Combined characterization of a new member of Marivita cryptomonadis strain LZ-15-2 isolated from cultivable phycosphere microbiota of highly toxic HAB dinoflagellate Alexandrium catenella LZT09.

During our conveying the microbial structures of phycosphere microbiota (PM) derived from diverse marine harmful algal bloom (HAB) dinoflagellates, a new rod-sharped, white-colored cultivable bacterial strain, designated as LZ-15-2, was isolated from the PM of highly toxic Alexandrium catenella LZT09. Phylogenetic analysis of 16S rRNA gene sequence indicated that strain LZ-15-2 belonged to the genus Marivita within the family Rhodobacteraceae, and demonstrated the highest gene similarity of 99.2% to M. cryptomonadis CL-SK44T, and less than 98.65% with other type strains of Marivita. Phylogenomic calculations on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the new isolate and M. cryptomonadis CL-SK44T were 99.86% and 99.88%, respectively. Genomic comparison of strain LZ-15-2 with available genomes of Marivita species further verified its taxonomic position within the genus of Marivita. Moreover, comparative genomics analysis showed a proximal similarity of strain LZ-15-2 with M. cryptomonadis CL-SK44T, and it also revealed an open pan-genome status based on constructed gene accumulation curves among Marivita members with 9,361 and 1,712 genes for the pan- and core-genome analysis, respectively. Based on combined polyphasic taxonomic characteristics, strain LZ-15-2 represents a new member of M. cryptomonadis, and proposed as a potential candidate for further exploration of the detailed mechanisms governing the dynamic cross-kingdom algae-bacteria interactions (ABI) between PM and their algal host LZT09.

Enhanced 2-keto-L-gulonic acid production by a mixed culture of Ketogulonicigenium vulgare and Bacillus megaterium using three-stage temperature control strategy.

As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32 °C and 29 °C, respectively, while the optimum temperature for 2-KLG production was 35 °C. We developed a TSTC process: the temperature was kept at 32 °C during the first 16 h of fermentation, then decreased to 29 °C for the following 14 h, and maintained at 35 °C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19 g/L/h and 92.91 ± 1.02 g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29 °C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36 h, 17.14% at 44 h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.

Bioactive Potential of Extracts of Labrenzia aggregata Strain USBA 371, a Halophilic Bacterium Isolated from a Terrestrial Source.

Previous studies revealed the potential of Labrenzia aggregata USBA 371 to produce cytotoxic metabolites. This study explores its metabolic diversity and compounds involved in its cytotoxic activity. Extracts from the extracellular fraction of strain USBA 371 showed high levels of cytotoxic activity associated with the production of diketopiperazines (DKPs). We purified two compounds and a mixture of two other compounds from this fraction. Their structures were characterized by 1D and 2D nuclear magnetic resonance (NMR). The purified compounds were evaluated for additional cytotoxic activities. Compound 1 (cyclo (l-Pro-l-Tyr)) showed cytotoxicity to the following cancer cell lines: breast cancer 4T1 (IC50 57.09 ± 2.11 µM), 4T1H17 (IC50 40.38 ± 1.94), MCF-7 (IC50 87.74 ± 2.32 µM), murine melanoma B16 (IC50 80.87 ± 3.67), human uterus sarcoma MES-SA/Dx5 P-pg (-) (IC50 291.32 ± 5.64) and MES-SA/Dx5 P-pg (+) (IC50 225.28 ± 1.23), and murine colon MCA 38 (IC50 29.85 ± 1.55). In order to elucidate the biosynthetic route of the production of DKPs and other secondary metabolites, we sequenced the genome of L. aggregata USBA 371. We found no evidence for biosynthetic pathways associated with cyclodipeptide synthases (CDPSs) or non-ribosomal peptides (NRPS), but based on proteogenomic analysis we suggest that they are produced by proteolytic enzymes. This is the first report in which the cytotoxic effect of cyclo (l-Pro-l-Tyr) produced by an organism of the genus Labrenzia has been evaluated against several cancer cell lines.

Characterization of a novel thermostable (S)-amine-transaminase from an Antarctic moderately-thermophilic bacterium Albidovulum sp. SLM16.

