Bacteria and bacteriophages are in a constant arms race to develop defence and anti-defence systems, respectively. Currently known phage-encoded anti-defence systems are specific to the activity of the targeted bacterial defence system. Here we identify a mechanism by which the T7 bacteriophage broadly counteracts bacterial defences using protein phosphorylation. Its kinase (T7K), which has been reported to redirect the function of a few host proteins1-5, is actually a hyperpromiscuous dual-specificity kinase that phosphorylates nearly all host and phage proteins during infection. The scale of phosphorylation vastly exceeds known phosphosites in Escherichia coli, has no sequence motif specificity and results in a higher proteome-wide phosphorylation density than mammalian cells with around 500 kinases. Stoichiometry analysis of phosphorylation sites revealed strong bias in T7K activity towards nucleic-acid-binding substrates mediated by its C-terminal DNA-binding domain. This highly stoichiometric phosphorylation enables the deactivation of DNA-targeting or DNA-containing bacterial defence systems. We provide mechanistic insights into how T7K weakens DNA-containing Retron-Eco9 through specific phosphorylation events, with single phosphomimetic mutations in key sites of the toxin abolishing defence. Moreover, by screening a large collection of E. coli strains, we provide evidence of broad anti-defence abilities of T7K in nature, as counteracted strains contain diverse bacterial defence systems. T7K homologues are found almost exclusively in phages, with hyperpromiscuous kinase activity probably being enabled by a divergent DFG-like motif in the catalytic centre.

Cryo-electron tomography has emerged as powerful technology for in-cell structural biology, and in combination with breakthroughs in protein structure prediction, offers a unique opportunity for illuminating functions of previously uncharacterized macromolecular complexes. Here, we used the genome-reduced bacterium Mycoplasma pneumoniae as a minimal cell model, and determined in-cell maps of an unknown complex located at the cell surface. By combining proteomics, structure prediction, and bioinformatics, we identified the complex to include the conserved Sec-translocon and an extracellular dome-like structure largely formed by three uncharacterized proteins (Mdps). The Mdps show structural homology to a periplasmic ATP-independent foldase, PrsA, with Mdp444 retaining key catalytic residues. Our study provides the first sub-nanometer resolution maps of the complete bacterial Sec-translocation machinery, depicting several conformational states that allow us to suggest a model for translocation initiation, and revealing its coordination with a novel, extracellular, protein-folding system. ### Competing Interest Statement The authors have declared no competing interest. Independent Research Fund Denmark, , 0164-00010A National Key Research and Development Program of China, , 2024YFA1306200 Deutsche Forschungsgemeinschaft, , 426290502 Chan Zuckerberg Initiative (United States), , 2021-234620

Abstract The Rcs signal transduction system is a phosphorelay responsible for sensing enterobacterial cell envelope stresses. In Escherichia coli, the Rcs system is required to survive treatment with A22 and mecillinam, antibiotics that perturb cell size. To test whether size changes are correlated with envelope damage and thereby sensed by the Rcs system, we tuned E. coli cell size via A22 treatment, mutations in the cell-shape determinant MreB, and mechanically confined growth. In all conditions, cell width was strongly correlated with Rcs activation, and RcsF, the outer-membrane-localized upstream component, was essential for responding to cell width changes. Several gene deletions that induce Rcs resulted in cells that were wider than wild-type. Cryo-electron microscopy revealed that the periplasm of a wide MreB mutant is ~3 nm thinner than in wild-type cells, bringing RcsF closer to the downstream, inner-membrane-localized components of the signaling cascade. Conversely, extending the RcsF linker region in wild-type cells by ~3 nm increased Rcs activity. Thus, we propose that the Rcs system responds to changes in cell width due to altered periplasmic thickness.

