Production of gold nanoparticles by biogenesis using bacteria

Diazotrophic cyanobacteria Anabaena sp. PCC 7120, four Nostoc strains, and two Azotobacter species (A. vinelandii and A. chroococcum) were found to produce gold nanoparticles (GNP) under nitrogen fixation conditions. GNP biogenesis occurred at AuHCl₄ concentrations from 0.1 to 1 mM. In the cultures of unicellular cyanobacteria Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 incapable of nitrogen fixation, no GNP were formed at the same concentrations of gold salts. The plasmon resonance band peak was located at 552 nm. This position is characteristic of spherical GNP 10 to 30 nm in size. Small amounts of GNP were also formed in the culture liquid supernatants of the tested nitrogen-fixing bacteria at AuHCl₄ concentrations from 0.25 to 0.5 mM.     

Bio-optical characterization of selected cyanobacteria strains present in marine and freshwater ecosystems

The optical properties, i.e., absorption and scattering spectra of ten strains of cyanobacteria from the Baltic Sea and Pomeranian lakes (Aphanizomenon flos-aquae KAC 15, Microcystis aeruginosa CCNP 1101, Anabaena sp. CCNP 1406, Synechocystis salina CCNP 1104, Phormidium sp. CCNP 1317, Nodularia spumigena CCNP 1401, Synechococcus sp. CCNP 1108, Nostoc sp. CCNP 1411, Cyanobacterium sp. CCNP 1105, Pseudanabaena cf. galeata CCNP 1312) grown under low light conditions were investigated. Moreover, the chlorophylls, carotenoids, and phycobilin composition as well as the size structure of chosen cyanobacteria were measured. Studied species revealed high diversity both in optical properties with the absorption spectra similarity index ranging from 0.67 to 0.94 and the pigment composition. The chlorophyll-specific absorption coefficient at 440 nm a _ph *(440) varied between 0.017 and 0.065 m² mg^?1. The influence of the package effect was only observed in the case of large filamentous cyanobacteria like N. spumigena or Nostoc sp. Interestingly, the package effect factor Q _a *(675) for large-celled Anabaena sp. was 0.92. Besides chlorophyll a, only echinenone, β-carotene, and phycocyanin were present in all analyzed cyanobacteria strains. Zeaxanthin, which is widely used as a marker pigment for cyanobacteria, was absent in the toxic N. spumigena and Anabaena sp., which are the species that occur in the Baltic Sea most frequently causing summer cyanobacterial blooms. The investigation also showed that the sample preservation technique can introduce some major errors within the absorption band affected by the phycocyanin absorption.     

A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase

Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.     

Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Antioxidant activity and phenolic profile in filamentous cyanobacteria: the impact of nitrogen

In the present study, ethanolic extracts of ten cyanobacterial strains cultivated under different nitrogen conditions were assessed for the phenolic content and antioxidant activity. The amount of detected phenolic compounds ranged from 14.86 to 701.69 μg g^?1 dry weight (dw) and HPLC-MS/MS analysis revealed gallic acid, chlorogenic acid, quinic acid, catechin, epicatechin, kaempferol, rutin and apiin. Only catechin, among the detected phenolics, was present in all the tested strains, while quinic acid was the most dominant compound in all the tested Nostoc strains. The results also indicated the possibility of increasing the phenolic content in cyanobacterial biomass by manipulating nitrogen conditions, such as in the case of quinic acid in Nostoc 2S7B from 70.83 to 594.43 μg g^?1 dw. The highest radical scavenging activity in DPPH assay expressed Nostoc LC1B with IC₅₀ value of 0.04?±?0.01 mg mL^?1, while Nostoc 2S3B with IC₅₀ =?9.47?±?3.61 mg mL^?1 was the least potent. Furthermore, the reducing power determined by FRAP assay ranged from 8.36?±?0.08 to 21.01?±?1.66 mg AAE g^?1, and it was significantly different among the tested genera. The Arthrospira strains exhibited the highest activity, which in the case of Arthrospira S1 was approximately twofold higher in comparison to those in nitrogen-fixing strains. In addition to this, statistical analysis has indicated that detected phenolics were not major contributor to antioxidant capacities of tested cyanobacteria. However, this study highlights cyanobacteria of the genera Nostoc, Anabaena, and Arthrospira as producers of antioxidants and phenolics with pharmacological and health-beneficial effects, i.e., quinic acid and catechin in particular.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Gram-negative bacilli-derived peptide antibiotics developed since 2000

Gram-negative bacilli such as Pseudomonas spp., Pseudoalteromonas sp., Angiococcus sp., Archangium sp., Burkholderia spp., Chromobacterium sp., Chondromyces sp., Cystobacter sp., Jahnella sp., Janthinobacterium sp., Lysobacter spp., Paraliomyxa sp., Photobacterium spp., Photorhabdus sp., Pontibacter sp., Ruegeria sp., Serratia sp., Sorangium sp., Sphingomonas sp., and Xenorhabdus spp. produce an enormous array of short peptides of 30 residues or fewer that are potential pharmaceutical drugs and/or biocontrol agents. The need for novel lead antibiotic compounds is urgent due to increasing drug resistance, and this review summarises 150 Gram-negative bacilli-derived compounds reported since 2000, including 40 cyclic lipopeptides from Pseudomonas spp.; nine aromatic peptides; eight glycopeptides; 45 different cyclic lipopeptides; 24 linear lipopeptides; eight thiopeptides; one lasso peptide; ten typical cyclic peptides; and five standard linear peptides. The current and potential therapeutic applications of these peptides, including structures and antituberculotic, anti-cyanobacterial, antifungal, antibacterial, antiviral, insecticidal, and antiprotozoal activities are discussed.     

