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Microbiology Spectrum Jun 2024Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial...
Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX in was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to BamB, which functions as a subunit of the β-barrel assembly machinery complex that is responsible for the assembly of β-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in , we introduced the gene into an ∆ mutant. Growth deficiencies in the ∆ mutant were restored when was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by deletion was restored upon expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in . As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCE is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in K12. Furthermore, we demonstrate that GOX1969 is capable of complementing knockout phenotypes in K12. Taken together, these findings enhance our understanding of physiology and expand the list of potential targets for future industrial strain design.
PubMed: 38916353
DOI: 10.1128/spectrum.01060-24 -
Microbiology Spectrum Jun 2024All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the...
All sulfur transfer pathways have generally a l-cysteine desulfurase as an initial sulfur-mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In , the housekeeping l-cysteine desulfurase IscS has several interaction partners, which bind at different sites of the protein. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron-sulfur (Fe-S) cluster assembly have been mapped, in addition to TusA, which is required for molybdenum cofactor biosynthesis and mnmsU34 tRNA modifications, and ThiI, which is involved in thiamine biosynthesis and sU8 tRNA modifications. Previous studies predicted that the sulfur acceptor proteins bind to IscS one at a time. TusA has, however, been suggested to be involved in Fe-S cluster assembly, as fewer Fe-S clusters were detected in a mutant. The basis for this reduction in Fe-S cluster content is unknown. In this work, we investigated the role of TusA in iron-sulfur cluster assembly and iron homeostasis. We show that the absence of TusA reduces the translation of , thereby leading to pleiotropic cellular effects, which we dissect in detail in this study.IMPORTANCEIron-sulfur clusters are evolutionarily ancient prosthetic groups. The ferric uptake regulator plays a major role in controlling the expression of iron homeostasis genes in bacteria. We show that a mutant is impaired in the assembly of Fe-S clusters and accumulates iron. TusA, therefore, reduces mRNA translation leading to pleiotropic cellular effects.
PubMed: 38916309
DOI: 10.1128/spectrum.00556-24 -
Disease Models & Mechanisms Jun 2024Survival for children with cancer has primarily improved over the past decades due to refinements in surgery, radiation and chemotherapy. Although these general... (Review)
Review
Survival for children with cancer has primarily improved over the past decades due to refinements in surgery, radiation and chemotherapy. Although these general therapies are sometimes curative, the cancer often recurs, resulting in poor outcomes for patients. Fusion-driven pediatric soft tissue sarcomas are genetically defined by chromosomal translocations that create a chimeric oncogene. This distinctive, almost 'monogenic', genetic feature supports the generation of animal models to study the respective diseases in vivo. This Review focuses on a subset of fusion-driven pediatric soft tissue sarcomas that have transgenic animal tumor models, which includes fusion-positive and infantile rhabdomyosarcoma, synovial sarcoma, undifferentiated small round cell sarcoma, alveolar soft part sarcoma and clear cell sarcoma. Studies using the animal models of these sarcomas have highlighted that pediatric cancers require a specific cellular state or developmental stage to drive tumorigenesis, as the fusion oncogenes cause different outcomes depending on their lineage and timing of expression. Therefore, understanding these context-specific activities could identify targetable activities and mechanisms critical for tumorigenesis. Broadly, these cancers show dependencies on chromatin regulators to support oncogenic gene expression and co-opting of developmental pathways. Comparative analyses across lineages and tumor models will further provide biological and therapeutic insights to improve outcomes for these children.
Topics: Animals; Humans; Sarcoma; Disease Models, Animal; Oncogene Proteins, Fusion; Child
PubMed: 38916046
DOI: 10.1242/dmm.050704 -
BioRxiv : the Preprint Server For... Jun 2024Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the...
