRA15/24/NF-κB-dependent transcription at the Ccr7 gene locus in primary macrophages. In vivo, adoptively transferred macrophages infected with wild-type T. gondii outcompeted macrophages infected with a GRA15/24 double mutant in migrating to secondary organs in mice. The data show that T. gondii, rather than being passively shuttled, actively promotes its dissemination by inducing a finely regulated pro-migratory state in parasitized human and murine phagocytes via co-operating polymorphic GRA effectors.

Importance: Intracellular pathogens can hijack the cellular functions of infected host cells to their advantage, for example, for intracellular survival and dissemination. However, how microbes orchestrate the hijacking of complex cellular processes, such as host cell migration, remains poorly understood. As such, the common parasite Toxoplasma gondii actively invades the immune cells of humans and other vertebrates and modifies their migratory properties. Here, we show that the concerted action of a number of secreted effector proteins from the parasite, principally GRA15 and GRA24, acts on host cell signaling pathways to activate chemotaxis. Furthermore, the protein effector GRA28 selectively acted on chromatin accessibility in the host cell nucleus to selectively boost host gene expression. The joint activities of GRA effectors culminated in pro-migratory signaling within the infected phagocyte. We provide a molecular framework delineating how T. gondii can orchestrate a complex biological phenotype, such as the migratory activation of phagocytes to boost dissemination.

During its cell cycle, the bacterium Caulobacter crescentus switches from a motile, free-living state, to a sessile surface-attached cell. During this coordinated process, cells undergo irreversible morphological changes, such as shedding of their polar flagellum and synthesis of an adhesive holdfast at the same pole. In this work, we used genetic screens to identify genes involved in the regulation of the transition from the motile to the sessile lifestyle. We identified a predicted hybrid histidine kinase that inhibits biofilm formation and promotes the motile lifestyle: HmrA (holdfast and motility regulator A). Genetic screens and genomic localization led to the identification of additional genes that form a putative phosphorelay pathway with HmrA. We postulate that the Hmr pathway acts as a rheostat to control the proportion of cells harboring a flagellum or a holdfast in the population. Further genetic analysis suggests that the Hmr pathway impacts c-di-GMP synthesis through the diguanylate cyclase DgcB pathway. Our results also indicate that the Hmr pathway is involved in the regulation of motile to sessile lifestyle transition as a function of various environmental factors: biofilm formation is repressed when excess copper is present and derepressed under non-optimal temperatures. Finally, we provide evidence that the Hmr pathway regulates motility and adhesion without modulating the transcription of the holdfast synthesis regulator HfiA.

Importance: Complex communities attached to a surface, or biofilms, represent the major lifestyle of bacteria in the environment. Such a sessile state enables the inhabitants to be more resistant to adverse environmental conditions. Thus, having a deeper understanding of the underlying mechanisms that regulate the transition between the motile and the sessile states could help design strategies to improve biofilms when they are beneficial or impede them when they are detrimental. For Caulobacter crescentus motile cells, the transition to the sessile lifestyle is irreversible, and this decision is regulated at several levels. In this work, we describe a putative phosphorelay that promotes the motile lifestyle and inhibits biofilm formation, providing new insights into the control of adhesin production that leads to the formation of biofilms.

The cell wall of monoderm bacteria consists of peptidoglycan and glycopolymers in roughly equal proportions and is crucial for cellular integrity, cell shape, and bacterial vitality. Despite the immense value of Streptomyces in biotechnology and medicine as antibiotic producers, we know very little about their cell wall biogenesis, composition, and functions. Here, we have identified the LCP-LytR_C domain protein CglA (Vnz_13690) as a key glycopolymer ligase, which specifically localizes in zones of cell wall biosynthesis in S. venezuelae. Reduced amount of glycopolymers in the cglA mutant results in enlarged vegetative hyphae and failures in FtsZ-rings formation and positioning. Consequently, division septa are misplaced leading to the formation of aberrant cell compartments, misshaped spores, and reduced cell vitality. In addition, we report our discovery that c-di-AMP signaling and decoration of the cell wall with glycopolymers are physiologically linked in Streptomyces since the deletion of cglA restores growth of the S. venezuelae disA mutant at high salt. Altogether, we have identified and characterized CglA as a novel component of cell wall biogenesis in Streptomyces, which is required for cell shape maintenance and cellular vitality in filamentous, multicellular bacteria.IMPORTANCEStreptomyces are our key producers of antibitiotics and other bioactive molecules and are, therefore, of high value for medicine and biotechnology. They proliferate by apical extension and branching of hyphae and undergo complex cell differentiation from filaments to spores during their life cycle. For both, growth and sporulation, coordinated cell wall biogenesis is crucial. However, our knowledge about cell wall biosynthesis, functions, and architecture in Streptomyces and in other Actinomycetota is still very limited. Here, we identify CglA as the key enzyme needed for the attachment of glycopolymers to the cell wall of S. venezuelae. We demonstrate that defects in the cell wall glycopolymer content result in loss of cell shape in these filamentous bacteria and show that division-competent FtsZ-rings cannot assemble properly and fail to be positioned correctly. As a consequence, cell septa placement is disturbed leading to the formation of misshaped spores with reduced viability.

