The Min system disassembles FtsZ-rings after septation in Bacillus subtilis and is localized to the nascent division plane and cell poles by the protein MinJ. The N-terminal region of MinJ contains transmembrane segments while the C-terminal region contains a PDZ domain, but its membrane topology and functional domains are poorly understood. Here, we empirically tested competing topology models and found that MinJ is a nine-pass transmembrane protein with an extracellular N terminus and a cytoplasmic C terminal PDZ domain. Deletion of the PDZ domain resembled a MinJ null mutation but retained polar localization and interaction with both MinD and DivIVA. Deletion of transmembrane segments 6 and 7 abolished polar localization and interaction with MinD and DivIVA but conferred a hyper-minicell phenotype that was additive in the absence of MinD. Structural prediction by AlphaFold3 suggests that segments 6 and 7 may constitute an unusual transmembrane β-sheet, making MinJ perhaps the first of its kind to have such a secondary structure in the plasma membrane. Finally, phylogenetic analyses and structural comparison showed that MinJ sequence and structure are highly conserved within the Firmicutes and tightly co-conserved with MinD and DivIVA with which it interacts. Our work clarifies the membrane topology and functional organization of MinJ and supports models in which MinJ contributes to bacterial cell division through mechanisms beyond canonical Min system regulation.

The mechanistic target of rapamycin (mTOR) signaling pathway is a central regulator of nutrient sensing, metabolism, growth, and stress adaptation in most eukaryotes. Malaria parasites of the genus Plasmodium, however, lack identifiable orthologues of mTOR kinase and its associated signaling complexes, raising the question of how these organisms coordinate environmental sensing with developmental progression and proliferation. Here, we discuss emerging evidence suggesting that Plasmodium has evolutionarily replaced the canonical mTOR signaling axis with a distributed regulatory architecture composed of parasite-specific nutrient sensors, kinase-based signaling pathways, translational repression mechanisms, and unconventional autophagy processes. Components such as KIN, PI3K, eIF2α kinases, eIF4E-interacting protein DOZI, and Maf1 collectively preserve many hallmark outputs of mTOR signaling, including metabolic adaptation, stress tolerance, developmental plasticity, and reversible quiescence. In addition, malaria parasites interact with host mTOR pathways during infection, potentially exploiting host-derived anabolic signaling environments to support intracellular development. These findings suggest that Plasmodium exemplifies an extreme case of signaling plasticity in eukaryotic evolution, demonstrating how essential mTOR-associated functions can be maintained despite the apparent loss of the pathway itself.

Outer surface protein A (OspA)-based Lyme disease vaccines elicit antibodies that inhibit transmission of infectious Borrelia burgdorferi spirochetes from ticks to humans, although the mechanism by which this occurs is unclear. Here we demonstrate using high-resolution, single-cell fluorescence video microscopy that B. burgdorferi motility is arrested within minutes by human OspA-specific monoclonal antibodies and polyclonal OspA antisera from mice immunized with a candidate OspA mRNA lipid nanoparticle vaccine.

Burkholderia thailandensis is a commonly used surrogate for pathogenic Burkholderia species, yet how it adapts to changes in extracellular pH remains poorly characterized. Using quantitative mass spectrometry, we report here the relative proteomes for cells exposed to a change in pH from 7.0 to either 6.0 or 8.0, conditions that may be encountered in the rhizosphere. Acute exposure to mildly acidic conditions resulted in four proteins being depleted while four accumulated based on an absolute log2-fold change of 0.4. By comparison, a transition to mildly alkaline conditions resulted in accumulation of 13 proteins and depletion of 14. Exposure to both pH 6.0 and pH 8.0 resulted in an overall reduction in the pathways linked to ribosome, flagellar assembly, and β-lactam resistance. Consistent with the observed pathway changes, both surface motility and resistance to certain β-lactams were greatly reduced at pH 6.0 and 8.0 compared to pH 7.0. Transcriptome changes at pH 8.0 primarily included global downregulation of genes associated with flagellar motility and chemotaxis, while genes encoding secondary metabolite biosynthetic enzymes were markedly upregulated. Taken together, our data afford insight into how B. thailandensis responds to physiologically relevant pH changes to achieve optimal niche adaptation, survival, and competitive fitness.