Zinc knuckle (ZCCHC) motif-containing proteins are present in unicellular and multicellular eukaryotes and most ZCCHC proteins with known functions participate in the metabolism of various classes of RNA, such as mRNAs, ribosomal RNAs, and microRNAs. The Arabidopsis ( ) genome encodes 69 ZCCHC-containing proteins, but the functions of most remain unclear. One of these proteins is CAX-INTERACTING PROTEIN 4 (CXIP4), which has been classified as a PTHR31437 family member, along with human SREK1-interacting protein 1 (SREK1IP1), which is thought to function in pre-mRNA splicing and RNA methylation. Metazoan SREK1IP1-like and plant CXIP4-like proteins only share a ZCCHC motif, and their functions remain almost entirely unknown. We studied two loss-of-function alleles of Arabidopsis , the first mutations in PTHR31437 family genes described to date: is likely null and shows early lethality, and is hypomorphic and viable, with pleiotropic morphological defects. The mutant exhibited deregulation of defense genes and upregulation of transcription factor encoding genes, some of which might explain its developmental defects. This mutant also exhibited increased intron retention events, and the specific functions of misspliced genes, such as those involved in "gene silencing by DNA methylation" and "mRNA polyadenylation factor" suggest that CXIP4 has additional functions. The CXIP4 protein localizes to the nucleus in a pattern resembling nuclear speckles, which are rich in splicing factors. Therefore, is required for plant survival and proper development, and mRNA maturation.
PubMed: 38915646
DOI: 10.1101/2024.06.06.597795 -
BioRxiv : the Preprint Server For... Jun 2024Antibacterial proteins inhibiting have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which...
Antibacterial proteins inhibiting have been identified in various phages and explored as antibiotic alternatives. Here, we isolated a phiKZ-like phage, Churi, which encodes 364 open reading frames. We examined 15 early-expressed phage proteins for their ability to inhibit bacterial growth, and found that gp335, closely related to phiKZ-gp14, exhibits antibacterial activity. Similar to phiKZ-gp14, recently shown to form a complex with the ribosome, we predict experimentally that gp335 interacts with ribosomal proteins, suggesting its involvement in protein translation. GFP-tagged gp335 clusters around the phage nucleus as early as 15 minutes post-infection and remains associated with it throughout the infection, suggesting its role in protein expression in the cell cytoplasm. CRISPR-Cas13-mediated deletion of gp355 reveals that the mutant phage has a prolonged latent period. Altogether, we demonstrate that gp335 is an antibacterial protein of nucleus-forming phages that associates with the ribosomes at the phage nucleus.
PubMed: 38915640
DOI: 10.1101/2024.06.15.599175 -
BioRxiv : the Preprint Server For... Jun 2024YabG is a sporulation-specific protease that is conserved among sporulating bacteria. YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB...
YabG is a sporulation-specific protease that is conserved among sporulating bacteria. YabG processes cortex destined proteins preproSleC into proSleC and CspBA to CspB and CspA. YabG also affects synthesis of spore coat/exosporium proteins CotA and CdeM. In prior work that identified CspA as the co-germinant receptor, mutations in were found which altered the co-germinants required to initiate spore germination. To understand how these mutations in the locus contribute to spore germination, we introduced these mutations into an isogenic background. Spores derived from (catalytically inactive), , and strains germinated in response to TA alone. Recombinantly expressed and purified preproSleC incubated with lysate expressing wild type YabG resulted in the removal of the pre sequence from preproSleC. Interestingly, only YabG showed any activity towards purified preproSleC. Mutation of the YabG processing site in preproSleC (R119A) led to YabG shifting its processing to R115 or R112. Finally, changes in expression under the mutant promoters were analyzed using a SNAP-tag and revealed expression differences at early and late stages of sporulation. Overall, our results support and expand upon the hypothesis that YabG is important for germination and spore assembly and, upon mutation of the processing site, can shift where it cleaves substrates.
PubMed: 38915615
DOI: 10.1101/2024.06.10.598338 -
BioRxiv : the Preprint Server For... Jun 2024Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent...
