Microbiological, Health and Comfort Aspects of Indoor Air Quality in a Romanian Historical Wooden Church.

Monitoring the indoor microclimate in old buildings of cultural heritage and significance is a practice of great importance because of the importance of their identity for local communities and national consciousness. Most aged heritage buildings, especially those made of wood, develop an indoor microclimate conducive to the development of microorganisms. This study aims to analyze one wooden church dating back to the 1710s in Romania from the microclimatic perspective, i.e., temperature and relative humidity and the fungal load of the air and surfaces. One further aim was to determine if the internal microclimate of the monument is favorable for the health of parishioners and visitors, as well as for the integrity of the church itself. The research methodology involved monitoring of the microclimate for a period of nine weeks (November 2020-January 2021) and evaluating the fungal load in indoor air as well as on the surfaces. The results show a very high contamination of air and surfaces (>2000 CFU/m3). In terms of fungal contamination, Aspergillus spp. (two different species), Alternaria spp., Cladosporium spp., Mucor spp., Penicillium spp. (two different species) and Trichopyton spp. were the genera of fungi identified in the indoor wooden church air and Aspergillus spp., Cladosporium spp., Penicillium spp. (two different species) and Botrytis spp. on the surfaces (church walls and iconostasis). The results obtained reveal that the internal microclimate not only imposes a potential risk factor for the parishioners and visitors, but also for the preservation of the wooden church as a historical monument, which is facing a crisis of biodeterioration of its artwork.

Spatio-temporal insights into microbiology of the freshwater-to-hypersaline, oxic-hypoxic-euxinic waters of Ursu Lake.

Ursu Lake is located in the Middle Miocene salt deposit of Central Romania. It is stratified, and the water column has three distinct water masses: an upper freshwater-to-moderately saline stratum (0-3 m), an intermediate stratum exhibiting a steep halocline (3-3.5 m), and a lower hypersaline stratum (4 m and below) that is euxinic (i.e. anoxic and sulphidic). Recent studies have characterized the lake's microbial taxonomy and given rise to intriguing ecological questions. Here, we explore whether the communities are dynamic or stable in relation to taxonomic composition, geochemistry, biophysics, and ecophysiological functions during the annual cycle. We found: (i) seasonally fluctuating, light-dependent communities in the upper layer (≥0.987-0.990 water-activity), a stable but phylogenetically diverse population of heterotrophs in the hypersaline stratum (water activities down to 0.762) and a persistent plate of green sulphur bacteria that connects these two (0.958-0.956 water activity) at 3-3.5 to 4 m; (ii) communities that might be involved in carbon- and sulphur-cycling between and within the lake's three main water masses; (iii) uncultured lineages including Acetothermia (OP1), Cloacimonetes (WWE1), Marinimicrobia (SAR406), Omnitrophicaeota (OP3), Parcubacteria (OD1) and other Candidate Phyla Radiation bacteria, and SR1 in the hypersaline stratum (likely involved in the anaerobic steps of carbon- and sulphur-cycling); and (iv) that species richness and habitat stability are associated with high redox-potentials. Ursu Lake has a unique and complex ecology, at the same time exhibiting dynamic fluctuations and stability, and can be used as a modern analogue for ancient euxinic water bodies and comparator system for other stratified hypersaline systems.

The impact of cation concentration on Microcystis (cyanobacteria) scum formation.

Cyanobacterial scums at the surface of the lakes are potentially harmful phenomena with increasing occurrence in the last decades, and the causes that lead to their formation are still an unresolved issue. In order to better understand what triggers the scums, we investigated the effect of several Mg2+ and Ca2+ ion concentrations in promoting them in eight Microcystis aeruginosa strains. The possibility to prevent scum formation by using the ion chelator EDTA was also explored. We found that in some strains the cell aggregation takes place under lower ion source concentrations (20 mM MgSO4 or CaCl2), while in others this phenomenon does not occur even at 60 mM concentration. The scum formation correlated to the amount of extracellular polymeric substances (between 234 and 351 µg/cell). EDTA failed to prevent the scum formation in most strains, and in turn it caused cell lysis followed by the release of cellular content into the culture medium. We emphasize the relevance of these results for cyanobacterial scum formation in the environment and we also suggest that controlling the salinity of the medium (by manipulating the ion concentration) is a potentially efficient method for biomass harvesting in large ponds/tanks.

Regulation of PSII function in Cyanothece sp. ATCC 51142 during a light-dark cycle.

