Plasmodium falciparum parasites cause the most virulent form of malaria, a disease that remains a major global health burden. The appearance of resistance to first line artemisinin-based therapies emphasizes the need to identify new parasite vulnerabilities to develop new therapeutics. Phosphoinositides are central regulators of membrane identity, vesicular trafficking, and signaling, and their synthesis depends on tightly controlled phosphatidylinositol transfer by Sec14-like phosphatidylinositol transfer proteins in many eukaryotes, yet their roles in P. falciparum remain poorly defined. Here, we analyzed six P. falciparum Sec14 domain-containing proteins: PfSec14-1 (PF3D7_0626400), PfSec14-2 (PF3D7_0629900), PfSec14-3 (PF3D7_0717100), PfSec14-4 (PF3D7_0920700), PfSec14-5 (PF3D7_1007200), and PfSec14-6 (PF3D7_1127600). Domain organization segregates these proteins into a BNIP-2 and Cdc42GAP homology (BCH) subfamily (PfSec14-3, PfSec14-5) and a canonical Sec14 subfamily (PfSec14-1, PfSec14-2, PfSec14-4, and PfSec14-6). Yeast complementation assays showed that PfSec14-1, PfSec14-4, and PfSec14-6 partially rescue growth of a temperature-sensitive sec14 mutant, suggesting phosphatidylinositol and phosphatidylcholine transfer activity. Gene disruption revealed that PfSec14-1 is important for asexual blood-stage proliferation, whereas PfSec14-2 is dispensable under standard culture conditions. In contrast, mislocalization of PfSec14-2 and PfSec14-4 using a knock-sideways approach did not impair asexual growth. Subcellular localization indicates distinct distributions for PfSec14-1, PfSec14-2, and PfSec14-4. Together, these findings reveal potential functional and spatial diversification of Sec14-like phosphatidylinositol transfer proteins in P. falciparum.

The mitochondrial contact site and cristae organizing system (MICOS) is a multiprotein complex that shapes crista junctions and maintains inner and outer membrane contacts. MICOS coordinates the assembly of electron transport chain complexes, a prerequisite for cellular respiration. Indeed, MICOS is lost in eukaryotes that dispensed with cellular respiration, suggesting that its assembly depends on the presence of an active respiratory chain. Trypanosoma brucei provides a unique system to test this hypothesis as its mitochondrion undergoes developmentally regulated remodeling. In the insect stage, the mitochondrion contains cristae with an active electron transport chain, whereas the mammalian bloodstream form possesses precursor cristae with stub-like morphology that lack respiratory activity. MICOS has been characterized in the insect stage but remains unexamined in the bloodstream form. Here, we demonstrate that all MICOS subunits assemble onto precursor cristae, retaining conserved interactions with both outer and inner membrane protein machineries. This is somewhat unexpected given the co-occurrence of MICOS with active cellular respiration in nature. Furthermore, we identify novel MICOS-associated proteins that are dispensable for its stability, suggesting auxiliary rather than core roles in MICOS function. Together, our findings establish that MICOS assembly precedes cellular respiratory competence and expand its interaction landscape in trypanosomatids.

In Escherichia coli, cell-to-cell variability in aerobic expression of trimethylamine N-oxide (TMAO) reductase serves as a bet-hedging strategy, enabling a subset of cells to continue growing upon a shift to anaerobic conditions in the presence of TMAO. This variability has been attributed to the low abundance of TorS and TorT signaling proteins, which along with TorR, regulate transcription of torCAD encoding TMAO reductase. Here, we characterize the divergent promoters that drive expression of the bipartite sensor and kinase, TorT and TorS. In vitro transcription analysis identified two transcription start sites for torS and one start site for torT, all located in close proximity. Notably, the RNA polymerase-σ[70]-10 element of the upstream torS promoter overlaps that of the torT promoter, defining it as a bidirectional promoter. We further demonstrate that these two promoters are subject to repression by the global regulator IscR, which binds to a site that partially overlaps the shared -10 elements. IscR binding antagonizes RNAP-σ[70] binding, limiting transcription of both promoters and contributing to low torS/torT expression. Finally, we show that a single base substitution within the overlapping -10 elements increases torS/torT transcription by at least 4-fold and abolishes cell-to-cell variability in torCAD expression, despite this promoter variant remaining sensitive to IscR-mediated repression. Together, these findings indicate that promoter strength and transcriptional repression are critical components for regulated bet-hedging, enabling E. coli to optimize energy metabolism in response to fluctuations in oxygen availability and TMAO levels.

