Molecular Microbiology

Inorganic polyphosphate (polyP) is a universally conserved biopolymer central to bacterial stress survival, yet understanding of its roles derives almost entirely from Gram-negative models in which polyP accumulates intracellularly following nutrient downshift. We examined polyP metabolism in the Gram-positive spore-forming bacterium Bacillus cereus using deletions of the polyP kinases PPK1 and PPK2 and the exopolyphosphatase PPX. Intracellular polyP synthesis required PPK1 and was opposed by PPX and PPK2: ppx mutants accumulated polyP in sporulation medium by 24 hours, ppx ppk2 double mutants accumulated more, and no ppk1 mutant accumulated any. A ppk1 ppx double mutant could not be generated, suggesting that unopposed PPK2 activity is lethal. Unlike Escherichia coli and Pseudomonas aeruginosa, B. cereus did not accumulate polyP after shift to minimal medium, increasing only modestly in stationary phase. Fluorescence and transmission electron microscopy localized intracellular polyP to electron-dense granules within ribosome-depleted cytoplasm. Cells bearing these granules remained membrane-intact yet failed to resume growth over 8 hours in rich medium, leading us to propose that polyP drives ribosome sequestration into condensates and a hibernation-like state. Unexpectedly, B. cereus also released close to 100{micro}M polyP extracellularly during late stationary phase, even in a ppk1 ppk2 mutant lacking both known synthetases. Extracellular polyP resisted hydrolysis by purified PPX even after deproteinization, indicating an atypical structure. Bacillus thuringiensis and Bacillus anthracis released similar amounts of extracellular polyP. Together these results identify two distinct polyP pools in the B. cereus group: a PPK1-dependent intracellular pool and an extracellular pool made by an uncharacterized pathway. ImportanceBacillus cereus is a spore-forming bacterium that causes foodborne illness and persists in soil and food-processing environments, where survival depends on managing phosphate and energy reserves during starvation. Inorganic polyphosphate (polyP), an ancient polymer used by nearly all cells to withstand stress, has been studied almost entirely as a molecule stored inside bacteria. We show that Bacillus cereus maintains two separate polyP pools. The internal pool is made by a known enzyme (PPK1) and is associated with dormant cells whose protein-making machinery appears to be packed away. The external pool is made without any known polyP-synthesizing enzyme, pointing to a novel polyP synthesis pathway that is shared with the close relatives Bacillus thuringiensis and Bacillus anthracis.

Onion tissues produce antimicrobial thiosulfinates after tissue damage and cellular decompartmentalization. Burkholderia gladioli pv. alliicola (Bga), a common onion pathogen, encodes a thiosulfinate tolerance gene (TTG) cluster that protects the bacterium during thiosulfinate exposure. Previous work showed that the TTG cluster contributes to foliar infection but has little effect on infection of onion bulb tissue. To further examine Bga-thiosulfinate interactions in foliar and bulb tissues, we used a thiosulfinate-responsive PaltR-Lux reporter strain to determine when and where Bga encounters thiosulfinates. In leaves, Bga-induced necrosis was associated with de-repression of the PaltR-Lux reporter and coincided with a contribution of the TTG cluster to bacterial population size, indicating thiosulfinate exposure during foliar infection. In contrast, TTG mutants and wild-type (WT) strains showed similar growth in scales, and PaltR-Lux signal declined as scale necrosis progressed, suggesting limited thiosulfinate exposure during bulb colonization. However, when necrosis was induced by the non-native toxin pantaphos, PaltR-Lux was de-repressed and recovery of the TTG mutant was reduced. These results indicate that Bga encounters thiosulfinates during foliar infection but largely avoids exposure during bulb infection. Preconditioning the TTG mutant in onion scale tissue did not alter its thiosulfinate sensitivity in vitro, arguing against an infection-associated thiosulfinate exclusion mechanism. In contrast, partial rescue of the TTG mutant by the WT strain in zone-of-inhibition co-plating assays suggests extracellular thiosulfinate detoxification. Together, these findings indicate that Bga detoxifies thiosulfinates released during bulb necrosis, limiting thiosulfinate exposure during onion bulb infection. The molecular basis for detoxification and tissue specificity remain unresolved.

