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1.
Environ Microbiol ; 25(12): 3387-3405, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37915109

RESUMO

In this study, we compared the genomes of three metal-resistant bacteria isolated from mercury-contaminated soil. We identified diverse and novel MGEs with evidence of multiple LGT events shaping their genomic structure and heavy metal resistance. Among the three metal-resistant strains, Sphingobium sp SA2 and Sphingopyxis sp SE2 were resistant to multiple metals including mercury, cadmium, copper, zinc and lead. Pseudoxanthomonas sp SE1 showed resistance to mercury only. Whole genome sequencing by Illumina and Oxford Nanopore technologies was undertaken to obtain comprehensive genomic data. The Sphingobium and Sphingopyxis strains contained multiple chromosomes and plasmids, whereas the Pseudoxanthomonas strain contained one circular chromosome. Consistent with their metal resistance profiles, the strains of Sphingobium and Sphingopyxis contained a higher quantity of diverse metal resistance genes across their chromosomes and plasmids compared to the single-metal resistant Pseudoxanthomonas SE1. In all three strains, metal resistance genes were principally associated with various novel MGEs including genomic islands (GIs), integrative conjugative elements (ICEs), transposons, insertion sequences (IS), recombinase in trio (RIT) elements and group II introns, indicating their importance in facilitating metal resistance adaptation in a contaminated environment. In the Pseudoxanthomonas strain, metal resistance regions were largely situated on a GI. The chromosomes of the strains of Sphingobium and Sphingopyxis contained multiple metal resistance regions, which were likely acquired by several GIs, ICEs, numerous IS elements, several Tn3 family transposons and RIT elements. Two of the plasmids of Sphingobium were impacted by Tn3 family transposons and ISs likely integrating metal resistance genes. The two plasmids of Sphingopyxis harboured transposons, IS elements, an RIT element and a group II intron. This study provides a comprehensive annotation of complex genomic regions of metal resistance associated with novel MGEs. It highlights the critical importance of LGT in the evolution of metal resistance of bacteria in contaminated environments.


Assuntos
Elementos de DNA Transponíveis , Mercúrio , Elementos de DNA Transponíveis/genética , Genoma Bacteriano/genética , Plasmídeos/genética , Ilhas Genômicas , Bactérias/genética
2.
BMC Genomics ; 23(1): 777, 2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-36443683

RESUMO

BACKGROUND: Bacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease. RESULTS: We have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160-170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection. CONCLUSIONS: Ultimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages.


Assuntos
Fagos Bacilares , Bacillus , Humanos , Bacillus/genética , Solo , Filogenia , Fagos Bacilares/genética , Recombinases , Tirosina
3.
Int J Syst Evol Microbiol ; 72(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36256446

RESUMO

The taxonomic status of two Gordonia strains, designated BEN371 and CON9T, isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia. The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9T represent a novel species of the genus Gordonia. The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9T (=DSM 43602T=JCM 35249T) as the type strain.


Assuntos
Actinobacteria , Bactéria Gordonia , Purificação da Água , Esgotos/microbiologia , RNA Ribossômico 16S/genética , Filogenia , DNA Bacteriano/genética , Composição de Bases , Técnicas de Tipagem Bacteriana , Análise de Sequência de DNA , Ácidos Graxos/química , Nucleotídeos
4.
Appl Microbiol Biotechnol ; 104(22): 9839-9852, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32974744

RESUMO

Amplicon sequence fingerprinting of communities in activated sludge systems have provided data revealing the true level of their microbial biodiversity and led to suggestions of which intrinsic and extrinsic parameters might affect the dynamics of community assemblage. Most studies have been performed in China and Denmark, and comparatively little information is available for plants in other countries. This study looked at how the communities of three plants in Victoria, Australia, treating domestic sewage changed with season. All were designed to remove nitrogen microbiologically. They were all located close together to minimise any influence that climate and demographics might have on their operation, and samples were taken at weekly intervals for 12 months. 16S rRNA amplicon sequencing revealed that each plant community was distinctively different to the others and changed over the 12-month sampling period. Many of the factors suggested in other similar studies to be important in determining community composition in activated sludge systems could not explain the changes noted here. The most likely influential factors were considered to be temperature and influent composition reflecting changes in dietary intake by the populations served by each plant, since in all three, the most noticeable changes corresponded to seasonal shifts. KEY POINTS: • Monitoring microbial communities in 3 wastewater treatment plants removing nitrogen • Temperature is the most influential factor in dynamic changes in community composition.


