Analysis of actinobacterial biodiversity in reservoir sediment and cave soil and screening of isolates for antimycobacterial activity

A total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL)

Ecological Strategies Behind the Selection of Cultivable Actinomycete Strains from the Yucatan Peninsula for the Discovery of Secondary Metabolites with Antibiotic Activity

The quest for novel natural products has recently focused on the marine environment as a source for novel microorganisms. Although isolation of marine-derived actinomycete strains is now common, understanding their distribution in the oceans and their adaptation to this environment can be helpful in the selection of isolates for further novel secondary metabolite discovery. This study explores the taxonomic diversity of marine-derived actinomycetes from distinct environments in the coastal areas of the Yucatan Peninsula and their adaptation to the marine environment as a first step towards novel natural product discovery. The use of simple ecological principles, for example, phylogenetic relatedness to previously characterized actinomycetes or seawater requirements for growth, to recognize isolates with adaptations to the ocean in an effort to select for marine-derived actinomycete to be used for further chemical studies. Marine microbial environments are an important source of novel bioactive natural products and, together with methods such as genome mining for detection of strains with biotechnological potential, ecological strategies can bring useful insights in the selection and identification of marine-derived actinomycetes for novel natural product discovery

Digitizing mass spectrometry data to explore the chemical diversity and distribution of marine cyanobacteria and algae.

Natural product screening programs have uncovered molecules from diverse natural sources with various biological activities and unique structures. However, much is yet underexplored and additional information is hidden in these exceptional collections. We applied untargeted mass spectrometry approaches to capture the chemical space and dispersal patterns of metabolites from an in-house library of marine cyanobacterial and algal collections. Remarkably, 86% of the metabolomics signals detected were not found in other available datasets of similar nature, supporting the hypothesis that marine cyanobacteria and algae possess distinctive metabolomes. The data were plotted onto a world map representing eight major sampling sites, and revealed potential geographic locations with high chemical diversity. We demonstrate the use of these inventories as a tool to explore the diversity and distribution of natural products. Finally, we utilized this tool to guide the isolation of a new cyclic lipopeptide, yuvalamide A, from a marine cyanobacterium

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus andCortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatantsof termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensionalgel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.Fil: Ben Guerrero, Emiliano. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Arneodo Larochette, Joel Demián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Bombarda Campanha, Raquel. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Oliveira, Patrícia Abrão de. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Labate, Mônica T. Veneziano. Universidade de Sao Paulo; BrasilFil: Cataldi, Thaís Regiani. Universidade de Sao Paulo; BrasilFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Labate, Carlos A.. Universidade de Sao Paulo; BrasilFil: Rodrigues, Clenilson Martins. Ministerio da Agricultura Pecuaria e Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; BrasilFil: Talia, Paola Monica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Actinobacteria from arctic and atlantic deep-sea sediments—biodiversity and bioactive potential

The deep-sea covers over 70% of the Earth’s surface and harbors predominantly uncharacterized bacterial communities. Actinobacteria are the major prokaryotic source of bioactive natural products that find their way into drug discovery programs, and the deep-sea is a promising source of biotechnologically relevant actinobacteria. Previous studies on actinobacteria in deep-sea sediments were either regionally restricted or did not combine a community characterization with the analysis of their bioactive potential. Here we characterized the actinobacterial communities of upper layers of deep-sea sediments from the Arctic and the Atlantic (Azores and Madeira) ocean basins, employing 16S rRNA metabarcoding, and studied the biosynthetic potential of cultivable actinobacteria retrieved from those samples. Metabarcoding analysis showed that the actinobacterial composition varied between the sampled regions, with higher abundance in the Arctic samples but higher diversity in the Atlantic ones. Twenty actinobacterial genera were detected using metabarcoding, as a culture-independent method, while culture-dependent methods only allowed the identification of nine genera. Isolation of actinobacteria resulted on the retrieval of 44 isolates, mainly associated with Brachybacterium, Microbacterium, and Brevibacterium genera. Some of these isolates were only identified on a specific sampled region. Chemical extracts of the actinobacterial isolates were subsequently screened for their antimicrobial, anticancer and anti-inflammatory activities. Extracts from two Streptomyces strains demonstrated activity against Candida albicans. Additionally, eight extracts (obtained from Brachybacterium, Brevibacterium, Microbacterium, Rhodococcus, and Streptomyces isolates) showed significant activity against at least one of the tested cancer cell lines (HepG2 and T-47D). Furthermore, 15 actinobacterial extracts showed anti-inflammatory potential in the RAW 264.4 cell model assay, with no concomitant cytotoxic response. Dereplication and molecular networking analysis of the bioactive actinobacterial extracts showed the presence of some metabolites associated with known natural products, but one of the analyzed clusters did not show any match with the natural products described as responsible for these bioactivities. Overall, we were able to recover taxonomically diverse actinobacteria with different bioactivities from the studied deep-sea samples. The conjugation of culture-dependent and -independent methods allows a better understanding of the actinobacterial diversity of deep-sea environments, which is important for the optimization of approaches to obtain novel chemically-rich isolates.info:eu-repo/semantics/publishedVersio