Phage D29 infects Mycobacterium smegmatis mc2 155 and has a non-canonical lysis cassette that encodes two endolysin proteins (Lysin A and Lysin B) and a single two transmembrane domain (TMD) protein, LysA2a similar to F1 cluster phage LysF1a. A 1TMD LysF1b homolog, LysA2b, is encoded by a gene found downstream of the tape measure. Exogenous expression of both LysA2 proteins in tandem is a cytotoxic to M. smegmatis. Deletion of lysA2a produces phages that are lysis competent with a 10-minute triggering delay and 30% plaque size reduction. Deletion of lysA2b results in severe lysis defects manifest by 70% reduced plaque size, delayed lysis timing and reduced burst size. Deletion of both lysA2 genes results in phages that are viable and show lysis phenotypes like the lysF1b deletion. Genetic complementation of lysA2b deleted phage with the lysF1b gene fully complements the lysis phenotypes but alters the triggering time to that of an F1 cluster phage. Energy poisons trigger lysis prematurely in all phages with lysA2 gene deletions. Lysis recovery mutants (LRM) isolated from phages lacking the lysA2b genes generate wild type plaque size and have point mutations that map to TMD1 or the C-terminal region of the lysA2a gene. LRMs isolated from phages lacking both lysA2 genes show premature lysis and have mutations that all map to residue C31 of a novel lipoprotein (gene 64). Deletion of gene 64 does not change wild type D29 lysis phenotypes or rescue the lysis defects of any of the lysA2 mutants. A fitness/competition assay shows that loss of the lysA2 genes imposes a substantial competitive fitness cost. These finding support a lysis regulatory network model where the 2TMD protein is maintained in an inactive state until activated by its cognate 1TMD lysis regulator and the lipoprotein has accessory function that may enhance lysis efficiency.

Contact with solid surfaces activates signaling pathways that promote biofilm formation in many bacteria. The alphaproteobacterium Caulobacter crescentus uses its flagellum to sense surfaces and responds by synthesizing an adhesive called the holdfast. The C. crescentus surface sensing pathway can be activated by mutating genes required for the assembly of the flagellum or genes required for chemotaxis. However, flagellar assembly and chemotaxis mutations activate distinct surface sensing pathways that differ in the activation of the diguanylate cyclase PleD. Here, we used a genome-wide screen to identify cmrA (CCNA_02061) as a crucial determinant of hyperadhesion in the chemotaxis mutant {Delta}cheYII. Genetic analysis showed that cmrA is important for activation of PleD in a context-specific manner. It is dispensable in wild-type and late-stage flagellar ({Delta}flgH) mutant backgrounds but promotes adhesion in early-stage flagellar assembly ({Delta}fliF), chemotaxis ({Delta}cheYII) and stator ({Delta}motB) mutant backgrounds. Fluorescently tagged CmrA displays a mostly cytoplasmic localization in genetic backgrounds where cmrA is dispensable for adhesion but localizes to the cell pole in backgrounds where it regulates adhesion. Structural modeling indicates that CmrA is a degenerate, catalytically inactive GGDEF/EAL domain containing protein, but cmrA alleles with mutated conserved c-di-GMP coordinating residues are unable to support hyperadhesion. Our results indicate that altering the directional switching of MotAB stators recruits CmrA to the cell pole where it activates PleD to drive surface adaptation. Ultimately, this work underscores the complexity of flagellar surface sensing by highlighting how the many rotational states of the motor stimulate distinct but overlapping c-di-GMP signaling pathways.

Functional type III secretion in Gram negative bacteria relies on precise substrate targeting and a strict order of secretion with early, intermediate, and late substrates. Type III secretion chaperones facilitate these processes by maintaining substrates in a partially unfolded, secretion-competent state and serving as order-specific targeting factors. Early needle filament assembling substrates are chaperoned by none or class III chaperones, intermediate translocator-type substrates by class II and late effector-type substrates by class I chaperones. In case of hydrophobic transmembrane effectors, chaperones may also serve to prevent erroneous mistargeting of these substrates to the bacterial inner membrane. Here, we characterized the Salmonella transmembrane effectors SseF and SseG and their chaperone SscB, encoded in the operon sscB-sseF-sseG, in order to gain a deeper understanding of the underlying molecular requirements of targeting of this special class of substrates. We show that the gene linkage of SscB and SseF is critical for these proteins stability and SseF secretion. Counterintuitively, SscB revealed to feature a class II chaperone structure with a class I chaperone function. Likewise, SseF and SseG harbour conserved, translocator-like chaperone-binding motifs (PXI/LXXP) but were secreted as late substrates, independent of the gatekeeper protein SsaL. These findings challenge the current chaperone classification and our understanding of the molecular basis of the hierarchy of substrate secretion. They show that chaperone structure is not a sufficient molecular determinant for the correct order of substrate secretion.

