Food Science

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.

ABSTRACT Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. “ Candidatus Methylomirabilis oxyfera” is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, “ Ca. Methylomirabilis oxyfera” encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by “ Ca. Methylomirabilis oxyfera” via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an “ Ca. Methylomirabilis oxyfera” enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either 13 CH 4 or [ 13 C]bicarbonate revealed that “ Ca. Methylomirabilis oxyfera” biomass and lipids became significantly more enriched in 13 C after incubation with 13 C-labeled bicarbonate (and unlabeled methane) than after incubation with 13 C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in “ Ca. Methylomirabilis oxyfera.” Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by “ Ca. Methylomirabilis oxyfera” bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by 13 C-depleted lipids.

ABSTRACT The tick-borne bacterium “ Candidatus Neoehrlichia mikurensis” has recently been recognized as a human pathogen. Together with Borrelia afzelii , it is one of the most common pathogens found in the tick Ixodes ricinus . Here, we compared the epidemiologies of “ Ca . Neoehrlichia mikurensis” and B. afzelii by longitudinal sampling from May to September in one of their most abundant vertebrate hosts, the bank vole ( Myodes glareolus ), using real-time PCR for detection and quantification. The prevalences of “ Ca . Neoehrlichia mikurensis” and B. afzelii were determined to be 19% (50/261) and 22% (56/261), respectively. The prevalence of “ Ca . Neoehrlichia mikurensis” increased significantly during the sampling season. The clearance rate of “ Ca . Neoehrlichia mikurensis” was significantly higher than that of B. afzelii . We found a high frequency of double infections; 46% of all samples infected with “ Ca . Neoehrlichia mikurensis” also had a coinfection with B. afzelii . The frequency of coinfections was significantly higher than expected from the prevalence of each pathogen. The high level of coinfections can be caused by interactions between the pathogens or might reflect variation in general susceptibility among voles.

ABSTRACT Bacterial endosymbionts of the pine bark adelgid, Pineus strobi (Insecta: Hemiptera: Adelgidae), were investigated using transmission electron microscopy, 16S and 23S rRNA-based phylogeny, and fluorescence in situ hybridization. Two morphologically different symbionts affiliated with the Gammaproteobacteria were present in distinct bacteriocytes. One of them (“ Candidatus Annandia pinicola”) is most closely related to an endosymbiont of Adelges tsugae , suggesting that they originate from a lineage already present in ancient adelgids before the hosts diversified into the two major clades, Adelges and Pineus . The other P. strobi symbiont (“ Candidatus Hartigia pinicola”) represents a novel symbiont lineage in members of the Adelgidae. Our findings lend further support for a complex evolutionary history of the association of adelgids with a phylogenetically diverse set of bacterial symbionts.

ABSTRACT A gammaproteobacterial facultative symbiont of the genus Rickettsiella was recently identified in the pea aphid, Acyrthosiphon pisum . Infection with this symbiont altered the color of the aphid body from red to green, potentially affecting the host's ecological characteristics, such as attractiveness to different natural enemies. In European populations of A. pisum , the majority of Rickettsiella -infected aphids also harbor another facultative symbiont, of the genus Hamiltonella . We investigated this Rickettsiella symbiont for its interactions with the coinfecting Hamiltonella symbiont, its phenotypic effects on A. pisum with and without Hamiltonella coinfection, and its infection prevalence in A. pisum populations. Histological analyses revealed that coinfecting Rickettsiella and Hamiltonella exhibited overlapping localizations in secondary bacteriocytes, sheath cells, and hemolymph, while Rickettsiella -specific localization was found in oenocytes. Rickettsiella infections consistently altered hosts' body color from red to green, where the greenish hue was affected by both host and symbiont genotypes. Rickettsiella - Hamiltonella coinfections also changed red aphids to green; this greenish hue tended to be enhanced by Hamiltonella coinfection. With different host genotypes, Rickettsiella infection exhibited either weakly beneficial or nearly neutral effects on host fitness, whereas Hamiltonella infection and Rickettsiella - Hamiltonella coinfection had negative effects. Despite considerable frequencies of Rickettsiella infection in European and North American A. pisum populations, no Rickettsiella infection was detected among 1,093 insects collected from 14 sites in Japan. On the basis of these results, we discuss possible mechanisms for the interaction of Rickettsiella with other facultative symbionts, their effects on their hosts' phenotypes, and their persistence in natural host populations. We propose the designation “ Candidatus Rickettsiella viridis” for the symbiont.

ABSTRACT “ Candidatus Cardinium hertigii” ( Bacteroidetes ) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “ Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “ Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola , a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “ Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “ Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “ Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “ Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “ Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “ Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.

ABSTRACTHistological analysis of gill samples taken from individuals ofLatris lineatareared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological,in situhybridization, and molecular analyses were performed to confirm the presence of this disease and discovered aChlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman's postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the orderChlamydiales. This new taxon formed a well-supported clade with “CandidatusParilichlamydia carangidicola” from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the orderChlamydiales, a new genus and species are proposed here for theChlamydia-like bacterium fromL. lineata, i.e., “CandidatusSimilichlamydia latridicola” gen. nov., sp. nov.

ABSTRACT The draft genome sequence of a single orange Beggiatoa (“ Candidatus Maribeggiatoa”) filament collected from a microbial mat at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) shows evidence of extensive genetic exchange with cyanobacteria, in particular for sensory and signal transduction genes. A putative homing endonuclease gene and group I intron within the 23S rRNA gene; several group II catalytic introns; GyrB and DnaE inteins, also encoding homing endonucleases; multiple copies of sequences similar to the fdxN excision elements XisH and XisI (required for heterocyst differentiation in some cyanobacteria); and multiple sequences related to an open reading frame (ORF) (00024_0693) of unknown function all have close non- Beggiatoaceae matches with cyanobacterial sequences. Sequences similar to the uncharacterized ORF and Xis elements are found in other Beggiatoaceae genomes, a variety of cyanobacteria, and a few phylogenetically dispersed pleiomorphic or filamentous bacteria. We speculate that elements shared among filamentous bacterial species may have been exchanged in microbial mats and that some of them may be involved in cell differentiation.

ABSTRACT The phylogenetic affiliation and physiological characteristics (e.g., K s and maximum specific growth rate [μ max ]) of an anaerobic ammonium oxidation (anammox) bacterium, “ Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “ Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.