Quantifying microbial utilization of petroleum hydrocarbons in salt-marsh sediments using the ^(13)C content of bacterial rRNA

Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we take a whole-community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon-isotopic abundance between a salt marsh ecosystem supported by the ^(13)C-enriched C4 grass, Spartina alterniflora, and the ^(13)C-depleted composition of petroleum to monitor changes in the ^(13)C content of biomass. Magnetic-bead capture methods for the selective recovery of bacterial RNA were used to monitor the ^(13)C content of bacterial biomass during a two-week experiment. The data show that by the end of the experiment, up to 26% of bacterial biomass derived from consumption of the freshly-spilled oil. The results contrast with the inertness of a nearby relict spill, which occurred in 1969 in West Falmouth, MA. Sequences of 16S rRNA genes from our experimental samples also were consistent with previous reports suggesting the importance of {gamma}- and {delta}-Proteobacteria and Firmicutes in the remineralization of hydrocarbons. The magnetic-bead capture approach makes it possible to quantify uptake of petroleum hydrocarbons by microbes in-situ. Although employed here at the Domain level, RNA-capture procedures can be highly specific. The same strategy could be used with genus-level specificity, something which is not currently possible using the ^(13)C content of biomarker lipids

The Algal Polysaccharide Ulvan and Carotenoid Astaxanthin Both Positively Modulate Gut Microbiota in Mice

The intestinal microbial community (microbiota) is dynamic and variable amongst individuals and plays an essential part in gut health and homeostasis. Dietary components can modulate the structure of the gut microbiota. In recent years, substantial efforts have been made to find novel dietary components with positive effects on the gut microbial community structure. Natural algal polysaccharides and carotenoids have been reported to possess various functions of biological relevance and their impact on the gut microbiota is currently a topic of interest. This study, therefore, reports the effect of the sulfated polysaccharide ulvan and the carotenoid astaxanthin extracted and purified from the aquacultured marine green macroalgae Ulva ohnoi and freshwater green microalgae Haematococcus pluvialis, respectively, on the temporal development of the murine gut microbiota. Significant changes with the increase in the bacterial classes Bacteroidia, Bacilli, Clostridia, and Verrucomicrobia were observed after feeding the mice with ulvan and astaxanthin. Duration of the treatments had a more substantial effect on the bacterial community structure than the type of treatment. Our findings highlight the potential of ulvan and astaxanthin to mediate aspects of host-microbe symbiosis in the gut, and if incorporated into the diet, these could assist positively in improving disease conditions associated with gut health

Investigating the effects of biochar and activated carbon amendment on the microbial community response in a volatile petroleum hydrocarbon - contaminated gravelly sand

