Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Alpha-subunit of the Chloroplast ATP Synthase of Tomato Reinforces the Resistance to Grey Mold and Broad-spectrum Resistance in Transgenic Tobacco

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed the cpATPase CF1 alpha subunit (AtpA) is a key protein involved in Clonostachys rosea (C. rosea)-induced resistance to the fungus Botrytis cinerea (B. cinerea) in tomato. Here, we show the expression level of tomato’s cpATPase CF1 alpha subunit gene (atpA) was up-regulated by B. cinerea and C. rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, the atpA-overexpressing tobacco showed an increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response (HR)-like reactions, balanced reactive oxygen species, alleviated damage to chloroplast ultra-structure of leaf cell, elevated levels of ATP content and cpATPase activity, enhanced expression of carbon metabolism-, photosynthesis-, and defense-related genes. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after their inoculation with B. cinerea. Additionally, overexpression of atpA gene conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, the α subunit of cpATPase is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection, in the process providing broad-spectrum resistance in plants.

Ralstonia solanacearum elicitor RipX Induces Defense Reaction by Suppressing the Mitochondrial atpA Gene in Host Plant

RipX of Ralstonia solanacearum is translocated into host cells by a type III secretion system and acts as a harpin-like protein to induce a hypersensitive response in tobacco plants. The molecular events in association with RipX-induced signaling transduction have not been fully elucidated. This work reports that transient expression of RipX induced a yellowing phenotype in Nicotiana benthamiana, coupled with activation of the defense reaction. Using yeast two-hybrid and split-luciferase complementation assays, mitochondrial ATP synthase F1 subunit α (ATPA) was identified as an interaction partner of RipX from N. benthamiana. Although a certain proportion was found in mitochondria, the YFP-ATPA fusion was able to localize to the cell membrane, cytoplasm, and nucleus. RFP-RipX fusion was found from the cell membrane and cytoplasm. Moreover, ATPA interacted with RipX at both the cell membrane and cytoplasm in vivo. Silencing of the atpA gene had no effect on the appearance of yellowing phenotype induced by RipX. However, the silenced plants improved the resistance to R. solanacearum. Moreover, qRT-PCR and promoter GUS fusion experiments revealed that the transcript levels of atpA were evidently reduced in response to expression of RipX. These data demonstrated that RipX exerts a suppressive effect on the transcription of atpA gene, to induce defense reaction in N. benthamiana.

Sensitive Detection of Soy (Glycine max) by Real-Time Polymerase Chain Reaction Targeting the Mitochondrial atpA Gene

Detection of trace amounts of allergens is essential for correct labeling of food products by the food industry. PCR-based detection methods currently used for this purpose are targeting sequences of DNA present in the cell nucleus. In addition to nuclear DNA, a substantial amount of mitochondrial DNA (mtDNA) copies are present in the cytoplasm of eukaryotic cells. The nuclear DNA usually consists of a set of DNA molecules present in two copies per cell, whereas mitochondrial DNA is present in a few hundred copies per cell. Thus, an increase in sensitivity can be expected when mtDNA is used as the target. In this study, we present a reporter probe-based real-time PCR method amplifying the mitochondrial gene of the alpha chain of adenosine triphosphate synthetase from soy. Increase in sensitivity was examined by determining the minimal amount of soy DNA detectable by mtDNA and nuclear DNA (nDNA) amplification. Additionally, the LOD of soy in a food matrix was determined for mtDNA amplification and compared to the LOD determined by nDNA amplification. As food matrix, a model spice spiked with soy flour was used. Sensitivity of PCR-based soy detection can be increased by using mtDNA as the target.

Hybrid origin of Athrotaxis laxifolia (Taxodiaceae) confirmed by random amplified polymorphic DNA analysis

Random amplified polymorphic DNA (RAPD) and single-strand conformation polymorphism (SSCP) analyses were employed for investigating genetic relationships of three Athrotaxis D.Don species. Twenty-nine RAPD primers produced 103 polymorphic bands. Principal component analysis revealed the genomic differentiation among three Athrotaxis species. Mean genetic distance (mean d) between A. selaginoides D.Don and A. cupressoides D.Don was 0.89. Mean d values were reduced to 0.42/0.54 between A. laxifolia Hook. and A. selaginoides/A. cupressoides, respectively. Intraspecific mean d of A. selaginoides and A. cupressoides were, respectively, 0.03 and 0.11. These values indicated that A. laxifolia, which is regarded as a hybrid between A. selaginoides and A. cupressoides, is genetically intermediate between A. selaginoides and A. cupressoides. This genetic characteristic and previously reported morphological characteristics suggest the hybrid origins of A. laxifolia. The genomic composition of A. laxifolia was estimated by the number of bands specific to A. selaginoides or A. cupressoides in order to determine the genomic contribution of these two species to its proposed hybrid, A. laxifolia. All of the five individuals investigated herein possessed genomes derived almost evenly from A. selaginoides and A. cupressoides. Furthermore, the pollen donor of A. laxifolia was determined by SSCP analysis of the atpA gene on chloroplast DNA. Because all of the five A. laxifolia possessed the A. selaginoides-type chloroplast genome, A. laxifolia would be a hybrid of A. selaginoides as a paternal parent and A. cupressoides as a maternal parent.