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Microbiology Spectrum Jun 2024Extracellular iodate reduction by spp. contributes to iodide generation in the biogeochemical cycling of iodine. However, there is a disagreement on whether spp. use...
Extracellular iodate reduction by spp. contributes to iodide generation in the biogeochemical cycling of iodine. However, there is a disagreement on whether spp. use different extracellular electron transfer pathways with dependence on electron donors in iodate reduction. In this study, a series of gene deletion mutants of MR-1 were created to investigate the roles of , and operons in iodate reduction. The iodate-reducing activity of the mutants was tested with lactate, formate, and H as the sole electron donors, respectively. In the absence of single- gene, iodate reduction efficiency of the mutants was only 12.9%-84.0% with lactate at 24 hours, 22.1%-85.9% with formate at 20 hours, and 19.6%-57.7% with H at 42 hours in comparison to complete reduction by the wild type. Progressive inhibition of iodate reduction was observed when the homolog from the operon was deleted in the single- gene mutants. This result revealed complementation of by at the single-gene level, indicating modularity of the extracellular electron transfer pathway encoded by operon. Under the conditions of all electron donors, significant inhibition of iodate reduction and accumulation of HO were detected for Δ. Collectively, these results demonstrated that the operon encodes an essential and modular iodate-reducing pathway without electron donor dependence in MR-1. The operon was involved in HO elimination with all electron donors. The findings in this study improved the understanding of molecular mechanisms underlying extracellular iodate reduction.IMPORTANCEIodine is an essential trace element for human and animals. Recent studies revealed the contribution of microbial extracellular reduction of iodate in biogeochemical cycling of iodine. Multiple reduced substances can be utilized by microorganisms as energy source for iodate reduction. However, varied electron transfer pathways were proposed for iodate reduction with different electron donors in the model strain MR-1. Here, through a series of gene deletion and iodate reduction experiments, we discovered that the operon was essential for iodate reduction with at least three electron donors, including lactate, formate, and H. The operon was first demonstrated to be capable of complementing the function of at single-gene level.
PubMed: 38916364
DOI: 10.1128/spectrum.00512-24 -
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 2024O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from and induces a massive...
UNLABELLED
O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that O1 isolated from stool is genetically similar to recovered from the upper intestinal tract.
IMPORTANCE
O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed genomes from stool are usually studied, but isolated from vomit may be more representative of where colonizes in the upper intestinal epithelium. may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in genetic diversity, and only in some patients. These results fill a gap in our understanding of the life cycle, transmission, and evolution.
PubMed: 38916318
DOI: 10.1128/spectrum.00785-24 -
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 -
MBio Jun 2024causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs...
UNLABELLED
causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against . BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system by analyzing libraries of mutants grown in the presence of BRI. In , BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. experiments show BRI significantly reduces survival inside macrophages and partially clears lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against . BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis.
IMPORTANCE
Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, and . There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against . BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of , acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against . We propose BRI as a new antifungal agent against cryptococcosis.
PubMed: 38916308
DOI: 10.1128/mbio.01031-24 -
BioRxiv : the Preprint Server For... Jun 2024Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the...
UNLABELLED
Efforts to cure BCR::ABL1 B cell acute lymphoblastic leukemia (Ph+ ALL) solely through inhibition of ABL1 kinase activity have thus far been insufficient despite the availability of tyrosine kinase inhibitors (TKIs) with broad activity against resistance mutants. The mechanisms that drive persistence within minimal residual disease (MRD) remain poorly understood and therefore untargeted. Utilizing 13 patient-derived xenograft (PDX) models and clinical trial specimens of Ph+ ALL, we examined how genetic and transcriptional features co-evolve to drive progression during prolonged TKI response. Our work reveals a landscape of cooperative mutational and transcriptional escape mechanisms that differ from those causing resistance to first generation TKIs. By analyzing MRD during remission, we show that the same resistance mutation can either increase or decrease cellular fitness depending on transcriptional state. We further demonstrate that directly targeting transcriptional state-associated vulnerabilities at MRD can overcome BCR::ABL1 independence, suggesting a new paradigm for rationally eradicating MRD prior to relapse. Finally, we illustrate how cell mass measurements of leukemia cells can be used to rapidly monitor dominant transcriptional features of Ph+ ALL to help rationally guide therapeutic selection from low-input samples.