Antigenic variation in Trypanosoma brucei relies on strict monoallelic expression of variant surface glycoprotein (VSG) genes from a single telomeric expression site (ES), a process sustained by the extranucleolar RNA polymerase I (Pol I) transcriptional body known as the expression site body (ESB). Although the ESB is essential for VSG expression, the mechanisms governing its assembly and maintenance remain poorly understood. Here, we identify SUMOylation as a central regulator of ESB organization and demonstrate that the balance between SUMO conjugation and deconjugation determines the transcriptional state of VSG expression sites. Ectopic expression of the SUMO protease TbSENP disrupted the highly SUMOylated nuclear focus associated with the active-ES, displaced Pol I from its extranucleolar compartment, and markedly increased VSG in situ switching frequency, indicating that continuous SUMOylation is required to preserve ESB integrity. Conversely, targeted recruitment of the SUMO-conjugating enzyme TbUBC9 to a silent ES locally restored SUMOylation, induced de novo formation of an extranucleolar Pol I compartment, activated transcription of the corresponding telomeric VSG gene, and generated stable antigenic switchers expressing the new surface coat. Local SUMOylation preceded Pol I redistribution, supporting a model in which SUMO-dependent interactions nucleate assembly of a transcriptionally competent ESB. Together, our findings identify SUMOylation as both a structural and regulatory determinant of nuclear organization in T. brucei and suggest that dynamic SUMO homeostasis governs the assembly, maintenance, and remodeling of this specialized transcriptional body. Significance StatementAntigenic variation in Trypanosoma brucei depends on the monoallelic expression of Variant Surface Glycoprotein (VSG) genes from a specialized RNA polymerase I transcriptional compartment known as the Expression Site Body (ESB). However, the molecular signals that govern transitions between active and silent expression sites have remained unknown. We show that SUMOylation acts as a reversible molecular switch: disruption of SUMO homeostasis dismantles ESB organization and promotes VSG switching, whereas localized SUMOylation is sufficient to nucleate a functional transcriptional compartment and activate a silent VSG expression site. Our findings establish SUMOylation as a central regulator of nuclear architecture and antigenic variation.

Protein phosphorylation is a central regulatory mechanism that enables organisms to adapt to changing environmental conditions. The hyperthermophilic archaeon Sulfolobus acidocaldarius encodes only two phosphatases: the dual-specificity phosphatase PTP and the serine/threonine phosphatase PP2A. PP2A has previously been implicated in archaellum regulation, and its deletion results in a hypermotile phenotype. Under starvation conditions, PP2A associates with a stress regulatory module comprising the archaellum repressors ArnA and ArnB, the universal stress protein UspA, and a GPN-loop GTPase. Here, we investigated PP2A-associated proteins under normal growth conditions and following UV-induced DNA damage. Pulldown experiments using a genomically HA-tagged PP2A strain identified a PP2A-associated basal regulatory module consisting of ArnA, ArnB, ArnE, and PTP, distinct from the previously described starvation-associated network. In addition, several proteins involved in the biogenesis and regulation of type IV pili co-purified with PP2A. Functional analyses using thermomicroscopy and electron microscopy revealed that deletion of {Delta}pp2a, {Delta}arnA, or {Delta}arnB abolishes Aap-pilus formation and twitching motility, demonstrating that the PP2A regulatory network controls both swimming and surface-associated motility. In contrast, the same network exerted only a modulatory effect on UV-induced cell aggregation. Together, our findings establish PP2A as a central regulator coordinating multiple archaeal surface structures through phosphorylation-dependent signaling.

Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call "developmental winners", are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology.

