Publications

4851

Abstract Anaerobic ammon ium oxidizing (anammox) bacteria oxidize ammonium and reduce nitrite, producing N2, and could play a major role in energy-optimized wastewater treatment. However, sensitivity to various environmental conditions and slow growth currently hinder their wide application. Here, we attempted to determine online the effect of environmental stresses on anammox bacteria by using an overnight batch activity test with whole cells, in which anammox activity was calculated by quantifying N2 production via headspace-pressure monitoring. A planktonic mixed culture dominated by “Candidatus Kuenenia stuttgartiensis” strain CSTR1 was cultivated in a 30-L semi-continuous stirring tank reactor. In overnight resting-cell anammox activity tests, oxygen caused strong inhibition of anammox activity, which was reversed by sodium sulfite (30 µM). The tested antibiotics sulfamethoxazole, kanamycin, and ciprofloxacin elicited their effect on a dose-dependent manner; however, strain CSTR1 was highly resistant to sulfamethoxazole. Anammox activity was improved by activated carbon and Fe2O3. Protein expression analysis from resting cells after anammox activity stimulation revealed that NapC/NirT family cytochrome c (KsCSTR_12840), hydrazine synthase, hydrazine dehydrogenase, hydroxylamine oxidase, and nitrate:nitrite oxidoreductase were upregulated, while a putative hydroxylamine oxidoreductase HAO (KsCSTR_49490) was downregulated. These findings contribute to the growing knowledge on anammox bacteria physiology, eventually leading to the control of anammox bacteria growth and activity in real-world application. Key Points • Sulfite additions can reverse oxygen inhibition of the anammox process • Anammox activity was improved by activated carbon and ferric oxide • Sulfamethoxazole marginally affected anammox activity Graphical abstract

AbstractOur knowledge of ciliate endosymbiont diversity greatly expanded over the past decades due to the development of characterization methods for uncultivable bacteria. Chlamydia‐like bacteria have been described as symbionts of free‐living amoebae and other phylogenetically diverse eukaryotic hosts. In the present work, a systematic survey of the bacterial diversity associated with the ciliate Euplotes octocarinatus strain Zam5b‐1 was performed, using metagenomic screening as well as classical full‐cycle rRNA approach, and a novel chlamydial symbiont was characterized. The metagenomic screening revealed 16S rRNA gene sequences from Polynucleobacter necessarius, three previously reported accessory symbionts, and a novel chlamydia‐like bacterium. Following the full‐cycle rRNA approach, we obtained the full‐length 16S rRNA gene sequence of this chlamydia‐like bacterium and developed probes for diagnostic fluorescence in situ hybridizations. The phylogenetic analysis of the 16S rRNA gene sequences unambiguously places the new bacterium in the family Rhabdochlamydiaceae. This is the first report of chlamydia‐like bacterium being found in Euplotes. Based on the obtained data, the bacterium is proposed as a new candidate genus and species: “Candidatus Euplotechlamydia quinta.”

Abstract Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O2 (~7%). The cave’s surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

AbstractSymbiotic bacteria alter host biology in numerous ways, including the ability to reproduce or vector disease. Deployment of symbiont control of vector borne disease has focused onWolbachiainteractions withAedesand is hampered inAnophelesby a lack of compatible symbioses. Previous screening found the symbiont ‘Ca. Tisiphia’ inAnopheles plumbeus, an aggressive biter and potential secondary vector of malaria parasites and West Nile virus. We screenAn. plumbeussamples collected over a ten-year period across Germany and use climate databases to assess environmental influence on incidence. We find a 95% infection rate that does not fluctuate with broad environmental factors. Microscopy suggests the infection is maternally inherited based on strong localisation throughout the ovaries. Finally, we assemble a high-quality draft genome of ‘Ca. Tisiphia’ to explore its phylogeny and potential metabolism. This strain is closely related to those found inCulicoidesmidges and shows similar patterns of metabolic potential.An. plumbeusprovides a viable avenue of symbiosis research in anopheline mosquitoes, which to date have one other proven infection of a heritable symbiont. Additionally, it provides future opportunity to study the impacts of ‘Ca. Tisiphia’ on natural and transinfected hosts, especially in relation to reproductive fitness and vector efficiency.

AbstractFour pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.