Cyanobacteria, as well as green algae and higher plants, have highly conserved photosynthetic machinery. Cyanothece sp. ATCC 51142 is a unicellular, aerobic, diazotrophic cyanobacterium that fixes N2 in the dark. In Cyanothece, the psbA gene family is composed of five members, encoding different isoforms of the D1 protein. A new D1 protein has been postulated in the literature, which blocks PSII during the night and allows the fixation of nitrogen. We present data showing changes in PSII function in cells grown in cycles alternating between 12 h of light and dark, respectively, at Cyanothece sp. ATCC 51142. Cyanothece sp. ATCC 51142 uses intrinsic mechanisms to protect its nitrogenase activity in a two-stage process. In Stage I, immediately after the onset of darkness, the cells lose photosynthetic activity in a reversible process, probably by dissociation of water oxidation complex from photosystem II via a mechanism that does not require de novo protein synthesis. In Stage II, a more severe disruption of photosystem II function occurs is in part protein synthesis dependent and it could be a functional signature of the presence of sentinel D1 in a limited number of reaction centers still active or not yet inactivated by the mechanism described in Stage I. This process of inhibition uses light as a triggering signal for both the inhibition of photosynthetic activity and recovery when light returns. The intrinsic mechanism of photosynthetic inactivation during darkness with the interplay of the two mechanisms requires further studies.

Contrasting taxonomic stratification of microbial communities in two hypersaline meromictic lakes.

Hypersaline meromictic lakes are extreme environments in which water stratification is associated with powerful physicochemical gradients and high salt concentrations. Furthermore, their physical stability coupled with vertical water column partitioning makes them important research model systems in microbial niche differentiation and biogeochemical cycling. Here, we compare the prokaryotic assemblages from Ursu and Fara Fund hypersaline meromictic lakes (Transylvanian Basin, Romania) in relation to their limnological factors and infer their role in elemental cycling by matching taxa to known taxon-specific biogeochemical functions. To assess the composition and structure of prokaryotic communities and the environmental factors that structure them, deep-coverage small subunit (SSU) ribosomal RNA (rDNA) amplicon sequencing, community domain-specific quantitative PCR and physicochemical analyses were performed on samples collected along depth profiles. The analyses showed that the lakes harbored multiple and diverse prokaryotic communities whose distribution mirrored the water stratification patterns. Ursu Lake was found to be dominated by Bacteria and to have a greater prokaryotic diversity than Fara Fund Lake that harbored an increased cell density and was populated mostly by Archaea within oxic strata. In spite of their contrasting diversity, the microbial populations indigenous to each lake pointed to similar physiological functions within carbon degradation and sulfate reduction. Furthermore, the taxonomy results coupled with methane detection and its stable C isotope composition indicated the presence of a yet-undescribed methanogenic group in the lakes' hypersaline monimolimnion. In addition, ultrasmall uncultivated archaeal lineages were detected in the chemocline of Fara Fund Lake, where the recently proposed Nanohaloarchaeota phylum was found to thrive.

Structure, mineralogy, and microbial diversity of geothermal spring microbialites associated with a deep oil drilling in Romania.

Modern mineral deposits play an important role in evolutionary studies by providing clues to the formation of ancient lithified microbial communities. Here we report the presence of microbialite-forming microbial mats in different microenvironments at 32°C, 49°C, and 65°C around the geothermal spring from an abandoned oil drill in Ciocaia, Romania. The mineralogy and the macro- and microstructure of the microbialites were investigated, together with their microbial diversity based on a 16S rRNA gene amplicon sequencing approach. The calcium carbonate is deposited mainly in the form of calcite. At 32°C and 49°C, the microbialites show a laminated structure with visible microbial mat-carbonate crystal interactions. At 65°C, the mineral deposit is clotted, without obvious organic residues. Partial 16S rRNA gene amplicon sequencing showed that the relative abundance of the phylum Archaea was low at 32°C (<0.5%) but increased significantly at 65°C (36%). The bacterial diversity was either similar to other microbialites described in literature (the 32°C sample) or displayed a specific combination of phyla and classes (the 49°C and 65°C samples). Bacterial taxa were distributed among 39 phyla, out of which 14 had inferred abundances >1%. The dominant bacterial groups at 32°C were Cyanobacteria, Gammaproteobacteria, Firmicutes, Bacteroidetes, Chloroflexi, Thermi, Actinobacteria, Planctomycetes, and Defferibacteres. At 49°C, there was a striking dominance of the Gammaproteobacteria, followed by Firmicutes, Bacteroidetes, and Armantimonadetes. The 65°C sample was dominated by Betaproteobacteria, Firmicutes, [OP1], Defferibacteres, Thermi, Thermotogae, [EM3], and Nitrospirae. Several groups from Proteobacteria and Firmicutes, together with Halobacteria and Melainabacteria were described for the first time in calcium carbonate deposits. Overall, the spring from Ciocaia emerges as a valuable site to probe microbes-minerals interrelationships along thermal and geochemical gradients.