N2-methylguanosine (m2G) is widely found at multiple positions in tRNAs across the three domains of life. Tryptophan tRNA from Thermococcus kodakarensis contains m2G at position 67. We previously proposed that the tRNA m2G methyltransferase Trm14 is responsible for m2G67 formation in tRNATrp from T. kodakarensis, although Trm14 was originally identified as the enzyme catalyzing m2G6 formation in tRNACys in Methanocaldococcus jannaschii. Thus, it remained unclear whether Trm14 could also methylate G67. Here, we characterized archaeal Trm14. Biochemical analyses using recombinant T. kodakarensis Trm14 revealed that the enzyme catalyzes m2G formation at positions 6 and 67 in T. kodakarensis tRNACys and tRNATrp transcripts, respectively. Mass spectrometric analyses demonstrated the loss of m2G6 and m2G67 in native tRNACys and tRNATrp, respectively, from a T. kodakarensis trm14 gene disruptant strain, providing direct evidence for the dual-site specificity of T. kodakarensis Trm14. The growth phenotype of the trm14 gene disruptant strain was comparable to that of the wild-type strain. In contrast, a trm14/trm11 double disruptant, in which trm11 encodes the tRNA m2G10/m22G10 methyltransferase, exhibited severe growth retardation at 95 {degrees}C. This suggests that m2G6/m2G67 and m2G10/m22G10 cooperatively contribute to cellular fitness at high temperatures. Biochemical analyses revealed that Trm14 methylates all 46 T. kodakarensis tRNA transcripts. Furthermore, we found that recombinant M. jannaschii Trm14 methylated both positions. In contrast, the bacterial ortholog TrmN modified only position 6 in tRNA. Overall, this study expands our understanding of archaeal Trm14 by demonstrating its broader substrate specificity and the physiological significance of these modifications under hyperthermophilic conditions.

A growing body of literature supports a critical role for nucleomodulins, proteins that traffic to host cell nuclei and manipulate nuclear processes, in intracellular bacterial pathogenesis. Here, we identify the Mycobacterium tuberculosis (Mtb) secreted protein Rv1075c as a nucleomodulin that targets a histone modifying protein complex in macrophages. We report that {Delta}Rv1075c Mtb infection elicits a blunted transcriptional response in inflammatory and lipid metabolism pathways and fails to induce foamy macrophage formation in the lungs of infected mice. Using an unbiased mass-spectrometry based approach, we found that Rv1075c interacts with components of the H3K4me3-depositing SET1 histone methyltransferase complex, and that this interaction is required for Rv1075c nuclear localization. Consistent with Rv1075c inhibiting SET1 activity, SET1 deficiency results in hyperinduction of inflammatory genes in activated macrophages. Together, these findings reveal a mechanism by which Mtb engages host chromatin machinery and support a model whereby Rv1075c exploits the SET1 complex to promote a host environment conducive to mycobacterial persistence.

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of alpha-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

Streptococcus pneumoniae is a major human respiratory pathogen. The bacterial 70S ribosome is a target of many clinically important antibiotics. The N-terminus of ribosomal protein bL27 extends into the peptidyl transferase center and contributes to the translation process. In Firmicutes, full length bL27 contains an 8-12 amino acid N-terminal extension that is absent from Gram-negative bacteria. This extension is cleaved by the protease Prp, which is absent from organisms lacking the extension. Prp-mediated cleavage of bL27 is essential in Staphylococcus aureus, and Prp has been proposed as a potential antibiotic target. Here, we show that in S. pneumoniae strain TIGR4, a {Delta}prp mutant remained viable, and produced ribosomes containing cleaved bL27, whereas deletion of prp was not tolerated in strain D39. These results suggested the presence of an alternate bL27-processing protease in TIGR4 that was absent from D39. Using a combination of genomics, proteomics and biochemical analyses, we identified this enzyme as the product of previously uncharacterized gene SP_1145, encoding a protease that we named Ribosome rescue protease (Rrp). SP_1145 is carried on a mobile genetic element that is present in strain TIGR4, but absent from D39. Our findings shed light on an alternative mechanism for bL27 maturation, and indicate that some strains of S. pneumoniae harbor horizontally acquired redundant pathways for this essential ribosome processing step.