Phages and bacteria are locked in a molecular arms race, with phage anti-defence proteins (ADPs) enabling them to evade bacterial immune systems. To streamline access to information on ADPs, we developed the Encyclopaedia of Viral Anti-DefencE Systems (EVADES), an online resource containing sequences, structures, protein family annotations, and mechanisms of action (MoA) for 257 ADPs. Through computational structural analysis we established putative MoAs for 21 uncharacterised ADPs. We demonstrate the utility of EVADES by exploring three hypotheses and demonstrate that: (i) ADPs acting as DNA mimics exhibit broad-spectrum activity against defences; (ii) protein sharing across defence systems enables multi-defence inhibitory activity of ADPs; and (iii) eukaryotic dsDNA viruses encode ADP homologs, suggesting conserved immune evasion strategies across domains. These results highlight the broader relevance of phage ADPs in understanding interactions between prokaryotic or eukaryotic viruses and their hosts. EVADES is freely accessible at . Highlights ### Competing Interest Statement The authors have declared no competing interest. European Union’s Horizon 2020 research and innovation programme, 945405

Bacteria use gene regulation to dynamically adapt to changes in their environment, including resistance to stress and the occupation of new niches. Gene expression is known to vary within a species pangenome, but the extent to which these changes could be explained by genetic variants in cis non-coding regions has so far been poorly investigated. Statistical genetics offers a hypothesis-free approach to this problem, as opposed to mechanistic models, which can be used only for reference isolates that are not representative of the whole species. In this study, we assembled two genomic and transcriptomic datasets for Escherichia coli (N=117) and Pseudomonas aeruginosa (N=413) and identified associations between genetic variants in cis non-coding regions and recorded gene expression variation. We identified at least one associated variant in up to 39% of the tested genes in both species. We partly validated the associations in-silico and in-vitro for E. coli , reinforcing the difficulty of identifying a single mechanism generating gene expression diversity. We then investigated the relevance of non-coding variants in explaining the variability in antimicrobial resistance in both species using two additional publicly available datasets, identifying a large number of these variants across antimicrobial compounds. This work confirms the role of genetic variation in often overlooked regions of bacterial genomes in influencing molecular and clinically relevant phenotypes. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 390874280 Hannover Biomedical Research School German Academic Exchange Service

Protein glycosylation regulates essential cellular processes such as signaling, adhesion and cell-cell interactions; however, dysregulated glycosylation is associated with diseases such as cancer. Here we introduce deep quantitative glycoprofiling (DQGlyco), a robust method that integrates high-throughput sample preparation, highly sensitive detection and precise multiplexed quantification to investigate protein glycosylation dynamics at an unprecedented depth. Using DQGlyco, we profiled the mouse brain glycoproteome, identifying 177,198 unique N-glycopeptides-25 times more than previous studies. We quantified glycopeptide changes in human cells treated with a fucosylation inhibitor and characterized surface-exposed glycoforms. Furthermore, we analyzed tissue-specific glycosylation patterns in mice and demonstrated that a defined gut microbiota substantially remodels the mouse brain glycoproteome, shedding light on the link between the gut microbiome and brain protein functions. Additionally, we developed a novel strategy to evaluate glycoform solubility, offering new insights into their biophysical properties. Overall, the in-depth profiling offered by DQGlyco uncovered extensive complexity in glycosylation regulation.

The gut microbiome impacts human health in direct and indirect ways. While many associations have been discovered between specific microbiome compositions and diseases, establishing causality, understanding the underlying mechanisms, and developing successful microbiome-based therapies require novel experimental approaches. In this opinion, we discuss how in vitro cultivation of diverse communities enables systematic investigation of the individual and collective functions of gut microbes. Up to now, the field has relied mostly on simple, bottom-up assembled synthetic communities or more complex, undefined stool-derived communities. Although powerful for dissecting interactions and mapping causal effects, these communities suffer either from ignoring the complexity, diversity, coevolution, and dynamics of natural communities or from lack of control of community composition. These limitations can be overcome in the future by establishing personalized culture collections from stool samples of different donors and assembling personalized communities to investigate native interactions and ecological relationships in a controlled manner.

Jumbo bacteriophages of the ϕKZ-like family assemble a lipid-based early phage infection (EPI) vesicle and a proteinaceous nucleus-like structure during infection. These structures protect the phage from nucleases and may create selective pressure for immunity mechanisms targeting this specific phage family. Here, we identify "jumbo phage killer" (Juk), a two-component immune system that terminates infection of ϕKZ-like phages, suppressing the expression of early phage genes and preventing phage DNA replication and phage nucleus assembly while saving the cell. JukA (formerly YaaW) rapidly senses the EPI vesicle by binding to an early-expressed phage protein, gp241, and then directly recruits JukB. The JukB effector structurally resembles a pore-forming toxin and destabilizes the EPI vesicle. Functional anti-ϕKZ JukA homologs are found across bacterial phyla, associated with diverse effectors. These findings reveal a widespread defense system that specifically targets early events executed by ϕKZ-like jumbo phages prior to phage nucleus assembly.

