Gene transfer to the desiccation-tolerant cyanobacterium Chroococcidiopsis

The coccoid cyanobacterium Chroococcidiopsis dominates microbial communities in the most extreme arid hot and cold deserts. These communities withstand constraints that result from multiple cycles of drying and wetting and/or prolonged desiccation, through mechanisms which remain poorly understood. Here we describe the first system for genetic manipulation of Chroococcidiopsis. Plasmids pDUCA7 and pRL489, based on the pDU1 replicon of Nostoc sp. strain PCC 7524, were transferred to different isolates of Chroococcidiopsis via conjugation and electroporation. This report provides the first evidence that pDU1 replicons can be maintained in cyanobacteria other than Nostoc and Anabaena. Following conjugation, both plasmids replicated in Chroococcidiopsis sp. strains 029, 057, and 123 but not in strains 171 and 584. Both plasmids were electroporated into strains 029 and 123 but not into strains 057, 171, and 584. Expression of P(psbA)-luxAB on pRL489 was visualized through in vivo luminescence. Efficiencies of conjugative transfer for pDUCA7 and pRL489 into Chroococcidiopsis sp. strain 029 were approximately 10(-2) and 10(-4) transconjugants per recipient cell, respectively. Conjugative transfer occurred with a lower efficiency into strains 057 and 123. Electrotransformation efficiencies of about 10(-4) electrotransformants per recipient cell were achieved with strains 029 and 123, using either pDUCA7 or pRL489. Extracellular deoxyribonucleases were associated with each of the five strains. Phylogenetic analysis, based upon the V6 to V8 variable regions of 16S rRNA, suggests that desert strains 057, 123, 171, and 029 are distinct from the type species strain Chroococcidiopsis thermalis PCC 7203. The high efficiency of conjugative transfer of Chroococcidiopsis sp. strain 029, from the Negev Desert, Israel, makes this a suitable experimental strain for genetic studies on desiccation tolerance.     

Hydrogen metabolism of the unicellular cyanobacterium Chroococcidiopsis thermalis ATCC29380

Abstract Hydrogenase was induced in the unicellular cyanobacterium Chroococcidiopsis thermalis ATCC29380 when grown aerobically in a medium lacking combined nitrogen. Nitrogenase, however, was only observed after incubation of cells in a microaerobic environment. Hydrogen evolution could not be detected under aerobic conditions, but upon transfer of cells to dark anaerobic conditions, large amounts of hydrogen were immediately produced. This hydrogen evolution was sensitive to light and oxygen but not to inhibitors of protein synthesis. The enzyme activity catalyzing the formation of hydrogen was not membrane-bound; some functional properties were characterized in cell-free extracts.     

Salt tolerance and polyphyly in the cyanobacterium Chroococcidiopsis (Pleurocapsales)

Chroococcidiopsis Geitler (Geitler 1933) is a genus of cyanobacteria containing desiccation and radiation resistant strains. Members of the genus live in habitats ranging from hot and cold deserts to fresh and saltwater environments. Morphology and cell division pattern have historically been used to define the genus. To better understand the evolution and ability of the Chroococcidiopsis genus to survive in diverse environments we investigated how salt tolerance varies among 15 strains previously isolated from different locations, and if salt tolerant strains are monophyletic to those isolated from freshwater and land environments. Four markers were sequenced from these 15 strains, the 16S rRNA, rbcL, desC1, and gltX genes. Phylogenetic trees were generated which identified a distinct clade of salt‐tolerant strains. This study demonstrates that the genus is polyphyletic based on saltwater and freshwater phenotypes. To understand the resistance to salt in more details, the strains were grown on a range of sea salt concentrations which demonstrated that the freshwater strains were salt‐intolerant whilst the saltwater strains required salt for growth. This study shows an increased resolution of the phylogeny of Chroococcidiopsis and provides further evidence that the genus is polyphyletic and should be reclassified to improve clarity in the literature.     

