Streptococcus mutans é o principal agente etiológico da cárie dental humana, uma doença infecciosa para a qual não há vacina disponível. A influência dos sistemas de transporte ABC na virulência bacteriana suporta seu uso como alvos vacinais. Em S. mutans a proteína ligadora de fosfato (PstS) influencia a expressão de fatores de virulência. A proposta deste trabalho foi caracterizar uma estratégia vacinal de mucosa anti-cárie baseada na proteína PstS como antígeno alvo. Inicialmente, a forma recombinante da proteína foi obtida em E. coli. A caracterização biofísica revelou uma estrutura secundária estável, semelhante a outras proteínas ligadoras e capaz de interagir com seu ligante. Epítopos antigênicos conservados foram identificados na proteína recombinante pela reatividade com soro anti-S. mutans. A proteína rPstS foi imunogênica por via sublingual, combinada ou não à LTK4R e anticorpos rPstS-específicos interferiram na colonização oral in vivo por S. mutans. Os resultados indicam que a proteína rPstS pode ser explorada em estratégias vacinais contra a cárie. / Streptococcus mutans is the main etiological agent of human dental caries, an infectious disease for which there is no vaccine available. The influence of ABC transport systems in bacterial virulence supports its use as vaccine targets. In S. mutans the phosphate binding protein (PstS) influences the expression of virulence traits. The purpose of this work was characterizing an anti-caries mucosal vaccine based on the PstS protein as target antigen. First, a recombinant form of the protein was obtained in E. coli. The biophysical characterization showed a stable secondary structure, similar to other binding proteins and able to interact with its ligand. Conserved antigenic epitopes were identified in the recombinant protein by reactivity with anti- S. mutans serum. The rPstS protein was immunogenic by the sublingual route in combination or not with LTK4R and rPstS-specific antibodies interfered with S. mutans oral colonization in vivo. The results indicated that the recombinant PstS protein can be exploited in vaccine strategies against dental caries.

Streptococcus mutans é o principal agente etiológico da cárie dental humana, uma doença infecciosa para a qual não há vacina disponível. A influência dos sistemas de transporte ABC na virulência bacteriana suporta seu uso como alvos vacinais. Em S. mutans a proteína ligadora de fosfato (PstS) influencia a expressão de fatores de virulência. A proposta deste trabalho foi caracterizar uma estratégia vacinal de mucosa anti-cárie baseada na proteína PstS como antígeno alvo. Inicialmente, a forma recombinante da proteína foi obtida em E. coli. A caracterização biofísica revelou uma estrutura secundária estável, semelhante a outras proteínas ligadoras e capaz de interagir com seu ligante. Epítopos antigênicos conservados foram identificados na proteína recombinante pela reatividade com soro anti-S. mutans. A proteína rPstS foi imunogênica por via sublingual, combinada ou não à LTK4R e anticorpos rPstS-específicos interferiram na colonização oral in vivo por S. mutans. Os resultados indicam que a proteína rPstS pode ser explorada em estratégias vacinais contra a cárie. / Streptococcus mutans is the main etiological agent of human dental caries, an infectious disease for which there is no vaccine available. The influence of ABC transport systems in bacterial virulence supports its use as vaccine targets. In S. mutans the phosphate binding protein (PstS) influences the expression of virulence traits. The purpose of this work was characterizing an anti-caries mucosal vaccine based on the PstS protein as target antigen. First, a recombinant form of the protein was obtained in E. coli. The biophysical characterization showed a stable secondary structure, similar to other binding proteins and able to interact with its ligand. Conserved antigenic epitopes were identified in the recombinant protein by reactivity with anti- S. mutans serum. The rPstS protein was immunogenic by the sublingual route in combination or not with LTK4R and rPstS-specific antibodies interfered with S. mutans oral colonization in vivo. The results indicated that the recombinant PstS protein can be exploited in vaccine strategies against dental caries.

