Rich Organic Medium Research Articles

Small-molecule autocatalytic networks are universal metabolic fossils.

Life and the genetic code are self-referential and so are autocatalytic networks made of simpler, small molecules. Several origins of life theories postulate autocatalytic chemical networks preceding the primordial genetic code, yet demonstration with biochemical systems is lacking. Here, small-molecule reflexively autocatalytic food-generated networks (RAFs) ranging in size from 3 to 619 reactions were found in all of 6683 prokaryotic metabolic networks searched. The average maximum RAF size is 275 reactions for a rich organic medium and 93 for a medium with a single organic cofactor, NAD. In the rich medium, all universally essential metabolites are produced with the exception of glycerol-1-p (archaeal lipid precursor), phenylalanine, histidine and arginine. The 300 most common reactions, present in at least 2732 RAFs, are mostly involved in amino acid biosynthesis and the metabolism of carbon, 2-oxocarboxylic acid and purines. ATP and NAD are central in generating network complexity, and because ATP is also one of the monomers of RNA, autocatalytic networks producing redox and energy currencies are a strong candidate niche of the origin of a primordial information-processing system. The wide distribution of small-molecule autocatalytic networks indicates that molecular reproduction may be much more prevalent in the Universe than hitherto predicted. This article is part of the theme issue 'Emergent phenomena in complex physical and socio-technical systems: from cells to societies'.

Association of aerobic anoxygenic phototrophs and zebra mussels, Dreissena polymorpha, within the littoral zone of Lake Winnipeg

While nutrient loading has affected all levels of Lake Winnipeg’s ecology, its greatest influence has likely been on the microbial community. In addition to eutrophication, zebra mussels (Dreissena polymorpha) have recently invaded the ecosystem and threaten food web dynamics. Their filter-feeding predation and association with bacteria, specifically phototrophs, was investigated. A sampling trip to Lake Winnipeg in October 2017, focused on the isolation, enumeration, and identification of aerobic anoxygenic phototrophs in littoral water, sediment, and tissues of mussels. Gimli, Patricia, and Grand beaches, separated by >15 km across the South Basin, had similar bacterial counts when cultivated on rich organic, BG-11, purple non-sulphur, and K2TeO3-supplemented media. Culture-based enumeration on rich organic medium revealed 1.74% of heterotrophs from littoral waters were aerobic anoxygenic phototrophs, and represented 13.98% within sediments. In contrast, 0.48, 1.15, and 0.16% of cultured heterotrophs were aerobic anoxygenic phototrophs within zebra mussel gill, gut, and gonadal tissues, respectively. High-throughput sequencing of bacterial 16S V4 rDNA maintained similar trends in respective bivalve organs, where 0.22, 1.13, and 0.20% of total 16S genes belonged to these phototrophs. Several Sphingomonadaceae isolates were recovered from gut tissues, all with filamentous morphology large enough for predation. Bioaccumulation of metals was also studied in D. polymorpha. All tested associated aerobic anoxygenic phototrophs were capable of resisting the metalloid oxide tellurite. The consistent distribution of aerobic anoxygenic phototrophs within microbial communities across Lake Winnipeg, and their predominance in the gut tissues of zebra mussels suggested bacterial consumption by this invasive species.