Cells mount elaborate responses to environmental stresses across multiple scales. In enterobacteria, the Rcs phosphorelay is activated by cell-envelope damage and by alterations to periplasmic thickness and cell width. To explore Rcs activation in the absence of external stresses, we investigated the physiological and morphological consequences of Rcs activation in Escherichia coli. We used an inducible variant of the lipoprotein RcsF that mislocalizes to the inner membrane (RcsFIM) and constitutively activates the Rcs system. RcsFIM expression slowed growth and reduced the cell length added per cell cycle, independent of Rcs-regulated capsule production. Concurrently, the concentration of the division protein FtsZ increased, and division rings formed closer together in filamentous cells. Depletion of the Rcs negative regulator IgaA also slowed growth and elevated FtsZ levels, indicating that IgaA is essential due to growth inhibition in its absence. In contrast, treatment with the Rcs-activating antibiotic A22 did not affect growth rate or FtsZ concentration and restored normal growth in cells in which the Rcs system was pre-induced with RcsFIM expression. Hence, the cellular response to A22 buffers the Rcs-dependent effect on cell growth. Together, our findings suggest that Rcs modulates cell division indirectly through growth-rate changes and that activation of the same pathway in different contexts can produce distinct physiological outcomes.IMPORTANCEBacteria must coordinate their growth rate, shape, and division to survive and flourish, yet how these cellular properties are maintained in the face of environmental stresses is poorly understood. Working with Escherichia coli, we show that activating the Rcs phosphorelay, an envelope stress-signaling system, in the absence of external stresses slows growth, shortens cells, and increases the concentration of the key division protein FtsZ, leading to more closely spaced division sites. Depleting the levels of IgaA, a regulator of the Rcs pathway, yielded similar phenotypes. However, activating Rcs via drug-induced cell-wall disruption did not affect growth rate, indicating that the physiological impact of this pathway depends on the context of activation. Our findings reveal links among cell growth, shape homeostasis, and cell envelope stress. Understanding this coupling further will provide new avenues to predict and modulate bacterial growth and physiology during stress.

De novo protein design is of fundamental interest to synthetic biology, with a plethora of computational methods of various degrees of generality developed in recent years. Here, we introduce AlphaDesign, a hallucination-based computational framework for de novo protein design developed with maximum generality and usability in mind, which combines AlphaFold with autoregressive diffusion models to enable rapid generation and computational validation of proteins with controllable interactions, conformations and oligomeric state without the requirement for class-dependent model re-training or fine-tuning. We apply our framework to design and systematically validate in vivo active inhibitors of a family of bacterial phage defense systems with toxic effectors called retrons, paving the way towards efficient, rational design of novel proteins as biologics.

Nitroxoline is a bacteriostatic quinoline antibiotic, known to form complexes with metals. Its clinical indications are limited to uncomplicated urinary tract infections, with a susceptibility breakpoint only available for Escherichia coli. Here, we test > 1000 clinical isolates and demonstrate a much broader activity spectrum and species-specific bactericidal activity, including Gram-negative bacteria for which therapeutic options are limited due to multidrug resistance. By combining genetic and proteomic approaches with direct measurement of intracellular metals, we show that nitroxoline acts as a metallophore, inducing copper and zinc intoxication in bacterial cells. The compound displays additional effects on bacterial physiology, including alteration of outer membrane integrity, which underpins nitroxoline's synergies with large-scaffold antibiotics and resensitization of colistin-resistant Enterobacteriaceae in vitro and in vivo. Furthermore, we identify conserved resistance mechanisms across bacterial species, often leading to nitroxoline efflux.