Amine-transaminases (ATAs) are enzymes that catalyze the reversible transfer of an amino group between primary amines and carbonyl compounds. They have been widely studied in the last decades for their application in stereoselective synthesis of chiral amines, which are one of the most valuable building blocks in pharmaceuticals manufacturing. Their excellent enantioselectivity, use of low-cost substrates and no need for external cofactors has turned these enzymes into a promising alternative to the chemical synthesis of chiral amines. Nevertheless, its application at industrial scale remains limited mainly because most of the available ATAs are scarcely tolerant to harsh reaction conditions such as high temperatures and presence of organic solvents. In this work, a novel (S)-ATA was discovered in a thermophilic bacterium, Albidovulum sp. SLM16, isolated from a geothermal Antarctic environmental sample, more specifically from a shoreline fumarole in Deception Island. The transaminase-coding gene was identified in the genome of the microorganism, cloned and overexpressed in Escherichia coli for biochemical characterization. The activity of the recombinant ATA was optimal at 65 °C and pH 9.5. Molecular mass estimates suggest a 75 kDa homodimeric structure. The enzyme turned out to be highly thermostable, maintaining 80% of its specific activity after 5 days of incubation at 50 °C. These results indicate that ATA_SLM16 is an excellent candidate for potential applications in biocatalytic synthesis. To the best of our knowledge, this would be the first report of the characterization of a thermostable (S)-ATA discovered by means of in vivo screening of thermophilic microorganisms.

Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica.

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.

Isolation and partial characterization of a new moderate thermophilic Albidovulum sp. SLM16 with transaminase activity from Deception Island, Antarctica

BACKGROUND: A moderately thermophilic, slightly halophilic, aerobic, Gram-stain negative, bacterial strain, SLM16, was isolated from a mixed of seawater-sand-sediment sample collected from a coastal fumarole located in Whalers Bay, Deception Island, Antarctica. The aim was to screen for thermophilic microorganisms able to degrade primary amines and search for amine transaminase activity for potential industrial application. RESULTS: Identification and partial characterization of the microorganism SLM16 were carried out by means of morphological, physiological and biochemical tests along with molecular methods. Cells of strain SLM16 were non-motile irregular rods of 1.5-2.5 µm long and 0.3-0.45 µm wide. Growth occurred in the presence of 0.5-5.5% NaCl within temperature range of 35-55 °C and pH range of 5.5-9.5, respectively. The DNA G+C composition, estimated from ftsY gene, was 66% mol. Phylogenetic analysis using de 16S rRNA gene sequence showed that strain SLM16 belongs to the marine bacterial genus Albidovulum. CONCLUSION: Strain SLM16 is a moderate thermophilic Gram negative microorganisms which belongs to the marine bacterial genus Albidovulum and is closely related to Albidovulum inexpectatum species based on phylogenetic analysis. Additionally, amine-transaminase activity towards the arylaliphatic amine α-methylbenzylamine was detected.

Metabolic versatility of a novel N2 -fixing Alphaproteobacterium isolated from a marine oxygen minimum zone.

The N2 -fixing (diazotrophic) community in marine ecosystems is dominated by non-cyanobacterial microorganisms. Yet, very little is known about their identity, function and ecological relevance due to a lack of cultured representatives. Here we report a novel heterotrophic diazotroph isolated from the oxygen minimum zone (OMZ) off Peru. The new species belongs to the genus Sagittula (Rhodobacteraceae, Alphaproteobacteria) and its capability to fix N2 was confirmed in laboratory experiments. Genome sequencing revealed that it is a strict heterotroph with a high versatility in substrate utilization and energy acquisition mechanisms. Pathways for sulfide oxidation and nitrite reduction to nitrous oxide are encoded in the genome and might explain the presence throughout the Peruvian OMZ. The genome further indicates that this novel organism could be in direct interaction with other microbes or particles. NanoSIMS analyses were used to compare the metabolic potential of S. castanea with single-cell activity in situ; however, N2 fixation by this diazotroph could not be detected at the isolation site. While the biogeochemical impact of S. castanea is yet to be resolved, its abundance and widespread distribution suggests that its potential to contribute to the marine N input could be significant at a larger geographical scale.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.(AU)

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production

Abstract A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

A plate method for rapid screening of Ketogulonicigenium vulgare mutants for enhanced 2-keto-l-gulonic acid production.

A new plate method was developed for rapid screening of Ketogulonicigenium vulgare mutants overproducing 2-keto-l-gulonic acid (2-KLG). The screening methodology took the advantage of the acidity caused by 2-KLG, which changes the color of bromothymol blue (pH indicator) from blue to yellow. Using the proposed method, a mutant, K. vulgare 65, was selected from 20,000 colonies produced by a strain subjected to spaceflight mutagenesis. When co-cultured with Bacillus megaterium 2980 in 20-L fermenters, K. vulgare 65 showed a high conversion rate (94.45%) of l-sorbose to 2-KLG. In contrast to the traditional screening method, this one significantly improved the frequency of obtaining positive mutants. The proposed plate screening method is cost-effective and easy to run and is thus useful for the isolation and screening of K. vulgare mutants overproducing 2-KLG.

Streptomyces effect on the bacterial microbiota associated to Crassostrea sikamea oyster.

AIM: To determine the composition and diversity of the microbiota associated to Crassostrea sikamea treated during 30 days with Streptomyces strains N7 and RL8. METHODS AND RESULTS: DNA was extracted from oysters followed by 16S rRNA gene amplification and pyrosequencing. The highest and lowest species diversity richness was observed in the initial and final control group, whereas Streptomyces-treated oysters exhibited intermediate values. Proteobacteria was the most abundant phylum (81·4-95·1%), followed by Bacteroidetes, Actinobacteria and Firmicutes. The genera Anderseniella, Oceanicola, Roseovarius, Ruegeria, Sulfitobacter, Granulosicoccus and Marinicella encompassed the core microbiota of all experimental groups. The genus Bacteriovorax was detected in all groups except in the final control and the depurated N7, whereas Vibrio remained undetected in all Streptomyces-treated groups. RL8 was the only group that harboured the genus Streptomyces in its microbiota. Principal component analysis showed that Streptomyces strains significantly changed oyster microbiota with respect to the initial and final control. CONCLUSIONS: Crassostrea sikamea treated with Streptomyces showed high species diversity and a microbiota composition shift, characterized by keeping the predator genus Bacteriovorax and decreasing the pathogenic Vibrio. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first culture-independent study showing the effect of Streptomyces over the oyster microbiota. It also sheds light about the potential use of Streptomyces to improve mollusc health and safety for consumers after the depuration process.

The discovery of stromatolites developing at 3570 m above sea level in a high-altitude volcanic lake Socompa, Argentinean Andes.

We describe stromatolites forming at an altitude of 3570 m at the shore of a volcanic lake Socompa, Argentinean Andes. The water at the site of stromatolites formation is alkaline, hypersaline, rich in inorganic nutrients, very rich in arsenic, and warm (20-24°C) due to a hydrothermal input. The stromatolites do not lithify, but form broad, rounded and low-domed bioherms dominated by diatom frustules and aragonite micro-crystals agglutinated by extracellular substances. In comparison to other modern stromatolites, they harbour an atypical microbial community characterized by highly abundant representatives of Deinococcus-Thermus, Rhodobacteraceae, Desulfobacterales and Spirochaetes. Additionally, a high proportion of the sequences that could not be classified at phylum level showed less than 80% identity to the best hit in the NCBI database, suggesting the presence of novel distant lineages. The primary production in the stromatolites is generally high and likely dominated by Microcoleus sp. Through negative phototaxis, the location of these cyanobacteria in the stromatolites is controlled by UV light, which greatly influences their photosynthetic activity. Diatoms, dominated by Amphora sp., are abundant in the anoxic, sulfidic and essentially dark parts of the stromatolites. Although their origin in the stromatolites is unclear, they are possibly an important source of anaerobically degraded organic matter that induces in situ aragonite precipitation. To the best of our knowledge, this is so far the highest altitude with documented actively forming stromatolites. Their generally rich, diverse and to a large extent novel microbial community likely harbours valuable genetic and proteomic reserves, and thus deserves active protection. Furthermore, since the stromatolites flourish in an environment characterized by a multitude of extremes, including high exposure to UV radiation, they can be an excellent model system for studying microbial adaptations under conditions that, at least in part, resemble those during the early phase of life evolution on Earth.

Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil.

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with alpha-Proteobacteria (Pseudovibrio sp.) and four with gamma-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Predominance of Roseobacter, Sulfitobacter, Glaciecola and Psychrobacter in seawater collected off Ushuaia, Argentina, Sub-Antarctica.

Bacterial diversity in sub-Antarctic seawater, collected off Ushuaia, Argentina, was examined using a culture independent approach. The composition of the 16S rRNA gene libraries from seawater and seawater contaminated with the water soluble fraction of crude oil was statistically different (P value 0.001). In both libraries, clones representing the Alphaproteobacteria, Gammaproteobacteria, the Cytophaga-Flavobacterium-Bacteroidetes group and unculturable bacteria were dominant. Clones associated with the genera Roseobacter, Sulfitobacter, Staleya, Glaciecola, Colwellia, Marinomonas, Cytophaga and Cellulophaga were common to both the libraries. However, clones associated with Psychrobacter, Arcobacter, Formosa algae, Polaribacter, Ulvibacter and Tenacibaculum were found only in seawater contaminated with hydrocarbons (Table 1). Further, the percentage of clones of Roseobacter, Sulfitobacter and Glaceicola was high in seawater (43%, 90% and 12% respectively) compared to seawater contaminated with hydrocarbons (35%, 4% and 9% respectively). One of the clones F2C63 showed 100% similarity with Marinomonas ushuaiensis a bacterium identified by us from the same site.

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella.

The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high-nutrient media, released algal-lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial-derived lytic activities are expressed only in the presence of high-nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.