Nitric oxide (·NO) is one of the toxic metabolites that bacteria can be exposed to within phagosomes. Gre factors, which are also known as transcript cleavage factors or transcription elongation factors, relieve back-tracked transcription elongation complexes by cleaving nascent RNAs, which allows transcription to resume after stalling. Here we discovered that loss of both Gre factors in Escherichia coli, GreA and GreB, significantly compromised ·NO detoxification due to ·NO-induced phenotypic heterogeneity in ΔgreAΔgreB populations, which did not occur in wild-type cultures. Under normal culturing conditions, both wild-type and ΔgreAΔgreB synthesized transcripts uniformly, whereas treatment with ·NO led to bimodal transcript levels in ΔgreAΔgreB that were unimodal in wild-type. Interestingly, exposure to another toxic metabolite of phagosomes, hydrogen peroxide (H₂O₂), produced analogous results. Furthermore, we showed that loss of Gre factors led to cheating under ·NO stress where transcriptionally deficient cells benefited from the detoxification activities of the transcriptionally proficient subpopulation. Collectively, these results show that loss of Gre factor activities produces phenotypic heterogeneity under ·NO and H₂O₂ stress that can yield cheating between subpopulations.IMPORTANCEToxic metabolite stress occurs in a broad range of contexts that are important to human health, microbial ecology, and biotechnology, whereas Gre factors are highly conserved throughout the bacterial kingdom. Here we discovered that loss of Gre factors in E. coli leads to phenotypic heterogeneity under ·NO and H₂O₂ stress, which we further show with ·NO results in cheating between subpopulations. Collectively, these data suggest that Gre factors play a role in coping with toxic metabolite stress, and that loss of Gre factors can produce cheating between neighbors.

Secretory (S) IgA antibodies against severe acute respiratory syndrome (SARS)-CoV-2 are induced in saliva and upper respiratory tract (URT) secretions by natural infection and may be critical in determining the outcome of initial infection. Secretory IgA1 (SIgA1) is the predominant isotype of antibodies in these secretions. Neutralization of SARS-CoV-2 is most effectively accomplished by polymeric antibodies such as SIgA. We hypothesize that cleavage of SIgA1 antibodies against SARS-CoV-2 by unique bacterial IgA1 proteases to univalent Fabα antibody fragments with diminished virus neutralizing activity would facilitate the descent of the virus into the lungs to cause serious disease and also enhance its airborne transmission to others. Recent studies of the nasopharyngeal microbiota of patients with SARS-CoV-2 infection have revealed significant increases in the proportions of IgA1 protease-producing bacteria in comparison with healthy subjects. Similar considerations might apply also to other respiratory viral infections including influenza, possibly explaining the original attribution of influenza to Haemophilus influenzae, which produces IgA1 protease.

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover.

Importance: Protein methylation in bacteria was first identified over 60 years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics data set encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis.

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.

Carboxysomes are proteinaceous organelles featuring icosahedral protein shells that enclose the carbon-fixing enzymes, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), along with carbonic anhydrase. The intrinsically disordered scaffolding protein CsoS2 plays a vital role in the construction of α-carboxysomes through bridging the shell and cargo enzymes. The N-terminal domain of CsoS2 binds Rubisco and facilitates Rubisco packaging within the α-carboxysome, whereas the C-terminal domain of CsoS2 (CsoS2-C) anchors to the shell and promotes shell assembly. However, the role of the middle region of CsoS2 (CsoS2-M) has remained elusive. Here, we conducted in-depth examinations on the function of CsoS2-M in the assembly of the α-carboxysome shell by generating a series of recombinant shell variants in the absence of cargos. Our results reveal that CsoS2-M assists CsoS2-C in the assembly of the α-carboxysome shell and plays an important role in shaping the α-carboxysome shell through enhancing the association of shell proteins on both the facet-facet interfaces and flat shell facets. Moreover, CsoS2-M is responsible for recruiting the C-terminal truncated isoform of CsoS2, CsoS2A, into α-carboxysomes, which is crucial for Rubisco encapsulation and packaging. This study not only deepens our knowledge of how the carboxysome shell is constructed and regulated but also lays the groundwork for engineering and repurposing carboxysome-based nanostructures for diverse biotechnological purposes.

Importance: Carboxysomes are a paradigm of organelle-like structures in cyanobacteria and many proteobacteria. These nanoscale compartments enclose Rubisco and carbonic anhydrase within an icosahedral virus-like shell to improve CO₂ fixation, playing a vital role in the global carbon cycle. Understanding how the carboxysomes are formed is not only important for basic research studies but also holds promise for repurposing carboxysomes in bioengineering applications. In this study, we focuses on a specific scaffolding protein called CsoS2, which is involved in facilitating the assembly of α-type carboxysomes. By deciphering the functions of different parts of CsoS2, especially its middle region, we provide new insights into how CsoS2 drives the stepwise assembly of the carboxysome at the molecular level. This knowledge will guide the rational design and reprogramming of carboxysome nanostructures for many biotechnological applications.