Cyclic di-GMP is ubiquitous in Bacteria, including members of the deepest branching phyla, but has not yet been detected in Archaea. Thus, whether cyclic di-GMP was present as a signaling nucleotide in the last universal common ancestor (LUCA) of Bacteria and Archaea remains unknown. In this work, bioinformatic analyses and structural modelling identified GGDEF domain proteins in archaeal isolates and encoded by metagenomes of confirmed archaeal origin. In particular, in bacterial model organisms, phenotypic and in vivo assays, in combination with catalytic mutants, suggest that selected archaeal GGDEF domain proteins possess diguanylate cyclase activity. These include the complex RECS-PAS/PAC-PocR-GGDEF-HD-GYP domain protein of Methanocella arvoryzae MRE50, a member of the Stenosarchaea order Methanocellales. While cyclic di-GMP signaling proteins are ubiquitous in Bacteria, their presence seems to be more sporadic in Archaea. It is currently unclear whether cyclic di-GMP signaling proteins have been lost in some lineages, secondarily introduced by horizontal gene transfer into others, or whether both scenarios have occurred.

Carbon catabolite repression (CCR) enables fungi to preferentially utilize favorable carbon sources through global transcriptional control. In filamentous fungi, CCR is classically attributed to the C2H2 zinc-finger repressor Mig1/CreA; however, residual repression following Mig1 disruption suggests additional regulatory layers in Talaromyces pinophilus (previously called Penicillium funiculosum NCIM1228). Here, we identify Mig3, a previously uncharacterized, Eurotiales-specific C2H2 transcription factor that functions redundantly with Mig1 in CCR. Mig3 contains conserved N-terminal zinc-finger motifs and a fungal transcription factor middle homology region and binds canonical Mig1 response elements with higher DNA affinity. Genetic analyses reveal that simultaneous loss of Mig1 and Mig3 is synthetically lethal, indicating an essential shared function beyond carbon repression. Integrative transcriptomic, chromatin immunoprecipitation sequencing, and proteomic analyses demonstrate that Mig1 and Mig3 directly regulate genes encoding mitochondrial and ribosomal components, including key factors involved in oxidative phosphorylation, while repressing genes required for alternative carbon metabolism. Loss of either repressor compromises expression of respiratory and ribosomal genes and confers hypersensitivity to mitochondrial and ribosomal inhibitors. In addition, both repressors physically associate with mitochondrial, ribosomal, and central metabolic proteins, suggesting coordination of transcriptional and posttranscriptional regulatory mechanisms. Together, our findings redefine fungal catabolite repressors as metabolic gatekeepers that couple CCR to mitochondrial and ribosome biogenesis, thereby controlling the metabolic switch between core cellular growth processes and enzyme secretion.

Fluorescence microscopy is a powerful tool to analyze the subcellular architecture, and long-term live-cell imaging permits the analysis of the dynamics of intracellular structures, such as membranes. Fluorescent labeling of the plasma membrane of Plasmodium, the causative agent of malaria, via membrane-resident proteins has been described, but proteins that broadly mark internal membranes are currently elusive. Alternatively, general membrane dyes can be employed to study membrane dynamics; however, we find that the membrane dye BODIPY TR Ceramide has adverse effects on cell viability during live-cell imaging. To overcome this limitation, we present IMMP (Internal Membrane Marker for Plasmodium), a 32-amino acid long peptide derived from P. falciparum PCNA1, which targets (fluorescent) protein tags to internal membranes for detection in live or fixed samples. Importantly, IMMP enables non-invasive, long-term live-cell imaging of internal membranes without affecting P. falciparum viability and is functional in blood stages of both the human malaria parasite P. falciparum and the rodent-infecting parasite P. berghei, as well as in P. berghei mosquito stages. IMMP is also compatible with advanced techniques such as expansion microscopy. Together, our results establish IMMP as a tool for internal membrane imaging in Plasmodium.