UNLABELLED
Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNA variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNA variant that misincorporates serine at valine codons (V➔S) and the other that misincorporates serine at threonine codons (TàS). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V➔S variant extended embryonic, larval, and pupal development whereas the T➔S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Mistranslating females also experienced extended lifespan whereas mistranslating male lifespan was unaffected. In addition, mistranslating flies from both sexes showed improved locomotion as they aged, suggesting delayed neurodegeneration. Therefore, although mistranslation causes detrimental effects, we demonstrate that mistranslation also has positive effects on complex traits such as lifespan and locomotion. This has important implications for human health given the prevalence of tRNA variants in humans.
PLAIN LANGUAGE SUMMARY
Mutant tRNA genes can cause mistranslation, the misincorporation of amino acids into proteins, and are associated with several human diseases. This study investigated the role of two tRNA variants that cause threonine-to-serine (T➔S) or valine-to-serine (V➔S) substitution. Both variants caused developmental delays and lethality in both sexes and increase prevalence of deformities in females. Surprisingly, female T➔S and V➔S flies experienced increased lifespan and mistranslating males and females showed improved locomotion. These results suggest that mistranslation has both positive and negative effects that depend on the tRNA variant and sex of the fly.
PubMed: 38915589
DOI: 10.1101/2024.06.11.598535 -
BioRxiv : the Preprint Server For... Jun 2024(or ) , the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While...
UNLABELLED
(or ) , the causative agent of Lyme disease, is a motile and invasive zoonotic pathogen, adept at navigating between its arthropod vector and mammalian host. While motility and chemotaxis are well established as essential for its enzootic cycle, the function of methyl-accepting chemotaxis proteins (MCPs) in the infectious cycle of remains unclear. In this study, we demonstrate that MCP5, one of the most abundant MCPs in , is differentially expressed in response to environmental signals as well as at different stages of the pathogen's enzootic cycle. Specifically, the expression of is regulated by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways, which are critical for the spirochete's colonization of the tick vector and mammalian host, respectively. Infection experiments with an mutant revealed that spirochetes lacking MCP5 could not establish infections in either C3H/HeN mice or Severe Combined Immunodeficiency (SCID) mice, which are defective in adaptive immunity, indicating the essential role of MCP5 in mammalian infection. However, the mutant could establish infection and disseminate in NOD SCID Gamma (NSG) mice, which are deficient in both adaptive and most innate immune responses, suggesting a crucial role of MCP5 in evading host innate immunity. In the tick vector, the mutants survived feeding but failed to transmit to mice, highlighting the importance of MCP5 in transmission. Our findings reveal that MCP5, regulated by the Rrp1 and Rrp2 pathways, is critical for the establishment of infection in mammalian hosts by evading host innate immunity and is important for the transmission of spirochetes from ticks to mammalian hosts, underscoring its potential as a target for intervention strategies.
SUMMARY
Lyme disease is the most commonly reported arthropod-borne illness in the US, Europe, and Asia. The causative agent of Lyme disease, , is maintained in an enzootic cycle involving arthropod vectors ( ticks) and rodent mammalian hosts. Understanding how moves within this natural cycle is crucial for developing new strategies to combat Lyme disease. The complex nature of the enzootic cycle necessitates sensory-guided movement in response to environmental stimuli. possesses a unique and intricate chemotaxis signaling system, with methyl-accepting chemotaxis proteins (MCPs) at its core. These proteins are responsible for sensing environmental signals and guiding bacterial movement toward or away from stimuli. This study found that one of the MCPs, MCP5, is highly expressed and differentially regulated during the enzootic cycle by the Hk1-Rrp1 and Rrp2-RpoN-RpoS pathways. MCP5 is crucial for mammalian infection, aiding in immune evasion and transmission from ticks to mammals, providing a foundation for further research into 's navigation within its hosts.
PubMed: 38915556
DOI: 10.1101/2024.06.10.598185 -
BioRxiv : the Preprint Server For... Jun 2024-Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure...
-Related Dilated Cardiomyopathy: Single-Cell Transcriptomics during Patient-derived iPSC Differentiation Support Cell type and Lineage-specific Dysregulation of Gene Expression and Development for Cardiomyocytes and Epicardium-Derived Cells with Lamin A/C Haploinsufficiency.
