Marine Proteobacteria metabolize glycolate via the ß-hydroxyaspartate cycle.

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments1. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria2-4, the further fate of this C2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the ß-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago5. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing-to our knowledge-the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

Detection of Borrelia miyamotoi in Ixodes ricinus ticks in Southern Moravia - risk for public health.

Borrelia miyamotoi is an emerging tick-borne pathogen. The vector is ticks of the Ixodes ricinus complex, causing relapsing fever- like disease and widespread in the temperate zone of the Northern Hemisphere. The present study documents the prevalence of B. miyamotoi in Ixodes ricinus ticks at four sites of the South Moravian Region. The spirochete B. miyamotoi was detected in a total of 10 samples from all four sites and all instars. The occurrence of B. miyamotoi in ticks in the Czech Republic poses a potential risk to public health.

Borrelia miyamotoi - another emerging tick-borne pathogen.

Borrelia miyamotoi is an emerging tick-borne pathogen phylogenetically belonging to spirochaetes causing relapsing fever. It is primarily transmitted by ticks from the Ixodes ricinus complex, similarly to borreliae causing Lyme borreliosis. Small rodents can serve as reservoir hosts. It is widespread in mild climate areas of the northern hemisphere, with constant low prevalence in ticks, in the range of units of percent. To date more than 200 human cases have been described including five cases of meningoencephalitis in immunocompromised patients. Clinical features of illness are non-specific, characterized by fever, fatigue, chills, headaches, muscles and joint pains. It can be treated with antibiotics. The diagnostic approach includes mainly PCR and serological methods. This review summarizes current knowledge on B. miyamotoi with an emphasis on taxonomy, ecology of vectors and reservoir hosts, geographical distribution, diagnosis and treatment of the disease. The review also highlights the need for an accurate determination of the etiology of the disease and its differentiation from Lyme borreliosis and human granulocytic anaplasmosis.

West Nile fever - autochthonous human cases in South Moravia in 2018 from the epidemiological perspective.

AIM: To analyse repeated detection of West Nile virus (WNV) in mosquitoes in South Moravia in correlation with the first documented autochthonous human cases of West Nile fever (WNF), focusing on epidemiological and environmental investigation. MATERIALS AND METHODS: Here we report case studies of five patients with autochthonous WNF without any travel history diagnosed in South Moravia in 2018, along with in-depth epidemiological and environmental investigation. The cases were classified as confirmed based on the case definition criteria established in Commission Implementing Decision (EU) 2018/945. RESULTS: Between July and September 2018, a total of five human cases of West Nile virus infection were suspected and subsequently confirmed by the National Reference Laboratory for Arboviruses in Ostrava. All mosquito suspensions from the area where the first case of WNV infection was diagnosed tested WNV negative. CONCLUSIONS: WNV circulation in South Moravia has been known since 1997 when the first human cases (probably caused by WNV lineage 1) were confirmed after floods. The presence of more serious neuroinvasive lineage 2 (WNV-2), now circulating in central and eastern Europe, was detected repeatedly in 2013, 2015, and 2016 in Cx. modestus and Cx. pipiens mosquitoes within the scope of targeted entomological surveillance. These findings were published in 2016-2018 in both the professional press and mass media. It was only a matter of time and of making the correct differential diagnosis in patients with non-specific neuroinfections before there were confirmed autochthonous human cases of WNF caused by WNV-2. These presumptions were corroborated by epidemiological investigations performed by the staff of the Regional Public Health Authority of the South Moravian Region and Academy of Sciences.

West Nile virus (lineage 2) detected for the first time in mosquitoes in Southern Bohemia: new WNV endemic area?

Here we report the first detection of lineage 2 of neuroinvasive West Nile virus (WNV-2) in mosquitoes collected in a fishpond area of the Trebon Basin in southern Bohemia during the 2018 mosquito season. A total of 6790 mosquito females belonging to the Culex modestus, Culex pipiens, and Coquillettidia richiardii species were investigated in 136 pools, and WNV RNA was detected in two of them. The WNV strain shares genetic homology with other WNV-2 strains isolated in southern Moravia as well as with those causing outbreaks in southern and central Europe. The results highlight the need for entomological surveillance of pathogenic arboviruses even in areas not yet affected (WNV-free areas). The South Bohemian Region (in addition to southern Moravia) is becoming another risk zone of autochthonous occurrence of West Nile fever in the Czech Republic.