The human gut microbiome is a key contributor to health, and its perturbations are linked to many diseases. Small-molecule xenobiotics such as drugs, chemical pollutants and food additives can alter the microbiota composition and are now recognized as one of the main factors underlying microbiome diversity. Mapping the effects of such compounds on the gut microbiome is challenging because of the complexity of the community, anaerobic growth requirements of individual species and the large number of interactions that need to be quantitatively assessed. High-throughput screening setups offer a promising solution for probing the direct inhibitory effects of hundreds of xenobiotics on tens of anaerobic gut bacteria. When automated, such assays enable the cost-effective investigation of a wide range of compound-microbe combinations. We have developed an experimental setup and protocol that enables testing of up to 5,000 compounds on a target gut species under strict anaerobic conditions within 5 d. In addition, with minor modifications to the protocol, drug effects can be tested on microbial communities either assembled from isolates or obtained from stool samples. Experience in working in an anaerobic chamber, especially in performing delicate work with thick chamber gloves, is required for implementing this protocol. We anticipate that this protocol will accelerate the study of interactions between small molecules and the gut microbiome and provide a deeper understanding of this microbial ecosystem, which is intimately intertwined with human health.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species also employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

Peptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogen Agrobacterium tumefaciens and found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions for A. tumefaciens LDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking in A. tumefaciens cell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.

By acquiring or evolving resistance to one antibiotic, bacteria can become cross-resistant to a second antibiotic, which further limits therapeutic choices. In the opposite scenario, initial resistance leads to collateral sensitivity to a second antibiotic, which can inform cycling or combinatorial treatments. Despite their clinical relevance, our knowledge of both interactions is limited. We used published chemical genetics data of the Escherichia coli single-gene deletion library in 40 antibiotics and devised a metric that discriminates between known cross-resistance and collateral-sensitivity antibiotic interactions. Thereby we inferred 404 cases of cross-resistance and 267 of collateral-sensitivity, expanding the number of known interactions by over threefold. We further validated 64/70 inferred interactions using experimental evolution. By identifying mutants driving these interactions in chemical genetics, we demonstrated that a drug pair can exhibit both interactions depending on the resistance mechanism. Finally, we applied collateral-sensitive drug pairs in combination to reduce antibiotic-resistance development in vitro. Resistance to one antibiotic can make bacteria resistant or sensitive to another antibiotic, opening paths for combinatorial treatments. This study presents an approach to systematically discover and understand such antibiotic relationships.

Pharmaceuticals can directly inhibit the growth of gut bacteria, but the degree to which such interactions manifest in complex community settings is an open question. Here, we compared the effects of 30 drugs on a 32-species synthetic community with their effects on each community member in isolation. While most individual drug-species interactions remained the same in the community context, communal behaviors emerged in 26% of all tested cases. Cross-protection during which drug-sensitive species were protected in community was 6 times more frequent than cross-sensitization, the converse phenomenon. Cross-protection decreased and cross-sensitization increased at higher drug concentrations, suggesting that the resilience of microbial communities can collapse when perturbations get stronger. By metabolically profiling drug-treated communities, we showed that both drug biotransformation and bioaccumulation contribute mechanistically to communal protection. As a proof of principle, we molecularly dissected a prominent case: species expressing specific nitroreductases degraded niclosamide, thereby protecting both themselves and sensitive community members.

Pharmaceuticals can directly inhibit the growth of gut bacteria, but the degree to which such interactions manifest in complex community settings is an open question. Here, we compared the effects of 30 drugs on a 32-species synthetic community with their effects on each community member in isolation. While most individual drug-species interactions remained the same in the community context, communal behaviors emerged in 26% of all tested cases. Cross-protection during which drug-sensitive species were protected in community was 6 times more frequent than cross-sensitization, the converse phenomenon. Cross-protection decreased and cross-sensitization increased at higher drug concentrations, suggesting that the resilience of microbial communities can collapse when perturbations get stronger. By metabolically profiling drug-treated communities, we showed that both drug biotransformation and bioaccumulation contribute mechanistically to communal protection. As a proof of principle, we molecularly dissected a prominent case: species expressing specific nitroreductases degraded niclosamide, thereby protecting both themselves and sensitive community members.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

All organisms must defend themselves against viral predators. This includes bacteria, which harbor immunity factors such as restriction-modification systems and CRISPR-Cas systems. More recently, a plethora of additional defense systems have been identified, revealing a richer, more sophisticated immune system than previously appreciated. Some of these newly identified defense systems have distant homologs in mammals, suggesting an ancient evolutionary origin of some facets of mammalian immunity. An even broader conservation exists at the level of how these immunity systems operate. Here, we focus at this level, reviewing key principles and high-level attributes of innate immunity in bacteria that are shared with mammalian immunity, while also noting key differences, with a particular emphasis on how cells sense viral infection.