The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. ...cerevisiae mitochondria can exist with partial (ρ− mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well‐suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.
The ease with which the unicellular yeast Saccharomyces cerevisiae can be manipulated genetically and biochemically has established this organism as a good model for the study of human mitochondrial ...diseases. The combined use of biochemical and molecular genetic tools has been instrumental in elucidating the functions of numerous yeast nuclear gene products with human homologs that affect a large number of metabolic and biological processes, including those housed in mitochondria. These include structural and catalytic subunits of enzymes and protein factors that impinge on the biogenesis of the respiratory chain. This article will review what is currently known about the genetics and clinical phenotypes of mitochondrial diseases of the respiratory chain and ATP synthase, with special emphasis on the contribution of information gained from pet mutants with mutations in nuclear genes that impair mitochondrial respiration. Our intent is to provide the yeast mitochondrial specialist with basic knowledge of human mitochondrial pathologies and the human specialist with information on how genes that directly and indirectly affect respiration were identified and characterized in yeast.
Mitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase ...consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the bona fide ring. We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymes.
Mitochondrial tRNAs are processed at their 5’ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel ...genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2.
The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.
Mta1p, Mta2p, Gep5 and Pet130p are necessary for proper processing of the mitochondrial tRNAs at their 5' ends. Display omitted
•Four uncharacterized genes are necessary for mitochondrial tRNA processing.•Northern analyses shown accumulation of tRNA precursor transcripts in the tested mutants.•These transcripts are also accumulated in rpm2 mutants, the yeast protein component of RNase P.•Mta1p is co-purified with Rpm2p, indicating their physical interaction.
Cytochrome oxidase (COX) is a hetero-oligomeric complex of the mitochondrial inner membrane that reduces molecular oxygen to water, a reaction coupled to proton transfer from the mitochondrial matrix ...to the intermembrane space. In the yeast Saccharomyces cerevisiae, COX is composed of 11–13 different polypeptide subunits. Here, using pulse labeling of mitochondrial gene products in isolated yeast mitochondria, combined with purification of tagged COX subunits and ancillary factors, we studied the Cox2p assembly intermediates. Analysis of radiolabeled Cox2p obtained in pulldown assays by native gel electrophoresis revealed the existence of several assembly intermediates, the largest of which had an estimated mass of 450–550 kDa. None of the other known subunits of COX were present in these Cox2p intermediates. This was also true for the several ancillary factors having still undefined functions in COX assembly. In agreement with earlier evidence, Cox18p and Cox20p, previously shown to be involved in processing and in membrane insertion of the Cox2p precursor, were found to be associated with the two largest Cox2p intermediates. A small fraction of the Cox2p module contained Sco1p and Coa6p, which have been implicated in metalation of the binuclear copper site on this subunit. Our results indicate that following its insertion into the mitochondrial inner membrane, Cox2p assembles as a stand-alone protein with the compositionally more complex Cox1p and Cox3p modules.
Product and byproducts of the citrus industry are destined mainly for export and they play a significant role in the Brazilian economy. The major threats to citrus production and business include ...weather conditions (abiotic stress) and plant disease epidemics (biotic stress), especially bacterial diseases. Historical examples of devastating bacterial diseases that have shaped and changed radically citrus production are found in many countries. It is important for citrus pathologists to become aware of the dimension of citrus business in terms of the global demand and consumption. For such, we gathered data from several sources and calculated the global consumption of Frozen Concentrate Orange Juice (FCOJ) 66° Brix equivalent, the main citrus industry product. A historical analysis of the 16 years (2013 to 2018) of the global consumption including total and
per capita
consumption of FCOJ was conducted. In addition, we summarized the consumer preferences regarding 100% juices, nectars, and juice drinks in the main market worldwide. The data depict the dynamics of consumption and the trends that drive consumer behavior in the food and fruit beverage market. We find that, despite a growth of 23.7% in the consumption of industrialized fruit drinks during the period, the consumption of 100% orange juice declined 22.78%. The drop in consumption of FCOJ has been accentuated in developed economies, such as Germany (− 44.75%), USA (− 42.41%), UK (− 26.80%), and France (− 14.34%). On the other hand, emerging economies have increased their consumption of FCOJ, as evidenced by BRICs plus Mexico (+ 75.52%). Food and beverage trends are leading different behaviors in the demand of orange juice globally, which tends to stabilize the global market. With this in mind, pathologists play a fundamental role in citrus chain, in order to guarantee that citrus suppliers continue providing good quality fruits to the industry and overcome the challenges of bacterial diseases.