Assuntos
Reatores Biológicos , Nitrogênio , Purificação da Água , Bactérias/genética , China , Desnitrificação , RNA Ribossômico 16S/genética , Esgotos , Vitória , Eliminação de Resíduos Líquidos , Águas Residuárias
5.
PLoS Pathog ; 13(10): e1006659, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28985225

RESUMO

Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.


Assuntos
Microtúbulos/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Desenvolvimento Sexual/fisiologia , Animais , Microscopia Eletrônica/métodos , Microtúbulos/ultraestrutura , Plasmodium falciparum/ultraestrutura , Transporte Proteico
6.
J Cell Sci ; 129(2): 406-16, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26675237

RESUMO

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.


Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Avaliação Pré-Clínica de Medicamentos , Resistência a Medicamentos , Sinergismo Farmacológico , Hemoglobinas , Humanos , Dose Letal Mediana , Leucina/análogos & derivados , Leucina/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/enzimologia , Proteólise , Proteínas de Protozoários/metabolismo
7.
Cell Microbiol ; 18(11): 1551-1569, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27019089

RESUMO

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex.


Assuntos
Eritrócitos/parasitologia , Proteínas de Membrana Transportadoras/fisiologia , Plasmodium falciparum/fisiologia , Proteoma/metabolismo , Proteínas de Protozoários/fisiologia , Células Cultivadas , Humanos , Complexos Multiproteicos/metabolismo , Domínios Proteicos , Mapas de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico
8.
Mol Microbiol ; 98(6): 1101-14, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26304012

RESUMO

The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.


Assuntos
Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Antígenos CD36/metabolismo , DNA de Protozoário , Membrana Eritrocítica/metabolismo , Eritrócitos/parasitologia , Técnicas de Silenciamento de Genes , Humanos , Mutação , Plasmodium falciparum/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Sequências Repetitivas de Ácido Nucleico , Fatores de Virulência/genética
9.
Cell Microbiol ; 15(8): 1401-18, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23421990

RESUMO

The human malaria parasite, Plasmodium falciparum, modifies the red blood cells (RBCs) that it infects by exporting proteins to the host cell. One key virulence protein, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), is trafficked to the surface of the infected RBC, where it mediates adhesion to the vascular endothelium. We have investigated the organization and development of the exomembrane system that is used for PfEMP1 trafficking. Maurer's cleft cisternae are formed early after invasion and proteins are delivered to these (initially mobile) structures in a temporally staggered and spatially segregated manner. Membrane-Associated Histidine-Rich Protein-2 (MAHRP2)-containing tether-like structures are generated as early as 4 h post invasion and become attached to Maurer's clefts. The tether/Maurer's cleft complex docks onto the RBC membrane at ~20 h post invasion via a process that is not affected by cytochalasin D treatment. We have examined the trafficking of a GFP chimera of PfEMP1 expressed in transfected parasites. PfEMP1B-GFP accumulates near the parasite surface, within membranous structures exhibiting a defined ultrastructure, before being transferred to pre-formed mobile Maurer's clefts. Endogenous PfEMP1 and PfEMP1B-GFP are associated with Electron-Dense Vesicles that may be responsible for trafficking PfEMP1 from the Maurer's clefts to the RBC membrane.


Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/patogenicidade , Transporte Proteico/fisiologia , Proteínas de Protozoários/fisiologia , Células Cultivadas , Membrana Eritrocítica/parasitologia , Membrana Eritrocítica/fisiologia , Eritrócitos/patologia , Proteínas de Fluorescência Verde , Interações Hospedeiro-Parasita/fisiologia , Humanos , Técnicas In Vitro , Plasmodium falciparum/fisiologia
10.
Amino Acids ; 46(12): 2759-66, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25200682