Marine primary productivity in large parts of the oceans is supported by nitrogen fixers (diazotrophs). Richelia, a genus of multiple closely related species of heterocystous filamentous cyanobacterial diazotrophs, are often found in symbioses with a few genera of diatoms. Several Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae, form stable host specific partnerships and these populations are important for the nitrogen (N) cycle, especially in the oligotrophic open oceans. In general, the gene nifH, which encodes the enzyme nitrogenase for N2 fixation, is used as a molecular marker for presence (DNA level) and activity (RNA level) of diazotrophs. However, evidence of cross-reactivity of the nifH assays between two of the Richelia species shows the risk of incorrect estimations of abundance and activity. Moreover, transcript abundance is rarely normalized to a housekeeping gene.  The aims of this work were to develop and assess new assays for the detection of three symbiotic Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae. These new assays targeted molecular markers that were indicative nutrient acquisition. For example, one assay targets a gene encoding the high affinity phosphate transporter (pstS), a second targets a gene involved in iron (Fe) transport (exbB) (indicator of P transport, Fe transport, respectively), and a third assays a housekeeping gene encoding Ribonuclease P protein (rnpA). All assays were used to estimate abundance and expression in lab and field based samples. The new assays have high species specificity as revealed by BlastN analyses and lab-based cross-hybridization assays. We applied the assays to samples from a lab-based experiment of the facultative symbiont R. rhizosoleniae RrhiSC01 in order to investigate the temporal dynamics of expression. This revealed periodic expression of nifH and pstS related to the photoperiod with higher expression in the early and late photoperiod, respectively. The peak of pstS expression in the late photoperiod is likely to support the high P requirement of both photosynthesis and N2 fixation. Given the temporal regulation of pstS (and nifH) expression shown here, one must consider these results when sampling and/or interpreting field studies. Moreover, a comparison of the Richelia draft genomes and environmental metagenomic assembled genomes (MAGs) show that gene copy number per genome of exbB and pstS varies between strains. Given that Richelia tend to live in low nutrient environments, including N, P, and often Fe, the increased copy number per genome for pstS and exbB could be suggestive/evidence of adaptation to constant nutrient limitation. In addition, the assays were applied to natural samples containing populations of R. intracellularis and R. euintracellularis collected in the North and South Atlantic Ocean from multiple depths (5-90 meters). The DNA based gene copy abundances showed higher estimates when nifH is used compared to rnpA, which is likely due to a higher degree of cross-reactivity in the nifH assays. Gene expression was present but low for all targets. However, expression of exbB and pstS was higher in surface water – where nutrients are expected to be depleted. Lastly, bulk fixation rates of N2 and carbon (C) showed that the activity was low and fixation rates did not increase with the addition of dissolved organic phosphate (DOP) (a 1:4 mixture of 2-aminoethylphosphonic acid and beta-glycerophosphate disodium salt hydrate). This could be due to patchiness and low abundance of diazotrophs and discrepancies between samples, seasonal variation or that the populations were not P limited. In summary, the new assays constitute a better option to interspecific detection of Richelia. However, more work is required to assess how the expression relates to limitation of nutrients and other external factors