Environmental bacteria have abundant signaling systems wired into complex gene regulatory networks to adapt to fluctuating conditions. In Myxococcus xanthus, starvation triggers a developmental program (specialized biofilm) that produces spore-filled multicellular fruiting bodies surrounded by a distinct quiescent state termed peripheral rods. Fruiting body structure as well as the proportion of cells following each fate can be tuned by a large repertoire of signaling proteins, including numerous orphan histidine kinases. Here, we focus on the histidine kinase TodK, which was previously demonstrated to influence developmental progression. We find that loss of TodK produces distinct developmental phenotypes that vary with environmental conditions. To quantify these effects, we developed an image-analysis pipeline that measures aggregation and fruiting body patterning during development on nutrient-limited agar. These analyses revealed the todK mutant prematurely aggregates particularly at the peripheries of the colony. Under submerged-culture conditions, initial production of aggregates was observed with wild-type timing, but these aggregates displayed accelerated transition to mature fruiting bodies. Overexpression of active TodK completely blocked fruiting body formation. Molecular analyses demonstrated that TodK overproduction suppressed expression of core developmental regulators including FruA and CsgA. Interestingly, protein accumulation of MrpC, a transcription factor necessary for expression of both FruA and CsgA, was not significantly perturbed. These data suggest TodK silences MrpC's ability to activate transcription of key developmental targets. Together, these findings establish TodK as a modulator of developmental progression and demonstrate how quantitative phenotyping approaches can reveal biologically meaningful functions for orphan histidine kinases whose mutant phenotypes might otherwise appear subtle.

YdcI, a LysR-type transcription regulator, has been associated with various phenotypes related to stress response and metabolic adaptation in Escherichia coli and Salmonella enterica. This work addresses regulation of specific target loci by YdcI as well as regulation of ydcI expression and YdcI synthesis in E. coli. DNA binding of YdcI to loci previously identified through ChIP-exo analysis in E. coli K12 was validated for 10 loci by electrophoretic mobility shift assays. Two of these loci, iraP and mqo, are regulated by YdcI as shown using promoter lacZ fusions, while regulation of the other loci is marginal, at least under the conditions tested. Our data suggest that YdcI acts as an H-NS antagonist at the H-NS repressed and (p)ppGpp-dependent stress-induced iraP promoter. Gene iraP encodes the RssB anti-adaptor protein IraP that prevents RssB-mediated targeting of RpoS for degradation. Gene mqo, which is repressed by YdcI, encodes the TCA cycle enzyme malate: quinone oxidoreductase. Other YdcI bound loci encode enzymes involved in the TCA cycle, as well as membrane transport proteins. Furthermore, the transcription of the ydcI gene itself is induced by the alarmone (p)ppGpp under phosphate stress conditions; it is not autoregulated, and a significant increase in YdcI protein levels occurs upon entry from early to mid-exponential growth phase. Taken together, aspects of regulation by YdcI and of ydcI expression as well as YdcI synthesis are presented that relate to its potential role in the response to abiotic stresses and control of metabolism in E. coli.

Slowly translated codons are overrepresented among the first approximately 30 codons of natural genes across all domains of life, yet the functional basis for this conserved feature remains incompletely understood. Using the Escherichia coli lacZ gene as a model, we previously showed that insertion of fast-translated codons into the early coding region dramatically reduces β-galactosidase production by increasing premature transcription termination and decreasing mRNA stability. Here we show that premature transcription termination events occur several nucleotides downstream of the fast-codon inserts, at positions coinciding with previously characterized Rho-dependent intragenic termination sites in lacZ. To exclude the possibility that the specific amino acid sequence of the inserts-rather than their translation speed-was responsible for termination, we constructed two additional lacZ variants with distinct fast-translated sequences, including one replacing the 30 earliest lacZ codons with their fastest synonymous alternatives. Both variants showed premature transcription termination of comparable magnitude to the original inserts, demonstrating that it is the high translation rate itself which causes premature termination. Analysis of a conservative set of seven high-confidence fast-translated codons across natural highly expressed genes and the synthetic constructs revealed that a run of consecutive fast codons is the feature that most clearly distinguishes the terminating sequences from natural genes. We propose that excessively rapid synthesis of the N-terminal part of a polypeptide may impair its proper entry into the ribosomal exit tunnel, thereby disrupting transcription-translation coupling and exposing downstream mRNA to Rho-dependent termination.

