Publications

4372

ABSTRACT Nitrification is a key process of the biogeochemical nitrogen cycle and of biological wastewater treatment. The second step, nitrite oxidation to nitrate, is catalyzed by phylogenetically diverse, chemolithoautotrophic nitrite-oxidizing bacteria (NOB). Uncultured NOB from the genus “ Candidatus Nitrotoga” are widespread in natural and engineered ecosystems. Knowledge about their biology is sparse, because no genomic information and no pure “ Ca . Nitrotoga” culture was available. Here we obtained the first “ Ca . Nitrotoga” isolate from activated sludge. This organism, “ Candidatus Nitrotoga fabula,” prefers higher temperatures (>20°C; optimum, 24 to 28°C) than previous “ Ca . Nitrotoga” enrichments, which were described as cold-adapted NOB. “ Ca . Nitrotoga fabula” also showed an unusually high tolerance to nitrite (activity at 30 mM NO 2 − ) and nitrate (up to 25 mM NO 3 − ). Nitrite oxidation followed Michaelis-Menten kinetics, with an apparent K m ( K m (app) ) of ~89 µM nitrite and a V max of ~28 µmol of nitrite per mg of protein per h. Key metabolic pathways of “ Ca . Nitrotoga fabula” were reconstructed from the closed genome. “ Ca . Nitrotoga fabula” possesses a new type of periplasmic nitrite oxidoreductase belonging to a lineage of mostly uncharacterized proteins. This novel enzyme indicates (i) separate evolution of nitrite oxidation in “ Ca . Nitrotoga” and other NOB, (ii) the possible existence of phylogenetically diverse, unrecognized NOB, and (iii) together with new metagenomic data, the potential existence of nitrite-oxidizing archaea. For carbon fixation, “ Ca . Nitrotoga fabula” uses the Calvin-Benson-Bassham cycle. It also carries genes encoding complete pathways for hydrogen and sulfite oxidation, suggesting that alternative energy metabolisms enable “ Ca . Nitrotoga fabula” to survive nitrite depletion and colonize new niches. IMPORTANCE Nitrite-oxidizing bacteria (NOB) are major players in the biogeochemical nitrogen cycle and critical for wastewater treatment. However, most NOB remain uncultured, and their biology is poorly understood. Here, we obtained the first isolate from the environmentally widespread NOB genus “ Candidatus Nitrotoga” and performed a detailed physiological and genomic characterization of this organism (“ Candidatus Nitrotoga fabula”). Differences between key phenotypic properties of “ Ca . Nitrotoga fabula” and those of previously enriched “ Ca . Nitrotoga” members reveal an unexpectedly broad range of physiological adaptations in this genus. Moreover, genes encoding components of energy metabolisms outside nitrification suggest that “ Ca . Nitrotoga” are ecologically more flexible than previously anticipated. The identification of a novel nitrite-oxidizing enzyme in “ Ca . Nitrotoga fabula” expands our picture of the evolutionary history of nitrification and might lead to discoveries of novel nitrite oxidizers. Altogether, this study provides urgently needed insights into the biology of understudied but environmentally and biotechnologically important microorganisms.

Carrot yellows disease has been associated for many years with the Gram-positive, insect-vectored bacteria, ‘Candidatus Phytoplasma’ and Spiroplasma citri. However, reports in the last decade also link carrot yellows symptoms with a different, Gram-negative, insect-vectored bacterium, ‘Ca. Liberibacter solanacearum’. Our study shows that to date ‘Ca. L. solanacearum’ is tightly associated with carrot yellows symptoms across Israel. The genetic variant found in Israel is most similar to haplotype D, found around the Mediterranean Basin. We further show that the psyllid vector of ‘Ca. L. solanacearum’, Bactericera trigonica, is highly abundant in Israel and is an efficient vector for this pathogen. A survey conducted comparing conventional and organic carrot fields showed a marked reduction in psyllid numbers and disease incidence in the field practicing chemical control. Fluorescent in situ hybridization and scanning electron microscopy analyses further support the association of ‘Ca. L. solanacearum’ with disease symptoms and show that the pathogen is located in phloem sieve elements. Seed transmission experiments revealed that while approximately 30% of the tested carrot seed lots are positive for ‘Ca. L. solanacearum’, disease transmission was not observed. Possible scenarios that may have led to the change in association of the disease etiological agent with carrot yellows are discussed. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Grapevine yellows diseases occur in cultivated grapevine (Vitis vinifera L.) on several continents, where the diseases are known by different names depending upon the identities of the causal phytoplasmas. In this study, phytoplasma strains associated with grapevine yellows disease (North American grapevine yellows [NAGY]) in vineyards of Pennsylvania were characterized as belonging to 16S ribosomal RNA (rRNA) gene restriction fragment length polymorphism group 16SrI (aster yellows phytoplasma group), subgroup 16SrI-B (I-B), and variant subgroup I-B*. The strains (NAGYI strains) were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico three-dimensional modeling of the SecY protein. Although the NAGYI strains are closely related to aster yellows (AY) phytoplasma strains and are classified like AY strains in subgroup I-B or in variant subgroup I-B*, the results from genotyping and protein modeling may signal ongoing evolutionary divergence of NAGYI strains from related strains in subgroup 16SrI-B.