A cyanobacterial β-actin-like protein,responsible for lichenized <Emphasis Type="Italic">Nostoc</Emphasis> sp. motility towards a fungal lectin

Lichenized Nostoc cells isolated from the lichen Peltigera canina develop chemotactic movement towards a lectin purified from the lichen thallus. Inhibitors of cytoskeleton dynamics, such as phalloidin, latrunculin A and blebbistatin, impede cell displacement. The inhibition of chemotaxis produced by the combined action of phalloidin and blebbistatin is largely reversed by GTP and its analogs, GTP(γ)S and GDP(β)S, as well as by cyclic AMP. Movement implies a rearrangement of the cytoskeleton causing cell polarity, which is, in turn, inhibited by phalloidin and latrunculin A, as revealed by confocal microscopy. F-actin fibers composing Nostoc cytoskeleton have been visualized by immunocytochemical techniques associated with transmission electron microscopy.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Isolation of identical nitrilase genes from multiple bacterial strains and real-time PCR detection of the genes from soils provides evidence of horizontal gene transfer

Bacterial enzymes capable of nitrile hydrolysis have significant industrial potential. Microbacterium sp. AJ115, Rhodococcus erythropolis AJ270 and AJ300 were isolated from the same location in England and harbour identical nitrile hydratase/amidase gene clusters. Strain AJ270 has been well studied due to its nitrile hydratase and amidase activity. R. erythropolis ITCBP was isolated from Denmark and carries a very similar nitrile hydratase/amidase gene cluster. In this study, an identical nitrilase gene (nit1) was isolated from the four strains, and the nitrilase from strain AJ270 cloned and expressed in Escherichia coli. Analysis of the recombinant nitrilase has shown it to be functional with activity demonstrated towards phenylacetonitrile. A real-time PCR TaqMan^® assay was developed that allowed nit1 detection directly from soil enrichment cultures without DNA extraction, with nit1 detected in all samples tested. Real-time PCR screening of isolates from these soils resulted in the isolation of nit1 and also very similar nitrilase gene nit2 from a number of Burkholderia sp. The genes nit1 and nit2 have also been detected in many bacteria of different genera but are unstable in these isolates. It is likely that the genes were acquired by horizontal gene transfer and may be widespread in the environment.     

Nitrogen fixing bacterial diversity in a tropical estuarine sediments

Microorganisms play a significant role in biogeochemical cycles, especially in the benthic and pelagic ecosystems. Role of environmental parameters in regulating the diversity, distribution and physiology of these microorganisms in tropical marine environment is not well understood. In this study, we have identified dinitrogen (N₂) fixing bacterial communities in the sediments by constructing clone libraries of nitrogenase (nifH) gene from four different stations in the Cochin estuary, along the southeastern Arabian Sea. N₂ fixing bacterial clones revealed that over 20 putative diazotrophs belong to alpha-, beta-, gamma-, delta- and epsilon- proteobacteria and firmicutes. Predominant genera among these were Bradyrhizobium sp. (α-proteobacteria), Dechloromonas sp. (β-proteobacteria); Azotobactor sp., Teredinibacter sp., Methylobacter sp., Rheinheimera sp. and Marinobacterium sp. (γ-proteobacteria); Desulfobacter sp., Desulfobulbus sp. and Desulfovibrio sp. (δ -proteobacteria); Arcobacter sp. and Sulfurospirillum sp. (ε-proteobacteria). Nostoc sp. was solely identified among the cyanobacterial phylotype. Nitrogen fixing Sulfate reducing bacteria (SRBs) such as Desulfobulbus sp., Desulfovibrio sp., Desulfuromonas sp., Desulfosporosinus sp., Desulfobacter sp., were also observed in the study. Most of the bacterial nifH sequences revealed that the identities of N₂ fixing bacteria were less than 95% similar to that available in the GenBank database, which suggested that the sequences were of novel N₂ fixing microorganisms. Shannon-Weiner diversity index of nifH gene ranged from 2.95 to 3.61, indicating an inflated diversity of N₂ fixing bacteria. Canonical correspondence analysis (CCA) implied positive correlation among nifH diversity, N₂ fixation rate and other environmental variables.     