PhD ThesisLaboratory batch and long-term column experiments were conducted to investigate the effects of wood-chip biochar and coal-derived activated carbon amendment on the microbiology of a volatile petroleum hydrocarbon (VPH) - contaminated gravelly sand. First, a stable isotope-labelled mono aromatic compound – toluene was used as a model VPH to gain insights into the mineralization of VPHs by soil microorganisms in the presence and absence of biochar or activated carbon. The biodegradation of a mixture of 12 VPHs was subsequently monitored in batch microcosms over a duration of 6-19 days by measuring headspace CO2 concentration. Further analysis was carried out by characterizing changes in the soil microbial community composition using next generation sequencing techniques – 454 pyrosequencing and Ion torrent sequencing. Increases in the levels of headspace CO2 in contaminated soil batches as compared to live and abiotic controls to which no VPHs were added indicated a stimulation of microbial activity in the batches through VPH addition. By fitting a maximum specific growth rate of 0.6 h-1 (in line with published rates), it was possible to match model predictions of 45CO2 and 44CO2 concentrations with the experimentally determined data. Half-saturation constants of 4.06 x 103 mgL-1, 7.76 x 102 mgL-1 and 1.83 x 102 mgL-1 were predicted for soil, soil & BC and soil & AC respectively, much higher than values reported in the literature. Differences in the half-saturation constant suggests that sorbent amendment affects the microbial ecology, by making microorganisms which can utilize substrates at lower concentrations more competitive. Yield coefficients (g biomass-C relative to g (biomass-C + CO2-C)) compared more closely in the nutrient (N & P) amended soils ranging from 4.83±0.46 in soil and biochar to 7.86±0.72 in unamended soil, than in the batches without nutrients, 4.1±3.1 in soil & BC, 17.7±5.2 in soil and 13.7±4.6 in soil & AC. Sorbent amendment thus reduced yield coefficients, thereby slowing the growth of VPH degrading biomass. Microbial community structure analysis revealed an increase in the relative abundance ranking of members of the genera Pseudomonas, Pseudoxanthomonas, and Arenimonas by up to 32 folds and in the families Nocardioidaceae and Pseudomonadaceae by at least 32 folds in sorbent amended and unamended soil batches and columns compared to their initial soil conditions. Consequently, amending soils with 2% BC or AC changed the biokinetics of VPH degradation by rendering VPHs less bioavailable, but did not appear to have any detrimental effects on the VPH degrading bacteria both in the short- and long-term, and may serve as a sustainable, cost-effective approach for enhancing the natural attenuation of VPHs in soil, thus addressing the challenge of petroleum hydrocarbon contamination.The Petroleum Technology Development Fund (PTDF), Nigeri

Integrating Shotgun Metagenomics, 16s Rrna Gene Metabarcoding and Culture Approaches: A Better Outlook for Functional Profiling of a Pah-Contaminated Soil

Understanding bacterial diversity and function is critical for designing bioremediation strategies. This research aimed to assess chronically hydrocarbon contaminated soil bacterial diversity and their aromatic compound degradation (ACD) potential by integrating shotgun metagenomic, 16S rRNA gene metabarcoding and culture approaches. While soil metabarcoding showed dominance of Proteobacteria, metagenomics indicated that 99,5% of the sequences were taxonomically assigned to Streptomycetales order and that almost all genes related to ACD were assigned to the latter. To inspect other phyla contribution to ACD, a functional prediction was delved, and two culture approaches were used. PICRUSt2 revealed that ACD pathways were mostly found in Alphaproteobacteria, Actinobacteria and Gammaproteobacteria classes. An enrichment culture (r-EFP) was obtained with pyrene as sole carbon and energy source and a bacterial strain (S19P6), identified as a member of Mycolicibacterium genus, was isolated. Both cultures demonstrated the ability to degrade more than 90% of the supplemented pyrene after 21 days of incubation. 16S rRNA gene metabarcoding and shotgun metagenomics approaches in r-EFP indicated predominance of Proteobacteria Phylum and the presence of genes responsible for the degradation of ACD mostly assigned to the predominant phyla. Complementing different methodologies enable the recognition of the metabolic potential of soil Proteobacteria related to ACD.Fil: Festa, Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Granada, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Irazoqui, José Matías. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Cuadros Orellana, Sara. Universidad Catolica de Maule; ChileFil: Quevedo, Claudio. Universidad Catolica de Maule; ChileFil: Coppotelli, Bibiana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Morelli, Irma Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentin