Publications

4985

Abstract Candidatus Liberibacter asiaticus (Las) has been reported to increase the susceptibility of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), to selected insecticides. Reduced general esterase activity in Las-infected, compared with uninfected, D. citri has been proposed as a possible explanation for this difference in insecticide susceptibility. The current study was conducted to quantify glutathione transferase (GST) and cytochrome P450 (general oxidase) activities in Las-infected D. citri to further explain the possible mechanisms for altered susceptibility to insecticides due to Las infection. GST and cytochrome P450 activities (indirectly through general oxidase levels) were quantified in Las-infected and uninfected D. citri nymphs and adults. Mean (±SEM) GST activity was significantly lower in Las-infected (468.23 ± 26.87 /µmol/min/mg protein) than uninfected (757.63 ± 59.46 µmol/min/mg protein) D. citri adults. Likewise, mean cytochrome P450 activity was significantly lower in Las-infected (0.23 ± 0.02 equivalent units [EU] cytochrome P450/mg protein) than uninfected (0.49 ± 0.05 EU cytochrome P450/mg protein) D. citri adults. Immature stages (second and fifth instars) were characterized by significantly lower GST activity than adults for uninfected D. citri. However, cytochrome P450 activity was significantly higher in second instar nymphs than adults and fifth-instar nymphs for uninfected D. citri. Lower activities of GST and general oxidase in Las-infected D. citri indicate that infection with Las alters D. citri physiology in a manner that could increase insecticide susceptibility. The reduced activities of these detoxifying enzymes due to Las infection may be explained by examining expression levels of associated genes in Las-infected and uninfected D. citri.

The anaerobic nitrite-reducing methanotroph ‘CandidatusMethylomirabilis oxyfera’ (‘Ca.M. oxyfera’) produces oxygen from nitrite by a novel pathway. The major part of the O2is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of ‘Ca.M. oxyfera’ harbours four sets of genes encoding terminal respiratory oxidases: two cytochromecoxidases, a third putativebo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV–visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by ‘Ca.M. oxyfera’, notably the membrane-boundbo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.

ABSTRACTOne emerging disease of grapevine in Europe is Bois noir (BN), a phytoplasmosis caused by “CandidatusPhytoplasma solani” and spread in vineyards by the planthopperHyalesthes obsoletus(Hemiptera: Cixiidae). Here we present the first full characterization of the bacterial community of this important disease vector collected from BN-contaminated areas in Piedmont, Italy. Length heterogeneity PCR and denaturing gradient gel electrophoresis analysis targeting the 16S rRNA gene revealed the presence of a number of bacteria stably associated with the insect vector. In particular, symbiotic bacteria detected by PCR with high infection rates in adult individuals fell within the “CandidatusSulcia muelleri” cluster in theBacteroidetesand in the “CandidatusPurcelliella pentastirinorum” group in theGammaproteobacteria, both previously identified in different leafhoppers and planthoppers. A high infection rate (81%) was also shown for another symbiont belonging to theBetaproteobacteria, designated the HO1-V symbiont. Because of the low level of 16S rRNA gene identity (80%) with the closest relative, an uncharacterized symbiont of the tickHaemaphysalis longicornis, we propose the new name “CandidatusVidania fulgoroideae.” Other bacterial endosymbionts identified inH. obsoletuswere related to the intracellular bacteriaWolbachia pipientis,Rickettsiasp., and “CandidatusCardinium hertigii.” Fluorescentin situhybridization coupled with confocal laser scanning microscopy and transmission electron microscopy showed that these bacteria are localized in the gut, testicles, and oocytes. As “Ca.Sulcia” is usually reported in association with other symbiotic bacteria, we propose that inH. obsoletus, it may occur in a bipartite or even tripartite relationship between “Ca.Sulcia” and “Ca.Purcelliella,” “Ca.Vidania,” or both.

Abstract Enhanced biological phosphorus removal (EBPR) activated sludge communities enriched in ‘Candidatus Accumulibacter’ relatives are widely used in wastewater treatment, but much remains to be learned about molecular-level controls on the EBPR process. The expression of genes found in the carbon and polyphosphate metabolic pathways in Accumulibacter was investigated using reverse transcription quantitative PCR. During a normal anaerobic/aerobic EBPR cycle, gene expression exhibited a dynamic change in response to external acetate, oxygen, phosphate concentrations and probably internal chemical pools. Anaerobic acetate addition induced expression of genes associated with the methylmalonyl-CoA pathway enabling the split mode of the tricarboxylic acid (TCA) cycle. Components of the full TCA cycle were induced after the switch to aerobic conditions. The induction of a key gene in the glyoxylate shunt pathway was observed under both anaerobic and aerobic conditions, with a higher induction by aeration. Polyphosphate kinase 1 from Accumulibacter was expressed, but did not appear to be regulated by phosphate limitation. To understand how Accumulibacter responds to disturbed electron donor and acceptor conditions, we perturbed the process by adding acetate aerobically. When high concentrations of oxygen were present simultaneously with acetate, phosphate-release was almost completely inhibited, and polyphosphate kinase 1 transcript abundance decreased. Genes associated with the methylmalonyl-CoA pathway were repressed and genes associated with the aerobic TCA cycle exhibited higher expression under this perturbation, suggesting that more acetyl-CoA was metabolized through the TCA cycle. These findings suggest that several genes involved in EBPR are tightly regulated at the transcriptional level.

Summary A magnetotactic bacterium, designated strain LO‐1, of the Nitrospirae phylum was detected and concentrated from a number of freshwater and slightly brackish aquatic environments in southern Nevada. The closest phylogenetic relative to LO‐1 is Candidatus Magnetobacterium bavaricum based on a 91.2% identity in their 16S rRNA gene sequence. Chemical and cell profiles of a microcosm containing water and sediment show that cells of strain LO‐1 are confined to the oxic–anoxic interface and the upper regions of the anaerobic zone which in this case, occurred in the sediment. This microorganism is relatively large, ovoid in morphology and usually biomineralizes three braid‐like bundles of multiple chains of bullet‐shaped magnetosomes that appeared to be enclosed in a magnetosome membrane. Cells of LO‐1 had an unusual three‐layered unit membrane cell wall and contained several types of inclusions, some of which are sulfur‐rich. Strain LO‐1 is motile by means of a single bundle of sheathed flagella and exhibits the typical ‘wobbling’ motility and helical swimming (‘flight’) path of the magnetotactic cocci. This study and reports from others suggest that LO‐1‐like organisms are widespread in sediments of freshwater to brackish natural aquatic environments.

Summary ‘ Candidatus Liberibacter spp.’ cause serious plant diseases. ‘ Candidatus Liberibacter asiaticus’, ‘ Ca. L. americanus’ and ‘ Ca. L. africanus’ are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. ‘ Candidatus Liberibacter solanacearum’ causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named ‘ Ca. Liberibacter europaeus’. It can bloom to high titres in the psyllid host, with more than 10 9 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that ‘ Ca. L. europaeus’ is present in the host midgut lumen, salivary glands and Malpighian tubules. ‘ Candidatus L. europaeus’ has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10 8 16S rRNA gene copies g −1 of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6‐month period. Despite liberibacters representing potential quarantine organisms, ‘ Ca. L. europaeus’, first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.