Endurance of the endolithic desert cyanobacterium Chroococcidiopsis under UVC radiation

Desert cyanobacteria of the genus Chroococcidiopsis are extremely resistant to desiccation and ionizing radiation. When an endolithic strain was exposed to UVC radiation cell lysis, genome damage, photosynthetic pigment bleaching and reduced photochemical performance occurred. Nevertheless, survivors were scored after UVC doses as high as 13?kJ/m² and their endurance ascribed to multicellular aggregates enveloped in thick envelopes, so that attenuated UVC radiation reached the inner cells. In addition, the accumulation of carotenoids contributed to UVC resistance by providing protection against oxidative stress. Finally, in survivors repair mechanisms were responsible for the recovery of the induced damage to genome and photosynthetic apparatus.     

Cytology of long-term desiccation in the desert cyanobacterium Chroococcidiopsis (Chroococcales)

Young and old cultures (up to 66 months) of two Chroococcidiopsis sp. strains isolated from the Negev desert, Israel, were examined by epifluorescence and electron microscopy. In old cultures, cell viability and autofluorescence were lower than in young cultures. An increase was seen with age in the polysaccharide content of the sheaths of nanocytes and nanocyte mother cells, and a decrease of phycobiliproteins was also seen. In the oldest cultures most of the cells were dead and in various stages of degeneration. Single living cells were scattered among the dead ones. No resting cells were formed in the oldest cultures, but many cell groups showed highly electron-dense sheaths and, in the cytoplasm, ribosomes and glycogen. These changes in cell structure may have a role in preventing water loss from the cell.     

H2-Uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758

The unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758 growing photoautotrophically synthesised a hydrogenase which catalysed an in vivo H₂ uptake in the oxyhydrogen reaction at a significant rate and showed only low level of in vitro MV-dependent H₂ evolution. The in vitro hydrogenase activity was not induced under microaerobic or nitrate-limiting conditions. Some correlation observed between the two activities indicated that the same enzyme may be involved in both H₂ uptake and H₂ evolution. Heterologous Southern hybridisations, using cyanobacterial hup and hox DNA fragments as probes, showed the presence of sequences similar to hox (encoding for a bidirectional hydrogenase) in C. thermalis CALU 758 with no indication for the presence of any sequences corresponding to an uptake hydrogenase. Further molecular experiments, using specific primers directed against different conserved regions of the large subunit (hoxH) of the bidirectional hydrogenase confirmed the presence of corresponding sequences in C. thermalis CALU 758. Low-stringency Southern hybridisations detected only one copy of hoxH within the genome of C. thermalis CALU 758.     

Purification of polysomes from a lysozyme- resistant desiccation-tolerant cyanobacterium

Six different techniques were compared for the extraction and purification of polysomes from cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584. Cells resisted treatment with lysozyme, and methods which relied upon ‘gentle lysis’ resulted in inefficient cell breakage and poor yields of polysomes. In contrast, the passage of cells through a French Pressure Cell achieved complete disruption of even the most resistant cell aggregates but only monosomes and ribosomal subunits were recovered. The grinding of cells with glass beads in the presence of neutral detergents was the most successful of all the methods tested and resulted in efficient cell lysis with high yields of polysomes. Treatment of the cells with acetone, at 0°C, prior to homogenization, also resulted in good yields of polysomes although the degree of cell breakage was less than when the cells were ground. The choice of the grinding material, and the extent of the grinding, were both critical for polysome extraction. Grinding of cells with alumina and sterile sand gave very efficient cell breakage but no polysomes were recovered. Excessive grinding with glass beads led to a progressive loss of intact polysomes and concomitant increase in 70 S monosomes and subunits in cell extracts.This study provides data on various physical treatments and buffer compositions which may be used effectively in the isolation and purification of polysomal RNA from highly resistant bacterial cells. A method which relies upon the grinding of cells in the presence of neutral detergents will permit further studies of gene expression in cells which resist methods of ‘gentle lysis’.     