Alpha- and beta-mannan utilization by marine Bacteroidetes.

Marine microscopic algae carry out about half of the global carbon dioxide fixation into organic matter. They provide organic substrates for marine microbes such as members of the Bacteroidetes that degrade algal polysaccharides using carbohydrate-active enzymes (CAZymes). In Bacteroidetes genomes CAZyme encoding genes are mostly grouped in distinct regions termed polysaccharide utilization loci (PULs). While some studies have shown involvement of PULs in the degradation of algal polysaccharides, the specific substrates are for the most part still unknown. We investigated four marine Bacteroidetes isolated from the southern North Sea that harbour putative mannan-specific PULs. These PULs are similarly organized as PULs in human gut Bacteroides that digest α- and ß-mannans from yeasts and plants respectively. Using proteomics and defined growth experiments with polysaccharides as sole carbon sources we could show that the investigated marine Bacteroidetes express the predicted functional proteins required for α- and ß-mannan degradation. Our data suggest that algal mannans play an as yet unknown important role in the marine carbon cycle, and that biochemical principles established for gut or terrestrial microbes also apply to marine bacteria, even though their PULs are evolutionarily distant.

West Nile virus (lineage 2) in mosquitoes in southern Moravia - awaiting the first autochthonous human cases.

Here we report repeated detection of lineage 2 West Nile virus (WNV-2) from Culex modestus and Cx. pipiens mosquitoes collected at fishponds in the Lednice-Valtice Area during the mosquito seasons 2015 and 2016. The WNV strains recovered share genetic homology with WNV strains isolated during an extensive monitoring in 2013 as well as with strains circulating in southern and central Europe at the same time. Repeated detection of WNV indicates its establishment in the area and also warns infection specialists and epidemiologists about possible emergence of human cases or even outbreaks of West Nile fever in the region.

Genomic and physiological analyses of 'Reinekea forsetii' reveal a versatile opportunistic lifestyle during spring algae blooms.

Gammaproteobacterial Reinekea spp. were detected during North Sea spring algae blooms in the years 2009-2012, with relative abundances of up to 16% in the bacterioplankton. Here, we explore the ecophysiology of 'R. forsetii' strain Hel1_31_D35 that was isolated during the 2010 spring bloom using (i) its manually annotated, high-quality closed genome, (ii) re-analysis of in situ data from the 2009-2012 blooms and (iii) physiological tests. High resolution analysis of 16S rRNA gene sequences suggested that 'R. forsetii' dominated Reinekea populations during these blooms. This was corroborated by retrieval of almost complete Hel1_31_D35 genomes from 2009 and 2010 bacterioplankton metagenomes. Strain Hel1_31_D35 can use numerous low-molecular weight substrates including diverse sugar monomers, and few but relevant algal polysaccharides such as mannan, α-glucans, and likely bacterial peptidoglycan. It oxidizes thiosulfate to sulfate, and ferments under anoxic conditions. The strain can attach to algae and thrives at low phosphate concentrations as they occur during blooms. Its genome encodes RTX toxin and secretion proteins, and in cultivation experiments Hel1_31_D35 crude cell extracts inhibited growth of a North Sea Polaribacter strain. Our data suggest that the combination of these traits make strain Hel1_31_D35 a versatile opportunist that is particularly competitive during spring phytoplankton blooms.

Description of Gramella forsetii sp. nov., a marine Flavobacteriaceae isolated from North Sea water, and emended description of Gramella gaetbulicola Cho et al. 2011.