Selection of proper reference genes for the cyanobacterium Synechococcus PCC 7002 using real-time quantitative PCR.

Synechococcus sp. PCC 7002 is known to be tolerant to most of the environmental factors in natural habitats of Cyanobacteria. Gene expression can be easily studied in this cyanobacterium, as its complete genome sequence is available. These properties make Synechococcus sp. PCC 7002 an appropriate model organism for biotechnological applications. To study the gene expression in Cyanobacteria, real-time quantitative PCR (qPCR) can be used, but as this is a highly sensitive method, data standardization is indicated between samples. The most commonly used strategy is normalization against internal reference genes. Synechococcus sp. PCC 7002 has not yet been evaluated for the best reference genes. In this work, six candidate genes were analyzed for this purpose. Cyanobacterial cultures were exposed to several stress conditions, and three different algorithms were used for ranking the reference genes: geNorm, NormFinder, and BestKeeper. Moreover, gene expression stability value M and single-control normalization error E were calculated. Our data provided a list of reference genes that can be used in qPCR experiments in Synechococcus sp. PCC 7002.

Archaeal and bacterial diversity in two hot spring microbial mats from a geothermal region in Romania.

The diversity of archaea and bacteria was investigated in two slightly alkaline, mesophilic hot springs from the Western Plain of Romania. Phylogenetic analysis showed a low diversity of Archaea, only three Euryarchaeota taxa being detected: Methanomethylovorans thermophila, Methanomassiliicoccus luminyensis and Methanococcus aeolicus. Twelve major bacterial groups were identified, both springs being dominated by Cyanobacteria, Chloroflexi and Proteobacteria. While at the phylum/class-level the microbial mats share a similar biodiversity; at the species level the geothermal springs investigated seem to be colonized by specific consortia. The dominant taxa were filamentous heterocyst-containing Fischerella, at 45 °C and non-heterocyst Leptolyngbya and Geitlerinema, at 55 °C. Other bacterial taxa (Thauera sp., Methyloversatilis universalis, Pannonibacter phragmitetus, Polymorphum gilvum, Metallibacterium sp. and Spartobacteria) were observed for the first time in association with a geothermal habitat. Based on their bacterial diversity the two mats were clustered together with other similar habitats from Europe and part of Asia, most likely the water temperature playing a major role in the formation of specific microbial communities that colonize the investigated thermal springs.

Cyanobacterial psbA gene family: optimization of oxygenic photosynthesis.

The D1 protein of Photosystem II (PSII), encoded by the psbA genes, is an indispensable component of oxygenic photosynthesis. Due to strongly oxidative chemistry of PSII water splitting, the D1 protein is prone to constant photodamage requiring its replacement, whereas most of the other PSII subunits remain ordinarily undamaged. In cyanobacteria, the D1 protein is encoded by a psbA gene family, whose members are differentially expressed according to environmental cues. Here, the regulation of the psbA gene expression is first discussed with emphasis on the model organisms Synechococcus sp. and Synechocystis sp. Then, a general classification of cyanobacterial D1 isoforms in various cyanobacterial species into D1m, D1:1, D1:2, and D1' forms depending on their expression pattern under acclimated growth conditions and upon stress is discussed, taking into consideration the phototolerance of different D1 forms and the expression conditions of respective members of the psbA gene family.

Transcription of a "silent" cyanobacterial psbA gene is induced by microaerobic conditions.

Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1'. Only three amino acid residues are consistently conserved in D1'. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.

The psbA gene family responds differentially to light and UVB stress in Gloeobacter violaceus PCC 7421, a deeply divergent cyanobacterium.