Psychrotrophic violacein-producing bacteria isolated from Lake Winnipeg, Canada

Violet and purple bacterial pigmentations are uncommon when considering strict aerobes. Here we report discovery of two novel isolates, P102 and P117 from the freshwater Lake Winnipeg, each with violet to deep purple colony colouration. The relationship between pigment production and growth was investigated under different conditions, including a variety of carbon sources, pH, and temperatures. The violet compounds were purified using polar organic solvents, and then structurally characterized via mass spectrometry and nuclear magnetic resonance to be violacein and deoxyviolacein. Strain P117 produced higher concentrations of the darker pigment deoxyviolacein, which resulted in a deeper hue of violet than in cultures of P102. Strain P102, synthesized violacein maximally in liquid rich organic medium at pH 8 and 20 °C, and had 99.3% sequence similarity to the 16S rDNA from Janthinobacterium lividum. Strain P117, related to Massilia violaceinigra sharing 99.2% 16S rDNA sequence similarity, also produced violacein at similar optimal conditions, but developed higher concentrations of pigment at 15 °C. Culture-based methods found that violacein producers composed 0.001% or 0.023% CFU of environmental heterotrophic populations using BG-11 and potato-based media, respectively. Culture-independent high-throughput ribosomal 16S V4 sequencing of environmental DNA was used to detect Gram-negative species known for their production of violacein. Of all bacterial and archaeal sequences present during the fall in littoral waters and sediment of Lake Winnipeg, 5.5% and 6.3%, respectively, belonged to species that have similar pigment, demonstrating the scarcity of violacein producers in this aquatic freshwater system.

Erythrobacter nanhaiensis sp. nov., A Novel Member of the Genus Erythrobacter Isolated from the South China Sea.

A Gram-negative, motile, rod-shaped, aerobic bacterial strain with tufty polar flagella, JLT1363T, was isolated from the South China Sea. The bacteria formed yellow colonies on rich organic medium. The major cellular fatty acids present in JLT1363T were C18:1 ω7c and/or C18:1 ω6c (36.06%), C17:1 ω6c (17.04%), C14:0 2-OH (9.85%), and C16:0 (8.09%). The genome size was ~3.12Mbps with a G+C content of 64.9%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JLT1363T fell within the genus Erythrobacter. The 16S rRNA gene sequence similarity between strain JLT1363T and the type strains of Erythrobacter species ranged from 95.0% (with Erythromicrobium ramosum) to 98.7% (with Erythrobacter lutimaris). The Average Nucleotide Identity (ANI) value between genome sequences of strain JLT1363T and Erythrobacter lutimaris KCTC 42109T was 82.2%. Strain JLT1363T lacked bacteriochlorophyll a, and the major polar lipids were sphingoglycolipids, diphosphatidylglycerol, phosphatidylcholine, and phosphatidylglycerol. Phylogenetic and phenotypic properties indicated that strain JLT1363T represents a novel species of the genus Erythrobacter, for which the name Erythrobacter nanhaiensis sp. nov. is proposed. The type strain is JLT1363T (=CGMCC 1.7293T = LMG 24872T).

Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae.

Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia) revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS) region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported.

Pelagibacterium nitratireducens sp.nov., A Marine Alphaproteobacterium Isolated from the East China Sea

A Gram-negative, rod-shaped, non-spore-forming aerobic bacterium, motile with a single polar flagellum, strain JLT2005(T), was isolated from surface seawater collected from the East China Sea and formed ivory white colonies on a rich organic medium. The strain was positive for catalase, oxidase, and urease. It grew in the presence of 0-12 % (w/v) NaCl (optimum 5 %), at 20-35 °C (optimum 25 °C), or at pH 6-10 (optimum pH 9). The major fatty acids (>10 %) were C(18:1)ω7c, C(19:0)ω8c cyclo, C(16:0), and C(18:0). The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and five unidentified glycolipids. Ubiquinone-10 and Ubiquinone-11 were present as the major quinones. The DNA G+C content was 74.3 mol%. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain JLT2005(T) belongs to the genus Pelagibacterium in the family Hyphomicrobiaceae, class Alphaproteobacteria. The closest neighbors were Pelagibacterium halotolerans B2(T) (98.7 % similarity) and Pelagibacterium luteolum 1_C16_27(T) (97.1 % similarity). DNA-DNA relatedness values of strain JLT2005(T) with P. halotolerans B2(T) and with P. luteolum 1_C16_27(T) were 31.6 and 25 %. Evidence from genotypic, chemotaxonomic, and phenotypic data shows that strain JLT2005(T) represents a novel species of the genus Pelagibacterium, for which the name Pelagibacterium nitratireducens sp. nov is proposed. The type strain is JLT2005(T) (=CGMCC 1.10829(T) =JCM 17767(T)).