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, showsunique responses to dehydration. These responses are: (i) lossof PSII activity in parallel with the loss of photosynthesis;(ii) loss of PSI activity; and (iii) dissipation of light energyabsorbed by pigment–protein complexes. In this study,the deactivation of PSII is shown to be important in avoidingphotoinhibition when the Calvin–Benson cycle is repressedby dehydration. Furthermore, our evidence suggests that dissipationof light energy absorbed by PSII blocks photoinhibition understrong light in dehydrated states.     

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H₂O₂ up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H₂O₂. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

     

Gloeocapsopsis AAB1, an extremely desiccation-tolerant cyanobacterium isolated from the Atacama Desert

The comprehensive study of microorganisms that evolved in the Atacama Desert, the driest and oldest on earth, may help to understand the key role of water for life. In this context, we previously characterized the microenvironment that allows colonization of the underside of quartzes in the Coastal Range of this desert by hypolithic microorganisms (Azua-Bustos et al. Microb Ecol 58:568–581, 2011). Now, we describe the biodiversity composition of these biofilms and the isolation from it of a new cyanobacterial strain. Based on morphologic and phylogenetic analyses, this isolate (AAB1) was classified as a new member of the Gloeocapsopsis genus. Physiological, morphological and molecular responses by isolate AAB1 show that this strain is extremely tolerant to desiccation. Our results also indicate that the isolate biosynthesizes sucrose and trehalose in response to this stressful condition. We identified two candidate genes involved in sucrose synthesis, namely sucrose 6-phosphate synthase and sucrose 6-phosphate phosphatase. Thus, the Gloeocapsopsis isolate AAB1 may represent a suitable model for understanding tolerance to low water availability.     

Novel water stress protein from a desiccation-tolerant cyanobacterium. Purification and partial characterization

A desiccation-tolerant cyanobacterium Nostoc commune accumulates a novel group of acidic proteins when colonies are subjected to repeated cycles of drying and rehydration. The proteins occur in high concentrations; they have isoelectric points between 4.3 and 4.8 and apparent molecular masses between 30 and 39 kDa. The purification of three of these proteins with molecular masses of 33, 37, and 39 kDa is described. The amino-terminal sequence of the 39-kDa protein is Ala-Leu-Tyr-Gly-Tyr-Thr-Ile-Gly-Glu. Peptide mapping of the 39- and the 33-kDa proteins, using different protease, gave similar patterns of digestion fragments. The amino acid compositions of the proteins isolated were similar, and each cross-reacted with a polyclonal antibody raised against the largest (39-kDa) protein. The results indicate that the microheterogeneity observed was generated by in vivo proteolysis of the 39-kDa protein. It is suggested that this protein is a water stress protein with a protective function on a structural level.     

Insights into the chaotropic tolerance of the desert cyanobacterium Chroococcidiopsis sp. 029 (Chroococcidiopsales,Cyanobacteria)

The mechanism of perchlorate resistance of the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated by assessing whether the pathways associated with its desiccation tolerance might play a role against the destabilizing effects of this chaotropic agent. During 3 weeks of growth in the presence of 2.4 mM perchlorate, an upregulation of trehalose and sucrose biosynthetic pathways was detected. This suggested that in response to the water stress triggered by perchlorate salts, these two compatible solutes play a role in the stabilization of macromolecules and membranes as they do in response to dehydration. During the perchlorate exposure, the production of oxidizing species was observed by using an oxidant-sensing fluorochrome and determining the expression of the antioxidant defense genes, namely superoxide dismutases and catalases, while the presence of oxidative DNA damage was highlighted by the over-expression of genes of the base excision repair. The involvement of desiccation-tolerance mechanisms in the perchlorate resistance of this desert cyanobacterium is interesting since, so far, chaotropic-tolerant bacteria have been identified among halophiles. Hence, it is anticipated that desert microorganisms might possess an unrevealed capability of adapting to perchlorate concentrations exceeding those naturally occurring in dry environments. Furthermore, in the endeavor of supporting future human outposts on Mars, the identified mechanisms might contribute to enhance the perchlorate resistance of microorganisms relevant for biologically driven utilization of the perchlorate-rich soil of the red planet.     