Sporulation in starved Bacillus subtilis depends on precise control of kinA, which encodes the major phosphorelay kinase that generates the threshold level of phosphorylated Spo0A (Spo0A~P) required for entry into sporulation. Here, we define the regulatory logic that shapes kinA expression and uncover structural features that influence Spo0A~P recruitment. First, we show that the kinA promoter integrates positive and negative Spo0A~P inputs through a composite three-site 0A-box architecture. Distinct affinities and cooperative interactions among the three sites generate a controlled output in which activating and repressive inputs are balanced during starvation, maintaining basal-to-moderate kinA transcription. Second, we identify intrinsic DNA bending as a sequence-independent mechanism that enhances Spo0A~P engagement, promoting low-specificity interactions that may contribute to transcriptional regulation and more broadly chromosome organization. Finally, we show that both the kinA promoter and KinA protein domains diverge between pathogenic and nonpathogenic Bacillus species, reflecting niche-specific evolutionary pressures on sporulation initiation. Together, these findings support an integrated model in which promoter architecture and DNA shape collectively tune Spo0A~P dependent regulation of kinA.

FliL is a conserved single-transmembrane protein implicated in stator function and load adaptation of bacterial flagellar motors. We analyzed the oligomeric organization and structural plasticity of the periplasmic domain of polar-flagellum FliL from Vibrio alginolyticus ([pof]FliLC) and compared it with detergent-solubilized full-length [pof]FliL. Recombinant proteins were purified by affinity and size-exclusion chromatography (SEC). SEC profiles indicated condition-dependent assemblies; [pof]FliLC eluted broadly at ~100 kDa and partially shifted to ~45 kDa on re-chromatography, whereas full-length [pof]FliL eluted as large, heterogeneous assemblies (~180 kDa). Negative-stain electron microscopy (EM) revealed abundant ~10-nm rings for [pof]FliLC but heterogeneous particles for full-length protein. Cryo-EM single-particle analysis of the [pof]FliLC yielded high-quality two-dimensional top/bottom-view class averages that show ring particles with clear subunit repeats; class averages correspond to apparent 10-, 11- and 12-fold stoichiometries, but severe preferred orientation and sample heterogeneity prevented reliable three-dimensional reconstruction. High-speed AFM imaged ~15 nm rings and captured transient subunit rearrangements and opening/closing events; representative AFM frames can be interpreted as 10-subunit-like, yet AFM contrast and tip convolution limited unambiguous subunit counting across the population. These complementary data indicate that the [pof]FliLC SPFH-like periplasmic domain forms flexible, dynamic ring oligomers whose stoichiometry and conformation are modulated by membrane association and experimental conditions. We propose that such plasticity enables FliL to act as an adaptable scaffold for stator engagement and mechanosensitive remodeling of the motor.

Bioluminescence in luminous Vibrionaceae is often regulated by quorum sensing, yet several Photobacterium strains emit light independently of cell density, suggesting alternative regulatory inputs. Here, we characterized DimLum, a spontaneous, stable low-luminescence mutant of Photobacterium kishitanii ANT-2200. RNA-seq showed a pronounced downregulation of the lux-rib operon, while whole-genome sequencing identified a 20-nucleotide deletion in tfoX predicted to truncate the C-terminal region of TfoX. Complementation with the wild-type tfoX gene restored lux-rib expression and bioluminescence, linking this mutation to the DimLum phenotype. The mutant also displayed a limited transcriptional response, including upregulation of a small set of genes such as a Cro/C1-type regulator. Together, these results identify TfoX as an important component of the regulatory network supporting bioluminescence in P. kishitanii.

