Malaria remains a significant global health problem, mainly due to Plasmodium falciparum, which is responsible for most fatal infections. Infected red blood cells (iRBCs) evade spleen clearance by adhering to endothelial cells (ECs), triggering capillary blockage, inflammation, endothelial dysfunction and altered vascular permeability, prompting an endothelial transcriptional response. The iRBC IT4var04/HBEC-5i model, where iRBCs present IT4var04 (VAR2CSA) on their surface, was used to analyse the effects of iRBC binding on ECs at different temperature (37°C vs. 40°C). Binding of non-infected RBCs (niRBCs) and fever alone altered the expression of hundreds of genes in ECs. Comparing the expression profile of HBEC-5i cells cultured either in the presence of iRBCs or in the presence of niRBCs revealed significant upregulation of genes linked to immune response, nucleosome assembly, NF-kappa B signaling, angiogenesis, and antiviral immune response/interferon-alpha/beta signaling. Raising the temperature to 40°C, simulating fever, led to further upregulation of many genes, particularly those involved in cytokine production and angiogenesis. In summary, the presence of iRBCs stimulates ECs, activating several immunological pathways and affecting antiviral (-parasitic) mechanisms and angiogenesis. Our data uncovered the induction of the interferon-alpha/beta signaling pathway in ECs in response to iRBCs.

Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab’s β-lactamase (Bla Mab) that mediates β-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the β-lactamase BlaZ. BlaR binds extracellular β-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mtb encodes homologs of BlaRI, that regulate the Mtb β-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaRIMtb in Mab and hypothesized that they regulate blaMab. Surprisingly, neither deletion of blaRIMab nor overexpression of only blaIMab altered blaMab expression or β-lactam susceptibility. However, BlaI Mab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaI Mtb. Prompted by evidence that respiration inhibitors including clofazimine (CFZ) induce the BlaI regulon in Mtb, we found that CFZ triggers induction of blaIRMab and its downstream regulon. Highlighting an important role for BlaRI Mab in adapting to disruptions in energy metabolism, constitutive repression of the BlaI Mab regulon rendered Mab highly susceptible to CFZ. In addition to our unexpected findings that BlaIR Mab does not regulate β-lactam resistance, this study highlights the novel role for mycobacterial BlaRI-type regulators in regulating electron transport and respiration.

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA) to be a membrane-bound nuclease. We show that EcnA is Ca 2+-activated, present on the cell surface, and essential for transformation. While EcnA is capable of degrading several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion.

In bacteria, faithful DNA segregation of chromosomes and plasmids is mainly mediated by ParABS systems. These systems, consisting of a ParA ATPase, a DNA binding ParB CTPase, and centromere sites parS, orchestrate the separation of newly replicated DNA copies and their intracellular positioning. Accurate segregation relies on the assembly of a high-molecular-weight complex, comprising a few hundreds of ParB dimers nucleated from parS sites. This complex assembles in a multi-step process and exhibits dynamic liquid-droplet properties. Despite various proposed models, the complete mechanism for partition complex assembly remains elusive. This study investigates the impact of DNA supercoiling on ParB DNA binding profiles in vivo, using the ParABS system of the plasmid F. We found that variations in DNA supercoiling does not significantly affect any steps in the assembly of the partition complex. Furthermore, physical modeling, leveraging ChIP-seq data from linear plasmids F, suggests that ParB sliding is restricted to approximately 2-Kbp from parS, highlighting the necessity for additional mechanisms beyond ParB sliding over DNA for concentrating ParB into condensates nucleated at parS. Lastly, explicit simulations of a polymer coated with bound ParB suggest a dominant role for ParB-ParB interactions in DNA compaction within ParB condensates.

In bacteria, the chromosome is folded into a structure called the nucleoid. This compaction must facilitate and maintain key biological processes such as gene expression and DNA transactions (replication, recombination, repair, segregation). Chromosome and chromatin 3D-organization in bacteria has been a puzzle for decades. Chromosome conformation capture coupled to deep sequencing (HiC) in combination with other ‘omics’ approaches has allowed dissection of the structural layers that shape bacterial chromosome organization, from DNA topology to global chromosome architecture. Here we review the latest findings in the field and discuss the main features of bacterial genome folding.

