The domestication of the probiotic bacterium Lactobacillus acidophilus

Lactobacillus acidophilus is a Gram-positive lactic acid bacterium that has had widespread historical use in the dairy industry and more recently as a probiotic. Although L. acidophilus has been designated as safe for human consumption, increasing commercial regulation and clinical demands for probiotic validation has resulted in a need to understand its genetic diversity. By drawing on large, well-characterised collections of lactic acid bacteria, we examined L. acidophilus isolates spanning 92 years and including multiple strains in current commercial use. Analysis of the whole genome sequence data set (34 isolate genomes) demonstrated L. acidophilus was a low diversity, monophyletic species with commercial isolates essentially identical at the sequence level. Our results indicate that commercial use has domesticated L. acidophilus with genetically stable, invariant strains being consumed globally by the human population

Biotechnological applications of functional metagenomics in the food and pharmaceutical industries

peer-reviewedMicroorganisms are found throughout nature, thriving in a vast range of environmental conditions. The majority of them are unculturable or difficult to culture by traditional methods. Metagenomics enables the study of all microorganisms, regardless of whether they can be cultured or not, through the analysis of genomic data obtained directly from an environmental sample, providing knowledge of the species present, and allowing the extraction of information regarding the functionality of microbial communities in their natural habitat. Function-based screenings, following the cloning and expression of metagenomic DNA in a heterologous host, can be applied to the discovery of novel proteins of industrial interest encoded by the genes of previously inaccessible microorganisms. Functional metagenomics has considerable potential in the food and pharmaceutical industries, where it can, for instance, aid (i) the identification of enzymes with desirable technological properties, capable of catalyzing novel reactions or replacing existing chemically synthesized catalysts which may be difficult or expensive to produce, and able to work under a wide range of environmental conditions encountered in food and pharmaceutical processing cycles including extreme conditions of temperature, pH, osmolarity, etc; (ii) the discovery of novel bioactives including antimicrobials active against microorganisms of concern both in food and medical settings; (iii) the investigation of industrial and societal issues such as antibiotic resistance development. This review article summarizes the state-of-the-art functional metagenomic methods available and discusses the potential of functional metagenomic approaches to mine as yet unexplored environments to discover novel genes with biotechnological application in the food and pharmaceutical industries.Science Foundation Ireland(SFI)Grant Number 13/SIRG/215

Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.España, MINECO CGL2017-83385-

The P3 platform: an approach and software system for developing diagrammatic model-based methods in design research

Many issues in design and design management have been explored by building models which capture the relationships between different aspects of the problem at hand. These models require computer support to construct and analyse. However, appropriate modelling tools can be time-consuming to develop in a research environment. Reflecting upon five design research projects, this paper proposes that such projects can be facilitated by recognising the iterative and tightly-coupled nature of research and tool development, and by attempting to minimise the effort of solution prototyping within this process. Our approach is enabled by a software platform which can be rapidly configured to implement many conceivable modelling approaches. This configurability is complemented by an emerging library of modelling and analysis approaches tailored to explore design process systems. The platform-based approach enables any mix of modelling concepts to be easily created. We propose it could thus help researchers to explore a wide range of questions without being constrained to existing conventions for modelling – or for model integration

Design and evaluation of 16S rRNA sequence based oligonucleotide probes for the detection and quantification of Comamonas testosteroni in mixed microbial communities

Background The β-proteobacterial species Comamonas testosteroni is capable of biotransformation and also biodegradation of a range of chemical compounds and thus potentially useful in chemical manufacturing and bioremediation. The ability to detect and quantify members of this species in mixed microbial communities thus may be desirable. Results We have designed an oligonucleotide probe for use in fluorescent in situ hybridization (FISH) and two pairs of PCR primers targeting a C. testosteroni subgroup. The FISH probe and one of the PCR primer pairs are suitable for quantification of C. testosteroni in mixed microbial communities using FISH followed by quantitative image analysis or real-time quantitative PCR, respectively. This has been shown by analysis of samples from an enrichment of activated sludge on testosterone resulting in an increase in abundance and finally isolation of C. testosteroni. Additionally, we have successfully used quantitative PCR to follow the C. testosteroni abundance during a laboratory scale wastewater bioaugmentation experiment. Conclusion The oligonucleotides presented here provide a useful tool to study C. testosteroni population dynamics in mixed microbial communities