HIGHLIGHTS
Relapse after remission on TKI can harbor mutations in ABL1, RAS, or neitherMutations and development-like cell state dictate fitness in residual diseaseCo-targeting cell state and ABL1 markedly reduces MRDBiophysical measurements provide an integrative, rapid measurement of cell state.
PubMed: 38915726
DOI: 10.1101/2024.06.06.597767 -
BioRxiv : the Preprint Server For... Jun 2024Proteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or...
UNLABELLED
Proteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or "clients," undergo sequential processing by Hsp40, Hsp70, and Hsp90 chaperones to achieve proper folding and functionality. Despite extensive characterization of post-translational modifications (PTMs) on Hsp70 and Hsp90, the modifications on Hsp40 remain less understood. This study aims to elucidate the role of lysine acetylation on the yeast Hsp40, Ydj1. By mutating acetylation sites on Ydj1's J-domain to either abolish or mimic constitutive acetylation, we observed that preventing acetylation had no noticeable phenotypic impact, whereas acetyl-mimic mutants exhibited various defects indicative of impaired Ydj1 function. Proteomic analysis revealed several Ydj1 interactions affected by J-domain acetylation, notably with proteins involved in translation. Further investigation uncovered a novel role for Ydj1 acetylation in stabilizing ribosomal subunits and ensuring translational fidelity. Our data suggest that acetylation may facilitate the transfer of Ydj1 between Ssa1 and Hsp82. Collectively, this work highlights the critical role of Ydj1 acetylation in proteostasis and translational fidelity.
AUTHOR SUMMARY
Cells require a suite of chaperone and co-chaperone proteins to maintain a healthy balance of functional proteins. A large number of modifications on chaperone and co-chaperone proteins have been identified, but their functional importance has not been fully explored. In this study, we identify acetylation sites on the yeast co-chaperone Ydj1 that impact its interactions with major chaperones and client proteins including those involved in protein synthesis. This work sheds light on how modifications on co-chaperones can also play an important role in the health of the proteome.
PubMed: 38915721
DOI: 10.1101/2024.06.13.598777 -
BioRxiv : the Preprint Server For... Jun 2024The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low...
The ability to deliver large transgenes to a single genomic sequence with high efficiency would accelerate biomedical interventions. Current methods suffer from low insertion efficiency and most rely on undesired double-strand DNA breaks. Serine integrases catalyze the insertion of large DNA cargos at attachment (att) sites. By targeting att sites to the genome using technologies such as prime editing, integrases can target safe loci while avoiding double-strand breaks. We developed a method of phage-assisted continuous evolution we call IntePACE, that we used to rapidly perform hundreds of rounds of mutagenesis to systematically improve activity of PhiC31 and Bxb1 serine integrases. Novel hyperactive mutants were generated by combining synergistic mutations resulting in integration of a multi-gene cargo at rates as high as 80% of target chromosomes. Hyperactive integrases inserted a 15.7 kb therapeutic DNA cargo containing Von Willebrand Factor. This technology could accelerate gene delivery therapeutics and our directed evolution strategy can easily be adapted to improve novel integrases from nature.
PubMed: 38915697
DOI: 10.1101/2024.06.10.598370 -
BioRxiv : the Preprint Server For... Jun 2024Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's DNA synthesis...
Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT's DNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT's intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also tested , revealing concordance between each variant's bias and the bias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders, applications of TdT, and further study of the biology of TdT.
PubMed: 38915690
DOI: 10.1101/2024.06.11.598561 -
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