The use of the fungus Dichomitus squalens for degradation in rotating biological contactor conditions

Biodegradation potential of Dichomitus squalens in biofilm cultures and rotating biological contactor (RBC) was investigated. The fungus formed thick biofilms on inert and lignocellulosic supports and exhibited stable activities of laccase and manganese peroxidase to reach 40–62 and 25–32% decolorization of anthraquinone Remazol Brilliant Blue R and heterocyclic phthalocyanine dyes, respectively. The decolorization ceased when glucose concentration dropped to 1mmoll−1. In RBC reactor, respective decolorizations of Remazol Brilliant Blue R and heterocyclic Methylene Blue and Azure B dyes (50mgl−1) attained 99%, 93%, and 59% within 7, 40 and 200h. The fungus exhibited tolerance to coliform and non-coliform bacteria on rich organic media, the inhibition occurred only on media containing tryptone and NaCl. The degradation efficiency in RBC reactor, capability to decolorize a wide range of dye structures and tolerance to bacterial stress make D. squalens an organism applicable to remediation of textile wastewaters.

Melanin synthesis by Sclerotinia sclerotiorum

We confirmed that the melanin produced by Sclerotinia sclerotiorum is a dihydroxynaphthalene (DHN). The specific DHN melanogenesis inhibitor test that uses tricyclazole at low levels (typically2–5 ppm) to cause a confirmatory appearance of soluble red-brown inhibition products does not work when analyzing melanin synthesis in the sclerotia of S. sclerotiorum. We demonstrated the presence of scytalone dehydratase, an enzyme specific to DHN melanogenesis, in melanized sclerotia and melanized nonsclerotial mycelia and observed formation of mycelial nonsclerotial melanin when the fungus was grown on the surface of sterilized dialysis membrane or in rich organic media. Nonsclerotial melanized hyphae in wild type and mutant strains showed the typical excretion of pigmented inhibition products of the DHN pathway in the presence of tricyclazole, and one of these products, 2-hydroxyjuglone, was identified by thin layer chromatography and spectroscopy. We report basic conditions for sclerotial melanin degradation by the white rot fungus Phanerochaete chrysosporium.

My sixty years in applied algology

I am greatly honored to present this Keynote address. My focus, even after sixty years, continues to be on the future of phycology, even though I now present mainly from the past. I am delighted to see biological and physical scientists and engineers coming together in the field of Applied Phycology. Our working together will result in many benefits for mankind. There is no doubt that Applied Phycology has a great future because it has the potential for more efficient use of solar energy than conventional agriculture, and because it is poised to reach still unimagined goals through both genetic and ecological engineering. As an engineer I have focused mainly on large algal mass culture systems and the efficient use of solar energy in wastewater treatment (Oswald, 1962, 1963). But now I can envision such future triumphs as the introduction of genes for sulfur amino acids into the presently deficient Spirulina genome. I am fascinated by Dr Bailey Green’s crusade to minimize energy use and greenhouse gas emissions using algae-based sewage treatment (Green, 1998), by Dr Joseph Weissman’s commercial production of shellfish fed mass cultured algae, and by Dr John Benemann’s vision of achieving very high productivities through physiological and genetic manipulations of the photosynthetic apparatus (Benemann, 1990), to mention just three examples currently underway by former students and colleagues. All of these and many more advances have resulted from combining biological and engineering knowledge. As a young civil engineer I was greatly surprised when I learned that the growth of one unit dry weight of algae is accompanied by release of over one and one half times as much dissolved molecular oxygen, a process powered by virtually free solar energy. How to use this low cost dissolved O2 in wastewater treatment and life support systems has consumed much of my, and many of my students’, ingenuity during the past half century. Over these years my main area of research has been the design and operation of largescale cultures of microalgae that grow commensally with bacteria in rich organic media such as domestic sewage or some industrial wastes. Not only is solar energy and the resulting ‘photosynthetic oxygenation’ nearly free, but also the nutrients in the wastewaters are free and often ideally suited for algal mass cultures. In these waste treatment ponds we only exert minimal control over the algal species that grow, but we can impose some limits through pond operations such as residence time, depth and mixing. For reasons best known to the algae themselves, we often find species of Chlorella, Scenedesmus, and Micractinium, although species of Euglena, Chlamydomonas and Oscillatoria may occur in ponds with excessive loadings or long residence times. In these wastewater-fertilized systems the role of algae is primarily for production of O2 to support bacterial growth, although nutrient uptake, adsorption of heavy metals and, indirectly, disinfection are also important functions of the algae in these systems. In the following I highlight some of the benefits of growing microalgae in wastewaters (Oswald 1978).