Small annotated open reading frames are the most neglected part of the functionally uncharacterised M. tuberculosis genome. We revisit Rv0810c, a 60-residue protein carrying the unknown-function domain DUF3073 (Pfam PF11273), flagged as an Actinobacteria-signature protein in 2006 but never studied since. Rv0810c is genuinely translated (detected in 11 of 16 M. tuberculosis proteomic datasets), with no significant human homologue, no neighbouring-gene overlap, and no CRISPR-interference polar effect on either flank. Residue-resolved confidence reveals a bipartite architecture: a rigid 33-residue module (pLDDT 91.9) followed by an extended, acidic, intrinsically disordered tail (radius of gyration 22.8 A against 11-12 A expected for a globular protein). The gene is under strong purifying selection (non-synonymous/synonymous ratio 0.86 against 1.93 among 74 size-matched controls, p=5.8x10-8), and its two commonest missense variants are each confined to one sub-lineage, indicating clonal expansion rather than relaxed constraint. DUF3073 is present without a single confirmed loss across 260 well-supported Actinomycetia genera. Despite this conservation, eight independent computational strategies, spanning sequence, structure, electrostatic-patch, embedding-similarity and homo-oligomerisation searches, converge on the same negative: no assignable fold, binding site, or functional neighbour in curated or uncurated sequence space. A phosphosite (Thr24), reproducibly reported by three laboratories, cannot be attributed to a kinase by chemical-genetic or sequence-motif evidence. The contradiction between predicted cytoplasmic topology and macrophage-secretory-fraction detection is narrowed, not resolved: ESX secretion, an immunodominant-epitope confound and host-induced transcription are excluded. Rv0810c exemplifies a class of genuinely uncharacterisable small proteins for which negative reporting, not a manufactured function, is the honest outcome.

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to front-line antibiotics has focused efforts on understanding how alternative therapies, such as the expanded use of gentamicin (Gen), might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for the treatment of gonorrheal infections, we previously used RNA-seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level and showed that expression of the putative HicA-HicB toxin-antitoxin (TA) system was increased in response to sub-lethal Gen. Importantly, loss of this TA system resulted in reduction of Ng biofilm formation in a strain specific manner. Focusing on this strain specificity, we found that the CpxR/CpxA two-component system (TCS) influences expression of the hicAB operon independently of HicB autoregulation. We now report that CpxR selectively binds to the hicAB operon to enhance expression of hicAB but does not interfere with binding of HicB to the promoter region. Furthermore, we show that single base pair differences in the intergenic region between hicA and hicB impact regulation by CpxR. Hence, the regulation of the HicAB TA in gonococcal strains is a highly coordinated response that can involve autoregulation by HicB and the CpxRA TCS. We propose that this dual regulatory scheme maximizes the ability of Ng to respond to Gen and hostile environmental conditions.

Nitrogen-fixing Nostoc species form symbiotic relationships with diverse plants, yet the role of specialized metabolites in these interactions remains poorly understood. Here, we identify a previously cryptic gene cluster coding for the biosynthesis of the lanthipeptide nostolanthin (nlt), which is rapidly induced upon physical contact between Nostoc punctiforme and the liverwort Blasia pusilla. Despite its robust transcriptional activation, nostolanthin remained undetectable in its native producer by conventional metabolomic analyses. Heterologous reconstitution of the biosynthesis showed that the lanthipeptide synthetase NltM produces a bicyclic class II lanthipeptide containing a non-cyclized dehydroamino acid together with a free cysteine residue. We show that this lanthipeptide undergoes covalent oligomerization into high-molecular-weight assemblies, a process favored under native cyanobacterial expression conditions. An antibody raised against the oligomeric peptide enabled the detection of secreted nostolanthin in cyanobacteria and revealed that it accumulates predominantly in an oligomeric form. Comparative genomics showed that nostolanthin belongs to a larger family of Nif11-type-lanthipeptide biosynthetic gene clusters, consistently associated with homologous two-component regulatory systems. Importantly, oligomeric, but not monomeric nostolanthin, accelerated the establishment of physical contact between Nostoc and B. pusilla. Together, these findings reveal oligomerization by covalent bond formation as a previously unrecognized mode of lanthipeptide maturation and identify nostolanthin as a host-responsive peptide that regulates the transition from a free-living to a symbiotic lifestyle.

