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Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium of medical significance. It typically exists as part of the normal flora of the human intestine but can cause severe infections in the healthcare setting due to its rapid acquisition of antibiotic resistance. Cultivating and extracting genomic DNA from this bacterium is crucial for downstream characterization and comparative analyses. To provide a standardized approach for growing K. pneumoniae in the laboratory setting, this collection of protocols provides step-by-step procedures for routine culturing, generating growth curves, storing bacteria, and extracting genomic DNA. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Reviving K. pneumoniae from frozen stocks Basic Protocol 2: Cultivating K. pneumoniae in rich growth medium Alternate Protocol: Cultivating in minimal liquid growth medium Basic Protocol 3: Enumerating K. pneumoniae colony forming units Basic Protocol 4: Growth curves Basic Protocol 5: Genomic DNA extraction Basic Protocol 6: Characterizing K. pneumoniae strains based on genomic sequence Basic Protocol 7: Storage of K. pneumoniae frozen stocks in glycerol Basic Protocol 8: Storage of K. pneumoniae in agar stabs.

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Klebsiella pneumoniae is a clinically significant, Gram-negative pathogen in which the production of extracellular polysaccharides is a key virulence factor. Extracellular polysaccharides such as the capsule and its mucoviscosity play a significant role in K. pneumoniae infection. In this article, we explain several standard protocols used to characterize the extracellular polysaccharides of K. pneumoniae. Several of these protocols are adapted specifically for K. pneumoniae and describe methods to purify and quantify the extracellular polysaccharide of K. pneumoniae. We also present a standardized protocol to quantify K. pneumoniae mucoviscosity, a unique feature of K. pneumoniae extracellular polysaccharide. These protocols will help create uniformity in standard protocols used in K. pneumoniae extracellular polysaccharide studies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extracellular polysaccharide isolation and purification Basic Protocol 2: Large-scale isolation and purification of extracellular polysaccharide Basic Protocol 3: Uronic acid quantification of extracellular polysaccharide Basic Protocol 4: Extracellular polysaccharide visualization by SDS-PAGE Basic Protocol 5: Klebsiella pneumoniae mucoviscosity measurement by sedimentation resistance assay Alternate Protocol 5: 96-well plate-based Klebsiella pneumoniae sedimentation resistance assay Support Protocol 5: Determination of plate to cuvette conversion factor.

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Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare-associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulating K. pneumoniae presents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation of K. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifying K. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing electrocompetent K. pneumoniae cells Alternate Protocol 1: Preparing electrocompetent K. pneumoniae cells for recombineering Basic Protocol 2: Transforming K. pneumoniae using electroporation Basic Protocol 3: Constructing isogenic mutants in K. pneumoniae using the λ Red recombinase system Support Protocol 1: Confirming a knockout via colony PCR Support Protocol 2: Verifying absence of secondary mutations Basic Protocol 4: Generating unmarked knockout mutants in K. pneumoniae using the pFLP plasmid Basic Protocol 5: Constructing a complementation vector for K. pneumoniae.

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Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

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Clonal cytopenias of undetermined significance (CCUS) are associated with an increased risk of developing a myelodysplastic syndrome (MDS); however, the mechanism and factors associated with evolution remain unclear. We propose that next-generation sequencing (NGS) of cytopenic cases with equivocal morphologic dysplasia will improve patient clinical care and that serial sequencing of such equivocal cases could identify the factors that predict evolution to MDS. We performed targeted NGS of samples from 193 individuals with confirmed or suspected MDS or MDS/myeloproliferative neoplasm, including sequential investigation for 28 individuals at the time of diagnosis and during follow-up. NGS facilitated the diagnosis of all suspicious cases as myeloid neoplasm (21%), CCUS (34%), or idiopathic cytopenias of undetermined significance (45%) when no variants were detected. We found that there was no significant difference in most measured clinical features or clonal phenotypes, such as cell counts, number of variants, variant allele frequencies, and overall survival, between CCUS and International Prognostic Scoring System-Revised-defined low-risk MDS. However, there was a significant difference in the types of variants between CCUS and low-risk MDS, with a significantly lower number of splicing factor mutations in CCUS cases (P < .001). Moreover, we observed an increased probability of evolution to MDS of individuals with CCUS compared with that in those with idiopathic cytopenias of undetermined significance over the first 5 years (P = .045). Our analyses revealed no conclusive pattern associating clonal expansion or the number of variants with the evolution of CCUS to MDS, perhaps further supporting the similarity of these diseases and the clinical importance of recognizing and formally defining CCUS as a category of precursor myeloid disease state in the next revision of the World Health Organization guidelines.

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RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

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Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.

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Dissolved organic matter (DOM) is a ubiquitous constituent of natural waters and is comprised of a variety of chemically heterogeneous molecular structures and functional groups. DOM is often considered to be a major ligand for metals in most natural waters and its reactivity is thought to be strongly dependent on its chemical composition and structure. In this study, a combination of UV/visible, emission excitation matrix fluorescence (EEM) and (1)H NMR spectroscopies were used to characterize DOM from the Athabasca River (Alberta, Canada). The chemical characterization of river DOM showed that the most upstream samples located in agricultural areas were blue-shifted and less aromatic and contained more hydrogens connected with oxygen functional groups than those in the wetland dominated area in the Athabasca oil sand deposit region. The presence of paramagnetic ions (Fe and Al) was not found to significantly affect the structural composition of DOM as revealed by (1)H NMR. Such change in the quality of DOM may have a profound impact on metal binding in the Athabasca River watershed.

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The Iridoviridae family are large viruses (∼120-200 nm) that contain a linear double-stranded DNA genome. The genomic size of Iridoviridae family members range from 105,903 bases encoding 97 open reading frames (ORFs) for frog virus 3 to 212,482 bases encoding 211 ORFs for Chilo iridescent virus. The family Iridoviridae is currently subdivided into five genera: Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus. Iridoviruses have been found to infect invertebrates and poikilothermic vertebrates, including amphibians, reptiles, and fish. With such a diverse array of hosts, there is great diversity in gene content between different genera. To understand the origin of iridoviruses, we explored the phylogenetic relationship between individual iridoviruses and defined the core-set of genes shared by all members of the family. In order to further explore the evolutionary relationship between the Iridoviridae family repetitive sequences were identified and compared. Each genome was found to contain a set of unique repetitive sequences that could be used in future virus identification. Repeats common to more than one virus were also identified and changes in copy number between these repeats may provide a simple method to differentiate between very closely related virus strains. The results of this paper will be useful in identifying new iridoviruses and determining their relationship to other members of the family.