Effect of environmental factors on the synthesis of scytonemin, a UV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.)

Abstract.The UV-screening pigment scytonemin is found in many species of ensheathed cyanobacteria. Past work has shown that the pigment is synthesized in response to exposure to UV-A irradiance. This study investigated the effect of other correlated stress factors including heat, osmotic and oxidative stress on the synthesis of scytonemin in a clonal cyanobacterial isolate ( Chroococcidiopsis sp.) from an epilithic desert crust. Stress experiments were carried out both in conjunction with UV-A irradiance and in isolation. Increases in both temperature and photooxidative conditions in conjunction with UV-A caused a synergistic increase in the rate of scytonemin production. In contrast, increased salt concentration under UV-A irradiance inhibited scytonemin synthesis. However, unlike the responses to temperature and oxidative stress, cells synthesized low levels of scytonemin under osmotic stress in the absence of scytonemin-inducing irradiance. These results suggest that scytonemin induction may be regulated as a part of a complex stress response pathway in which multiple environmental signals affect its synthesis.     

Ionizing-Radiation Resistance in the Desiccation-Tolerant Cyanobacterium Chroococcidiopsis

The effect of X-ray irradiation on cell survival, induction, and repair of DNA damage was studied by using 10 Chroococcidiopsis strains isolated from desert and hypersaline environments. After exposure to 2.5 kGy, the percentages of survival for the strains ranged from 80 to 35%. In the four most resistant strains, the levels of survival were reduced by 1 or 2 orders of magnitude after irradiation with 5 kGy; viable cells were recovered after exposure to 15 kGy but not after exposure to 20 kGy. The severe DNA damage evident after exposure to 2.5 kGy was repaired within 3 h, and the severe DNA damage evident after exposure to 5 kGy was repaired within 24 h. The increase in trichloroacetic acid-precipitable radioactivity in the culture supernatant after irradiation with 2.5 kGy might have been due to cell lysis and/or an excision process involved in DNA repair. The radiation resistance of Chroococcidiopsis strains may reflect the ability of these cyanobacteria to survive prolonged desiccation through efficient repair of the DNA damage that accumulates during dehydration.     

Mass spectrometry and spectroscopic characterization of a tetrameric photosystem I supercomplex from Leptolyngbya ohadii,a desiccation-tolerant cyanobacterium

Cyanobacteria inhabiting desert biological soil crusts face the harsh conditions of the desert. They evolved a suite of strategies toward desiccation-hydration cycles mixed with high light irradiations, etc. In this study we purified and characterized the structure and function of Photosystem I (PSI) from Leptolyngbya ohadii, a desiccation-tolerant desert cyanobacterium. We discovered that PSI forms tetrameric (PSI-Tet) aggregate. We investigated it by using sucrose density gradient centrifugation, clear native PAGE, high performance liquid chromatography, mass spectrometry (MS), time-resolved fluorescence (TRF) and time-resolved transient absorption (TA) spectroscopy. MS analysis identified the presence of two PsaB and two PsaL proteins in PSI-Tet and uniquely revealed that PsaLs are N-terminally acetylated in contrast to non-modified PsaL in the trimeric PSI from Synechocystis sp. PCC 6803. Chlorophyll (Chl) a fluorescence decay profiles of the PSI-Tet performed at 77 K revealed two emission bands at ～690 nm and 725 nm with the former appearing only at early delay time. The main fluorescence emission peak, associated with emission from the low energy Chls a, decays within a few nanoseconds. TA studies demonstrated that the 725 nm emission band is associated with low energy Chls a with absorption band clearly resolved at ～710 nm at 77 K. In summary, our work suggests that the heterogenous composition of PsaBs and PsaL in PSI-Tet is related with the adaptation mechanisms needed to cope with stressful conditions under which this bacterium naturally grows.     