Gloeobacter violaceus PCC 7421 is a slow-growing cyanobacterium which lacks thylakoid membranes, but whose five-membered psbA gene family encodes three isoform variants of the PsbA (D1) reaction center protein of Photosystem II. Under standard culture conditions Gloeobacter exhibits photosystem II electron transport, but several clear modifications in the redox potential of key cofactors bound by the PsbA protein are manifested in the flash-fluorescence characteristics. In other cyanobacteria dynamic expression of multiple psbA genes and turnover of PsbA isoforms is critical to counter excitation stress. We found that each of Gloeobacter's five psbA genes is expressed, with transcript abundances spanning 4.5 orders of magnitude. psbAI (glr2322) and psbAII (glr0779), encoding identical PsbA:2 form proteins, are constitutively expressed and dominate the psbA transcript pool under control conditions. psbAIII (gll3144) was strongly induced under photoinhibitory high irradiance stress, thereby contributing to a large increase in the psbA transcript pool that allowed cells to maintain their PsbA protein pools and then recover from irradiance stress, within one cellular generation. In contrast, under comparable photoinhibition provoked by UVB the cells were unable to maintain their psbA transcript and PsbA protein pools, and showed limited subsequent recovery. psbAIV (glr1706) and psbAV (glr2656), encoding two divergent PsbA isoforms, showed consistent trace expression but were never quantitatively significant contributors to the psbA transcript pool.

FtsH protease is required for induction of inorganic carbon acquisition complexes in Synechocystis sp. PCC 6803.

Cyanobacteria possess a complex CO(2)-concentrating mechanism (CCM), which is induced by low inorganic carbon conditions. To investigate the involvement of proteases in the processes of induction and degradation of the CCM complexes, we studied the FtsH2 (DeltaSlr0228) and Deg-G (DeltaSlr1204/DeltaSll1679/DeltaSll1427) protease mutants of Synechocystis sp. PCC 6803. WT and protease mutant cells were grown under high CO(2) and then shifted to low CO(2), followed by a proteome analysis of the membrane protein complexes. Interestingly, in the FtsH2 protease mutant, inducible CCM complexes were not detected upon shift to low CO(2), whereas the Deg-G mutant behaved like WT. Also the transcripts of the inducible CCM genes and their regulator ndhR failed to accumulate upon shift of FtsH2 mutant cells from high to low CO(2), indicating that the regulation by the FtsH2 protease is upstream of NdhR. Moreover, functional photosynthesis was shown a prerequisite for induction of CCM in WT at low CO(2), possibly via generation of oxidative stress, which was shown here to enhance the expression of inducible CCM genes even at high CO(2) conditions. Once synthesized, the CCM complexes were not subject to proteolytic degradation, even when dispensable upon a shift of cells to high CO(2).

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803.

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a DeltaFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in DeltaDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in DeltaFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the DeltaDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.

Cyanobacterial psbA families in Anabaena and Synechocystis encode trace, constitutive and UVB-induced D1 isoforms.

Cyanobacteria cope with UVB induced photoinhibition of Photosystem II by regulating multiple psbA genes to boost the expression of D1 protein (in Synechocystis sp. PCC6803), or to exchange the constitutive D1:1 protein to an alternate D1:2 isoform (in Synechococcus sp. PCC7942). To define more general patterns of cyanobacterial psbA expression, we applied moderately photoinhibitory UVB to Anabaena sp. PCC7120 and tracked the expression of its five psbA genes. psbAI, encoding a D1:1 protein isoform characterized by a Gln130, represented the majority of the psbA transcript pool under control conditions. psbAI transcripts decreased upon UVB treatment but the total psbA transcript pool increased 3.5 fold within 90 min as a result of sharply increased psbAII, psbAIV and psbAIII transcripts encoding an alternate D1:2 protein isoform characterized by Glu130, similar to that of Synechococcus. Upon UVB treatment the relaxation of flash induced chlorophyll fluorescence showed a characteristic acceleration of a decay phase likely associated with the exchange from the D1:1 protein isoform encoded by psbAI to the alternate D1:2 isoform encoded by psbAIV, psbAII and psbAIII. Throughout the UVB treatment the divergent psbA0 made only a trace contribution to the total psbA transcript pool. This suggests a similarity to the divergent psbAI gene from Synechocystis, whose natural expression we demonstrate for the first time at a trace level similar to psbA0 in Anabaena. These trace-expressed psbA genes in two different cyanobacteria raise questions concerning the functions of these divergent genes.