Toxoplasmosis caused by the Apicomplexan parasite Toxoplasma gondii is a significant health threat to immunocompromised individuals and newborns. This parasite has a complex life cycle that is well controlled to achieve optimal transmission and pathogenesis. Acute phase of the disease is caused by rapid proliferation of tachyzoites, which has a highly coordinated and tightly regulated cell cycle to allow parasite propagation. Tachyzoite cell cycle has five partially overlapping phases (G1, S, G2, M and C) with distinct gene expression patterns and cellular activities, yet the underlying regulatory mechanisms are not well understood. In this study, we show that the AP2 family transcription factor AP2XI-3 has important roles in regulating the cell cycle to progress through the G1 phase. Depletion of AP2XI-3 resulted in cell cycle retention at G1, leading to growth inhibition of tachyzoites. RNA-Seq and CUT&Tag analyses revealed that AP2XI-3 regulates the transcription of genes involved in nucleic acid metabolism, RNA biogenesis and processing, which are consistent with the cellular activities of G1 phase in preparing biomass for cell cycle progression. Moreover, AP2XI-3 binds to the conserved DNA motif, TRP-2, a sequence widely distributed in the promoters of genes that exhibit peak expression during the G1 phase. Together, these findings underscore the essential role of AP2XI-3 in the tight regulation of the G1 phase, highlighting its potential as a therapeutic target for drug development.

Malaria parasites undergo extensive replication in hepatocytes before initiating the pathogenic blood stage of infection. Although this clinically silent phase is essential for parasite expansion, the molecular mechanisms regulating late liver stage maturation remain incompletely understood. Here, we characterize a previously unannotated protein (PBANKA_0620800), which we designate liver stage maturation protein (LSMP). LSMP was expressed during schizont, sporozoite, and liver stages and localized predominantly to the parasite cytosol. Using reverse genetic approaches, we generated LSMP KO parasites and examined their development across the parasite life cycle. Loss of LSMP did not affect asexual blood-stage replication or mosquito-stage development, including ookinete formation, oocyst development, and sporozoite formation. LSMP KO sporozoites invaded hepatocytes normally but exhibited pronounced defects during late liver stage development, including reduced nuclear replication, impaired merozoite formation, and delayed rupture of the parasitophorous vacuole membrane. Together, our findings demonstrate that LSMP is important for malaria liver stage maturation.

Autophagy is a highly conserved degradative and recycling pathway essential for maintaining cellular homeostasis. Although its molecular machinery is well characterized in yeast and mammalian systems, it is less studied in the early-diverging apicomplexan parasite Plasmodium, the causative agent of malaria. Plasmodium possesses a reduced yet functional repertoire of autophagy-related (ATG) proteins, suggesting adaptations of this pathway to parasite-specific biology. Among these, ATG8, a ubiquitin-like protein, has emerged as a central marker and key effector of plasmodial autophagy. Its branched localization and association with the relict plastid (apicoplast) membrane indicate roles beyond canonical degradative autophagy, particularly in organelle maintenance and biogenesis. ATG7, an essential E1-like enzyme, activates ATG8 and facilitates its lipidation, thereby regulating organelle turnover and development. This process is further supported by a conserved conjugation system involving ATG3 (E2-like enzyme) and the ATG12, ATG5, ATG16 complex, functioning as a ligase to enable ATG8 membrane association. ATG4, a cysteine protease, is critical for recycling lipidated ATG8 and maintaining its cytosolic pool, while the homolog Otu can partially compensate for its function. ATG18 also plays an important role in apicoplast biogenesis and maintenance. Collectively, these findings highlight both canonical and non-canonical roles of autophagy proteins in Plasmodium, driving metabolic reprogramming, intracellular remodeling, and stage-specific differentiation, and support their potential as targets for new antimalarial therapies.

Legionella pneumophila utilizes the Icm/Dot secretion system to translocate > 330 effectors into host cells, yet the regulatory mechanisms controlling the expression of many effector-encoding genes (EEGs) remain unknown. Here, we identify and characterize LexR1 (lpg2524), a LuxR-type transcriptional regulator that controls EEG expression in L. pneumophila and other Legionella species. In L. pneumophila, LexR1 directly activates the expression of four EEGs (lem26, lem16, ravW, and legC1) by binding a conserved inverted repeat located upstream of these genes. We demonstrate that LexR1 activity is stimulated by a nutrient-derived ligand present in casamino acids, and orthologs from other Legionella species exhibit similar casamino acid dependent activation of their target genes. Genomic analyses identified 29 genes containing the LexR1 regulatory element in the seven Legionella species containing LexR1, most of which encode validated effectors or proteins harboring common effector domains. Many of these genes are shared among the species and appear to have co-transferred with their regulatory regions via horizontal gene transfer. LexR1 expression is positively regulated by RpoS and repressed by Fis, linking it to the broader effector regulatory network. Together, these findings identify LexR1 as a regulator of EEGs that coordinates nutrient sensing with effector gene expression across the Legionella genus.

The rise of multidrug-resistant (MDR) bacterial pathogens, including uropathogenic Escherichia coli (UPEC), highlights the urgent need for alternative treatment strategies to restore antibiotic efficacy. The silver-ruthenium antimicrobial AGXX exerts potent bactericidal effects through the production of reactive oxygen species (ROS); however, its potential synergy with antibiotics has not been thoroughly investigated. Here, we show that sublethal concentrations of AGXX strongly enhance aminoglycoside-mediated killing across a diverse panel of Gram-negative and Gram-positive MDR clinical isolates, including highly aminoglycoside-resistant strains. Combinational treatments significantly reduced the effective concentrations of gentamicin, tobramycin, kanamycin, and amikacin required to kill bacteria. Mechanistic analyses revealed that AGXX/aminoglycoside co-treatments induce pronounced intracellular ROS accumulation, resulting in an imbalanced proteostasis due to extensive protein aggregation and DNA damage. Scavenging ROS abolished synergistic killing, establishing oxidative imbalance as the primary driver of the synergy between both antimicrobials. We further identified polyphosphate as a key bacterial defense mechanism that mitigates ROS accumulation, proteotoxicity, and genotoxic stress during combinational treatment. Moreover, AGXX-aminoglycoside synergy was preserved in an artificial urine medium and across clinical UPEC isolates, underscoring its relevance to urinary tract infections. Together, these findings position AGXX as a potent aminoglycoside adjuvant that restores antibiotic efficacy through ROS-driven macromolecular damage, supporting its development for combination therapies against MDR bacterial infections.

Menstrual toxic shock syndrome (mTSS) is a life-threatening disease caused by the Staphylococcus aureus superantigen TSST-1. At menstruation, the typical acidic vaginal environment rises to near neutral pH, which allows for enhanced TSST-1 production. However, the regulatory network which alters toxin production in response to pH is unknown, despite the importance of this cue in the vaginal environment. To mimic the vaginal environment, we used Vaginally Defined Medium to assess TSST-1 promoter activity in the mTSS strain S. aureus MN8 and discovered a significant upregulation of tst expression occurring at pH 4.5 in a low glucose environment, referred to here as the acidic virulence surge. This increased expression was observed in several regulatory mutant backgrounds tested, including in the absence of SaeS, which has previously been thought to be required for TSST-1 production. Recent studies in non-mTSS S. aureus strains have identified GraXRS as a pH sensor, in addition to its function in cationic antimicrobial peptide sensing. We therefore hypothesized that GraXRS alters TSST-1 expression at low pH. Deletion of the sensor-kinase graS gene resulted in the loss of TSST-1 surge at pH 4.5, indicating that GraXRS is required for the acidic virulence surge. We also found expression from the SaeRS P1 and SarA promoters to be significantly attenuated in the ∆graS background. At low pH, the absence of GraS resulted in reduced T cell activation from S. aureus supernatants when compared to other regulatory mutants, suggesting that GraXRS is the dominant activator at pH 4.5. Finally, we developed an in vivo murine model to measure tst expression using luciferase expression. Our results demonstrate a complex sequence of events that occur in response to changes in pH and further suggests that GraXRS is the main activator of TSST-1 expression at low pH.

Borrelia burgdorferi (Bb), the etiological agent of Lyme disease, lacks the genes for de novo riboflavin biosynthesis and depends on host-derived riboflavin to generate flavin cofactors FMN and FAD. Riboflavin salvage by Bb has demonstrated importance to infection and metabolic homeostasis, though the critical roles of flavin cofactors to central metabolism are not well defined. Here, we demonstrate that riboflavin availability directly controls glycolytic flux through regulation of intracellular redox balance. Disruption of riboflavin uptake by deletion of gene bb0318 encoding the putative ATPase component of the riboflavin transporter resulted in impaired glycolysis as indicated by reduced intracellular pyruvate and a perturbed NADH/NAD+ ratio driven by faulty NAD+ regeneration. We identified coenzyme A disulfide reductase (CoADR) as a key flavin-dependent mediator of glycolytic flux. A Δcdr mutant mimicked the glycolytic dysregulation of the Δbb0318 mutant, but was not rescued by exogenous riboflavin, placing BbCoADR downstream of riboflavin salvage. Biochemical analyses of recombinant BbCoADR demonstrated FAD-dependent NADH oxidase activity. Targeted mutagenesis of predicted FAD-binding residues impaired cofactor binding and NADH oxidase activity, further confirming the dependence of BbCoADR on FAD. Together, this work reveals a direct link between riboflavin acquisition and core metabolic processes driven by BbCoADR-mediated flavin-dependent redox metabolism.

Rho is a conserved, ATP-dependent RNA translocase that terminates transcription at hundreds of sites across bacterial genomes. Although the molecular mechanism of Rho-dependent termination is well characterized, its spatial interplay with RNA polymerase (RNAP) within bacterial cells remains elusive. To address this question, we constructed intragenic, in-frame fusions inserting mCherry or sfGFP 48 amino acid residues downstream of the N-terminus of Rho in Salmonella. Strikingly, mCherry-but not sfGFP-renders the first 48 residues of Rho dispensable. RhoΔ48::mCherry is viable in single copy and exhibits wild-type termination activity in vitro, whereas the full-length Rho::sfGFP fusion, although viable, slows growth and shows strongly reduced activity. Structured illumination microscopy (SIM) revealed that, despite these functional differences, both constructs exhibit similar localisation patterns relative to fluorescently tagged RNAP in single cells. During exponential growth, both Rho and RNAP form discrete clusters, but with markedly distinct spatial organisations: RNAP clusters associate with the nucleoid, whereas Rho is distributed throughout the cell body. This spatial partitioning persists in stationary phase, where RNAP becomes diffusely associated with a compacted nucleoid while Rho accumulates at the cell periphery. The widespread distribution of Rho at cytoplasmic locations is unexpected and suggests participation in cellular functions beyond its canonical role in transcription termination.

The Escherichia coli fabH gene encodes the 3-ketoacyl-ACP synthase III that initiates fatty acid synthesis. Deletion of the fabH gene results in severely limited fatty acid synthesis and tiny cells that are unusually sensitive to antibiotics. Genetic investigations identified the yiiD gene, now called madA, that encodes a malonyl-ACP decarboxylase that suppresses the ∆fabH phenotype, but only when madA is multicopy. We selected vancomycin-resistant suppressor derivatives of a ∆fabH strain. Although such chromosomal mutations were generally rare and weak, suppressor strain s1 (a single C-T transition) restored both wild-type growth, high-level vancomycin resistance, and wild-type fatty acid synthesis by creation of a new promoter within the coding sequence of a gene upstream of madA. This provides a caveat to the extensive effort to develop FabH inhibitors as antibacterial drugs.