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Bacterial interactions are vital for adapting to changing environments, with quorum sensing (QS) systems playing a central role in coordinating behaviors through small signaling molecules. The RRNPPA family is the prevalent QS systems in Bacillota and mediating communication through secreted oligopeptides, which are processed into active pheromones by extracellular proteases. Notably, in several cases the propeptides show the presence of multiple putative pheromones within their sequences, which has been proposed as a mechanism to diversify peptide-receptor specificity and potentially facilitate new functions. However, neither the processes governing the maturation of propeptides containing multiple pheromones, nor their functional significance has been evaluated. Here, using 2 Rap systems from bacteriophages infecting Bacillus subtilis that exhibit different types of pheromone duplication in their propeptides, we investigate the maturation process and the molecular and functional activities of the produced pheromones. Our results reveal that distinct maturation processes generate multiple mature pheromones, which bind to receptors with varying affinities but produce identical structural and biological responses. These findings add additional layers in the complexity of QS communication and regulation, opening new possibilities for microbial social behaviors, highlighting the intricate nature of bacterial interactions and adaptation.
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Capsules are long-chain carbohydrate polymers that envelop the surfaces of many bacteria, protecting them from host immune responses. Capsule biosynthesis enzymes are potential drug targets and valuable biotechnological tools for generating vaccine antigens. Despite their importance, it remains unknown how structurally variable capsule polymers of Gram-negative pathogens are linked to the conserved glycolipid anchoring these virulence factors to the bacterial membrane. Using Actinobacillus pleuropneumoniae as an example, we demonstrate that CpsA and CpsC generate a poly(glycerol-3-phosphate) linker to connect the glycolipid with capsules containing poly(galactosylglycerol-phosphate) backbones. We reconstruct the entire capsule biosynthesis pathway in A. pleuropneumoniae serotypes 3 and 7, solve the X-ray crystal structure of the capsule polymerase CpsD, identify its tetratricopeptide repeat domain as essential for elongating poly(glycerol-3-phosphate) and show that CpsA and CpsC stimulate CpsD to produce longer polymers. We identify the CpsA and CpsC product as a wall teichoic acid homolog, demonstrating similarity between the biosynthesis of Gram-positive wall teichoic acid and Gram-negative capsules.
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The discovery of antimicrobials with novel mechanisms of action is crucial to tackle the foreseen global health crisis due to antimicrobial resistance. Bacterial two-component signaling systems (TCSs) are attractive targets for the discovery of novel antibacterial agents. TCS-encoding genes are found in all bacterial genomes and typically consist of a sensor histidine kinase (HK) and a response regulator. Due to the conserved Bergerat fold in the ATP-binding domain of the TCS HK and the human chaperone Hsp90, there has been much interest in repurposing inhibitors of Hsp90 as antibacterial compounds. In this study, we explore the chemical space of the known Hsp90 inhibitor scaffold 3,4-diphenylpyrazole (DPP), building on previous literature to further understand their potential for HK inhibition. Six DPP analogs inhibited HK autophosphorylation in vitro and had good antimicrobial activity against Gram-positive bacteria. However, mechanistic studies showed that their antimicrobial activity was related to damage of bacterial membranes. In addition, DPP analogs were cytotoxic to human embryonic kidney cell lines and induced the cell arrest phenotype shown for other Hsp90 inhibitors. We conclude that these DPP structures can be further optimized as specific disruptors of bacterial membranes providing binding to Hsp90 and cytotoxicity are lowered. Moreover, the X-ray crystal structure of resorcinol, a substructure of the DPP derivatives, bound to the HK CheA represents a promising starting point for the fragment-based design of novel HK inhibitors. IMPORTANCE: The discovery of novel antimicrobials is of paramount importance in tackling the imminent global health crisis of antimicrobial resistance. The discovery of novel antimicrobials with novel mechanisms of actions, e.g., targeting bacterial two-component signaling systems, is crucial to bypass existing resistance mechanisms and stimulate pharmaceutical innovations. Here, we explore the possible repurposing of compounds developed in cancer research as inhibitors of two-component systems and investigate their off-target effects such as bacterial membrane disruption and toxicity. These results highlight compounds that are promising for further development of novel bacterial membrane disruptors and two-component system inhibitors.