RESUMO

Melittin (MLT) is a lytic peptide with a broad spectrum of activity against both eukaryotic and prokaryotic cells. To understand the role of proline and the thiol group of cysteine in the cytolytic activity of MLT, native MLT and cysteine-containing analogs were prepared using solid phase peptide synthesis. The antimicrobial and cytolytic activities of the monomeric and dimeric MLT peptides against different cells and model membranes were investigated. The results indicated that the proline residue was necessary for antimicrobial activity and cytotoxicity and its absence significantly reduced lysis of model membranes and hemolysis. Although lytic activity against model membranes decreased for the MLT dimer, hemolytic activity was increased. The native peptide and the MLT-P14C monomer were mainly unstructured in buffer while the dimer adopted a helical conformation. In the presence of neutral and negatively charged vesicles, the helical content of the three peptides was significantly increased. The lytic activity, therefore, is not correlated to the secondary structure of the peptides and, more particularly, on the propensity to adopt helical conformation.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Meliteno/farmacologia , Sequência de Aminoácidos , Antibacterianos/síntese química , Antibacterianos/química , Bactérias/efeitos dos fármacos , Células HeLa , Humanos , Meliteno/síntese química , Meliteno/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Relação Estrutura-Atividade
11.
Res Microbiol ; 175(5-6): 104202, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38582389

RESUMO

The Burkholderia cepacia complex (Bcc) is a group of increasingly multi-drug resistant opportunistic bacteria. This resistance is driven through a combination of intrinsic factors and the carriage of a broad range of conjugative plasmids harbouring virulence determinants. Therefore, novel treatments are required to treat and prevent further spread of these virulence determinants. In the search for phages infective for clinical Bcc isolates, CSP1 phage, a PRD1-like phage was isolated. CSP1 phage was found to require pilus machinery commonly encoded on conjugative plasmids to facilitate infection of Gram-negative bacteria genera including Escherichia and Pseudomonas. Whole genome sequencing and characterisation of one of the clinical Burkholderia isolates revealed it to be Burkholderia contaminans. B. contaminans 5080 was found to contain a genome of over 8 Mbp encoding multiple intrinsic resistance factors, such as efflux pump systems, but more interestingly, carried three novel plasmids encoding multiple putative virulence factors for increased host fitness, including antimicrobial resistance. Even though PRD1-like phages are broad host range, their use in novel antimicrobial treatments shouldn't be dismissed, as the dissemination potential of conjugative plasmids is extensive. Continued survey of clinical bacterial strains is also key to understanding the spread of antimicrobial resistance determinants and plasmid evolution.


Assuntos
Bacteriófagos , Complexo Burkholderia cepacia , Plasmídeos , Plasmídeos/genética , Complexo Burkholderia cepacia/virologia , Complexo Burkholderia cepacia/genética , Complexo Burkholderia cepacia/isolamento & purificação , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/classificação , Especificidade de Hospedeiro , Sequenciamento Completo do Genoma , Conjugação Genética , Fatores de Virulência/genética , Infecções por Burkholderia/microbiologia , Humanos , Genoma Viral , Genoma Bacteriano , Burkholderia/genética , Burkholderia/virologia
12.
Viruses ; 15(2)2023 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-36851720

RESUMO

Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages.


Assuntos
Bacteriófagos , Podoviridae , Bacteriófagos/genética , Japão , Filogenia , Bases de Dados Factuais
13.
BMC Genom Data ; 24(1): 49, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37658299

RESUMO

OBJECTIVES: Providencia is a genus of gram-negative bacteria within the order Enterobacterales, closely related to Proteus and Morganella. While ubiquitous in the environment, some species of Providencia, such as P. rettgeri and P. stuartii, are considered emerging nosocomial pathogens and have been implicated in urinary tract infection, gastrointestinal illness, and travelers' diarrhea. Given their intrinsic resistance to many commonly used antibiotics, this study aimed to isolate and sequence bacteriophages targeting a clinical P. rettgeri isolate. DATA DESCRIPTION: Here we report the complete genome sequence of three novel Providencia phages, PibeRecoleta, Stilesk and PatoteraRojo, which were isolated against a clinical P. rettgeri strain sourced from a patient in a metropolitan hospital in Victoria, Australia. The three phages contain dsDNA genomes between 60.7 and 60.9 kb in size and are predicted to encode between 72 and 73 proteins. These three new phages, which share high genomic similarity to two other Providencia phages previously isolated on P. stuartii, serve as important resources in our understanding about Providencia bacteriophages and the potential for future phage-based biotherapies.