Objectives
The present study aimed to assess the antimicrobial and antiadhesion behavior of quercetin‐loaded chitosan nanoparticles in Escherichia coli and Staphylococcus aureus multidrug‐resistant ...isolates.
Methods
The ionic gelation method was used to prepare chitosan nanoparticles loaded with quercetin. The antimicrobial and antibiofilm effects were observed by minimum inhibitory concentration (MIC), plate count, crystal violet assay, and the matrix exopolysaccharide dosages. The nanoparticles coated in silicone urethral catheters were evaluated by crystal violet assay and plating count method.
Results
MIC ranged from 6.25 to 12.5 mg/ml. A reduction of at least 3.6 log CFU/ml and 6.2 log CFU/ml for, respectively, E. coli and S. aureus isolates was observed (p < 0.05). Under subinhibitory concentration (3.1 mg/ml) it was found a reduction of microbial adhesion and exopolysaccharide dosages in respectively 83.3% and 75% of the bacterial samples. The coated silicone urethral catheters showed a reduction of adhered cells in 25% of the isolates and biomass decreasing in 91.6% of them (p < 0.05).
Conclusions
The quercetin nanoparticles provided antimicrobial and antiadhesion effects in multidrug‐resistant isolates.
The timely release of SARS-CoV-2 first genomic sequences allowed the identification of the etiologic agent and development of diagnostic protocols. Genomic sequencing was a crucial step in generating ...data for driving laboratory response and detections of SARS-CoV-2 since the start of the COVID-19 pandemic. Because of all the progression and achievements that timely release of genetic sequence data represents in the public health response, the Pan American Health Organization (PAHO) in collaboration with countries' public health laboratories, started implementation of a network for strengthening the Latin America and Caribbean (LAC) region on timely generation of SARS-CoV-2 genomic data. Here we describe the implementation of the COVID-19 Genomic Surveillance Regional Network in the Americas region during the beginning of the pandemic. The establishment of this network has strengthened laboratory response capacity at the country level, as well as facilitated timely release of SARS-CoV-2 genomic information to be used to complement the multiple response strategies for COVID-19 pandemic mitigation. As genomic epidemiology is useful for guiding public health decisions on outbreak and response, we also analysed the first SARS-CoV-2 genomic sequence data from countries of the Latin America and Caribbean Region.
Mitochondrial translation normally requires formylation of the initiator tRNA‐met, a reaction catalyzed by the enzyme formyltransferase, Fmt1p and MTFMT in Saccharomyces cerevisiae and human ...mitochondria, respectively. Yeast fmt1 mutants devoid of Fmt1p, however, can synthesize all mitochondrial gene products by initiating translation with a non‐formylated methionyl‐tRNA. Yeast synthetic respiratory‐deficient fmt1 mutants have uncovered several factors suggested to play a role in translation initiation with non‐formylated methionyl‐tRNA. Here, we present evidence that Msc6p, a member of the pentatricopeptide repeat (PPR) motif family, is another essential factor for mitochondrial translation in fmt1 mutants. The PPR motif is characteristic of RNA‐binding proteins found in chloroplasts and plant and fungal mitochondria, and is generally involved in RNA stability and transport. Moreover, in the present study, we show that the respiratory deficiency of fmt1msc6 double mutants can be rescued by overexpression of the yeast mitochondrial initiation factor mIF‐2, encoded by IFM1. The role of Msc6p in translational initiation is further supported by pull‐down assays showing that it transiently interacts with mIF‐2. Altogether, our data indicate that Msc6p is an important factor in mitochondrial translation with an auxiliary function related to the mIF‐2‐dependent formation of the initiation complex.
In wild‐type (wt), yeast mitochondrial translation initiation normally requires the recognition of formylated fMet‐tRNAfMet by the mitochondrial initiation factor 2 (mIF‐2). Msc6p, a protein with pentatricopeptide repeat motifs, is necessary for mitochondrial translation initiation in yeast mutants devoid of the enzyme formyltransferase (∆fmt1). Our data indicate that Msc6p is an important auxiliary factor for mIF‐2‐dependent initiation of translation on mitoribosomes
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