Fluidity is an inherent property of biological membranes and its maintenance (homeoviscous adaptation) is important for optimal functioning of membrane-associated processes. The fluidity of bacterial cytoplasmic membrane increases with temperature or an increase in the proportion of unsaturated fatty acids and vice versa. We found strains deficient in the synthesis of guanine nucleotide analogues (p)ppGpp and lacking FadR, a transcription factor involved in fatty acid metabolism exhibited growth defect that was rescued by an increase in growth temperature or unsaturated fatty acid content. The strain lacking (p)ppGpp was sensitive to genetic or chemical perturbations that decrease the proportion of unsaturated fatty acids over saturated fatty acids. Microscopy showed the growth defect was associated with cell filamentation and lysis and rescued by combined expression of cell division genes ftsQ, ftsA and ftsZ from plasmid. The results implicate (p)ppGpp in the positive regulation of cell division during loss of membrane fluidity. To our knowledge, this is the first report of a (p)ppGpp mediated regulation needed for adaptation to membrane fluidity loss in bacteria.

In E. coli K-12, absence of dephospho-PtsN, is thought to cause an L-leucine sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val) sensitive acetohyrodxyacid synthase I (AHAS I), that renders residual AHAS activity susceptible to inhibition by Leu and K+.  This is thought to lead to AHAS insufficiency and a requirement for isoleucine (Ile). Herein we provide an alternate mechanism for LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS, indicate that impaired expression of the ilvBN operon jointly caused by the absence of dephospho-PtsN and the presence of Leu coupled to Leu mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α-ketobutyrate level, mediate elevation of ilvBN expression, and alleviate the LeuS. The requirement of dephospho-PtsN as a positive regulator of ilvBN expression, may buffer Ile biosynthesis against Leu mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realised in certain environments that impair AHAS function.

DNA viruses recognize viral DNA and package it into virions. Specific recognition is needed to distinguish viral DNA from host cell DNA. The λ-like phages are interesting and good models to examine viral DNA packaging, and the Escherichia coli phage λ has been extensively studied. Gifsy-1 is a Salmonella phage largely genetically isolated from coliphage λ due to their different host ranges. In this present work, Gifsy-1’s DNA packaging specificity was studied using a Gifsy-1-λ hybrid phage with the Gifsy-1 recognition elements – the TerS protein’s winged helix-turn-helix DNA binding motif and cosB, the TerS binding site. In vivo packaging studies showed Gifsy-1 packages λ DNA at ⁓50% and λ packages Gifsy-1 DNA at ⁓ 30% efficiency, and thus share the same DNA packaging specificity. N15 is a third phage that shares phage λ’s packaging specificity. Here N15 is shown to package Gifsy-1 DNA. Phage 21 fails to package λ and Gifsy-1 DNAs; the efficiencies are 0.01% and 1%, resp. A known incompatibility between the 21 helix-turn-helix motif and cosB λ is also proposed to account for the inability of 21 to package Gifsy-1 DNA.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated in the course of DNA replication, presumably acting as Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR mutations are tightly controlled by genome maintenance functions including RecA. In this study, we investigate the roles of DprA, RecFOR and RecBCD, which are cytoplasmic functions that load DNA single-strands with RecA. All three functions suppress SPDIR mutations in wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA alone, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest an antimutational function for DprA and offer an explanation for the ubiquity of dprA in the genomes of non-transformable bacteria.

Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease. The parasite has developed various mechanisms to get through its intricate life cycle and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector’s digestive tract as an epimastigote form, encountering fluctuating nutrient availability and oxidative stress caused by the digestion of red blood cells from the mammalian host blood meal. To unravel how the parasite’s metabolism adapts to these changing conditions, we conducted an analysis of the chemical species present in epimastigote forms. This involved comparing cultured parasites with those subjected to nutritional deficiency or oxidative stress using untargeted metabolomics. We looked at twenty-one samples: seven biological copies of parasites that were actively growing, seven samples that were put in a medium without nutrients for three hours, and seven samples that were treated with glucose oxidase for thirty minutes to make H 2O 2 continuously. Importantly, in all conditions, parasite viability was maintained when the samples were collected. Upon nutrient removal, we observed a substantial decrease in amino acids and carbohydrate metabolites, accompanied by the accumulation of fatty acids and steroids, with the predominance of inositol and sphingolipid metabolism, along with a simultaneous decrease in the levels of H 2O 2. In the presence of H 2O 2, a significant rise in components of the pentose pathway and specific amino acids such as methionine and serine occurred, along with pathways related to an increase in antioxidant species metabolism such as ribulose 5-phosphate and glyceric acid. Conversely, fatty acid and steroid levels decrease. We found no common increase in metabolites or lipids. In contrast, eight species (succinic acid, glutamic acid, valine, 2-hydroxyisocaproic acid, alanine, indolelactic acid, proline, and lanosterol) were consumed under both stresses. These findings underscore the rapid and distinct enrichment responses in amino acids, lipids, and carbohydrates required to cope with each different environmental condition. This information not only enhances our understanding of T. cruzi’s metabolic flexibility but also opens avenues for studying cellular metabolism changes in diverse environments used by the parasite.