Taurine is an abundant sulfur-containing metabolite with diverse roles in cellular physiology across many organisms, yet its biosynthesis and biological functions remain largely unexplored in fungi. Here, we provide evidence for endogenous taurine production in the major human fungal pathogen Candida albicans and identify Csd1, a cysteine sulfinic acid decarboxylase (CSAD)-related protein, as a major determinant of this process. Loss of CSD1 nearly abolished intracellular taurine and caused extensive remodeling of sulfur metabolism, including cysteine accumulation and altered abundance of methionine-cycle metabolites. Consistent with these metabolic defects, csd1 cells exhibited impaired growth and increased sensitivity to cysteine, oxidative and osmotic stresses, elevated temperature, reactive sulfur species, and the antifungal drugs amphotericin B and caspofungin. Exogenous taurine selectively rescued a subset of these phenotypes, indicating that CSD1 loss causes both taurine-dependent and broader metabolic defects. Csd1 was also required for normal hyphal morphogenesis, and csd1 cells displayed markedly attenuated virulence in a Galleria mellonella systemic infection model. Comparative sequence analysis revealed conservation of key features of the pyridoxal 5'-phosphate-dependent catalytic machinery shared with mammalian and bacterial CSADs, together with divergence within the predicted substrate-recognition pocket. Our genetic data further suggest that taurine production in C. albicans differs from the canonical metazoan cysteine sulfinic acid pathway and may involve branched or redundant routes. Together, these findings establish endogenous taurine production as a new facet of fungal sulfur metabolism and identify Csd1-dependent metabolism as an important contributor to sulfur homeostasis, stress adaptation, morphogenesis, and pathogenic fitness in C. albicans.

Human-targeted non-antibiotic drugs are increasingly recognized for their intrinsic antibacterial activity, yet Gram-negative pathogens such as Acinetobacter baumannii exhibit substantial tolerance to these compounds. This tolerance is largely attributed to restricted outer membrane permeability and the activity of multidrug efflux systems. While Resistance Nodulation Division (RND) transporters have been extensively studied, the contribution of individual Major Facilitator Superfamily (MFS) transporters to non-antibiotic drug tolerance remains poorly understood. Here, we investigated H0N29_04330, designated Drug Resistance Transporter A (DrtA), a Bcr/CflA subfamily MFS transporter, to define its substrate specificity and contribution to antibiotic and non-antibiotic drug tolerance. DrtA was highly conserved across the A. calcoaceticus-baumannii complex and exhibited broad substrate specificity when heterologously expressed in an efflux-deficient Escherichia coli background, conferring resistance to benzalkonium, ethidium bromide, phenicols, and the antineoplastic agent mitomycin C. Intriguingly, drtA expression increased E. coli susceptibility to the antifolate compounds methotrexate and aminopterin, suggesting that DrtA may recognize folate-related metabolites rather than function as a dedicated antifolate transporter. In contrast, loss of drtA in its native A. baumannii host primarily impaired tolerance to mitomycin C, highlighting a context-dependent physiological role influenced by the extensive functional redundancy among A. baumannii efflux systems. Site-directed mutagenesis further identified M18 and the membrane-embedded protonatable residue D26 as critical determinants of DrtA transport activity and substrate recognition. Together with previous characterization of CraA, our findings demonstrate that Bcr/CflA subfamily MFS transporters contribute to protection against structurally diverse human-targeted compounds and expand the functional landscape of efflux-mediated intrinsic tolerance beyond conventional antibiotic resistance.