Assuntos
Bacteriófagos , Disenteria , Humanos , Diarreia/genética , Diarreia/terapia , Providencia/genética , Viagem , Bacteriófagos/genética , Hospitais Urbanos , Vitória
14.
Microbiol Spectr ; 11(3): e0533222, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37199610

RESUMO

The Burkholderia cepacia complex is a group of opportunistic pathogens that cause both severe acute and chronic respiratory infections. Due to their large genomes containing multiple intrinsic and acquired antimicrobial resistance mechanisms, treatment is often difficult and prolonged. One alternative to traditional antibiotics for treatment of bacterial infections is bacteriophages. Therefore, the characterization of bacteriophages infective for the Burkholderia cepacia complex is critical to determine their suitability for any future use. Here, we describe the isolation and characterization of novel phage, CSP3, infective against a clinical isolate of Burkholderia contaminans. CSP3 is a new member of the Lessievirus genus that targets various Burkholderia cepacia complex organisms. Single nucleotide polymorphism (SNP) analysis of CSP3-resistant B. contaminans showed that mutations to the O-antigen ligase gene, waaL, consequently inhibited CSP3 infection. This mutant phenotype is predicted to result in the loss of cell surface O-antigen, contrary to a related phage that requires the inner core of the lipopolysaccharide for infection. Additionally, liquid infection assays showed that CSP3 provides suppression of B. contaminans growth for up to 14 h. Despite the inclusion of genes that are typical of the phage lysogenic life cycle, we saw no evidence of CSP3's ability to lysogenize. Continuation of phage isolation and characterization is crucial in developing large and diverse phage banks for global usage in cases of antibiotic-resistant bacterial infections. IMPORTANCE Amid the global antibiotic resistance crisis, novel antimicrobials are needed to treat problematic bacterial infections, including those from the Burkholderia cepacia complex. One such alternative is the use of bacteriophages; however, a lot is still unknown about their biology. Bacteriophage characterization studies are of high importance for building phage banks, as future work in developing treatments such as phage cocktails should require well-characterized phages. Here, we report the isolation and characterization of a novel Burkholderia contaminans phage that requires the O-antigen for infection, a distinct phenotype seen among other related phages. Our findings presented in this article expand on the ever-evolving phage biology field, uncovering unique phage-host relationships and mechanisms of infection.


Assuntos
Bacteriófagos , Complexo Burkholderia cepacia , Burkholderia , Bacteriófagos/genética , Antígenos O/análise , Complexo Burkholderia cepacia/genética , Burkholderia/genética
15.
mSphere ; 8(4): e0023323, 2023 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-37366629

RESUMO

Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag the P. falciparum orthologs of two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). We localize both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in P. falciparum, they are not required for degradation of nonsense transcripts. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.


Assuntos
Malária , Plasmodium falciparum , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Regulação da Expressão Gênica , RNA Mensageiro/genética
16.
Microbiome ; 10(1): 12, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35074003

RESUMO

BACKGROUND: Understanding how elevated atmospheric CO2 (eCO2) impacts on phosphorus (P) transformation in plant rhizosphere is critical for maintaining ecological sustainability in response to climate change, especially in agricultural systems where soil P availability is low. METHODS: This study used rhizoboxes to physically separate rhizosphere regions (plant root-soil interface) into 1.5-mm segments. Wheat plants were grown in rhizoboxes under eCO2 (800 ppm) and ambient CO2 (400 ppm) in two farming soils, Chromosol and Vertosol, supplemented with phytate (organic P). Photosynthetic carbon flow in the plant-soil continuum was traced with 13CO2 labeling. Amplicon sequencing was performed on the rhizosphere-associated microbial community in the root-growth zone, and 1.5 mm and 3 mm away from the root. RESULTS: Elevated CO2 accelerated the mineralization of phytate in the rhizosphere zones, which corresponded with increases in plant-derived 13C enrichment and the relative abundances of discreet phylogenetic clades containing Bacteroidetes and Gemmatimonadetes in the bacterial community, and Funneliformis affiliated to arbuscular mycorrhizas in the fungal community. Although the amplicon sequence variants (ASVs) associated the stimulation of phytate mineralization under eCO2 differed between the two soils, these ASVs belonged to the same phyla associated with phytase and phosphatase production. The symbiotic mycorrhizas in the rhizosphere of wheat under eCO2 benefited from increased plant C supply and increased P access from soil. Further supportive evidence was the eCO2-induced increase in the genetic pool expressing the pentose phosphate pathway, which is the central pathway for biosynthesis of RNA/DNA precursors. CONCLUSIONS: The results suggested that an increased belowground carbon flow under eCO2 stimulated bacterial growth, changing community composition in favor of phylotypes capable of degrading aromatic P compounds. It is proposed that energy investments by bacteria into anabolic processes increase under eCO2 to level microbial P-use efficiencies and that synergies with symbiotic mycorrhizas further enhance the competition for and mineralization of organic P. Video Abstract.