-Related Dilated Cardiomyopathy (DCM) is an autosomal-dominant genetic condition with cardiomyocyte and conduction system dysfunction often resulting in heart failure or sudden death. The condition is caused by mutation in the Lamin A/C ( ) gene encoding Type-A nuclear lamin proteins involved in nuclear integrity, epigenetic regulation of gene expression, and differentiation. Molecular mechanisms of disease are not completely understood, and there are no definitive treatments to reverse progression or prevent mortality. We investigated possible mechanisms of -Related DCM using induced pluripotent stem cells derived from a family with a heterozygous splice-site mutation. We differentiated one mutant iPSC line derived from an affected female (Patient) and two non-mutant iPSC lines derived from her unaffected sister (Control) and conducted single-cell RNA sequencing for 12 samples (4 Patient and 8 Control) across seven time points: Day 0, 2, 4, 9, 16, 19, and 30. Our bioinformatics workflow identified 125,554 cells in raw data and 110,521 (88%) high-quality cells in sequentially processed data. Unsupervised clustering, cell annotation, and trajectory inference found complex heterogeneity: ten main cell types; many possible subtypes; and lineage bifurcation for Cardiac Progenitors to Cardiomyocytes (CM) and Epicardium-Derived Cells (EPDC). Data integration and comparative analyses of Patient and Control cells found cell type and lineage differentially expressed genes (DEG) with enrichment to support pathway dysregulation. Top DEG and enriched pathways included: 10 genes and RNA polymerase II transcription in Pluripotent cells (PP); and TGF Beta/BMP signaling, sarcomere gene subsets and cardiogenesis, and EMT in CM; and epigenetic regulation and and mTORC1 signaling in EPDC. Top DEG also included: and other X-linked genes, six imprinted genes: , , , , , , and enriched gene sets in metabolism, proliferation, and homeostasis. We confirmed Lamin A/C haploinsufficiency by allelic expression and Western blot. Our complex Patient-derived iPSC model for Lamin A/C haploinsufficiency in PP, CM, and EPDC provided support for dysregulation of genes and pathways, many previously associated with Lamin A/C defects, such as epigenetic gene expression, signaling, and differentiation. Our findings support disruption of epigenomic developmental programs as proposed in other disease models. We recognized other factors influencing epigenetics and differentiation; thus, our approach needs improvement to further investigate this mechanism in an iPSC-derived model.
PubMed: 38915555
DOI: 10.1101/2024.06.12.598335 -
BioRxiv : the Preprint Server For... Jun 2024In eukaryotic post-replicative mismatch repair, MutS homologs (MSH) detect mismatches and recruit MLH complexes to nick the newly replicated DNA strand upon activation...
In eukaryotic post-replicative mismatch repair, MutS homologs (MSH) detect mismatches and recruit MLH complexes to nick the newly replicated DNA strand upon activation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. Biasing MLH endonuclease activity to the newly replicated DNA strand is crucial for repair. In reconstituted assays, PCNA is loaded at pre-existing discontinuities and orients the major MLH endonuclease Mlh1-Pms1/MLH1-PMS2 (yeast/human) to nick the discontinuous strand. newly replicated DNA transiently contains discontinuities which are critical for efficient mismatch repair. How these discontinuities are preserved as strand discrimination signals during the window of time where mismatch repair occurs is unknown. Here, we demonstrate that yeast Mlh1-Pms1 uses ATP binding to recognize DNA discontinuities. This complex does not efficiently interact with PCNA, which partially suppresses ATPase activity, and prevents dissociation from the discontinuity. These data suggest that in addition to initiating mismatch repair by nicking newly replicated DNA, Mlh1-Pms1 protects strand discrimination signals, aiding in maintaining its own strand discrimination signposts. Our findings also highlight the significance of Mlh1-Pms1's ATPase activity for inducing DNA dissociation, as mutant proteins deficient in this function become immobilized on DNA post-incision, explaining phenotypes.
PubMed: 38915520
DOI: 10.1101/2024.06.13.598860