Effect of cell lipid composition on the formation of nonspecific antibiotic resistance in alkanotrophic rhodococci

The antibiotic resistance and lipid composition of rhodococci grown in rich organic media with gaseous or liquidn-alkanes were studied. Hydrocarbon-grown rhodococci exhibited an increased resistance to a wide range of antibiotics (aminoglycosides, linkosamides, macrolides, β-lactams, and aromatic compounds). The enhanced antibiotic resistance of rhodococci grown onn-alkanes correlated with an increased content of total cell lipids (up to 14–28%) and saturated straight-chain fatty acids (C16:0, C18:0, C21:0) and was accompanied by the appearance of cardiolipin and phosphatidylglycerol in cells. These lipid compounds are supposed to promote the formation of nonspecific antibiotic resistance in rhodococci by decreasing the permeability of their cell envelope to antibiotics.

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.

Bacteriophage of Haemophilus influenzae: I. Differences between infection by whole phage, extracted phage DNA and prophage DNA extracted from lysogenic cells

A recombination-deficient strain of Haemophilus influenzae is infected with DNA from phage HP 1 c1 much less efficiently than is the wild type, although DNA uptake and whole-phage infection are equally efficient in the two strains. When the cells have been made competent in rich organic medium, the efficiency of transfection of wild-type cells by phage DNA, but not by DNA from lysogenic cells, increases with increasing concentration of DNA. However, in the rec − strain, or in wild-type cells made competent in a defined medium, the transfection efficiency is independent of concentration. Dimeric forms of phage DNA separated by sedimentation velocity transfect the wild-type cell made competent in the organic medium more efficiently than monomer DNA, but they do not transfect the rec − strain or wild-type cells made competent in a defined medium. It is concluded that recombination is required for most wild-type transfections in the rich medium, either dimer molecules recombining with themselves or monomer molecules combining with each other, to convert damaged molecules to single intact molecules that have two cohesive ends and are capable of replication. Following whole phage infection, lysogenization readily occurs; but we have detected no lysogenization after transfection with either phage DNA or DNA from lysogenic cells.

The Survival of Starter Organisms in Concentrated Suspensions

Summary: Two representative lactic acid bacteria were grown in a rich organic medium, the cells were harvested by centrifugation, and the organisms were suspended in skimmilk at concentrations of 25–55 × 109/ml. The pH was adjusted to different values and the suspensions were stored, with and without the addition of 20% (v/v) glycerol, at 4° and ‐ 20°.Both organisms survived better at pH 7°0 than at pH 5°0. Glycerol protected the cells at the lower pH value but offered no benefit at neutrality. At 4° about half the cells died within 5 or 6 weeks. At ‐ 20°, however, there was no appreciable loss of viability or acid producing ability up to 9 months at pH 7°0. Suspensions stored for 9 months at ‐ 20° produced excellent cultured buttermilk within the normal incubation period from an inoculum comparable to that used for a fresh culture. Cells harvested early in the maximum stationary phase of the growth cycle were more active than older cells, and they survived better than older cells during storage in the frozen state.