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Antibacterianos , Reposicionamiento de Medicamentos , Proteínas HSP90 de Choque Térmico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Humanos , Antibacterianos/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Pirazoles/farmacología , Pirazoles/química , Histidina Quinasa/antagonistas & inhibidores , Histidina Quinasa/metabolismo , Histidina Quinasa/genética , Histidina Quinasa/química , Bacterias Grampositivas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células HEK293RESUMEN
The small, 78-residue long, regulator SipA interacts with the non-bleaching sensor histidine kinase (NblS). We have solved the solution structure of SipA on the basis of 990 nuclear Overhauser effect- (NOE-) derived distance constraints. The average pairwise root-mean-square deviation (RMSD) for the twenty best structures for the backbone residues, obtained by CYANA, was 1.35 ± 0.21 Å, and 1.90 ± 0.16 Å when all heavy atoms were considered (the target function of CYANA was 0.540 ± 0.08). The structure is that of a ß-II class protein, basically formed by a five-stranded ß-sheet composed of antiparallel strands following the arrangement: Gly6-Leu11 (ß-strand 1), which packs against Leu66-Val69 (ß-strand 5) on one side, and against Gly36-Thr42 (ß-strand 2) on the other side; Trp50-Phe54 (ß-strand 3); and Gly57-Leu60 (ß-strand 4). The protein is highly mobile, as shown by measurements of R1, R2, NOE and ηxy relaxation parameters, with an average order parameter (
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Early life represents a critical window for metabolic, cognitive and immune system development, which is influenced by the maternal microbiome as well as the infant gut microbiome. Antibiotic exposure, mode of delivery and breastfeeding practices modulate the gut microbiome and the reservoir of antibiotic resistance genes (ARGs). Vertical and horizontal microbial gene transfer during early life and the mechanisms behind these transfers are being uncovered. In this review, we aim to provide an overview of the current knowledge on the transfer of antibiotic resistance in the mother-infant dyad through vertical and horizontal transmission and to highlight the main gaps and challenges in this area.
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Microbioma Gastrointestinal , Microbiota , Lactante , Humanos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Microbioma Gastrointestinal/genética , Microbiota/genética , Transferencia de Gen HorizontalRESUMEN
Phages can use a small-molecule communication arbitrium system to coordinate lysis-lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin-antitoxin system MazE-MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis-lysogeny by domesticating and fine-tuning a phage-defence mechanism.
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Fagos de Bacillus , Lisogenia , Fagos de Bacillus/fisiología , Péptidos/metabolismo , Muerte CelularRESUMEN
Arbitrium-coding phages use peptides to communicate and coordinate the decision between lysis and lysogeny. However, the mechanism by which these phages establish lysogeny remains unknown. Here, focusing on the SPbeta phage family's model phages phi3T and SPß, we report that a six-gene operon called the "SPbeta phages repressor operon" (sro) expresses not one but two master repressors, SroE and SroF, the latter of which folds like a classical phage integrase. To promote lysogeny, these repressors bind to multiple sites in the phage genome. SroD serves as an auxiliary repressor that, with SroEF, forms the repression module necessary for lysogeny establishment and maintenance. Additionally, the proteins SroABC within the operon are proposed to constitute the transducer module, connecting the arbitrium communication system to the activity of the repression module. Overall, this research sheds light on the intricate and specialized repression system employed by arbitrium SPß-like phages in making lysis-lysogeny decisions.
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Bacteriófagos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Lisogenia , Péptidos/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, by infecting cells via the interaction of its spike protein (S) with the primary cell receptor angiotensin-converting enzyme (ACE2). To search for inhibitors of this key step in viral infection, we screened an in-house library of multivalent tryptophan derivatives. Using VSV-S pseudoparticles, we identified compound 2 as a potent entry inhibitor lacking cellular toxicity. Chemical optimization of 2 rendered compounds 63 and 65, which also potently inhibited genuine SARS-CoV-2 cell entry. Thermofluor and microscale thermophoresis studies revealed their binding to S and to its isolated receptor binding domain (RBD), interfering with the interaction with ACE2. High-resolution cryoelectron microscopy structure of S, free or bound to 2, shed light on cell entry inhibition mechanisms by these compounds. Overall, this work identifies and characterizes a new class of SARS-CoV-2 entry inhibitors with clear potential for preventing and/or fighting COVID-19.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Triptófano/farmacología , Triptófano/metabolismo , Enzima Convertidora de Angiotensina 2/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Microscopía por Crioelectrón , Unión ProteicaRESUMEN
Bacterial capsules have critical roles in host-pathogen interactions. They provide a protective envelope against host recognition, leading to immune evasion and bacterial survival. Here we define the capsule biosynthesis pathway of Haemophilus influenzae serotype b (Hib), a Gram-negative bacterium that causes severe infections in infants and children. Reconstitution of this pathway enabled the fermentation-free production of Hib vaccine antigens starting from widely available precursors and detailed characterization of the enzymatic machinery. The X-ray crystal structure of the capsule polymerase Bcs3 reveals a multi-enzyme machine adopting a basket-like shape that creates a protected environment for the synthesis of the complex Hib polymer. This architecture is commonly exploited for surface glycan synthesis by both Gram-negative and Gram-positive pathogens. Supported by biochemical studies and comprehensive 2D nuclear magnetic resonance, our data explain how the ribofuranosyltransferase CriT, the phosphatase CrpP, the ribitol-phosphate transferase CroT and a polymer-binding domain function as a unique multi-enzyme assembly.
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Infecciones por Haemophilus , Vacunas contra Haemophilus , Haemophilus influenzae tipo b , Lactante , Niño , Humanos , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/prevención & control , Vacunas contra Haemophilus/metabolismo , Cápsulas Bacterianas/metabolismo , Bacterias GramnegativasRESUMEN
Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI's repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. In silico and in vitro analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life. IMPORTANCE Stl, the Staphylococcus aureus pathogenicity island (SaPI) master repressor, targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. This fascinating phage-SaPI arms race is exemplified by the Stl from SaPIbov1 which targets phage dimeric and trimeric dUTPases (Duts), structurally unrelated proteins with identical functions in the phages. By solving the structure of the Stl in complex with a prokaryotic (M. tuberculosis) and a eukaryotic (human) trimeric Dut, we showed that Stl has developed a sophisticated substrate mimicry strategy to target trimeric Duts. Since all these Duts present identical catalytic mechanisms, Stl is able to interact with Duts from different kingdoms. In addition, in silico modeling with monomeric Dut supports the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor.
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Bacteriófagos , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Pirofosfatasas/genética , Islas Genómicas , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Antifungal proteins (AFPs) are promising antimicrobial compounds that represent a feasible alternative to fungicides. Penicillium expansum encodes three phylogenetically distinct AFPs (PeAfpA, PeAfpB and PeAfpC) which show different antifungal profiles and fruit protection effects. To gain knowledge about the structural determinants governing their activity, we solved the crystal structure of PeAfpB and rationally designed five PeAfpA::PeAfpB chimeras (chPeAFPV1-V5). Chimeras showed significant differences in their antifungal activity. chPeAFPV1 and chPeAFPV2 improved the parental PeAfpB potency, and it was very similar to that of PeAfpA. chPeAFPV4 and chPeAFPV5 showed an intermediate profile of activity compared to the parental proteins while chPeAFPV3 was inactive towards most of the fungi tested. Structural analysis of the chimeras evidenced an identical scaffold to PeAfpB, suggesting that the differences in activity are due to the contributions of specific residues and not to induced conformational changes or structural rearrangements. Results suggest that mannoproteins determine protein interaction with the cell wall and its antifungal activity while there is not a direct correlation between binding to membrane phospholipids and activity. This work provides new insights about the relevance of sequence motifs and the feasibility of modifying protein specificity, opening the door to the rational design of chimeras with biotechnological applicability.
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Fungicidas Industriales , Penicillium , Antifúngicos/farmacología , Antifúngicos/metabolismo , Fungicidas Industriales/farmacología , Hongos , Frutas , Relación Estructura-ActividadRESUMEN
The emergence of drug-resistant Mycobacterium tuberculosis strains highlights the need to discover anti-tuberculosis drugs with novel mechanisms of action. Here we discovered a mycobactericidal strategy based on the prodrug activation of selected chemical derivatives classified as nitronaphthofurans (nNFs) mediated by the coordinated action of the sigH and mrx2 genes. The transcription factor SigH is a key regulator of an extensive transcriptional network that responds to oxidative, nitrosative, and heat stresses in M. tuberculosis. The nNF action induced the SigH stress response which in turn induced the mrx2 overexpression. The nitroreductase Mrx2 was found to activate nNF prodrugs, killing replicating, non-replicating and intracellular forms of M. tuberculosis. Analysis of SigH DNA sequences obtained from spontaneous nNF-resistant M. tuberculosis mutants suggests disruption of SigH binding to the mrx2 promoter site and/or RNA polymerase core, likely promoting the observed loss of transcriptional control over Mrx2. Mutations found in mrx2 lead to structural defects in the thioredoxin fold of the Mrx2 protein, significantly impairing the activity of the Mrx2 enzyme against nNFs. Altogether, our work brings out the SigH/Mrx2 stress response pathway as a promising target for future drug discovery programs.
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Antibacterianos , Mycobacterium tuberculosis , Profármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Respuesta al Choque Térmico/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Profármacos/farmacología , Regiones Promotoras Genéticas , Transcripción Genética , Antibacterianos/farmacologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1010631.].
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Mobile genetic elements control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here, we describe an unprecedented repression-derepression mechanism involved in the transfer of Staphylococcus aureus pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor StlSaPI1 presents a unique tetrameric conformation never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of StlSaPI1, preventing the binding of the repressor to its cognate StlSaPI1 sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterize a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature.
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Islas Genómicas , Fagos de Staphylococcus , Islas Genómicas/genética , Fagos de Staphylococcus/genética , Staphylococcus aureus/genéticaRESUMEN
In Firmicutes, important processes such as competence development, sporulation, virulence, and biofilm formation are regulated by cytoplasmic quorum sensing (QS) receptors of the RRNPPA family using peptide-based communication. Although these systems regulate important processes in a variety of bacteria, their origin and diversification are poorly understood. Here, we integrate structural, genomic, and phylogenetic evidence to shed light on RRNPPA protein origin and diversification. The family is constituted by seven different subfamilies with different domain architectures and functions. Among these, three were found in Lactobacillales (Rgg, ComR, and PrgX) and four in Bacillales (AimR, NprR, PlcR, and Rap). The patterns of presence and the phylogeny of these proteins show that subfamilies diversified a long time ago, resulting in key structural and functional differences. The concordance between the distribution of subfamilies and the bacterial phylogeny was somewhat unexpected, since many of the subfamilies are very abundant in mobile genetic elements, such as phages, plasmids, and phage-plasmids. The existence of diverse propeptide architectures raises intriguing questions about their export and maturation. It also suggests the existence of diverse roles for the RRNPPA systems. Some systems encode multiple pheromones on the same propeptide or multiple similar propeptides, suggesting that they act as "chatterers." Many others lack pheromone genes and may be "eavesdroppers." Interestingly, AimR systems without associated propeptide genes were particularly abundant in chromosomal regions not classed as prophages, suggesting that either the bacterium or other mobile elements are eavesdropping on phage activity. IMPORTANCE Quorum sensing (QS) is a mechanism of bacterial communication, coordinating important decisions depending on bacterial population. QS regulates important processes not only in bacterial behavior but also in genetic mobile elements and host-guest interactions. In Firmicutes, the most important family of QS receptors is the RRNPPA family. Despite the importance of such systems in microbiology, we know little about RRNPPA origin and diversification. In this work, the combination of sequence analysis and structural biology allowed us to identify a very large number of novel systems but also to class of them in functional families and thereby study of their origin and functional diversification. Moreover, peptide pheromone analysis revealed new and intriguing mechanisms of communication, such as "eavesdropper" systems which only listen for the pheromone and "chatterers" that take control of the communication in their microenvironment.
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Proteínas Bacterianas , Percepción de Quorum , Filogenia , Proteínas Bacterianas/metabolismo , Percepción de Quorum/fisiología , Genómica , Evolución Molecular , Péptidos/metabolismo , Feromonas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.
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COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , COVID-19/genética , Antecedentes Genéticos , Humanos , Mutación , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Receptores Virales/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The arbitrium system is employed by phages of the SPbeta family to communicate with their progeny during infection to decide either to follow the lytic or the lysogenic cycle. The system is controlled by a peptide, AimP, that binds to the regulator AimR, inhibiting its DNA-binding activity and expression of aimX. Although the structure of AimR has been elucidated for phages SPß and phi3T, there is still controversy regarding the molecular mechanism of AimR function, with two different proposed models for SPß. In this study, we deepen our understanding of the system by solving the structure of an additional AimR that shows chimerical characteristics with the SPß receptor. The crystal structures of this AimR (apo, AimP-bound and DNA-bound) together with in vitro and in vivo analyses confirm a mechanism of action by AimP-induced conformational restriction, shedding light on peptide specificity and cross regulation with relevant biological implications.
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Fagos de Bacillus , Bacteriófagos , Fagos de Bacillus/genética , Bacteriófagos/metabolismo , Comunicación , ADN/metabolismo , Lisogenia , Péptidos/químicaRESUMEN
We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.
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Evolución Molecular , Mutación , Fenotipo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes/inmunología , COVID-19/virología , Europa (Continente) , Variación Genética , Genoma Viral , Humanos , Pruebas de Neutralización , SARS-CoV-2/inmunologíaRESUMEN
Glycolipids are prominent components of bacterial membranes that play critical roles not only in maintaining the structural integrity of the cell but also in modulating host-pathogen interactions. PatA is an essential acyltransferase involved in the biosynthesis of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. We demonstrate by electron spin resonance spectroscopy and surface plasmon resonance that PatA is an integral membrane acyltransferase tightly anchored to anionic lipid bilayers, using a two-helix structural motif and electrostatic interactions. PatA dictates the acyl chain composition of the glycolipid by using an acyl chain selectivity "ruler." We established this by a combination of structural biology, enzymatic activity, and binding measurements on chemically synthesized nonhydrolyzable acylcoenzyme A (CoA) derivatives. We propose an interfacial catalytic mechanism that allows PatA to acylate hydrophobic PIMs anchored in the inner membrane of mycobacteria, through the use of water-soluble acyl-CoA donors.
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Some Bacillus-infecting bacteriophages use a peptide-based communication system, termed arbitrium, to coordinate the lysis-lysogeny decision. In this system, the phage produces AimP peptide during the lytic cycle. Once internalized by the host cell, AimP binds to the transcription factor AimR, reducing aimX expression and promoting lysogeny. Although these systems are present in a variety of mobile genetic elements, their role in the phage life cycle has only been characterized in phage phi3T during phage infection. Here, using the B. subtilis SPß prophage, we show that the arbitrium system is also required for normal prophage induction. Deletion of the aimP gene increased phage reproduction, although the aimR deletion significantly reduced the number of phage particles produced after prophage induction. Moreover, our results indicated that AimR is involved in a complex network of regulation and brought forward two new players in the SPß lysis-lysogeny decision system, YopN and the phage repressor YopR. Importantly, these proteins are encoded in an operon, the function of which is conserved across all SPß-like phages encoding the arbitrium system. Finally, we obtained mutant phages in the arbitrium system, which behaved almost identically to the wild-type (WT) phage, indicating that the arbitrium system is not essential in the laboratory but is likely beneficial for phage fitness in nature. In support of this, by possessing a functional arbitrium system, the SPß phage can optimize production of infective particles while also preserving the number of cells that survive after prophage induction, a strategy that increases phage persistence in nature.