Recirculating aquaculture systems (RAS) are promising candidates for the sustainable development of the aquaculture industry. A current limitation of RAS is the production and potential accumulation of nitrogenous wastes, ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−), which could affect fish health and welfare. In a previous experiment, we have demonstrated that the marine anammox bacteria Candidatus Scalindua was a promising candidate to treat the wastewater (WW) of marine, cold-water RAS. However, the activity of the bacteria was negatively impacted after a direct exposure to RAS WW. In the current study, we have further investigated the potential of Ca. Scalindua to treat marine RAS WW in a three-phase experiment. In the first phase (control, 83 days), Ca. Scalindua was fed a synthetic feed, enriched in NH4+, NO2− and trace element (TE) mix. Removal rates of 98.9% and 99.6% for NH4+ and NO2−, respectively, were achieved. In the second phase (116 days), we gradually increased the exposure of Ca. Scalindua to nitrogen-enriched RAS WW over a period of about 80 days. In the last phase (79 days), we investigated the needs of TE supplementation for the Ca. Scalindua after they were fully acclimated to 100% RAS WW. Our results show that the gradual exposure of Ca. Scalindua resulted in a successful acclimation to 100% RAS WW, with maintained high removal rates of both NH4+ and NO2− throughout the experiment. Despite a slight decrease in relative abundance (from 21.4% to 16.7%), Ca. Scalindua remained the dominant species in the granules throughout the whole experiment. We conclude that Ca. Scalindua can be successfully used to treat marine RAS WW, without the addition of TE, once given enough time to acclimate to its new substrate. Future studies need to determine the specific needs for optimal RAS WW treatment by Ca. Scalindua at pilot scale.

Huanglongbing (HLB) is one of the most destructive diseases threatening citriculture worldwide. This disease has been associated with α-proteobacteria species, namely Candidatus Liberibacter. Due to the unculturable nature of the causal agent, it has been difficult to mitigate the disease, and nowadays a cure is not available. MicroRNAs (miRNAs) are key regulators of gene expression, playing an essential role in abiotic and biotic stress in plants including antibacterial responses. However, knowledge derived from non-model systems including Candidatus Liberibacter asiaticus (CLas)-citrus pathosystem remains largely unknown. In this study, small RNA profiles from Mexican lime (Citrus aurantifolia) plants infected with CLas at asymptomatic and symptomatic stages were generated by sRNA-Seq, and miRNAs were obtained with ShortStack software. A total of 46 miRNAs, including 29 known miRNAs and 17 novel miRNAs, were identified in Mexican lime. Among them, six miRNAs were deregulated in the asymptomatic stage, highlighting the up regulation of two new miRNAs. Meanwhile, eight miRNAs were differentially expressed in the symptomatic stage of the disease. The target genes of miRNAs were related to protein modification, transcription factors, and enzyme-coding genes. Our results provide new insights into miRNA-mediated regulation in C. aurantifolia in response to CLas infection. This information will be useful to understand molecular mechanisms behind the defense and pathogenesis of HLB.

AbstractBackgroundLake Baikal, the world’s deepest freshwater lake, contains important numbers ofCandidatusPatescibacteria (formerly CPR) in its deepest reaches. However, previously obtained CPR metagenome-assembled genomes recruited very poorly indicating the potential of other groups being present. Here, we have applied for the first time a long-read (PacBio CCS) metagenomic approach to analyze in depth theCa.Patescibacteria living in the bathypelagic water column of Lake Baikal at 1600 m.ResultsThe retrieval of nearly complete 16S rRNA genes before assembly has allowed us to detect the presence of a novel and a likely endemic group ofCa.Patescibacteria inhabiting bathypelagic Lake Baikal. This novel group seems to possess extremely high intra-clade diversity, precluding complete genomes' assembly. However, read binning and scaffolding indicate that these microbes are similar to otherCa.Patescibacteria (i.e. parasites or symbionts), although they seem to carry more anabolic pathways, likely reflecting the extremely oligotrophic habitat they inhabit. The novel bins have not been found anywhere, but one of the groups appears in small amounts in an oligotrophic and deep alpine Lake Thun. We propose this novel group be named Baikalibacteria.ConclusionThe recovery of 16S rRNA genes via long-read metagenomics plus the use of long-read binning to uncover highly diverse “hidden” groups of prokaryotes are key strategies to move forward in ecogenomic microbiology. The novel group possesses enormous intraclade diversity akin to what happens withCa.Patescibacteria at the interclade level, which is remarkable in an environment that has changed little in the last 25 million years.

Cable bacteria are centimeters-long filamentous bacteria that oxidize sulfide in anoxic sediment layers and reduce oxygen at the oxic-anoxic interface, connecting these reactions via electron transport. The ubiquitous cable bacteria have a major impact on sediment geochemistry and microbial communities. This includes diverse bacteria swimming around cable bacteria as dense flocks in the anoxic zone, where the cable bacteria act as chemotactic attractant. We hypothesized that flocking only appears when cable bacteria are highly abundant and active. We set out to discern the timing and drivers of flocking over 81 days in an enrichment culture of the freshwater cable bacterium Candidatus Electronema aureum GS by measuring sediment microprofiles of pH, oxygen, and electric potential as a proxy of cable bacteria activity. Cable bacterial relative abundance was quantified by 16S rRNA amplicon sequencing, and microscopy observations to determine presence of flocking. Flocking was always observed at some cable bacteria, irrespective of overall cable bacteria rRNA abundance, activity, or sediment pH. Diverse cell morphologies of flockers were observed, suggesting that flocking is not restricted to a specific, single bacterial associate. This, coupled with their consistent presence supports a common mechanism of interaction, likely interspecies electron transfer via electron shuttles. Flocking appears exclusively linked to the electron conducting activity of the individual cable bacteria.