The RND efflux pump FemT encoded by SAUSA300_2213 of Staphylococcus aureus USA300 is co-transcribed with femX which has an essential role in synthesizing the Lipid II precursor of peptidoglycan. Anticipating that this arrangement reflects a critical accessory role for femT, we constructed USA300{Delta}femT to assess its function. Although growth of USA300{Delta}femT in tryptic soy broth (TSB) was not impaired, transcriptomic data revealed a mild cellular stress response, accompanied by reduced expression of ohyA and crt genes involved in fatty acid metabolism and carotenoid lipid synthesis respectively. Accordingly, USA300{Delta}femT exhibited impaired growth on exposure to saturated and unsaturated fatty acids, and exposure to subinhibitory 50 {micro}M palmitic acid promoted accumulation of reactive oxygen species, reduced respiratory activity, and altered membrane function and morphology. The transcriptome of cells grown under this condition revealed strongly attenuated expression of ohyA and crt, and several genes required for oxidative and anaerobic respiration, concomitant with strongly enhanced expression of several stress response pathways. Cellular metabolites were also profoundly altered. Finally, lipidomic analysis of USA300{Delta}femT exposed to oleic acid revealed increased incorporation of oleic acid into phosphatidylglycerol, accompanied by a significant reduction in undecaprenol C55 lipid carrier, and respiratory quinones MK-7 and MK-8. Our data are consistent with a role for FemT in maintaining cellular lipid homeostasis by promoting efflux of isoprenoid and carotenoid lipids that are prone to oxidative damage, including C55 and menaquinones that undergo cyclic reactions in peptidoglycan synthesis and electron transport. IMPORTANCEThe FemT efflux pump of S. aureus is co-expressed in an operon with femX encoding an essential enzyme needed to complete the synthesis of peptidoglycan precursor Lipid II. Although this alluded to a specific role for FemT in supporting peptidoglycan synthesis, our data are instead consistent with a general role in efflux of cellular isoprenoids and carotenoid lipids that are susceptible to oxidation during routine cellular functions. Consequently, S. aureus became strongly dependent on FemT function when exogenous host-derived fatty acids were being actively metabolized. This represents a significant advance in our understanding of the role of an RND efflux pump in supporting routine growth-related functions of S. aureus and exposes a function that could be targeted to impair S. aureus growth on exposure to host-derived fatty acids.

Intracellular pathogens localize to various niches in the host cells to avoid immune detection. However, very little is known about bacteria that enter the host nuclei. Here we report that Burkholderia thailandensis, a facultative intracellular pathogen, can enter eukaryotic nuclei and replicate. Nuclear invasion events were rare, occurring 1 in 500-1,000 infected cells, and inhibition of cell division further reduced the frequency of these events. Moreover, we show that nuclear entry requires actin tail motility, although it is independent of other virulence factors such as the Type III Secretion System, Type VI Secretion System-5, and flagella motility. Inactivation of actin tail motility by deleting bimA or inhibition of actin polymerisation by cytochalasin D abolished nuclear entry. Surprisingly, we observed that accumulation of B. thailandensis in the nucleus activated assembly of the Type VI Secretion Systems-5. We further show that Shigella flexneri also enters nucleus in an actin polymerization dependent mechanism. Together, we show that actin tail forming intracellular pathogens occasionally localize to the nucleus, and while this largely requires host cell division, it may provide pathogens with a protective niche in certain mitotically active cells, such as skin, gut or epithelial cells.

The type VI secretion system (T6SS) is a major weapon used by Pseudomonas aeruginosa to antagonize competing bacteria through the delivery of a diverse repertoire of toxic effectors. Although several P. aeruginosa T6SS effectors target bacterial membranes, the mechanisms underlying membrane disruption remain poorly characterized. Here, we study TpeX (PA5265), an accessory T6SS effector from P. aeruginosa PAO1 with structural similarity to the VasX pore-forming effector of Vibrio cholerae. We show that membrane-targeted TpeX exerts a bactericidal activity in Escherichia coli, resulting in dissipation of the membrane potential and loss of membrane integrity. The TpeX C-terminal region containing the predicted colicin-like transmembrane domain is sufficient to confer toxicity, although with reduced activity, supporting its role as the membrane-disrupting module. TpeX also oligomerizes upon membrane targeting, forming at least dimers, and structural modelling predicts a membrane-embedded pore compatible with the observed permeabilization phenotype. We further identify TpiX (PA5264), the protein encoded by the downstream gene, as the cognate immunity protein, which partially protects cells from TpeX toxicity and interacts with TpeX. Finally, AlphaFold 3 modelling predicts an interaction between TpeX and the HcpB-VgrG6 T6SS spike, suggesting a possible mechanism for effector recruitment and delivery. Together, our results identify TpeX as a bactericidal, colicin-like pore-forming T6SS effector whose membrane activity is controlled by a cognate immunity protein, thereby expanding the repertoire of membrane-targeting weapons used by P. aeruginosa in interbacterial competition.