Comparison of photosynthetic performances of marine picocyanobacteria with different configurations of the oxygen-evolving complex

The extrinsic PsbU and PsbV proteins are known to play a critical role in stabilizing the Mn₄CaO₅ cluster of the PSII oxygen-evolving complex (OEC). However, most isolates of the marine cyanobacterium Prochlorococcus naturally miss these proteins, even though they have kept the main OEC protein, PsbO. A structural homology model of the PSII of such a natural deletion mutant strain (P. marinus MED4) did not reveal any obvious compensation mechanism for this lack. To assess the physiological consequences of this unusual OEC, we compared oxygen evolution between Prochlorococcus strains missing psbU and psbV (PCC 9511 and SS120) and two marine strains possessing these genes (Prochlorococcus sp. MIT9313 and Synechococcus sp. WH7803). While the low light-adapted strain SS120 exhibited the lowest maximal O₂ evolution rates (P_max per divinyl-chlorophyll a, per cell or per photosystem II) of all four strains, the high light-adapted strain PCC 9511 displayed even higher P^Chl_max and P^PSII_max at high irradiance than Synechococcus sp. WH7803. Furthermore, thermoluminescence glow curves did not show any alteration in the B-band shape or peak position that could be related to the lack of these extrinsic proteins. This suggests an efficient functional adaptation of the OEC in these natural deletion mutants, in which PsbO alone is seemingly sufficient to ensure proper oxygen evolution. Our study also showed that Prochlorococcus strains exhibit negative net O₂ evolution rates at the low irradiances encountered in minimum oxygen zones, possibly explaining the very low O₂ concentrations measured in these environments, where Prochlorococcus is the dominant oxyphototroph.     

Novel approach for the ammonium removal by simultaneous heterotrophic nitrification and denitrification using a novel bacterial species co-culture

Agricultural activities lead excessive emission of ammonia nitrogen in the environment and can profoundly interfere the equilibrium of the natural ecosystems leading to their contamination. Actually, the biological purification of wastewaters is the most adopted technique thanks to its several advantages such as high performance and low energy consumption. For this reason, two novel strains of Alcaligenes sp. S84S3 and Proteus sp. S19 genus were isolated from an activated sludge and applied in the treatment of ammonium and nitrite in aqueous solution. Under the optimum operating conditions of temperature (30 °C), pH (7), carbon substrate (2 g/L of glucose) and duration of incubation time (69 h), the strain Alcaligenes sp. S84S3 could oxidize 65 % of the ammonium as high as 272.72 mg-NH₄ ⁺/L. Moreover, during 48 h, the nitrate reduction rate performed by the strain Proteus S19 was about 99 % without production of nitrite intermediate (negligible concentration). Moreover, the coculture of the strains Alcaligenes sp. S84S3 and Proteus sp. S19 could eliminate 65.83 % of the ammonium ions without production of toxic forms of nitrogen oxides during a short time of incubation (118 h) at the same operational conditions with providing the aeration in the first treatment phase. The coculture of our isolated strains is assumed to have a good potential for nitrification and denitrification reactions applied in the treatment of wastewater containing ammonium, nitrite and nitrate. As a result, we can consider that the mixed culture is a practical method in the treatment of high-strength ammonium wastewater with reducing of sludge production.     

New <Emphasis Type="Italic">Penicillium</Emphasis> and <Emphasis Type="Italic">Talaromyces</Emphasis> species from honey,pollen and nests of stingless bees

Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and β-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.     

Identification of two cyanobacterial strains isolated from the Kotel’nikovskii hot spring of the Baikal rift

Two cyanobacterial strains, Pseudanabaena sp. 0411 and Synechococcus sp. 0431, were isolated from a sample collected in the Kotel’nikovskii hot spring of the Baikal rift. According to the results of light and transmission electron microscopy, as well as of the phylogenetic analysis of the 16S rRNA gene, these cyanobacteria were classified as Pseudanabaena sp. nov. and Synechococcus bigranulatus Skuja. The constructed phylogenetic tree shows that the studied strains are positioned in the clades of cyanobacteria isolated from hydrothermal vents of Asia and New Zealand, separately from marine and freshwater members of these genera, including those isolated from Lake Baikal.     

Suppression of Settlement of Larvae of Fouling Organism by Water-Soluble Substances from Epibiotic Bacteria

Twenty-nine bacterial strains were isolated from the surface of the green alga Ulva reticulata, the soft coral Dendronephthya sp., and the sponge Haliclona sp. The bacterial species Vibrio alginolyticus, Vibrio sp. 4, an unidentified α-Proteobacterium, Vibrio sp. 7, Pseudoalteromonas sp. 2, and Pseudoalteromonas sp. 4 were found to suppress the larval settlement of the polychaete Hydroides elegans (Haswell, 1883) and the bryozoan Bugula neritina (Linnaeus, 1758). Aqueous extracts of five bacteria (all those named above except Pseudoalteromonas sp. 2) prevented larval settlement. Bacteria V. alginolyticus, Vibrio sp. 4, and an unidentified α-Proteobacterium were first discovered to produce high-molecular substances (>100 kDa) preventing larval settlement. Their activity was inhibited by amylase treatment, while trypsin and papain did not influence their activity. The data obtained proved that bacteria from the surface of the number of marine organisms excrete water-soluble sacchariferous compounds preventing larval settlement.