Strain KT0803T was isolated from coastal eutrophic surface waters of Helgoland Roads near the island of Helgoland, North Sea, Germany. The taxonomic position of the strain, previously known as 'Gramella forsetii' KT0803, was investigated by using a polyphasic approach. The strain was Gram-stain-negative, chemo-organotrophic, heterotrophic, strictly aerobic, oxidase- and catalase-positive, rod-shaped, motile by gliding and had orange-yellow carotenoid pigments, but was negative for flexirubin-type pigments. It grew optimally at 22-25 °C, at pH 7.5 and at a salinity between 2-3 %. Strain KT0803T hydrolysed the polysaccharides laminarin, alginate, pachyman and starch. The respiratory quinone was MK-6. Polar lipids comprised phosphatidylethanolamine, six unidentified lipids and two unidentified aminolipids. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c and iso-C17 : 1ω7c, with smaller amounts of iso-C15 : 0 2-OH, C15 : 0, anteiso-C15 : 0 and C17 : 1ω6c. The G+C content of the genomic DNA was 36.6 mol%. The 16S rRNA gene sequence identities were 98.6 % with Gramella echinicola DSM 19838T, 98.3 % with Gramella gaetbulicola DSM 23082T, 98.1 % with Gramella aestuariivivens BG-MY13T and Gramella aquimixticola HJM-19T, 98.0 % with Gramella lutea YJ019T, 97.9 % with Gramella portivictoriae DSM 23547T and 96.9 % with Gramella marina KMM 6048T. The DNA-DNA relatedness values were <35 % between strain KT0803T and type strains with >98.2 % 16S rRNA gene sequence identity. Based on the chemotaxonomic, phenotypic and genomic characteristics, strain KT0803T has been assigned to the genus Gramella, as Gramella forsetii sp. nov. The type strain is KT0803T (=DSM 17595T=CGMCC 1.15422T). An emended description of Gramella gaetbulicolaCho et al. 2011 is also proposed.

Francisella tularensis prevalence and load in Dermacentor reticulatus ticks in an endemic area in Central Europe.

A total of 7778 host-seeking adult Dermacentor reticulatus (Ixodida: Ixodidae) ticks were examined for the prevalence of Francisella tularensis holarctica (Thiotrichales: Francisellaceae) in a natural focus of tularaemia in the floodplain forest-meadow ecosystem along the lower reaches of the Dyje (Thaya) river in South Moravia (Czech Republic) between 1995 and 2013. Ticks were pooled (10 specimens per pool) and their homogenates inoculated subcutaneously in 4-week-old specific pathogen-free mice. Dead mice were sectioned, their spleens cultivated on thioglycollate-glucose-blood agar and impression smears from the spleen, liver and heart blood were Giemsa-stained. Sixty-four pools were positive for F. tularensis: the overall minimum infection rate (MIR) was 0.82%. Overall MIRs for the 4714 female and 3064 male D. reticulatus examined were 0.89 and 0.72%, respectively; MIRs fluctuated across years between 0.0 and 2.43%. The estimated bacterial load in infected ticks varied from 0.84 to 5.34 log10 infectious F. tularensis cells per tick (i.e. from about seven to 220 000 cells). Ticks with low loads were more prevalent; more than 1000 infectious cells were detected in 24 ticks (0.3% of all ticks and 37.5% of infected ticks). Monitoring of D. reticulatus for the presence and cell numbers of F. tularensis may be a valuable tool in the surveillance of tularaemia.

Polysaccharide utilisation loci of Bacteroidetes from two contrasting open ocean sites in the North Atlantic.

Marine Bacteroidetes have pronounced capabilities of degrading high molecular weight organic matter such as proteins and polysaccharides. Previously we reported on 76 Bacteroidetes-affiliated fosmids from the North Atlantic Ocean's boreal polar and oligotrophic subtropical provinces. Here, we report on the analysis of further 174 fosmids from the same libraries. The combined, re-assembled dataset (226 contigs; 8.8 Mbp) suggests that planktonic Bacteroidetes at the oligotrophic southern station use more peptides and bacterial and animal polysaccharides, whereas Bacteroidetes at the polar station (East-Greenland Current) use more algal and plant polysaccharides. The latter agrees with higher abundances of algae and terrigenous organic matter, including plant material, at the polar station. Results were corroborated by in-depth bioinformatic analysis of 14 polysaccharide utilisation loci from both stations, suggesting laminarin-specificity for four and specificity for sulfated xylans for two loci. In addition, one locus from the polar station supported use of non-sulfated xylans and mannans, possibly of plant origin. While peptides likely represent a prime source of carbon for Bacteroidetes in open oceans, our data suggest that as yet unstudied clades of these Bacteroidetes have a surprisingly broad capacity for polysaccharide degradation. In particular, laminarin-specific PULs seem widespread and thus must be regarded as globally important.

[Human hantavirus diseases - still neglected zoonoses?]. / Onemocnení cloveka zpusobená hantaviry - stále opomíjené zoonózy?