Cyclic nucleotides, the photosynthetic apparatus and response to a UV-B stress in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyclic nucleotides cAMP and cGMP are ubiquitous signaling molecules that mediate many adaptative responses in eukaryotic cells. Cyanobacteria present the peculiarity among the prokaryotes of having the two types of cyclic nucleotide. Cellular homeostasis requires both cyclases (adenylyl/guanylyl, for their synthesis) and phosphodiesterases (for their degradation). Fully segregated null mutants have been obtained for the two genes, sll1624 and slr2100, which encode putative cNMP phosphodiesterases. We present physiological evidence that the Synechocystis PCC 6803 open reading frame slr2100 could be a cGMP phosphodiesterase. In addition, we show that Slr2100, but not Sll1624, is required for the adaptation of the cells to a UV-B stress. UV-B radiation has deleterious effects for photosynthetic organisms, in particular on the photosystem II, through damaging the protein structure of the reaction center. Using biophysical and biochemical approaches, it was found that Slr2100 is involved in the signal transduction events which permit the repair of the UV-B-damaged photosystem II. This was confirmed by quantitative reverse transcriptase-PCR analyses. Altogether, the data point to an important role for cGMP in signal transduction and photoacclimation processes during a UV-B stress.

Protective effect of supplemental low intensity white light on ultraviolet-B exposure-induced impairment in cyanobacterium Spirulina platensis: formation of air vacuoles as a possible protective measure.

Intact trichomes of Spirulina platensis were exposed to 1-5 h of low (0.2 mW cm(-2)) or high (0.6 mW cm(-2)) intensity UV-B (280-320 nm) radiation, alone or with photosynthetically active radiation (PAR) of supplemental 50 muE m(-2) s(-1) white light (WL). The mitigating effect of supplemental WL on UV-B induced alterations in Spirulina were investigated by monitoring time-dependent change in photosystem (PS) II mediated O(2) evolution, absorption, circular dichroism (CD) spectra, and ultrastructure. At low intensity, UV-B induced loss in PS II-catalyzed O(2) evolution, but caused no change in the absorption spectrum. At high intensity, UV-B caused a decrease in absorption by phycobilisomes (PBsomes), which was only partly prevented by the presence of low-intensity supplemental WL. The CD spectral analysis revealed that UV-B exposure caused time-dependent enhancement of the negative psi-type bands at 452 and 689 nm, reflecting alterations in the macroaggregation of chlorophyll-protein complexes. This enhancement of negative PS II-type bands was substantially arrested by the presence of supplemental WL exposure, even when UV-B exposure was continued for 5 h. These changes in UV-B-induced CD spectrum suggest alterations in the antenna structure of Spirulina involving both PBsomes and Chlorophyll a. Thus, supplemental low intensity WL arrests, to large extent, the macroaggregation of pigment-protein complexes. Furthermore, the electron micrographs of Spirulina revealed that UV-B exposure caused disorganization of the cellular ultrastructure, while the inclusion of supplemental WL enhanced the formation of air vacuoles in Spirulina. We suggest that the formation of vacuoles by supplemental WL is a protective feature against UV-B.

The interaction of visible and UV-B light during photodamage and repair of Photosystem II.

In order to understand the mechanism of photodamage induced by solar radiation under natural conditions, we studied the interaction of visible and ultraviolet-B light in the inactivation and repair of the Photosystem II complex by using oxygen evolution and flash-induced chlorophyll fluorescence measurements. In isolated spinach thylakoids and Synechocystis 6803 cells, in which de novo protein synthesis is blocked by lincomycin, photodamage of Photosystem II by visible and UV-B light is characterized by linear semilogarithmic inactivation curves for both separate and combined illumination protocols. The extent of PS II inactivation obtained after combined illumination can be well simulated by assuming independent damaging events induced by visible and UV-B photons. In intact Synechocystis cells capable of protein repair, simultaneous illumination by visible and UV-B light impairs Photosystem II activity to a smaller extent than expected from the independent damaging events. This protective effect is pronounced at low visible light (130 muE m(-2) s(-1)), but becomes negligible at high intensities (1300 muE m(-2) s(-1)). Exposure of intact Synechocystis 6803 cells to direct sunlight leads to a rapid inactivation of PS II, accompanied by the accumulation of donor side inhibited centers. This phenomenon, which shows the impairment of the manganese cluster of water oxidation was not observed when the ultraviolet components of sunlight were filtered out. We conclude that visible and UV-B photons inactivate PS II via non-interacting mechanisms, which affect different target sites. In intact cells, the two spectral regions do interact, and results in synergistically enhanced protein repair capacity when UV-B radiation is accompanied by low intensity visible light, which provides protection against photodamage. However, this ameliorating effect becomes insignificant at high light intensities characteristic of direct sunlight.