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)

Isolierung und Charakterisierung von Myxobakterien und anderen seltenen Meeresbakterien und deren Multilocus-Sequenzanalyse der indonesischen Biodiversität

Indonesia is a country with high potential in biodiversity exploration, especially for microorganisms. This study used samples collected from the islands of Java, Sulawesi, and Bali under the Germany-Indonesia Antiinfectives Cooperation (GINAICO) project. We have isolated 59 strains of gliding bacteria, 98% of which are myxobacteria while the remaining 2% is made up of other gliding bacteria. In this research, we have identified a rare myxobacterium and a novel non-myxobacterial strain from our selected samples. Finally, we confirmed the effectiveness of housekeeping genes as a core method in analysing the degree of relatedness between different strains. The strain Soce1964KM was found to be closest to the rare genus Sorangium and strain 1932KM was identified as a novel genus in the family Flammeovirgaceae. Both results are according to the analysis of its 16S rRNA gene sequence and whole-genome sequencing analysis for strain 1932KM. We also performed screening for antiinfectives against Escherichia coli, Candida albicans and Staphylococcus aureus. Strain Soce1964KM has the highest activity against Staphylococcus aureus and Candida albicans. Analysis of the methanol extract of Soce1964KM through the chromatogram from High-Pressure Liquid Chromatography (HPLC) and High-Resolution Electrospray Ionisation Mass Spectrometry (HRESIMS)–in addition to the database in Myxobase–showed that the extract contains a known compound, namely Disorazol. We found 1932KM to be a novel strain after an in-depth examination of this strain’s phenotype and genotype as part of a polyphasic taxonomy analysis. Also, we conducted a gene cluster analysis and bioassay against pathogenic bacteria and statistical analysis of 16S rRNA gene sequences from this strain with the statistical tool PAST. Bioinformatic tools, such as Prokka, RAST, AntiSmash, NP.searcher, PubChem, SMART and Molinspiration, were used for an in-depth analysis of the whole-genome sequence. Strain 1932KM has also obtained a deposition number from the Indonesian Culture Collections, namely InaCC B1242 with the proposed name of Balibacter flavus gen.nov.sp.nov. Meanwhile, we used the housekeeping genes pgm, pyrG and rpoB to analyse the relatedness between the genera Corallococcus and Myxococcus. These housekeeping genes were used in this study as the core to analyse the similarities between the isolated strains and the type strains. The analysis involved the reconstruction of a phylogenetic tree and its polymorphisms. Bioinformatics tools supported these analyses. These analyses showed that the method is suitable for preliminary analysis in the search for novel species and/or subspecies.Indonesien ist ein Land mit hohem Potenzial für die