Hantavirus disease is the most common rodent-borne viral infection in the Czech Republic, with a mean annual incidence of 0.02 cases per 100 000 population and specific antibodies detected in 1% of the human population. Four hantaviruses (Puumala, Dobrava-Belgrade, Tula, and Seewis) circulate in this country, of which Puumala virus (responsible for a mild form of hemorrhagic fever with renal syndrome called nephropathia epidemica) and Dobrava-Belgrade virus (causing haemorrhagic fever with renal syndrome) have been proven to cause human disease. The aim of this study is to provide a comprehensive review of the hantaviruses occurring in the Czech Republic, based on the literature published during the past three decades, including their geographical distribution and clinical symptoms. The recent detection of Tula virus in an immunocompromised person as well as reports of Seoul virus infections in Europe highlight the possible emergence of neglected hantavirus infections in the foreseeable future.

Distribution of a consortium between unicellular algae and the N2 fixing cyanobacterium UCYN-A in the North Atlantic Ocean.

The globally abundant, uncultured unicellular cyanobacterium UCYN-A was recently discovered living in association with a eukaryotic cell closely related to a prymnesiophyte. Here, we established a double CAtalysed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH) approach to identify both partners and provided quantitative information on their distribution and abundance across distinct water masses along a transect in the North Atlantic Ocean. The N2 fixation activity coincided with the detection of UCYN-A cells and was only observed in oligotrophic (< 0.067 NO3(-) µM and < 0.04 PO4(3-) µM) and warm (> 18°C) surface waters. Parallel 16S ribosomal RNA gene analyses among unicellular diazotrophs indicated that only UCYN-A cells were present. UCYN-A cells were associated with an algal partner or non-associated using the double CARD-FISH approach. We demonstrated that UCYN-A cells living in association with Haptophyta were the dominant form (87.0 ± 6.1%), whereas non-associated UCYN-A cells represented only a minor fraction (5.2 ± 3.9%). Interestingly, UCYN-A cells were also detected living in association with unknown single-celled eukaryotes in small amounts (7.8 ± 5.2%), presumably Alveolata. The proposed ecological niche of UCYN-A as an oligotrophic, mesophilic and obligate symbiotic nitrogen-fixing microorganism is evident for the North Atlantic Ocean.

Isolation of Tahyna Virus (California Encephalitis Group) From Anopheles hyrcanus (Diptera, Culicidae), a Mosquito Species New to, and Expanding in, Central Europe.

Two strains of Tahyna virus (TAHV; Orthobunyavirus, Bunyaviridae) were isolated from 4,568 (92 pools) female Anopheles hyrcanus Pallas (Diptera, Culicidae) mosquitoes collected on the fishponds in South Moravia (Czechland, central Europe) during July-August 2013. This is the first isolation of TAHV from An. hyrcanus in Europe. An. hyrcanus is a species new to Czechland since 2007; its population density was very high in the year 2013 at these ponds. The virus isolation procedure was based on intracerebral inoculation of newborn mice; moreover, the positive pools were also tested by polymerase chain reaction and found to contain TAHV RNA. An. hyrcanus, feeding preferentially on mammals including humans, may be a new potential vector for TAHV in Europe.

West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North?

We report the detection and isolation of four almost identical strains of West Nile virus (WNV) lineage 2from Culex modestus mosquitoes collected at three fish ponds in South Moravia, Czech Republic, during August 2013. Phylogenetic analysis demonstrated that the Czech WNV strains isolated are closely related to Austrian, Italian and Serbian strains reported in 2008,2011 and 2012, respectively. Our findings show the current northernmost range of lineage 2 WNV in Europe.

Seroprevalence study of hepatitis E virus infection in two districts of the Czech Republic.

The aim of this work was to retrospectively examine serum samples from 230 people living in two districts in the Czech Republic in year 1989 (Znojmo and Jihlava) for the IgG antibodies against hepatitis E virus (HEV). For examination, commercial ELISA kit DIA.PRO was used and 13 seropositive persons were found, i.e. 5.7% of those examined. The seropositivity rate was higher in women (6.8%) than in men (3.7%), but the difference was not statistically significant. The age of examined persons did not reveal a significant effect on the seropositivity rate: mean age of seropositive subjects was 38.0 years while that of seronegative persons was 39.4 years.

Proteomic insight into arabinogalactan utilization by particle-associated Maribacter sp. MAR_2009_72.

Arabinose and galactose are major, rapidly metabolized components of marine particulate and dissolved organic matter. In this study, we observed for the first time large microbiomes for the degradation of arabinogalactan and report a detailed investigation of arabinogalactan utilization by the flavobacterium Maribacter sp. MAR_2009_72. Cellular extracts hydrolysed arabinogalactan in vitro. Comparative proteomic analyses of cells grown on arabinogalactan, arabinose, galactose, and glucose revealed the expression of specific proteins in the presence of arabinogalactan, mainly glycoside hydrolases (GH). Extracellular glycan hydrolysis involved five alpha-l-arabinofuranosidases affiliating with glycoside hydrolase families 43 and 51, four unsaturated rhamnogalacturonylhydrolases (GH105) and a protein with a glycoside hydrolase family-like domain. We detected expression of three induced TonB-dependent SusC/D transporter systems, one SusC, and nine glycoside hydrolases with a predicted periplasmatic location. These are affiliated with the families GH3, GH10, GH29, GH31, GH67, GH78, and GH115. The genes are located outside of and within canonical polysaccharide utilization loci classified as specific for arabinogalactan, for galactose-containing glycans, and for arabinose-containing glycans. The breadth of enzymatic functions expressed in Maribacter sp. MAR_2009_72 as response to arabinogalactan from the terrestrial plant larch suggests that Flavobacteriia are main catalysts of the rapid turnover of arabinogalactans in the marine environment.

Genes for laminarin degradation are dispersed in the genomes of particle-associated Maribacter species.

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world's annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endo-laminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization.

Particle-attached bacteria act as gatekeepers in the decomposition of complex phytoplankton polysaccharides.

BACKGROUND: Marine microalgae (phytoplankton) mediate almost half of the worldwide photosynthetic carbon dioxide fixation and therefore play a pivotal role in global carbon cycling, most prominently during massive phytoplankton blooms. Phytoplankton biomass consists of considerable proportions of polysaccharides, substantial parts of which are rapidly remineralized by heterotrophic bacteria. We analyzed the diversity, activity, and functional potential of such polysaccharide-degrading bacteria in different size fractions during a diverse spring phytoplankton bloom at Helgoland Roads (southern North Sea) at high temporal resolution using microscopic, physicochemical, biodiversity, metagenome, and metaproteome analyses. RESULTS: Prominent active 0.2-3 µm free-living clades comprised Aurantivirga, "Formosa", Cd. Prosiliicoccus, NS4, NS5, Amylibacter, Planktomarina, SAR11 Ia, SAR92, and SAR86, whereas BD1-7, Stappiaceae, Nitrincolaceae, Methylophagaceae, Sulfitobacter, NS9, Polaribacter, Lentimonas, CL500-3, Algibacter, and Glaciecola dominated 3-10 µm and > 10 µm particles. Particle-attached bacteria were more diverse and exhibited more dynamic adaptive shifts over time in terms of taxonomic composition and repertoires of encoded polysaccharide-targeting enzymes. In total, 305 species-level metagenome-assembled genomes were obtained, including 152 particle-attached bacteria, 100 of which were novel for the sampling site with 76 representing new species. Compared to free-living bacteria, they featured on average larger metagenome-assembled genomes with higher proportions of polysaccharide utilization loci. The latter were predicted to target a broader spectrum of polysaccharide substrates, ranging from readily soluble, simple structured storage polysaccharides (e.g., laminarin, α-glucans) to less soluble, complex structural, or secreted polysaccharides (e.g., xylans, cellulose, pectins). In particular, the potential to target poorly soluble or complex polysaccharides was more widespread among abundant and active particle-attached bacteria. CONCLUSIONS: Particle-attached bacteria represented only 1% of all bloom-associated bacteria, yet our data suggest that many abundant active clades played a pivotal gatekeeping role in the solubilization and subsequent degradation of numerous important classes of algal glycans. The high diversity of polysaccharide niches among the most active particle-attached clades therefore is a determining factor for the proportion of algal polysaccharides that can be rapidly remineralized during generally short-lived phytoplankton bloom events. Video Abstract.

Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.