Rules for the classification of antibiotic-producing actinomycetes.

One of the most important tasks in our present day science of antibiotics is the establishment of a completely rational taxonomy and nomenclature for antibiotic-producing organisms. A purposeful search for antibiotic substances is possible only with good knowledge of the species which produce them. A great many antibiotic-producing actinomycetes have been described recently. Investigators often give new species names without a strict taxonomic analysis, ignoring the existing rules of nomenclature and classification, and without adequate analysis and appropriate comparison of a culture with previously described ones. One important deficiency of many publications in microbial taxonomy is the extremely poor description of cultures. Many authors often confine themselves to two or three insignificant phrases or do not give any information on the antibiotic-producing abilities of species described. Comparison of described species is complicated by the fact that published investigations characterize species under unlike conditions. Some investigators use only rich organic media, others use simple defined media with an inorganic source of nitrogen, etc. Great confusion is created as a result of such neglect of classification rules; it becomes more and more difficult to evaluate published material and often it is even impossible to make use of it. Therefore frequently different species of organisms are described under the same name, or the same species designation is given to cultures belonging to different species. We have encountered such inhomogeneity and species mixtures in the study of strains received from different laboratories in America, England, Japan, and other countries under the name of Actinomyces griseus, Actinomyces lavendulae, Actinomyces fradiae, Actinomyces erythreus, Actinomyces olivaceus, and Actinomyces globis-

PERIODICITY OF MITOSIS AND CELL DIVISION IN THE EUGLENINEAE

1. The periodicity of mitosis and cell division has been investigated in 15 green and 5 colorless species of the Euglenineae.2. Green species in biphasic culture under natural light conditions have mitosis confined to the dark period. Mitosis begins one to two hours after the onset of darkness, each species having a predictable percentage of cells dividing each night. There is a threshold period at the beginning of the dark period after which mitosis cannot be inhibited by light. The mitotic rhythm is exogenous, being removed by growth in artificial light or darkness (resulting in no mitosis), or in a rich organic medium (resulting in continuous mitosis at a constant rate).3. Colorless species in biphasic culture under any light conditions have an irregular mitotic periodicity, bursts of mitosis occurring at any time of the clock and alternating with periods in which mitosis is almost absent.4. It is suggested that the presence or absence of regular or irregular mitotic periodicity is related to the different modes and rates of nutrition of green and colorless species in various conditions of light and darkness, and in various media.

The Isolation and Preliminary Study of Some Physiological Characteristics of the Predominating Flora from the Rumen of Cattle and Sheep

Summary 1.New techniques for studying the microbiology of the rumen are described. These include a direct slide count technique, a cultural procedure for isolating the predominating flora, a method for obtaining a sample under anaerobic conditions, and methods for the determination of urea utilization and cellulose digestion by the predominating bacteria. 2.The direct slide count technique made use of nigrosine, a negative stain, which reduced to a minimum inaccuracies caused by artifacts. 3.When good cultural conditions prevailed and a fistulated animal was used, slide and cultural counts agreed closely. Material obtained by stomach tube gave consistently lower bacterial counts than samples taken directly from a rumen fistula. To isolate the predominating flora, the most important feature of the cultural technique was the maintenance of anaerobic conditions from the time of sampling until growth was obtained. A rich organic medium with a pH of 6.0 was used and cultures were incubated at 38° C. 4.It was shown by these methods that bacteria are present in the rumen contents of both sheep and cattle in numbers of about 100 billion per gram of fresh material. 5.Bacteria isolated from the highest dilutions of rumen contents from both sheep and cattle were tested for their ability to utilize urea and break down cellulose, and many of these cultures proved capable of performing these functions in vitro. 6.A few of the bacteria isolated from the highest dilutions of rumen contents were studied and were found to be mostly Gram-positive, non-motile anaerobic rods. A small number of cocci were cultured.