Publications

4859

Summary ‘ Candidatus Methylomirabilis oxyfera’ is a denitrifying methanotroph that performs nitrite‐dependent anaerobic methane oxidation through a newly discovered intra‐aerobic pathway. In this study, we investigated the response of a M. oxyfera enrichment culture to oxygen. Addition of either 2% or 8% oxygen resulted in an instant decrease of methane and nitrite conversion rates. Oxygen exposure also led to a deviation in the nitrite to methane oxidation stoichiometry. Oxygen‐uptake and inhibition studies with cell‐free extracts displayed a change from cytochrome c to quinol as electron donor after exposure to oxygen. The change in global gene expression was monitored by deep sequencing of cDNA using Illumina technology. After 24 h of oxygen exposure, transcription levels of 1109 (out of 2303) genes changed significantly when compared with the anoxic period. Most of the genes encoding enzymes of the methane oxidation pathway were constitutively expressed. Genes from the denitrification pathway, with exception of one of the putative nitric oxide reductases, norZ2 , were severely downregulated. The majority of known genes involved in the vital cellular functions, such as nucleic acid and protein biosynthesis and cell division processes, were downregulated. The alkyl hydroperoxide reductase, ahpC , and genes involved in the synthesis/repair of the iron–sulfur clusters were among the few upregulated genes. Further, transcription of the pmoCAB genes of aerobic methanotrophs present in the non‐ M. oxyfera community were triggered by the presence of oxygen. Our results show that oxygen‐exposed cells of M. oxyfera were under oxidative stress and that in spite of its oxygenic capacity, exposure to microoxic conditions has an overall detrimental effect.

Symptoms of abnormal proliferation of shoots resulting in formation of witches’-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group (‘Candidatus Phytoplasma pruni’-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described ‘Ca. Phytoplasma ’ species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Ca. Phytoplasma ’ species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, ‘Candidatus Phytoplasma sudamericanum’.

Carrot (Daucus carota) plants with symptoms resembling those associated with the carrot psyllid Trioza apicalis and the bacterium “Candidatus Liberibacter solanacearum” (1–4) were observed in 70% of commercial fields in southern Sweden in August 2011, with approximately 1 to 45% symptomatic plants per field. T. apicalis, a pest of carrot in northern and central Europe, including Sweden, can cause as much as 100% crop loss and is associated with “Ca. L. solanacearum” (1–4). Symptoms on affected plants include leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (3). Carrot plant and psyllid samples were collected from fields in the province of Halland. Total DNA was extracted from petiole and root tissues of 33 symptomatic and 16 asymptomatic plants (cvs. Nevis and Florida), with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,3). DNA was also extracted from 155 psyllids (3). DNA samples were tested by PCR using primer pairs OA2/OI2c (5′'-GCGCTTATTTTTAATAGGAGCGGCA-3′/5′-GCCTCGCGACTTCGCAACCCAT-3′) and CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′/5′-TATATCTATCGTTGCACCAG-3′), to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Ca. L. solanacearum” (2,3). A 1,168-bp 16S rDNA fragment was detected in the DNA from all 33 symptomatic and two asymptomatic plants, and a 668-bp rplJ/rplL fragment was amplified from the DNA of all 33 symptomatic and four asymptomatic plants, indicating the presence of liberibacter. DNA from 23 and 49 psyllid samples yielded similar amplicons with OA2/OI2c and CL514F/R primer pairs, respectively. Amplicons from the DNA of four carrot roots and three T. apicalis with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the 14 amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequences from carrot (GenBank Accession No. JN863095) and T. apicalis (GenBank Accession No. NJ863096) showed 100% identity to those of “Ca. L. solanacearum” previously amplified from carrot (GU373048 and GU373049) and T. apicalis (GU477254 and GU477255) from Finland (2,3). The rplJ/rplL consensus sequences from carrot (GenBank Accession No. JN863093) and T. apicalis (GenBank Accession No. JN863094) were 99% identical to the sequences of rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene from carrots in Finland (GU373050 and GU373051). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with carrot and T. apicalis in Sweden. The disease associated with this bacterium caused millions of dollars in losses to potato and several other solanaceous crops in North and Central America and New Zealand (1). This plant pathogen is also associated with significant economic damage to carrot crops observed in Finland (2,3). References: (1) J. E. Munyaneza. Southwest. Entomol. 35:471, 2010. (2) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (3) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.

Abstract Bat flies of the family Nycteribiidae are known for their extreme morphological and physiological traits specialized for ectoparasitic blood-feeding lifestyle on bats, including lack of wings, reduced head and eyes, adenotrophic viviparity with a highly developed uterus and milk glands, as well as association with endosymbiotic bacteria. We investigated Japanese nycteribiid bat flies representing 4 genera, 8 species and 27 populations for their bacterial endosymbionts. From all the nycteribiid species examined, a distinct clade of gammaproteobacteria was consistently detected, which was allied to endosymbionts of other insects such as Riesia spp. of primate lice and Arsenophonus spp. of diverse insects. In adult insects, the endosymbiont was localized in specific bacteriocytes in the abdomen, suggesting an intimate host–symbiont association. In adult females, the endosymbiont was also found in the cavity of milk gland tubules, which suggests uterine vertical transmission of the endosymbiont to larvae through milk gland secretion. In adult females of Penicillidia jenynsii, we discovered a previously unknown type of symbiotic organ in the Nycteribiidae: a pair of large bacteriomes located inside the swellings on the fifth abdominal ventral plate. The endosymbiont genes consistently exhibited adenine/thymine biased nucleotide compositions and accelerated rates of molecular evolution. The endosymbiont genome was estimated to be highly reduced, ∼0.76 Mb in size. The endosymbiont phylogeny perfectly mirrored the host insect phylogeny, indicating strict vertical transmission and host–symbiont co-speciation in the evolutionary course of the Nycteribiidae. The designation ‘Candidatus Aschnera chinzeii’ is proposed for the endosymbiont clade.