Translation of messenger RNA (mRNA) in bacteria occurs in the steps of initiation, elongation, termination and ribosome recycling. The initiation step comprises multiple stages and uses a special transfer RNA (tRNA) called initiator tRNA (i-tRNA), which is first aminoacylated and then formylated using methionine and N 10-formyl-tetrahydrofolate (N 10-fTHF), respectively. Both methionine and N 10-fTHF are produced via one-carbon metabolism, linking translation initiation with active cellular metabolism. The fidelity of i-tRNA binding to the ribosomal peptidyl-site (P-site) is attributed to the structural features in its acceptor stem, and the highly conserved three consecutive G-C base pairs (3GC pairs) in the anticodon stem. While the acceptor stem region is important in formylation of the amino acid attached to i-tRNA and its initial binding to the P-site, the 3GC pairs are crucial in transiting i-tRNA through various stages of initiation. We utilized the feature of 3GC pairs to investigate the nuanced layers of scrutiny that ensure fidelity of translation initiation through i-tRNA abundance and its interactions with the components of the translation apparatus. We discuss the importance of i-tRNA in the final stages of ribosome maturation, as also the roles of the Shine-Dalgarno sequence, ribosome heterogeneity, initiation factors, ribosome recycling factor and coevolution of the translation apparatus in orchestrating a delicate balance between the fidelity of initiation and/or its leakiness to generate proteome plasticity in cells to confer growth fitness advantages in response to the dynamic nutritional states.

YbeX of Escherichia coli, a member of the CorC protein family, is encoded in the same operon with ribosome-associated proteins YbeY and YbeZ. Here, we report the involvement of YbeX in ribosomal metabolism. The ΔybeX cells accumulate distinct 16S rRNA degradation intermediates in the 30S particles and the 70S ribosomes. E. coli lacking ybeX has a lengthened lag phase upon outgrowth from the stationary phase. This growth phenotype is heterogeneous at the individual cell level and especially prominent under low extracellular magnesium levels. The ΔybeX strain is sensitive to elevated growth temperatures and to several ribosome-targeting antibiotics that have in common the ability to induce the cold shock response in E. coli. Although generally milder, the phenotypes of the ΔybeX mutant overlap with those caused by ybeY deletion. A genetic screen revealed partial compensation of the ΔybeX growth phenotype by the overexpression of YbeY. These findings indicate an interconnectedness amongst the ybeZYX operon genes, highlighting their roles in ribosomal assembly and/or degradation.

The human digestive system harbors a vast diversity of commensal bacteria and maintains a symbiotic relationship with them. However, imbalances in the gut microbiota, known as dysbiosis, accompany various diseases, such as inflammatory bowel diseases (IBDs) and colorectal cancers (CRCs), which have a significant impact on the well-being of people globally. Glycosylation of the mucus layer is a key factor that plays a critical role in maintaining the homeostatic environment in the gut. This review delves into the ways in which the gut microbiota, gut epithelial barrier, and immune cells work together to establish a balanced gut environment. Specifically, the role of glycosylation in regulating immune cell responses and mucus metabolism in this process is examined. Additionally, the review explores various modulatory approaches used to maintain or restore the functional gut microbiota.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is required to maintain the membrane’s biophysical properties and fluidity. The malaria parasite Plasmodium can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver stage development. However, when scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which normally initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective preerythrocytic immunity.

Super-resolution fluorescence microscopy techniques developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These techniques have the potential to study bacterial macromolecular complexes such as secretion systems with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy in molecular bacteriology with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of bacterial molecular machines in action.

Apicomplexan parasites comprise significant pathogens of humans, livestock, and wildlife, but also represent a diverse group of eukaryotes with interesting and unique cell biology. Study of cell biology in apicomplexan parasites is complicated by their small size, and historically this has required the application of cutting-edge microscopy techniques to investigate fundamental processes like mitosis or cell division in these organisms. Recently, a technique called expansion microscopy has been developed, which rather than increasing instrument resolution like most imaging modalities, physically expands a biological sample >4-fold. In only a few years since its development, a derivative of expansion microscopy known as ultrastructure-expansion microscopy (U-ExM) has been widely adopted and proven extremely useful for studying cell biology of Apicomplexa. Here we review the insights into apicomplexan cell biology that have been enabled through the use of U-ExM, with a specific focus on Plasmodium, Toxoplasma, and Cryptosporidium. Further, we summarise emerging expansion microscopy modifications and modalities and forecast how these may influence the field of parasite cell biology in future.

The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low phosphate low magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, a known activator of SPI-2. Here we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC ( i.e., tktD, tktE) of this bacterium. Transketolases contribute to the non-redox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR binding sites between stm2344 and pagR.