to better understand the activity of these biogeochemically important populations. / I stora delar av haven stöds marin primärproduktion av kvävefixerare (diazotrofer). Richelia är ett släkte med ett flertal närbesläktade arter av heterocysta, filamentösa och kvävefixerande cyanobakterier som lever i symbios med ett fåtal kiselalger. Dessa är viktiga för kvävecykeln, särskilt i de oligotrofa (kvävefattiga) öppna haven. Genen nifH som kodar för det kvävefixerande enzymet nitrogenas, brukar användas som en molekylär markör för att detektera diazotrofa populationer och dess genuttryck används som en indikator för kvävefixering. Det finns emellertid tecken på att nifH ger felaktiga uppskattningar av dessa populationers abundans och aktivitet. Dessutom, när nifH-uttryck används som ett mått på aktivitet är det endast kvävefixering man observerar. Andra faktorer som begränsar förekomsten av dessa organismer, t.ex. brist på järn eller fosfor, kan inte avläsas. Dessutom har uttrycket av nifH sällan normaliserats till en stabilt uttryckt gen vilket gör jämförelser svåra. Syftet med detta arbete var att utveckla och utvärdera nya kvantitativ realtidspolymeraskedjereaktion (qPCR)-analyser för specifik detektion av identitet, abundans och aktivitet av tre symbiotiska Richelia-arter: R. euintracellularis, R. intracellularis och R. rhizosoleniae. De nya analyserna är inriktade på generna som kodar för högaffinitetsfosfattransportören pstS och biopolymertransportproteinet exbB (indikator för P-transport respektive Fe-transport). Genen som kodar för ribonukleas P-protein, rnpA, en gen med stabilt uttryck, användes för att uppskatta abundans och för att normalisera uttrycket av de andra målgenerna.  BlastN-analyser och labb-baserade korshybridiseringsanalyser visar att de nya analyserna har hög artspecificitet. Vi tillämpade analyserna på en kultur av den fakultativa symbionten R. rhizosoleniae RrhiSC01 för att undersöka det periodiska uttrycksmönster av målgenerna kopplat till tid på dygnet. Resultatet visade på ett periodiskt uttryck av nifH och pstS relaterat till fotoperioden med högre uttryck i den tidiga, respektive sena fotoperioden.  Det ökade uttrycket av pstS i den sena fotoperioden stödjer sannolikt det höga P-kravet för både fotosyntes och N2-fixering. Den tidsmässiga regleringen av pstS-uttryck som visas här, behöver man ta hänsyn till vid provtagning och tolkning av fältstudier. ​​ Dessutom visar en jämförelse av Richelia-genom att genkopiantalet för exbB och pstS varierar mellan stammar av Richelia. Med tanke på att Richelia tenderar att leva i miljöer med låga nivåer av näringsämnen, inklusive kväve, fosfor och ofta järn, kan det ökade antalet kopior per genom av pstS och exbB ett tecken på anpassning till konstant näringsbegränsning. Analyserna tillämpades även på naturliga populationer av R. intracellularis och R. euintracellularis i norra och södra Atlanten. DNA-baserad estimering av abundans (genkopior) visar på en högre uppskattning när nifH används som markör jämfört med rnpA - troligtvis på grund av en högre grad av korsreaktivitet i nifH-analyserna. Genuttryck var närvarande men lågt för alla målgener i norra och södra Atlanten. Uttrycket av exbB och pstS var dock högre i ytvatten – där halter av näringsämnen förväntas vara låga. Slutligen var kväve- och kolfixering i planktonsamhället låg och ökade inte vid tillsats av fosfor, vilket kan bero på ojämn fördelning av celler, låg förekomst av diazotrofer och diskrepanser mellan prover, säsongsvariationer eller att populationerna inte var primärt begränsade av fosfor. Sammanfattningsvis utgör de nya analyserna ett bättre alternativ för specifik detektion av Richelia. Det krävs dock mer arbete för att bedöma hur uttrycket påverkas av  näringsbrist och yttre faktorer och för att bättre förstå aktiviteten hos dessa ovärderliga populationer.