Erforschung der biologischen Vielfalt, insbesondere für Mikroorganismen. In dieser Studie wurden Proben verwendet, die auf den Inseln Java, Sulawesi, und Bali im Rahmen des Projekts GINAICO (Deutschland-Indonesien Antiinfectives Cooperation) gesammelt wurden. Wir haben 59 Stämme gleitender Bakterien isoliert, von denen 98% Myxobakterien sind, während die restlichen 2% aus anderen gleitenden Bakterien bestehen. In dieser Studie haben wir aus unseren ausgewählten Proben ein seltenes Myxobakterium und einen neuartigen nicht-myxobakteriellen Stamm identifiziert. Schließlich bestätigten wir die Wirksamkeit von Housekeeping-Genen als Kernmethode bei der Analyse des Grads der Verwandtschaft zwischen verschiedenen Stämmen. Es wurde festgestellt, dass der Stamm Soce1964KM der seltenen Gattung Sorangium am nächsten kommt, und der Stamm 1932KM wurde als neuartige Gattung in der Familie der Flammeovirgaceae identifiziert. Beide Ergebnisse entsprechen der Analyse seiner 16S-rRNA-Gensequenz und der Gesamtgenomsequenzierungsanalyse für den Stamm 1932KM. Wir führten auch ein Screening auf Antiinfektiva gegen Escherichia coli, Candida albicans und Staphylococcus aureus durch. Der Stamm Soce1964KM hat die höchste Aktivität gegen Staphylococcus aureus und Candida albicans. Die Analyse des Methanolextrakts von Soce1964KM mittels Chromatogramms aus Hochdruckflüssigchromatographie (HPLC) und hochauflösender Elektrospray-Ionisationsmassenspektrometrie (HRESIMS) - zusätzlich zur Datenbank in Myxobase - zeigte, dass der Extrakt eine bekannte Verbindung enthält, nämlich Disorazol. Nach einer eingehenden Untersuchung des Phänotyps und Genotyps dieses Stammes im Rahmen einer mehrphasigen Taxonomieanalyse stellten wir fest, dass 1932KM ein neuartiger Stamm ist. Außerdem führten wir mit dem statistischen Tool PAST eine Genclusteranalyse und einen Bioassay gegen pathogene Bakterien sowie eine statistische Analyse der 16S-rRNA-Gensequenzen dieses Stammes durch. Bioinformatische Werkzeuge wie Prokka, RAST, AntiSmash, NP.searcher, PubChem, SMART und Molinspiration wurden für eine eingehende Analyse der gesamten Genomsequenz verwendet. Der Stamm 1932KM hat auch eine Hinterlegungsnummer aus den indonesischen Kultursammlungen erhalten, nämlich InaCC B1242 mit dem vorgeschlagenen Namen Balibacter flavus gen.nov.sp.nov. In der Zwischenzeit haben wir die Housekeeping-Gene pgm, pyrG und rpoB verwendet, um die Verwandtschaft zwischen den Gattungen Corallococcus und Myxococcus zu analysieren. Diese Housekeeping-Gene wurden in dieser Studie als Kern verwendet, um die Ähnlichkeiten zwischen den isolierten Stämmen und den Typstämmen zu analysieren. Die Analyse umfasste die Rekonstruktion eines phylogenetischen Baums und seiner Polymorphismen. Bioinformatik-Tools unterstützten diese Analysen. Diese Untersuchungen zeigten, dass die Methode für eine vorläufige Analyse bei der Suche nach neuen Arten und / oder Unterarten geeignet ist