Assuntos
Microbiota , Rizosfera , Dióxido de Carbono/metabolismo , Microbiota/genética , Fósforo , Filogenia , Microbiologia do Solo , Triticum/metabolismo
17.
Microbiol Spectr ; 10(4): e0152422, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35862970

RESUMO

Streptococcus pneumoniae (the pneumococcus) is a human pathogen of global importance, classified into serotypes based on the type of capsular polysaccharide produced. Serotyping of pneumococci is essential for disease surveillance and vaccine impact measurement. However, the accuracy of serotyping methods can be affected by previously undiscovered variants. Previous studies have identified variants of serotype 14, a highly invasive serotype included in all licensed vaccine formulations. However, the potential of these variants to influence serotyping accuracy and evade vaccine-induced protection has not been investigated. In this study, we screened 1,386 nasopharyngeal swabs from children hospitalized with acute respiratory infection in Papua New Guinea for pneumococci. Swabs containing pneumococci (n = 1,226) were serotyped by microarray to identify pneumococci with a divergent serotype 14 capsule locus. Three serotype 14 variants ('14-like') were isolated and characterized further. The serotyping results of these isolates using molecular methods varied depending on the method, with 3/3 typing as nontypeable (PneumoCaT), 3/3 typing as serotype 14 (seroBA), and 2/3 typing as serotype 14 (SeroCall and quantitative PCR). All three isolates were nontypeable by phenotypic methods (Quellung and latex agglutination), indicating the absence of capsule. Illumina and nanopore sequencing were employed to examine their capsule loci and revealed unique mutations. Lastly, when incubated with sera from vaccinated individuals, the 14-like isolates evaded serotype-specific opsonophagocytic killing. Our study highlights the need for phenotypic testing to validate serotyping data derived from molecular methods. The convergent evolution of capsule loss underscores the importance of studying pneumococcal population biology to monitor the emergence of pneumococci capable of vaccine escape, globally. IMPORTANCE Pneumococcus is a pathogen of major public health importance. Current vaccines have limited valency, targeting a subset (up to 20) of the more than 100 capsule types (serotypes). Precise serotyping methods are therefore essential to avoid mistyping, which can reduce the accuracy of data used to inform decisions around vaccine introduction and/or maintenance of national vaccination programs. In this study, we examine a variant of serotype 14 (14-like), a virulent serotype present in all currently licensed vaccine formulations. Although these 14-like pneumococci no longer produce a serotype 14 capsule, widely used molecular methods can mistype them as serotype 14. Importantly, we show that 14-like pneumococci can evade opsonophagocytic killing mediated by vaccination. Despite the high accuracy of molecular methods for serotyping, our study reemphasizes their limitations. This is particularly relevant in situations where nonvaccine type pneumococci (e.g., the 14-likes in this study) could potentially be misidentified as a vaccine type (e.g., serotype 14).


Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Criança , Humanos , Papua Nova Guiné/epidemiologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas , Sorogrupo , Sorotipagem/métodos , Streptococcus pneumoniae/genética
18.
Microbiol Resour Announc ; 10(35): e0047821, 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34472973

RESUMO

Enterobacter asburiae NCR1 is a plant growth-promoting rhizobacterium isolated from the rhizosphere of Carpobrotus rossii. We report the draft genome sequence of E. asburiae strain NCR1, which revealed many genes facilitating beneficial interactions with plant hosts.

19.
Microbiol Resour Announc ; 10(34): e0048721, 2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34435860

RESUMO

Enterobacter mori is an important plant pathogen. Here, we report the draft genome sequence of the plant-associated strain Enterobacter mori NSE2, which was found to harbor genes for promotive and pathogenic interactions with plants.

20.
PLoS One ; 16(9): e0257102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34492081

RESUMO

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.


Assuntos
Bacteriófagos/fisiologia , Biofilmes , Klebsiella/virologia , Cápsulas Bacterianas/metabolismo , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/isolamento & purificação , Bacteriófagos/ultraestrutura , Códon/genética , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Especificidade de Hospedeiro , Klebsiella/genética , Viabilidade Microbiana , Fases de Leitura Aberta/genética , Filogenia , Proteômica
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