A mesocosm experiment was carried out in a Norwegian fjord near Bergen in May 2006, with the main objective being the study of the effects of increasing concentrations of atmospheric CO2 (and associated effects such as increased acidification) on blooms of natural marine coastal plankton. Three mesocosms were bubbled with CO2(g) to achieve a high (~700ppm) CO2 concentration (pH ~7.8) to simulate predicted future conditions as a result of rising atmospheric CO2 concentrations. Another three mesocosms were treated as controls and bubbled with ambient air to represent a near pre-industrial scenario (atmospheric CO2 concentration ~300ppm, surface seawater pH ~8.15). Blooms in the mesocosms were stimulated by the addition of nutrients at a near-Redfield ratio ([N:P] ≈ [16:1]), and scientific measurements and analyses were carried out over the course of the blooms for approximately one month. Of particular interest in this study were the autotrophic plankton. The diversity and activities of these microorganisms under the two treatments was therefore investigated. By designing and using new degenerate primers specifically targeting ‘Green-type’ (Form IA and IB), ‘Red-type’ (Form IC and ID) and Form II RuBisCO, analysis of primary producers was carried out using PCR and either gDNA or cDNA (mRNA) templates from key time points spanning the complete duration of the blooms throughout the mesocosm experiment. Over 1250 novel RuBisCO large subunit sequences have been fully annotated and deposited in the NCBI GenBank® database. These sequences revealed distinct changes in the diversity of primary producers both over the courses of the blooms and between treatments. Particularly striking was the effect of acidification on the community structure of the eukaryotic picoplankton, Prasinophytes. A clade of prasinophytes closely related to Micromonas pusilla showed a distinct preference for the high CO2 conditions; a laboratory-based experiment confirmed the high tolerance of Micromonas pusilla to lower pH. Conversely, a clade related to Bathycoccus prasinos was almost entirely excluded from the high CO2 treatments. Clades of form II RuBisCO-containing dinoflagellates were also abundant throughout the experiment in both treatments. The high similarity of some of these clades to the toxin-producing species Heterocapsa triquetra and Gonyaulax polyedra, and apparent high tolerance of some clades to high CO2 conditions, is perhaps cause for concern in a high CO2 world and demands further research. In parallel with the RubisCO work, new primers were designed that target the gene encoding the Fe protein of nitrogenase (NifH). 82 Bergen genomic nifH sequences have been annotated and submitted to GenBank®. These sequences include those from organisms related to Alpha, Beta, and Gammaproteobacteria, and Cluster II and Cluster III sequences that align most closely with anaerobic Bacteria, Gram positive, and/or sulphur-reducing Bacteria. The biggest surprise, however, was the apparent abundance and significance of a Rhodobacter sphaeroides-like microorganism throughout the duration of the experiment in both treatments. Whilst this clade was unsurprisingly absent in the RuBisCO cDNA libraries, all but two of 128 nifH cDNA clones analysed were identical to the gene from Rhodobacter sphaeroides. This shows that this clade was potentially fixing N2 throughout the entire experiment, even in the presence of combined N added to both sets of mesocosms at the start of the experiment. A group of Rhodobacter sphaeroides-like microorganisms present at Bergen may therefore have been an unexpected source of new N during the experiment and contributed to the maintenance of the mesocosm communities as nutrients became depleted. One organism dominated the autotrophic communities after the blooms in both treatments. Synechococcus spp. Form IA rbcL clones most closely related to the coastal strain Synechococcus sp. strain CC9902 were recovered throughout the experiment but were particularly numerous toward the end of the experiment and dominated the “Green-type” libraries at this time. Initially, rbcL clones from these cyanobacteria were mostly derived from the ambient CO2 mesocosms but were equally distributed between treatments by the end of the experiment. This suggests that cyanobacteria related to strain CC9902 may be less tolerant of elevated CO2 (which was greatest at the beginning rather than the end of the experiment). However, despite the mesocosms being Pi-limited at the end of the experiment, several Synechococcus species (including those related to strain CC9902 and another coastal strain, CC9311) thrived. Following on from this observation, Pi uptake and assimilation mechanisms in a Synechococcus species were investigated in the laboratory. This led to the sequencing and characterisation of a pstS gene from the marine cyanobacterium Synechococcus sp. WH 8103. Unlike conventional pstS, it was discovered that the pstS II gene in this organism is constitutively expressed and unresponsive to or only weakly regulated by Pi supply. The use of PstS/pstS as a marker for P-limitation in natural samples, therefore, should be interpreted with caution.