Actinomycete biodiversity assessed by culture-based and metagenomic investigations of three distinct samples in Cape Town, South Africa

All of the samples used for actinobacterial isolation were subjected to a culture-independent (metagenomic) study. The results provided an explanation for why no actinomycetes were found in the aquatic samples, as all of the sequenced clones were shown to be most closely related to uncultured bacteria. In the terrestrial sample, a total of 120 clones were obtained and all were sent for sequencing

Screening environmental actinobacteria for antimycobacterial antibiotics and characterisation of Kribbella stellenboschensis sp. nov

Soil was collected from a compost heap in a Mowbray suburban garden and a compost heap in a Plumstead suburban garden. The soil and ‘worm tea’ of a vermiculture farm from the same Mowbray suburban garden were also sampled. Using four different types of media (7H9, CZ, ISP2 and GOT) 135 isolates were putatively identified as actinobacteria based on colony morphology. These isolates were screened for antimycobacterial activity against the test bacterium Mycobacterium aurum A+. A Kribbella strain, isolated and identified by an intern in the lab, and a Micromonospora strain, isolated and identified during the authors Honours project, were also screened for antimycobacterial activity. Sixty-four (64) actinobacterial isolates displayed moderate antibiotic activity or higher (ZOI >1001 mm2 ) based on the standard overlay method. Kribbella strain SK5 displayed very strong antimycobacterial activity (3309 mm2 ). Forty (40) of the actinobacterial strains that exhibited moderate/strong/very strong antimycobacterial activity and/or had interesting morphological features were selected for genus identification via a standard nucleotide-nucleotide blastn analysis of their 16S rRNA gene sequences. Thirty-one (31) strains were identified as Streptomyces species, six strains were identified as Micromonospora species, one strain was identified as a Nocardia species, one strain was identified as a Kitasatospora species, and one strain was identified as a member of the genus Tsukamurella. These isolates were subjected to phylogenetic analysis using the partial 16S rRNA gene sequences. Based on analysis of the 16S rRNA gene sequences, Streptomyces strain PR10 was found to be the most interesting of the Streptomyces isolates and should be pursued as a novel species (99.7% sequence similarity to the top blastn hit and less than 98.8% sequence similarity from the third blastn hit onwards). Further analysis of the gyrase subunit B (gyrB) gene sequence of the Kitasatospora isolate (strain PR3) revealed that the isolate is more closely related to members of the genus Streptomyces. Further evidence to support the assignment of strain PR3 to the genus Streptomyces (rather than Kitasatospora) is that it has two Streptomyces-specific gyrB gene indels signatures. Tsukamurella strain G4 was noted for characterisation as a novel species. The potential for seven isolates to produce ansamycin, glycopeptide, non-ribosomal peptide, and/or TypeII polyketide antibiotics was determined by detection of antibiotic biosynthetic gene clusters using PCR. Strain M27 demonstrated the potential to produce all the aforementioned antibiotics. Strain Y10 demonstrated the potential to produce a non-ribosomal peptide antibiotic. Strains PR10, PR28, PR47 and UK1 demonstrated the potential to produce Type-II polyketide and non-ribosomal peptide antibiotics. The PCR products were sequenced and analysed via blastn to compare them to the known antibiotic biosynthetic gene sequences in the GenBank database. The non-ribosomal peptide synthetase (NRPS) A domain sequences were analysed using the NRPSpredictor2 software to identify the A domain substrate specificity Solvent extraction was done on the broth cultures of Streptomyces strains PR3, UK1 and Y30 and Kribbella strain SK5 to isolate the antimycobacterial compounds. It was found that the cell mass extract of the three Streptomyces isolates had active compounds against M. aurum A+. The culture broth extract of the Kribbella isolate was found to have an active compound against M. aurum A+ and Staphylococcus aureus ATCC 25923. One-dimensional and two-dimensional TLC of the culture broth extract from strain SK5 revealed that a single compound was active against M. aurum A+ and S. aureus ATCC 25923. Nocardamine was purified from the culture broth extract of strain SK5 by Mr Kojo Acquah (PhD student, Department of Chemistry, University of Cape Town). In a side-by-side spot bioautography analysis of the purified nocardamine and the strain SK5 culture broth extract, it was found that the active compound in the culture broth extract was not nocardamine, because nocardamine only had activity against M. aurum A+ while the culture broth extract had activity against M. aurum A+ and S. aureus ATCC 25923. Using the polyphasic taxonomic approach, Kribbella strain SK5 was tentatively characterised as a novel species, for which the name Kribbella stellenboschensis sp. nov. is proposed. The closest phylogenetic relatives were identified as the type strains of Kribbella aluminosa, Kribbella karoonensis, Kribbella pittospori, Kribbella shriazensis, ‘Kribbella sindirgiensis’ and ‘Kribbella soli’. Genetic distances of 0.030 and 0.016 were calculated for ‘K. soli’ and ‘Kribbella sindirgiensis’, respectively, for the concatenated gene sequence of five housekeeping genes (gyrB, rpoB, recA, relA, and atpD). Thus, DNA-DNA hybridisation (DDH) will need to be carried out to confirm that strain SK5 is a separate species. Phenotypic differences were observed between strain SK5 and all the type strains of the most closely related species. Chemotaxonomically, strain SK5 possessed the key characters definitive of the genus Kribbella: i) MK9(H4) as the major menaquinone; ii) LL-diaminopimelic acid as the diagnostic diamino acid; iii) anteiso-C15:0 and iso-C16:0 as the major fatty acids (>10%); and iv) phosphatidylcholine in the polar lipid profile