Although α-1,3-glucan represents a major polymer in cell walls of filamentous plant pathogenic fungi, its role in vegetative and pathogenic development is poorly understood. The maize anthracnose fungus Colletotrichum graminicola differentiates elaborate appressoria on the leaf surface and exhibits a hemibiotrophic lifestyle after host invasion. Promoter-eGFP-fusion analyses of all three α-1,3-glucan synthase (AGS) genes, designated as AGS1, AGS2, and AGS3, suggest that AGS3 is expressed in vegetative and pathogenic hyphae, whereas expression of AGS1 and AGS2 was not, or barely, detectable. Targeted gene deletions yielded single and double deletion mutants, as well as triple mutants, which lacked the entire repertoire of α-1,3-glucan synthase genes. Conidia of mutants lacking AGS3 developed multiple germ tubes, and vegetative hyphae had cell walls containing strongly reduced amounts of alkali-soluble glucose, showed hyperbranching, reduced growth rates on all substrata tested, and hypersensitivity to the cell wall-perturbing agents Caspofungin and Nikkomycin Z. Moreover, mutants lacking AGS3 exhibited strong virulence defects and, intriguingly, biotrophic hyphae of these mutants that differentiated in planta were severely misshapen. Increased cell wall pore diameters, increased mobility of uncharged dextran-FITC conjugates through cell walls of AGS3-deficient mutants and lack of PAMP-triggered immune responses in maize leaves inoculated with these mutants suggest that α-1,3-glucan does not play a role in masking of fungal cell walls and immune evasion, but rather controls mobility of fungal cell wall-modifying enzymes and cell wall integrity.

Lactobacillus crispatus dominance in the vaginal microbiome is associated with beneficial health outcomes, yet strain-level variation and its implications remain poorly understood. Here, we resolve the genomic context of three glycosyltransferase gene fragments (GT1-3) previously linked with dysbiotic states. Long-read resequencing revealed that GT1-3 are part of a ~18.7 kb Wzx/Wzy-dependent cell wall polysaccharide (CWPS) locus, containing several IS256-family transposases. Serial propagation in vitro produced isolates with 4.1 kb excised via a composite transposon encompassing the GT3, UDP-galactopyranose mutase, flippase, and hypothetical protein, demonstrating structural plasticity. Transmission electron microscopy showed a ~20%-25% thinner peptidoglycan layer in the derived strains, while FT-IR and monosaccharide analysis indicated no gross changes. Molecular dynamics simulations suggest that GT3 contributes to the structural stability of the glycosyltransferase complex, without compromising catalytic function. Together, these findings establish the CWPS locus as a mobile, structurally plastic element that directly influences cell wall architecture in L. crispatus.

Actin superfamily members are critical for the biology of eukaryotes and archaea. Actin-related proteins (Arps) are a subgroup within the actin superfamily and play essential roles in trafficking, replication and motility. The genome of the malaria parasite Plasmodium contains a set of Arps unique to apicomplexans, termed actin-like proteins (Alps). However, the importance and specific roles of many of these Alps in Plasmodium progression are not yet understood. Here, we determined the functional contribution of Plasmodium berghei Alp3 and Alp5a (recently relabelled as Arp3) by generation of knock-out (KO) lines and their subsequent characterisation across different life cycle stages. Deletion of either Alp did not affect blood stage growth, gametogenesis and ookinete gliding motility. However, deletion of Alp5a led to smaller and fewer oocysts as well as severely impaired sporozoite formation. The Alp3KO line had highly reduced oocyst loads compared to wild-type parasites. This striking decrease was due to impaired ookinete penetration of the mosquito midgut epithelium. Our study shows that both Alp3 and Alp5a are indispensable for Plasmodium transmission at different steps of initial mosquito infection, provides insights into the role of specific unique members of the actin superfamily during parasite progression and the requirements for efficient midgut penetration.

The archaeal tubulin-like cytoskeletal protein CetZ1 is required for rod-cell morphogenesis during the development of motility in Haloferax volcanii. This is expected to improve swimming speed and directionality. Here, we found that deletion of cetZ1 or expression of a GTPase-defective mutant caused the expected defects in cell shape, and also resulted in a substantial defect in the assembly of the motility machinery, including the archaellum base marker protein ArlD1, the chemotaxis sensory array adapter CheW1, and signal transducer CheY. Interestingly, moderate overexpression of cetZ1 (by 2-3 fold) reduced motility and the assembly and polar placement of the motility machinery without detectably affecting the rod shape of motile cells. We also investigated deletion of the conserved paralog cetZ2, which caused no detected defects in swimming or rod shape. However, overexpression of cetZ2 caused mild hyper-motility, whereas the cetZ2 GTPase-defective mutant reduced motility; these effects were dependent on the presence of cetZ1, suggesting that the altered cetZ2 might interfere with CetZ1's role in motility. Finally, we saw that a functional CetZ1-mTurquoise2 fusion strongly localized at the poles of mature motile cells, where it partially co-localized with the motility machinery markers. Overall, our results suggest that roles of CetZ1 in cell shape or potentially the organisation and structure of the cell poles influence the polar assembly of the motility machinery.

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.