Deactivation of photosynthetic activities is triggered by loss of a small amount of water in a desiccation-tolerant cyanobacterium, Nostoc commune

Changes in photosynthetic activities under hypertonic conditions were studied in a terrestrial, highly desiccation-tolerant cyanobacterium, Nostoc commune, and in some desiccation-sensitive cyanobacteria. The amounts of water sustained in the colony matrix outside the N. commune cells and the cellular solute concentration were estimated by measuring the water potential, and the solute concentration was supposed to correspond to around 0.22 M sorbitol. Incubation of the colonies in 0.8 M sorbitol solution inhibited the energy transfer from the phycobilisome (PBS) anchor to PSII core complexes. At higher sorbitol concentrations, light energy absorbed by PSI, PSII, and PBS was dissipated to heat. PSI and cyclic electron flow around PSI was also deactivated by hypertonic treatment. Fv/Fm and (Fm'-F)/Fm' values started to decrease at 0.6 and 0.3 M sorbitol and reached zero at 1.0 and 0.8 M, respectively. Decreases in these two fluorescence parameters corresponded to the decreases in PSII fluorescence (F695) and photosynthetic CO2 fixation, respectively. The intensity of delayed light emission started to decrease at 1.0 M sorbitol and became negligible at 4.0 M. Comparing these changes in N. commune with those in desiccation-sensitive species, we found that N. commune cells actively deactivates photosynthetic systems on sensing water loss.     

Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis.

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.     

Cadmium-mediated resistance to metals and antibiotics in a cyanobacterium

Summary Cadmium-resistant strains of the cyanobacterium Nostoc calcicola were isolated through the step-wise transfer of the organism to higher levels of the metal. One of the Cd-resistant strains (Cd^r–10) showed cross-resistance to antibiotics like neomycin (1 g/ml), chloramphenicol (3 g/ml) but not to streptomycin. The Cd-resistant strain also tolerated elevated levels of metals such as zinc (20 ppm) and mercury (1 ppm). The stability of the metal-resistance required the presence of Cd²⁺ ions in the growth medium. It is suggested that metal resistance may also be determined by gene(s) on the antibiotic resistance plasmids in cyanobacteria.     

Seasonal changes in photosynthesis in the desiccation-tolerant fern Polypodium virginianum

Summary Seasonal changes in photosynthesis were examined in the desiccation-tolerant fern Polypodium virginianum growing in a forest understory along cliff edges of the Niagara Escarpment in southern Ontario, Canada. For plants growing in situ, the photosynthetic response to irradiance was examined on a seasonal basis, to determine the degree to which the utilization of light changed over the growing season. Experiments were executed on control plants, on previously desiccated then rehydrated plants, and on continuously hydrated plants to determine if prior desiccation influenced the response to light. Soil and xylem water potential and temperature were monitored and used as covariates in analyses. The results showed that carbon gain in the spring greatly exceeded that of any other season. Despite this, there was little change in the photosynthetic response to light on a seasonal basis even though plants were exposed to highly variable and highly limited light most of the time. Prior desiccation had a slight influence on photosynthetic rate but not on other photosynthetic parameters such as the light compensation point and L_half. Temperature was a significant seasonal covariate and additional experiments conducted in the laboratory showed that the response of photosynthesis to temperature was broad. Xylem water potential was correlated with seasonal changes in relative humidity. The results suggest that P. virginanum persists in hostile cliff-edge habitats by being able to exploit high-light periods in the spring and by thereafter maintaining a low but relatively constant rate of carbon despite prior exposure to fluctuating supplies of light and water.